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H2S-mediated protein sulfhydration in stress reveals metabolic reprogramming

Posted in Biochemical pathways, Curation, Disease Biology, Metabolism, Sepsis, Signaling, Stress Disorders, Systemic Inflammatory Response Related Disorders, tagged -SH group, adaptive compensatory glycolysis, cancer cells, Cysteine, endoplasmic reticulum stress (ER stress), H2S, metabolic reprogramming, metabolic switch, pancreatic beta cell, stress response, sulfhydration, TCA cycle, Warburg Effect on November 26, 2015| Leave a Comment »

H2S-mediated protein sulfhydration in stress reveals metabolic reprogramming

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Quantitative H₂S-mediated protein sulfhydration reveals metabolic reprogramming during the Integrated Stress Response

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>Xing-HuangGao,

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>DawidKrokowski,

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>Bo-JhihGuan,

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>IlyaBederman,

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>MithuMajumder, et al.
eLife 2015;10.7554/eLife.10067

http://elifesciences.org/content/early/2015/11/23/eLife.10067 http://dx.doi.org/10.7554/eLife.10067

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.

Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.

The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).

In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.

Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.

The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.

Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.

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brown adipocyte protein CIDEA promotes lipid droplet fusion

Posted in Biochemical pathways, Embryology, Enzyme Induction, Lipidomics, Lipids, Metabolism, Proteins, Proteomics, Signaling, tagged Biophysics and structural biology, brown fat, Cell Biology, CIDEA, Human, lipid biosynthesis, Lipid Deposit (LD) membrane, lipolyis, Mouse, post translational modifications, regulation of metabolism, Research article, S. cerevisiae, TAG flux direction, triacylglycerides on November 26, 2015| Leave a Comment »

brown adipocyte protein CIDEA promotes lipid droplet fusion

Larry H. Bernstein, MD, FCAP, Curator

LPBI

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding

Parker, Nicholas T Ktistakis, Ann M Dixon, Judith Klein-Seetharaman, Susan Henry, Mark Christian Dirk Dormann, Gil-Soo Han, Stephen A Jesch, George M Carman, Valerian Kagan, et al.

eLife 2015;10.7554/eLife.07485 http://dx.doi.org/10.7554/eLife.07485

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

Evolutionary pressures for survival in fluctuating environments that expose organisms to times of both feast and famine have selected for the ability to efficiently store and release energy in the form of triacyclglycerol (TAG). However, excessive or defective lipid storage is a key feature of common diseases such as diabetes, atherosclerosis and the metabolic syndrome (1). The organelles that are essential for storing and mobilizing intracellular fat are lipid droplets (LDs) (2). They constitute a unique cellular structure where a core of neutral lipids is stabilized in the hydrophilic cytosol by a phospholipid monolayer embedding LD-proteins. While most mammalian 46 cells present small LDs (<1 Pm) (3), white (unilocular) adipocytes contain a single giant LD occupying most of their cell volume. In contrast, brown (multilocular) adipocytes hold multiple LDs of lesser size, increasing the LD surface/volume ratio which facilitates the rapid consumption of lipids for adaptive thermogenesis (4).

The exploration of new approaches for the treatment of metabolic disorders has been stimulated by the rediscovery of active brown adipose tissue (BAT) in adult humans (5, 6) and by the induction of multilocular brown-like cells in white adipose tissue (WAT) (7). The multilocular morphology of brown adipocytes is a defining characteristic of these cells along with expression of genes such as Ucp1. The acquisition of a unilocular or multilocular phenotype is likely to be controlled by the regulation of LD growth. Two related proteins, CIDEA and CIDEC promote LD enlargement in adipocytes (8-10), with CIDEA being specifically found in BAT. Together with CIDEB, they form the CIDE (cell death-inducing DFF45-like effector) family of LD-proteins, which have emerged as important metabolic regulators (11).

Different mechanisms have been proposed for LD enlargement, including in situ neutral lipid synthesis, lipid uptake and LD-LD coalescence (12-14). The study of CIDE 62 proteins has revealed a critical role in the LD fusion process in which a donor LD progressively transfers its content to an acceptor LD until it is completely absorbed (15). However, the underlying mechanism by which CIDEC and CIDEA facilitate the interchange of triacylglycerol (TAG) molecules between LDs is not understood. In the present study, we have obtained a detailed picture of the different steps driving this LD enlargement process, which involves the stabilization of LD pairs, phospholipid binding, and the permeabilization of the LD monolayer to allow the transference of lipids.

CIDEA expression mimics the LD dynamics observed during the differentiation of brown adipocytes

Phases of CIDEA activity: LD targeting, LD-LD docking and LD growth

A cationic amphipathic helix in C-term drives LD targeting

The amphipathic helix is essential for LD enlargement

LD-LD docking is induced by the formation of CIDEA complexes

CIDEC differs from CIDEA in its dependence on the N-term domain

CIDEA interacts with Phosphatidic Acid

PA is required for LD enlargement

The Cidea gene is highly expressed in BAT, induced in WAT following cold exposure (46), and is widely used by researchers as a defining marker to discriminate brown or brite adipocytes from white adipocytes (7, 28). As evidence indicated a key role in the LD biology (47) we have characterized the mechanism by which CIDEA promotes LD enlargement, which involves the targeting of LDs, the docking of LD pairs and the transference of lipids between them. The lipid transfer step requires the interaction of CIDEA and PA through a cationic amphipathic helix. Independently of PA-binding, this helix is also responsible for anchoring CIDEA in the LD membrane. Finally, we demonstrate that the docking of LD pairs is driven by the formation of CIDEA complexes involving the N-term domain and a C-term interaction site.

CIDE proteins appeared during vertebrate evolution by the combination of an ancestor N-term domain and a LD-binding C-term domain (35). In spite of this, the full process of LD enlargement can be induced in yeast by the sole exogenous expression of 395 CIDEA, indicating that in contrast to SNARE-triggered vesicle fusion, LD fusion by lipid transference does not require the coordination of multiple specific proteins (48). Whereas vesicle fusion implicates an intricate restructuring of the phospholipid bilayers, LD fusion is a spontaneous process that the cell has to prevent by tightly controlling their phospholipid composition (23). However, although phospholipid-modifying enzymes have been linked with the biogenesis of LDs (49, 50), the implication of phospholipids in physiologic LD fusion processes has not been previously described.

Complete LD fusion by lipid transfer can last several hours, during which the participating LDs remain in contact. Our results indicate that both the N-term domain and a C-term dimerization site (aa 126-155) independently participate in the docking of LD pairs by forming trans interactions (Fig. 7). Certain mutations in the dimerization sites that do not eliminate the interaction result in a decrease on the TAG transference efficiency, reflected on the presence of small LDs docked to enlarged LDs. This suggests that in addition to stabilizing the LD-LD interaction, the correct conformation of the 409 CIDEA complexes is necessary for optimal TAG transfer. Furthermore, the formation of stable LD pairs is not sufficient to trigger LD fusion by lipid transfer. In fact, although LDs can be tightly packed in cultured adipocytes, no TAG transference across neighbour LDs is observed in the absence of CIDE proteins (15), showing that the phospholipid monolayer acts as a barrier impermeable to TAG. Our CG-MD simulations indicate that certain TAG molecules can escape the neutral lipid core of the LD and be integrated within the aliphatic chains of the phospholipid monolayer. This could be a transition state 416 prior to the TAG transference and our data indicates that the docking of the amphipathic helix in the LD membrane could facilitate this process. However, the infiltrated TAGs in LD membranes in the presence of mutant helices, or even in the absence of docking, suggests that this is not enough to complete the TAG transference.

To be transferred to the adjacent LD, the TAGs integrated in the hydrophobic region of the LD membrane should cross the energy barrier defined by the phospholipid polar heads, and the interaction of CIDEA with PA could play a role in this process, as suggested by the disruption of LD enlargement by the mutations preventing PA-binding (K167E/R171E/R175E) and the inhibition of CIDEA after PA depletion. The minor effects observed with more conservative substitutions in the helix, suggests that the presence of positive charges is sufficient to induce TAG transference by attracting anionic phospholipids present in the LD membrane. PA, which requirement is indicated by our PA-depletion experiments, is a cone-shaped anionic phospholipid which could locally destabilize the LD monolayer by favoring a negative membrane curvature incompatible with the spherical LD morphology (51). Interestingly, while the zwitterion PC, the main component of the monolayer, stabilizes the LD structure (23), the negatively charged PA promote their coalescence (29). This is supported by our CD-MD results which resulted in a deformation of the LD shape by the addition of PA. We propose a model in which the C-term amphipathic helix positions itself in the LD monolayer and interacts with PA molecules in its vicinity, which might include trans interactions with PA in the adjacent LD. The interaction with PA disturbs the integrity of the phospholipid barrier at the LD-LD interface, allowing the LD to LD transference of TAG molecules integrated in the LD membrane (Fig. 7). Additional alterations in the LD composition could be facilitating TAG transference, as differentiating adipocytes experience a reduction in saturated fatty acids in the LD phospholipids (52), and in their PC/PE ratio (53) which could increase the permeability of the LD membranes, and we previously observed that a change in the molecular structures of TAG results in an altered migration pattern to the LD surface (32).

During LD fusion by lipid transfer, the pressure gradient experienced by LDs favors TAG flux from small to large LDs (15). However, the implication of PA, a minor component of the LD membrane, could represent a control mechanism, as it is plausible that the cell could actively influence the TAG flux direction by differently regulating the levels of PA in large and small LDs, which could be controlled by the activity of enzymes such as AGPAT3 and LIPIN-1J (13, 30). This is a remarkable possibility, as a switch in the favored TAG flux direction could promote the acquisition of a multilocular phenotype and facilitate the browning of WAT (24). Interestingly, Cidea mRNA is the LD protein- encoding transcript that experiences the greatest increase during the cold-induced process by which multilocular BAT-like cells appear in WAT (24). Furthermore, in BAT, cold exposure instigates a profound increase in CIDEA protein levels that is independent of transcriptional regulation (54). The profound increase in CIDEA is coincident with elevated lipolysis and de novo lipogenesis that occurs in both brown and white adipose tissues after E-adrenergic receptor activation (55). It is likely that CIDEA has a central role in coupling these processes to package newly synthesized TAG in LDs for subsequent lipolysis and fatty acid oxidation. Importantly, BAT displays high levels of glycerol kinase activity (56, 57) that facilitates glycerol recycling rather than release into the blood stream, following induction of lipolysis (58), which occurs in WAT. Hence, the reported elevated glycerol released from cells depleted of CIDEA (28) is likely to be a result of decoupling lipolysis from the ability to efficiently store the products of lipogenesis in LDs and therefore producing a net increase in detected extracellular glycerol. This important role of CIDEA is supported by the marked depletion of TAG in the BAT of Cidea null mice following overnight exposure to 4 °C (28) and our findings that CIDEA-dependent LD enlargement is maintained in a lipase negative yeast strain.

Cidea and the genes that are required to facilitate high rates of lipolysis and lipogenesis are associated with the “browning” of white fat either following cold exposure (46) or in genetic models such as RIP140 knockout WAT (59). The induction of a brown- like phenotype in WAT has potential benefits in the treatment and prevention of metabolic disorders (60). Differences in the activity and regulation of CIDEC and CIDEA could also be responsible for the adoption of unilocular or multilocular phenotypes. In addition to their differential interaction with PLIN1 and 5, we have observed that CIDEC is more resilient to the deletion of the N-term than CIDEA, indicating that it may be less sensitive to regulatory posttranslational modifications of this domain. This robustness of CIDEC activity together with its potentiation by PLIN1, could facilitate the continuity of the LD enlargement in white adipocytes until the unilocular phenotype is achieved. In contrast, in brown adipocytes expressing CIDEA the process would be stopped at the multilocular stage for example due to post-translational modifications that modulate the function or stability of the protein or alteration of the PA levels in LDs.

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matrix metalloproteinases role in vision

Posted in Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Curation, Neuroscience, Vision, tagged Mouse, Neuroscience, Research article on November 26, 2015| Leave a Comment »

matrix metalloproteinases role in vision

Larry H. Bernstein, MD, FCAP, LPBI

Optimal level activity of matrix metalloproteinases is critical for adult visual plasticity in the healthy and stroke-affected brain

” data-author-inst=”UniversityofGttingenGermany”>JustynaPielecka-Fortuna,

” data-author-inst=”UniversityofGttingenGermany”>EvgeniaKalogeraki,

” data-author-inst=”UniversityMedicalCenterGermany”>Michal G Fortuna,

” data-author-inst=”UniversityofGttingenGermany”>SiegridLöwe

eLife 2015;10.7554/eLife.11290 http://elifesciences.org/content/early/2015/11/26/eLife.11290 http://dx.doi.org/10.7554/eLife.11290

The ability of the adult brain to undergo plastic changes is of particular interest in medicine, especially regarding recovery from injuries or improving learning and cognition. Matrix metalloproteinases (MMPs) have been associated with juvenile experience-dependent primary visual cortex (V1) plasticity, yet little is known about their role in this process in the adult V1. Activation of MMPs is a crucial step facilitating structural changes in a healthy brain; however, upon brain injury, upregulated MMPs promote the spread of a lesion and impair recovery. To clarify these seemingly opposing outcomes of MMPs-activation, we examined the effects of MMPs-inhibition on experience-induced plasticity in healthy and stoke-affected adult mice. In healthy animals, 7-day application of MMPs-inhibitor prevented visual plasticity. Additionally, treatment with MMPs-inhibitor once but not twice following stroke rescued plasticity, normally lost under these conditions. Our data imply that a fine balance of MMPs-activity is crucial for adult visual plasticity to occur.

Neuroplasticity is the ability of the brain to adapt both structurally and functionally to changing patterns of activity induced by the environment or intrinsic factors. In the clinical setting, plasticity is important for tissue repair and neural network rewiring, necessary for recovery and optimal post-injury brain function. The primary visual cortex (V1) is a widely used model region for studying sensory plasticity in young vs. adult brains (Hofer et al., 2006; Espinosa and Stryker, 2012; Levelt and Hübener, 2012). In mammals, V1 consists of a monocular and a binocular zone; neurons in the binocular zone respond to stimulation of both eyes but are dominated by the contralateral eye in rodents (Dräger, 1975, 1978). This ocular dominance (OD) can be modified in an experience-dependent manner, by depriving one eye of pattern vision for several days (known as monocular deprivation or MD), as originally observed by Wiesel and Hubel more than 50 years ago (Wiesel and Hubel, 1965). OD-plasticity has become one of the major paradigms for studying cortical plasticity. In standard-cage raised mice, OD-plasticity in binocular V1 is most pronounced in 4- 30 week-old animals; reduced, yet present, in early adulthood; and absent in animals 31 older than 110 days of age (Lehmann and Löwel, 2008). In 4-week-old mice, 3-4 days of MD are sufficient to induce a significant OD-shift towards the open eye (juvenile OD-plasticity), while 7 days of MD are needed in 3-month-old animals (adult 34 OD-plasticity) (Gordon and Stryker, 1996; Sawtell et al., 2003; Frenkel and Bear, 2004; Mrsic-Flogel et al., 2007; Sato and Stryker, 2008). Although the experimental paradigm is rather similar, molecular mechanisms underlying “juvenile” and “adult” OD-plasticity are different: in juvenile mice, OD-shifts are mostly mediated by 38 reductions in deprived eye responses while adult plasticity is predominantly mediated by an increase in open eye responses in V1 (Hofer et al., 2006; Espinosa and Stryker, 2012; Levelt and Hübener, 2012).

Activity driven modifications in neuronal circuits can be facilitated by degradation of the extracellular matrix (ECM) (Pizzorusso et al., 2002; de Vivo et al., 2013), which 4 provides structural and biochemical support for the cells (Frischknecht and Gundelfinger, 2012). Structural and molecular composition of the ECM changes during development, becoming denser and more rigid in the mature brain (Frischknecht and Gundelfinger, 2012; de Vivo et al., 2013). This structural composition is partially regulated by a family of zinc dependent endopeptidases, the matrix metalloproteinases (MMPs), and their enzymatic activity is crucial for proper development, function and maintenance of neuronal networks (Milward et al., 2007; Huntley, 2012). A recent study in juvenile rats showed that pharmacological inhibition of MMPs with a broad spectrum inhibitor during the MD-period did not influence the reduction of the closed-eye responses induced by 3 days of MD, yet it prevented the potentiation of the nondeprived eye responses in V1 after 7 days of MD (Spolidoro et al., 2012). Whether MMPs are involved in adult OD-plasticity, for which mechanisms are believed to be different (Hofer et al., 2006; Sato and Stryker, 2008; Ranson et al., 2012), is not yet known, and clarifying this point was the first goal of this study.

In addition to MMPs involvement in healthy brain function, their excessive activity can also be detrimental (Agrawal et al., 2008; Huntley, 2012). Both human and animal studies found upregulated activity of MMPs following inflammation, infectious diseases or brain trauma (Agrawal et al., 2008; Rosell and Lo, 2008; Morancho et al., 2010; Vandenbroucke and Libert, 2014), and pharmacological inhibition of MMPs shortly after brain injuries reduced infarct sizes and prompted better recovery (Romanic et al., 1998; Lo et al., 2002; Gu et al., 2005; Wang and Tsirka, 2005; Yong, 2005; Morancho et al., 2010; Chang et al., 2014; Vandenbroucke and Libert, 2014). Stroke can influence synaptic activities within the area directly affected by it, and also in a broader area surrounding the lesion (Witte et al., 2000; Murphy and Corbett, 2009). Likewise, impairments in experience-dependent plasticity after a cortical stroke also have been observed in distant brain regions (Jablonka et al., 2007; Greifzu et al., 2011): in 3-month-old mice, a focal stroke in the primary somatosensory cortex (S1) prevented both V1-plasticity and improvements of the 5 spatial frequency and contrast thresholds of the optomotor reflex of the open eye (Greifzu et al., 2011). Interestingly, some MMPs were shown to be upregulated within the first 24 hours after focal stroke (Cybulska-Klosowicz et al., 2011; Liguz-Lecznar et al., 2012). Thus, the second goal of our study was to test whether balancing the upregulated MMPs resulting from a focal stroke in S1 would rescue visual plasticity.

Using a combination of in vivo optical imaging of intrinsic signals and behavioral vision tests in adult mice, we observed that application of the broad spectrum MMPs-inhibitor GM6001 during the 7-day MD-period prevented both OD-plasticity and enhancements of the optomotor response of the open eye. In addition, a single treatment after the S1-stroke rescued plasticity in both paradigms, whereas treatment with the inhibitor two times diminished plasticity-promoting effect. Together, these data reveal a crucial role of MMPs in adult visual plasticity and suggest that MMPs-activity has to be within a narrow window for experience-induced plasticity to occur.

Inhibition of MMPs prevents experience-induced adult visual cortex plasticity

Inhibition of MMPs prevented experience-enabled improvements in visual capabilities

Brief inhibition of MMPs rescued experience-induced visual cortex plasticity after stroke

Inhibition of MMPs after induction of a cortical lesion rescued experience-induced improvements in visual capabilities in adult mice

The objective of this study was to examine if MMPs are crucial for adult visual plasticity, and if inhibition of their upregulation following cortical stroke may be beneficial for rescuing lost plasticity. A combination of in vivo optical imaging and behavioral vision tests revealed that balanced MMPs activity is essential for adult visual plasticity to occur in the healthy and stroke-affected brain. In healthy adult mice, MMPs-inhibition with GM6001 prevented both ocular dominance plasticity and experience-driven improvements of the optomotor reflex of the open eye after MD, indicating that MMPs activation is required for normal adult plasticity. In addition, blockade of elevated MMPs-activity after a cortical stroke rescued compromised plasticity. Specifically, a single but not two-times treatment with GM6001 after a cortical PT-lesion in the neighboring S1 region fully rescued experience-dependent ocular dominance plasticity in adult V1, which is normally lost under these conditions.

These observations suggest that MMPs-activity has to be within a narrow window to allow visual plasticity: if MMPs-activity is downregulated (with inhibitors) or upregulated (after stroke) experience-induced plastic changes are compromised. In a healthy brain, the enzymatic activity of MMPs has plasticity promoting effects (Milward et al., 2007; Huntley, 2012). Consequently to increased neuronal activity, activated MMPs can lessen physical barriers (loosen up the ECM) or via the degradation of certain receptors influence signaling pathways. Such changes within neuronal cells and their synapses thereby alter the structure and activity of neuronal networks (Milward et al., 2007; Huntley, 2012). Involvement of MMPs in synaptic circuit remodeling has been mainly studied in the hippocampus, yet their abundant expression in the central nervous system (CNS) indicates a much broader function. For instance, MMP9-deficient mice showed modestly reduced experience-dependent plasticity in the barrel cortex (Kaliszewska et al., 2012) and MMP3-deficient mice displayed abnormal neuronal morphology in the visual cortex and impaired plasticity induced by long-term monocular enucleation (Aerts et al., 2014). Here, we used a different approach to probe the role of MMPs in experience-induced changes in the visual system: we performed the experiments in wild type mice and treated them with the broad-spectrum inhibitor GM6001 or vehicle during 7 days of MD. Consistent with previous findings (Gordon and Stryker, 1996; Sawtell et al., 2003; Sato and Stryker, 2008), the OD-shift of vehicle-treated mice was mediated by open-eye potentiation. In contrast, there was no change in the open nor in the closed eye responses and hence no OD-plasticity in V1 after MD in GM6001-treated adult mice. This is in line with recent observations from juvenile rats, in which chronic treatment with GM6001 also prevented open eye potentiation after 7 days of MD (Spolidoro et al., 2012). However, treatment in this study only partially prevented the OD-shift, as there was no effect on the reduction of deprived eye responses (Spolidoro et al., 2012). A reduction in deprived eye responses in V1 is mostly observed in juvenile rodents after 3-4 days of MD (Gordon and Stryker, 1996), unless different raising conditions are used such as enriched environment or running wheel (Greifzu et al., 2014; 323 Kalogeraki et al., 2014). In adult, standard-cage raised mice, 6-7 days of MD are necessary for significant OD-shifts and changes are mainly mediated by increases of open eye responses in V1 (Gordon and Stryker, 1996; Sawtell et al., 2003; Sato and 326 Stryker, 2008). MMP9 activity was suggested in the potentiation of the open eye responses in juvenile rats, as treatment with GM6001 significantly reduced MMP9-mRNA expression only in the hemisphere where structural changes took place (Spolidoro et al., 2012). Since we observed a full blockade of plasticity after GM6001- treatment, it would be of interest to determine whether MMP9 is crucial for open-eye potentiation also in the adult brain. MMP9 has been widely investigated in various plasticity paradigms (Milward et al., 2007; Frischknecht and Gundelfinger, 2012; Huntley, 2012; Tsilibary et al., 2014) and one of the molecules shown to stimulate MMP9 secretion and expression in vitro and in vivo is tumor necrosis factor alpha (TNFalpha) (Hanemaaijer et al., 1993; Candelario-Jalil et al., 2007). TNFalpha signaling was found to play an important role in the open eye potentiation in juvenile (Kaneko et al., 2008), but not in adult V1 plasticity (Ranson et al., 2012), and since our data reveal that MMPs are indispensable for adult V1 plasticity, MMPs-activation in the adult brain is most likely not dependent on TNF alpha signaling. Accordingly, this adds to the notion that juvenile and adult V1 plasticity depend on different molecular mechanisms (Hofer et al., 2006; Sato and Stryker, 2008; Ranson et al., 342 2012). Together, our new data demonstrate a vital role of MMPs for adult visual cortex plasticity, in particular for the increase of open eye responses in V1 after MD, and notably expand the previous studies from juvenile rats.

Under normal conditions, MMPs-activity supports healthy brain development and function; yet a different outcome of MMPs action has been described for diseased brain (Agrawal et al., 2008). Under pathophysiological conditions like inflammation, infection or stroke, uncontrolled MMPs driven proteolysis can lead to negative consequences (Rosenberg et al., 1996; Rosenberg, 2002; Agrawal et al., 2008). Excessive MMPs-activity after stroke caused blood brain barrier disruption, upregulation of inflammatory mediators, excitotoxicity and eventually cell death; (Romanic et al., 1998; Lo et al., 2002; Gu et al., 2005; Wang and Tsirka, 2005; Yong, 354 2005; Morancho et al., 2010; Chang et al., 2014; Vandenbroucke and Libert, 2014). Recent studies reported increased enzymatic MMP9 activity within 24 hours after a PT-stroke, and application of a broad spectrum MMPs-inhibitor (FN-439) applied at the time of stroke induction, partially rescued impaired barrel cortex plasticity (Cybulska-Klosowicz et al., 2011; Liguz-Lecznar et al., 2012). Here we tested whether inhibition of upregulated MMPs-activity (resulting from PT-stroke) may rescue cortical plasticity also when the treatment starts after lesion induction. Indeed, a single GM6001-treatment after PT in the neighboring S1 fully rescued plasticity in both of our experimental paradigms, OD- and interocular plasticity. Importantly, successful treatment did not have to start immediately after stroke induction (1h) because treatment 24h after stroke had the same beneficial effect, highlighting the therapeutic potential of brief MMPs-inhibition for stroke recovery. Beneficial treatment was, however, dependent on the number of injections: a single but not two-times injection of the MMPs-inhibitor rescued OD-plasticity. The reduced plasticity- promoting effect in V1 of mice treated twice with GM6001 is likely due to too excessive MMPs-downregulation, which in turn interfered with MMPs facilitation of MD-induced plasticity. Consistent with this interpretation, it was reported that a particular dosage, timing as well as duration of MMPs-inhibition mattered for reduciton of lesion sizes after intracerebral hemorrhage, blood brain barrier permability or neurovascular remodeling in post-stroke period (Wang and Tsirka, 2005; Zhao et al., 2006; Sood et al., 2008; Chang et al., 2014). Together with the results from healthy animals, our data suggest that the plasticity-promoting effects of MMPs are dependent on a well-balanced level of activation, and if that balance is disturbed, experience-induced plastic changes are compromised.

There are several plausible mechanisms by which MMPs inhibition rescues OD- plasticity after stroke in S1. Stroke influences brain function in numerous ways e.g., causing inflammation and apoptosis, or disrupting the tightly regulated balance of neuronal inhibition and excitation (Neumann-Haefelin et al., 1995; Schiene et al., 1996; Witte and Stoll, 1997; Que et al., 1999a; Que et al., 1999b; Witte et al., 2000) also in perilesional areas (Murphy and Corbett, 2009). One of the major consequences of ischemic damage is an elevated level of the neurotransmitter glutamate, leading to excitotoxicity and neuronal death (Lai et al., 2014). On the other hand, stroke can lead to increased tonic inhibition in the peri-infarct zone, and reducing this inhibition can promote functional recovery (Clarkson et al., 2010). In addition, focal ischemia can induce spreading depression within ipsilateral cortex (Schroeter et al., 1995) and a recent study showed that this phenomenon 391 upregulated MMPs, leading to a breakdown of the blood brain barrier, edema, and vascular leakage, which was suppressed by GM6001 treatment (Gursoy-Ozdemir et al., 2004). Thus, it is likely that treatment with GM6001 shortly after the stroke – as in the present study – downregulated MMPs, thus reduced spreading depression, improved disturbed excitation/inhibition balance and allowed plastic changes to 396 occur.

Although we observed clear functional rescue of OD-plasticity after GM6001- treatment, there was no apparent effect on the lesion size: the PT-lesions in GM6001-treated mice were not smaller compared to vehicle-injected mice. This is in line with a recent observation, that a different broad-spectrum MMPs-inhibitor (FN- 143) did not attenuate brain damage resulting from photothrombosis, but partially rescued barrel cortex plasticity (Cybulska-Klosowicz et al., 2011). The present results, together with the above mentioned study, are not easy to reconcile with findings where MMPs-inhibitors reduced the volume of a brain injury (Gu et al., 2005; Wang and Tsirka, 2005; Vandenbroucke and Libert, 2014). The difference might arise from the method used for stroke induction, dosage of inhibitors, timing and duration of the treatment and severity of the lesion.

Behavioral vision tests additionally revealed an involvement of MMPs for interocular 410 plasticity during MD. The optomotor reflex is known to be mediated by subcortical pathways (Giolli et al., 2005). While visual capabilities measured by optomotry mainly reflect the properties of the retinal ganglion cells that project to these subcortical structures (Douglas et al., 2005), daily testing in the optomotor setup after MD induces a cortex-dependent and experience-enabled enhancement of spatial vision through the open eye (Prusky et al., 2006). Although inflammation was shown to interfere with the experience-enabled optomotor changes (Greifzu et al., 2011), little is known about the cellular origins or signaling pathways responsible for this plasticity paradigm. Here, we found that daily application of the MMPs-inhibitor during MD prevented enhancements in both the spatial frequency and contrast sensitivity thresholds of the optomotor reflex of the open eye, while vehicle-treated control mice displayed the typical experience-enabled optomotor improvements. On the other hand, treatment of mice with the same inhibitor once or twice following cortical stroke rescued the impaired optomotor improvements. Thus, in contrast to the OD-plasticity results, rescue of optomotor improvements was present regardless of the duration of the treatment, adding to the idea that separate mechanisms and different neuronal circuits are responsible for OD- and interocular plasticity (Greifzu et al., 2011; Kang et al., 2013; Greifzu et al., 2014). Together our results establish a novel function of MMPs in experience-enabled enhancements of the optomotor reflex of the open eye after MD in adult mice.

In conclusion, our present data highlight a critical role of MMPs in adult visual plasticity. They further suggest that upregulation of MMPs-activity shortly after a cortical lesion compromises experience-induced visual plasticity, which in turn can be rescued by brief MMPs-inhibition. Precise regulation of MMPs-activity therefore seems to be essential for facilitating plasticity in the adult brain and offers new opportunities in treatment and recovery for stroke. It remains to be determined which particular MMPs account for the present results.

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Flu Virus Transmission

Posted in Viral diseases, tagged influenza A virus, influenza transmission on November 26, 2015| Leave a Comment »

Flu Virus Transmission

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Mystery behind flu virus transmission solved

lu virus, influenza virus, soft palate

The epidemiological success of flu viruses relies on successful airborne transmission from person to person. But the viral properties governing the airborne transmission of flu viruses are complex. A new study reveals that the soft palate at the back of the roof of the mouth plays a key role in the flu viruses’ ability to transmit through air. Previous research had shown that airborne transmissibility is dependent on the viral surface hemagluttinin (HA) glycoprotein’s ability to bind to receptors on human respiratory cells. Some viral strains bind better to alfa 2-6 glycan receptors found primarily in humans and other mammals while others are better suited to bind alfa 2-3 glycan receptors found in birds.

In the current study, researchers made 4 mutations in the HA protein of the flu virus which made it better suited to bind the bird receptors than the human receptors. They then used this strain to infect ferrets that are often used as models of human influenza infection. In theory the mutated virus should not have spread but it traveled through the air just as well as the wild type virus strain. Upon sequencing the virus genome, the scientists found that it had undergone a genetic reversion that allowed its HA protein to bind to the bird as well as human receptors. This experiment validated that gain of binding to the human receptor is critical for aerosol transmission. On examining the different parts of the respiratory tract, scientists discovered that viruses that genetically reverted were most abundantly found in the soft palate. The researchers are next trying to figure out how this genetic reversion takes place and why particularly in the soft palate. They hypothesize that the viruses outcompete each other in the soft palate from which they can spread by packaging themselves into mucus droplets produced by cells in the soft palate.

From a pandemic point of view, this study enables the systematic evaluation of highly transmissible viruses. The findings published in Nature will enable scientists better understand how the flu virus develops airborne transmissibility while helping monitor strains that acquire the potential to cause Influenza outbreaks.

The soft palate is an important site of adaptation for transmissible influenza viruses (Sep 2015)

Lakdawala SS¹, Jayaraman A², Halpin RA³, Lamirande EW¹, Shih AR¹, Stockwell TB³, Lin X³, Simenauer A³, Hanson CT, et al.
Nature. 2015 Oct 1; 526(7571):122-5. http://dx.doi.org:/10.1038/nature15379. Epub 2015 Sep 23. http://www.ncbi.nlm.nih.gov/pubmed/26416728

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

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Malaria Protein Anti-cancer Activity

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Curation, Embryology, Genomic Expression, Malaria, Proteins, Proteomics, tagged anti-cancer activity, malaria in pregnancy, malaria protein, vaccine on November 26, 2015| Leave a Comment »

Malaria Protein Anti-cancer Activity

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Malaria vaccine may cure cancer

http://www.genscript.com/protein_news.htm

Researchers trying to develop an anti-malaria vaccine in pregnant women have stumbled upon an unexpected discovery that could potentially transform cancer therapy. They revealed that the carbohydrate that the malaria parasite attaches itself to – an oncofetal chondroitin sulfate – which is expressed in the placenta is the same as the one displayed on proteoglycans in cancerous cells.

By conjugating diphtheria toxin to recombinant malaria protein VAR2CSA (rVAR2), the researchers were able to inhibit in vivo tumor growth and metastasis. The study demonstrates how a parasite-derived protein can be exploited to target a common but complex, cancer-associated modification. The researchers have already tested thousands of samples from brain tumors to leukemia and found that the malaria protein is able to inhibit more than 90% of all types of tumors. While this approach seems very promising a major drawback is that this treatment cannot be applied to pregnant women. Since the rVAR2-conjugated toxin cannot distinguish placenta from tumor, it would inhibit placenta growth as well. The research team wants to conduct human testing but the earliest possible scenario they predict would be in 4 years.

Malaria vaccine may cure cancer

Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein.

Salanti A¹, Clausen TM², Agerbæk MØ³, Al Nakouzi N⁴, Dahlbäck M⁵, Oo HZ⁴, Lee S⁶, Gustavsson T⁵, Rich JR⁷, Hedberg BJ⁷, Mao Y⁸, Barington L⁵, Pereira MA⁵, LoBello J⁹, et al. Author information

Cancer Cell. 2015 Oct 12;28(4):500-14. http://dx.doi.org:/ 10.1016/j.ccell.2015.09.003.

Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA.

Resende M¹, Nielsen MA, Dahlbäck M, Ditlev SB, Andersen P, Sander AF, Ndam NT, Theander TG, Salanti A. Author information

Malar J. 2008 Jun 6;7:104. doi: 10.1186/1475-2875-7-104.

BACKGROUND:

Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

METHODS:

To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

RESULTS:

The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

CONCLUSION:

Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.

AM (Pregnancy Associated Malaria) is a major health problem in malaria-endemic areas and on a world basis it affects millions of pregnant women and their offspring. The presence of parasites in the placenta of pregnant women can have serious consequences for both mother and child including: maternal anaemia, premature delivery, low birth weight and increased infant mortality [1]. In malaria endemic areas, children acquire clinical immunity after multiple infections, and adults are in general protected against malaria. Women who have acquired immunity against malaria during childhood become susceptible to malaria during pregnancy due to novel parasite phenotypes expressing unique antigens not encountered during childhood infections [2,3]. In areas of high parasite transmission PAM mainly affects primigravidae since immunity is acquired as a function of gravidity [1]. Protective antibodies target proteins expressed on the surface of infected erythrocytes (IE), which mediate binding to syncytiotrophoblasts. By this process, the parasite is not filtered through the spleen and thus avoids exposure to effector mechanisms, which clear erythrocytes infected with late blood stage parasite from circulation [4]. The best characterized surface protein is thePlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) [5,6], which is encoded by the polymorphic var gene family containing 50–60 copies per parasite genome [7]. The PfEMP1 family constitutes high-molecular proteins of 200–400 kDa, which are highly polymorphic. Different PfEMP1 molecules have different receptor specificities, therefore switching between expression of various var genes in a mutually exclusive manner allows the parasite to modify its adhesion properties (reviewed in [8]). PfEMP1 proteins include three to seven Duffy-binding-like (DBL) domains, which belong to a parasite adhesion-domain super-family present in erythrocyte invasion ligands called: erythrocyte-binding ligands (EBL). Antibodies against PfEMP1 can interfere with parasite binding and the successive acquisition of a broad range of PfEMP1 antibodies is important for the acquisition of immunity during childhood [9–13]. Several molecules such as ICAM-1 [14], VCAM-1 [15], thrombospondin [16], CD36 [17], and chondroitin sulfate A (CSA) [18,19] have been identified as host receptors for PfEMP1. In the placenta IE exclusively bind to the glycosaminoglycan CSA [19,20]. The parasite protein mediating IE adhesion to CSA in the placenta is a distinct member of the PfEMP1 protein family, named VAR2CSA [21]. High levels of anti-VAR2CSA antibodies are correlated with favourable birth outcome and they are acquired as a function of parity [22]. Disruption of the var2csa gene causes the loss of the IE’s ability to bind CSA [23]. VAR2CSA is a large IE surface-expressed antigen consisting of six DBL domains with a total estimated molecular mass of 350 kDa. The ultimate aim of PAM vaccine development is to define a VAR2CSA construct capable of eliciting antibodies that inhibit binding of IEs to CSA. However, several of the VAR2CSA domains have in vitro affinity to CSA [24–26] and this complicates vaccine design. It is thus of high priority to define the minimal epitopes within each domain and inter-domains that have affinity to CSA.

Phage display is a strong and widely used tool for mapping protein ligand interactions and has in several studies been used to define adhesive parts of proteins present on the surface of different organisms causing infectious diseases (reviewed in [27]). Phage display has also been extensively used in malaria research. For vaccine development Casey and others [28], used phage display to isolate a phage-derived peptide that mimic an important epitope of AMA-1 and had the ability to induce functionally protective antibodies. Lanzillotti and others [29], used a phage display library to search for P. falciparum encoded motifs involved in erythrocyte invasion, and identified regions in EBA-175 and Ebl-1 like proteins binding to receptors on the human erythrocyte. EBA and Ebl belong to the same super family of Duffy-binding-like proteins as the DBL domains from VAR2CSA. We were thus encouraged to use this technique to search for CSA-binding motifs in VAR2CSA. In this study a phage display library was constructed based on the exon1 coding region of VAR2CSA. The library was biopanned on different sources of glycosaminoglycans (GAG) including: immobilized bovine CSA; immobilized proteoglycans purified from placentas – CSPG; CSA-expressing CHO cells, and BeWo cells derived from the human placental syncytiotrophoblasts. Five regions of VAR2CSA potentially involved in in vivo parasite sequestration were identified and are thus potential candidate components of a multivalent PAM vaccine.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430714/bin/1475-2875-7-104-3.jpg

A DBL2 peptide binds to CSA and not to CSC. (A) Peptide binding assay to CSA: P2b (red) binds to CSA (■) in a peptide concentration-specific manner and not to the plate (▲). The control peptide (blue) does not bind to neither the plate (×) nor CSA (◆). (B) Peptide binding assay to CSC: P2b (red) does not bind to either CSC (■) or to the plate (▲). The control peptide (blue) does not bind to neither the plate (×) nor CSC (◆).

Multiple linear VAR2CSA regions have affinity for proteoglycans

The exon1 of var2csa has 9171 bp and was PCR amplified and cloned into the T7select 415-1b phage vector. The plasmid was propagated and the insert containing the whole var2csa exon1 was cut out and digested with DNase1 to generate 200 bp fragments. The var2csa fragments were used to create a T7 Phage display library as described in the material and methods section. This vector is described to display 415 copies of peptides on the surface of the T7 capsid [31].

To ensure that all parts of VAR2CSA exon1 were present in this constructed library a number of clones were sequenced before biopanning. No sequences were overrepresented and sequences belonging to all six DBL domains were present in the library (Figure (Figure1A,1A, black). The var2csa phage display library was biopanned four rounds on: CHO cells (two independent assays); human placental choriocarcinoma cell line BeWo (four independent assays); bovine CSA (two independent assays); biotinylated CSA (two independent assays) and chondroitin sulfate proteoglycans of human placenta (CSPG) (two independent assays). Control biopannings were done on ELISA plates coated with BSA (Figure (Figure1A,1A, Blocking Bf, blue). From each assay 15 clones were sequenced. Figure Figure11 shows the frequency by which different phages expressing particular VAR2CSA regions were identified after the different types of biopanning. Biopanning on CSA resulted in enrichment of a single region (20% of all phages) corresponding to a peptide sequence in DBL3 (Figure (Figure1A,1A, green). CSA coated directly on ELISA plates might not be very efficient; therefore the biopanning was subsequently repeated using biotinylated CSA (bCSA). This resulted in enrichment of phages representing sequences present in DBL1, DBL2 and DBL4 (Figure (Figure1A,1A, red). In addition, biopanning on human placental CSPG resulted in enrichment of phages representing DBL4 and DBL5 sequences (Figure (Figure1A,1A, yellow). However, the DBL4 sequence was also present in 18% of the control BSA biopannings (Figure (Figure1A,1A, blue). To further identify proteoglycan-binding regions, the VAR2CSA phage display library was biopanned on BeWo and CHO cells expressing CSPG and CSA, respectively (Figure (Figure1B).1B). Enrichment of phages expressing peptides from DBL2, DBL3, DBL4 and DBL5 was seen. Again the DBL4 sequence was also detected in the phages from the control biopanning. DBL2 and DBL3 phages binding to the GAG expressing cells contained VAR2CSA sequences that overlapped with the sequences identified by the CSA biopanning. The DBL5 sequence overlapped with the clone identified by CSPG biopanning. In summary, five relatively short stretches of VAR2CSA appeared to bind soluble GAGs as well as GAGs expressed on cells. No none-DBL regions (i.e. NTS, ID1 or ID2) were identified as GAG binding.

Figure 1

Frequency of identifed phages sorted according to the identity of the VAR2CSA region and the method of biopanning. The var2csaphage display library was biopanned four rounds on each of the following: biotinylated CSA (A, red); Chondroitin sulfate proteoglycans …

Mapping the phage display selected regions on models of VAR2CSA DBL domains and comparing with the previous described surface-expressed epitopes

Structural models of VAR2CSA 3D7 DBL domains have previously been produced using the solved DBL structures in EBA-175 and Pkα-DBL as templates [36]. As part of previous work we identified regions on native VAR2CSA, which were accessible to antibodies [26,36]. In the current study, the CSA-binding regions defined by the phage display screening were mapped onto the models and compared to the previous findings (Figure (Figure2).2). Interestingly, in DBL2, DBL3 and DBL5 there was a high degree of overlap between the CSA-binding regions and the surface-exposed regions (Figure (Figure2,2, green). Peptides residing in DBL1 and DBL4 did not map to the predicted surface exposed regions. All mapped regions, except the DBL1 region, mapped to the S2 subdomain of the DBL domains. The CSA-binding regions mapped in DBL2 and DBL5 are in close vicinity to the chemokine-binding site region of Pkα-DBL Duffy [37].

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Structural models of VAR2CSA DBL domains showing surface-exposed regions and GAG binding regions. The surface-exposed epitopes previously determined by depleting female IgG plasma on parasites expressing VAR2CSA are shown in blue. The GAG binding regions identified by phage display assays are shown in yellow. The overlap of surface-exposed regions and GAG binding regions is shown in green.

……..

Malaria parasites causing PAM have been shown to bind to glycosaminoglycans in the intevillous space of the placenta. These parasites also bind specifically to bovine CSA [18], CSPG [38] as well as BeWo [39] and CHO cells having CSPG on the surface [18]. The binding between the parasite and the placental CSPG is most likely mediated through the parasite expressed protein, VAR2CSA. VAR2CSA is a large multidomain protein and for vaccine development it is important to define which regions of VAR2CSA are responsible for the interaction with CSPG. In the present study a var2csa phage display library was biopanned on five different CSA containing targets (bovine CSA, bovine bCSA, human placental CSPG, BeWo and CHO cells) in 12 independent experiments. Five regions of VAR2CSA repeatedly showed affinity for the different CSA preparations. The CSA-binding peptides identified with the phage display approach were based on the linear sequence of VAR2CSA. However, the CSA binding region might be conformational and involve peptides from several domains. It is preferable that results obtained by phage display assays are confirmed by showing that peptides corresponding to the identified regions also possess binding capacity. The var2csa phage display library used in this study was constructed from DNA fragments of 100–150 bp, and the corresponding peptides are thus 34–75 aa. These long peptides were difficult to synthetize and were unstable in solution and we thus had to divide some of the phage regions into several synthetic peptides. Furthermore, the structure of peptides in solution might be very different from peptides bound to a phage. These factors could explain why only one out of seven synthetic peptides could have its binding to CSA confirmed.

DBL2, DBL3 and DBL5 domains of VAR2CSA have previously been shown to bind to CSA [24–26] and the surface-exposed regions within these domains have been mapped [26,36]. Three of the five peptides are located on surface-exposed parts of the previously described CSA-binding domains and two of these peptides map to regions on the DBL domains, which are in close proximity to the ligand-binding region of Pk-alfa-DBL [37]. These findings show an agreement between two independent approaches, which strengthens the present results. No CSA-binding epitopes were found in the highly polymorphic DBL6 domain, which previously has been shown to bind CSA indicating the presence of conformational CSA binding regions in this domain [25].

Phage display was used to identify GAG binding linear regions of VAR2CSA. Five regions located in five different domains were found to have affinity for both immobilized CSA and CSA expressed on the surface of cells. The most frequently observed GAG binding phages mapped to DBL2, 3, 4 and DBL5, and except DBL4 all these domains have been shown to bind CSA in vitro. These results are supported by data published by Andersen and others [36], demonstrating that the phage display defined CSA-binding regions in DBL2, 3, 5 all locate to areas of VAR2CSA that appear to be exposed on the native molecule. The DBL2 CSA binding peptide showed specific binding to CSA and affinity-purified antibodies against the same phage display identified region reacted with the surface of infected erythrocytes. This work is the first step in defining small regions of VAR2CSA, which can be used in an adhesion blocking sub-unit vaccine protecting pregnant women against PAM.

Brabin BJ. An analysis of malaria in pregnancy in Africa. Bull World Health Organ. 1983;61:1005–1016. [PMC free article] [PubMed]
Fried M, Nosten F, Brockman A, Brabin BJ, Duffy PE. Maternal antibodies block malaria. Nature.1998;395:851–852. doi: 10.1038/27570. [PubMed] [Cross Ref]
Ricke CH, Staalsoe T, Koram K, Akanmori BD, Riley EM, Theander TG, Hviid L. Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A. J Immunol. 2000;165:3309–3316. [PubMed]
David PH, Hommel M, Miller LH, Udeinya IJ, Oligino LD. Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc Natl Acad Sci USA. 1983;80:5075–5079. doi: 10.1073/pnas.80.16.5075. [PMC free article][PubMed] [Cross Ref]
Smith JD, Chitnis CE, Craig AG, Roberts DJ, Hudson-Taylor DE, Peterson DS, Pinches R, Newbold CI, Miller LH. Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell. 1995;82:101–110. doi: 10.1016/0092-8674(95)90056-X. [PMC free article] [PubMed] [Cross Ref]

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Life, connections and striving

Posted in Patient Experience, Patient Outlook, tagged about death and dying, fighting cancer, striving, writer's block on November 26, 2015| Leave a Comment »

Life, connections and striving

Larry H. Bernstein, MD, FCAP, Curator

LPBI

I have been writing about life, illness, and end of life experiences. I am recalling the untimely death of my mother in her 50s of Linitus Plastica. Then I recalled my cousin, Robert E. Liss, who was a reporter for the Miami Herald who came down with Hairy Cell leukemia and sought a cure. He wrote a book about the experience that would be a read for medical students. He died of his illness in 1980, leaving a wife and three children. I had not seen him since our youth, but I see his sister, Barbara, and my only living aunt, Bernice, who is 95. I accidentally came across a cousin he had on the Liss side who loves photography a few years ago, as I also have done photography and darkroom work some years ago, which complicated my recent move to be near a daughter, her husband and grandson. I am reminded of what I missed in seeing my terrific children growing up. I have been totally absorbed in Medicine for so many years that retiring was difficult. My surgical colleague, now deceased, once told me that all of his colleagues died in their boots. That may be a passing generation.

I admit that I am somewhat off the topic. I’ll return to a brief picture of Bobby Liss, author of a “Fading Rainbow”. He and his were of a more activist generation, despite the fact that they were within a decade of my birth, my mother coming to US in 1941 at 18 years age with her 11 year old sister. They settled in Cleveland where that side of my family lived. Bobby and Barbara’s mother married an airforce pilot who had served in the Asian campaign, and they settled in Chautauqua, New York, where my family visited when we were children. I remember my father hitting a deer in the travel in upstate New York.

Fading Rainbow: A Reporter’s Last Story
by Robert E. Liss

Renee‘s review

Oct 03, 10

Unfortunately, Bob Liss died before completing this book and it shows. He’s an amazing writer and it’s obvious this book would have been a masterpiece had he been able to complete it himself. His wife, Bonnie, did a good job finishing it, but I’ve never yet read a book that was started by one author and completed by another that was a great book. But it’s still worth reading. Facing one’s own death is never easy, but Bob (my cousin, by the way) has an amazing outlook and I feel like I really learned a lot. Plus, it’s an easy, quick, engrossing read.

http://ecx.images-amazon.com/images/I/41m3bSv-S1L._SX373_BO1,204,203,200_.jpg

Fading rainbow: A reporter’s last story Hardcover – 1980

by Robert E Liss (Author)

Published by Methuen

ISBN 10: 0416006310 ISBN 13: 9780416006315

Leukemia – Biography. | Journalists

For architect Bonnie Holmberg, writing began with tragedy. Diagnosed with an incurable form of leukemia, Holmberg’s first husband, Miami Herald reporter Robert Liss, had written most of Fading Rainbow, a book about his experience with a terminal illness. When he died before the book was finished, Holmberg completed his last few chapters in 1983, and the writing bug bit.

As the head of corporate design for now-defunct Eastern Airlines, she wrote her next book, Cruising at 30,000 Feet, aboard planes, writing about her life as a new widow and mother of three school-age boys (two of whom are now writers).

These days, Holmberg, 60, is remarried, retired, and a guide-in-training at Monticello, working under first-place winner David Ronka. Neither knew the other had entered.

Her winning entry, “Felonious Monk,” she says, was inspired by a friend who had put her home in her son’s name.

“I thought, ‘Oh gosh, what could go wrong there?'” she says. Fortunately for that friend, nothing terrible happened, but the thought stayed with Holmberg– and a recent Charlottesville bank robbery offered further inspiration.

The judges were drawn to her “felonious but strangely empathetic central character,” suggesting that the story’s only flaw was “a sense that the ending may best serve as the end of a beginning!”

They must have ESP (or else Jefferson really was whispering secrets from beyond). It turns out that “Monk” is her first short story, and it’s actually a part of her third novel. The first two, she laughs, “no one seems to want.”

She keeps her spirits up in a writing support group– an idea she suggests to anyone who wants to get into writing.

“It’s really given me a lot of encouragement,” she says, “kind of like AA.”

Bonnie Holmberg

Anyone who’s ever started a sentence with “I’m too old to…” should take a few pointers from first-place winner David Ronka.

“I was 49 or 50 when I went back for the MFA,” says Ronka, a long-time government worker who earned his graduate degree from University of Massachusetts-Amherst where he studied fiction under famed novelist John Edgar Wideman.

These days, Ronka, 61, is a historic interpreter at Monticello, but writing remains a passion.

“It’s something I can be doing when I’m 90,” he says.

His winning story, “What Can’t be Cured,” explores death through the eyes of a man whose marriage has also expired– but just might be resurrected.

Judges offered glowing praise.

“By putting an interesting twist on some recognizable male emotions, the author delivers present conflict and resolution in a light, but sincere vision of a man willing to admit his mistakes and try again,” they wrote. Ronka’s compliance with the contest rules, they said, earned him high marks as well.

So just how does one come up with the idea for a winning story?

“By observing, listening, asking myself constantly, ‘What if?'” says Ronka, whose inspiration for this story– originally a 61-page novella– came when a good friend passed away.

So what’s his favorite part of the story?

“The opening line is a pretty good hook, if I do say so myself,” he laughs. Getting readers interested immediately is “pretty essential,” he explains, “so I’m pleased with that.”

David Ronka
PHOTO BY JEN FARIELLO

http://www.songlyrics.com/jenny-wilson/like-a-fading-rainbow-lyrics/

Oh, no birds that flockin’ round my feet
No pockets full of grain, of crumbs
The wiener cake, the soft ice
Sweet
Sweet
Sweet

So I left my fading life
I left my hands with an open door
Left it like an open sore
I couldn’t stop the wind from flowin’

So I left my fading life
I left my hands with an open door
Left it like an open sore
I couldn’t stop the blood from flowin’

So I left my fading life
I left my hands with an open door
I left like a fading rainbow

So I left my fading life
I left my hands with an open door
I left like a fading rainbow

Oh, I left my fading life
I left my hands with an open door
I left like a fading rainbow

Jacobson, Richard
Fear and loathing on tenure trail

I do not know if she took my advice. Although I

offered to act for her, she left my office in the company

of her husband, who was still angrily demanding that

any letter contain the declaration that she had really

earned tenure. She never returned my calls.

The effect of an adverse decision is shattering. I

have noticed that rejected professors typically become

rather careless following the decision: they cross

streets without looking, they speak indiscreetly. One

client, following each of three adverse decisions, would

accidentally drop a pot of hot coffee on his hand or

foot. This symbolically suicidal behavior must be a

comment on the awful event: either it is an internaliza-

tion of the rejection-if you reject me, I reject

myself-or else it is a kind of dramatic reproach, as if to

say, if this is what you think of me, look at how great a

result you caused.

My advice to those I assist is nearly always that the

most important thing they can do is exactly what they

did before. They should go on with their work of

teaching and writing, if possible more energetically

than ever. This serves several purposes: it not only

diverts the mind from the powerful sense of grievance,

but it also confirms the identity of the client as teacher

and scholar. They have been put through a symbolic

execution, and it is up to them to carry out a symbolic

resurrection of their professional and personal vitality.

While I have always given this advice-and taken it

in my own case-I have only recently been able to ar-

ticulate the reasons for it. While I was writing the first

draft of this essay, I read a book written by a college

friend with whom I had lost touch, and which was

published posthumously (Fading Rainbow, Methuen

1980). Robert Liss was a very good writer who had

never quite achieved what he hoped. At the time he

learned he had a rare form of leukemia, he was a

reporter for the Miami Herald. He found a way of

transcending his pain and fear when he remembered

what he was, first and foremost: a writer and a jour-

nalist. So he spent the time remaining to him in-

vestigating his disease and writing about it in an in-

spiring and utterly truthful book.

If the basis of dispute is ultimately the fear of an-

nihilation, of a loss of the self, Liss teaches us that the

way to transcend the fear is through regaining your

Self. The effort may not change the external reality,

but it can alter the more important one. Courage is also

a denial of death.

Why do I bother with this’ business of helping

grievants? In most cases they do not pay me, and it

carries certain disutilities in my personal and

professional relations. The Authorities do not thank

me for it-although arguably my activity helps

legitimize their own. Being at odds with one’s world is

an extremely uncomfortable feeling: one grows

suspicious of other people, and one feels oddly guilty

about challenging Authority.

In one sense I think this kind of work is my own ef-

fort to achieve transcendence. I take pride in doing it

well, and most of those I have assisted have won sub-

stantial concessions, despite the conventional wisdom

that you can’t win these cases.

When the client soberly determines to undertake the

struggle to reverse the adverse decision, knowing the

immense stamina required, we both must face the in-

tensity of self-justifying response.

Family Hospice Care:
Pre-planning and Care Guide

harry@legacies.ca

In the twenty years since this book was first published, hundreds of thousands of patients, family members, professional and volunteer care providers have learned the basic fundamentals of providing physical, emotional, spiritual and information supports.
People need to relatively pain free and alert for as long as they can. The hospice philosophy of care is about living life to its fullest before you die. That is not what typically happens for people near the end of their lives. Their physical pain is often not controlled well. That is inexcusable. No one need suffer unbearable pain. No one.
Many people hope that their last weeks and months will be filled with compassionate medical support, well-informed and caring family and friends, and information on how to live life fully. That is what excellent hospice care is all about. That is what Family Hospice Care is all about.
Harry van Bommel helped his mother, father and grandfather to live at home until they died. He has helped countless others through his writing, speaking, teaching and one-to-one support turn an end-of-life experience into something to be treasured rather than feared. His detailed suggestions help people take some control of the roller coaster ride of emotions, feelings and experiences.
The journey at the end-of-life will have moments of frustration, anger, tears, despair and overwhelming fear. That is too often the only experiences people have. Family Hospice Care is a tool that helps you minimize these negative experiences while providing specific ideas so that you can also experience profound moments of love, laughter, joy, retelling of stories, bonding with family and friends and care providers. Like birth, death can be an incredible opportunity to review your own life and its direction and find out the wisdom of all ages: it is our relationships with others that matter most at these times. Living fully until you die provides an opportunity to nurture those relationships to an even greater degree.

Study Finds Shu Gan Liang Xue Herbal Formula Has Breast Cancer Anti Tumor Effect

Posted in Uncategorized on June 25, 2014 |

larryhbern:

interesting finding. Of course, you won’t find a large scale study for a medicinal that is regulated as a FOOD. Whatever the cost, if the side effect were insignificant, it would be a challenge to pharma, but the cost is not picked up by insurance.

read-
Fading Rainbow
A Reporter’s Last Story
by Robert E. Liss
Methuen, Inc. 1980
Leukemia – biography
ISBN: 0-416-00631-0

Reporter for Miami Herald, merit scholar graduate from Brandeis U., father of 3, develops Hairy Cell Leukemia, going out like a “failing rainbow”, tells the story of tests, hospitalizations, treatments, pain. Story completed by his wife – Bonnie Liss.

Renee Liss

But what really stuck with me, what really is making a difference in my life, the thing I need to remind myself of every single say is this: One of the characters goes to see a published author speak about his latest novel. They end up having a brief affair and she suggests one of his books for the group and he attends to meeting. In the course of speaking about his process, he tells the group that he used to putter around the house all day waiting for inspiration and it never came. So now he sits down at his desk at nine every morning and just starts. And the words come and he writes twenty pages a day.

So I’m trying something similar to that. Since I have a full-time job that is not writing a novel, I can’t sit down at nine every morning and just write all day. But I have decided to dedicate a minimum of one hour per day to it. I’ve done that for three days now and plan to continue today.

My friend Amy over at Mrs. Thor is in a similar situation — trying to get inspired and trying to make significant changes in her personal and creative life. So we’ve started our own private little writing group, though I don’t know that’s the appropriate term. We’re going to speak on the phone once a week and set goals for ourselves (like my writing an hour a day) and then check in by e-mail each day on whether we met the goals. It’s no pressure, but it’s still being accountable to someone else and hopefully inspired by the other person’s progress.

So far, I’ve added 3,100 words to my novel and rewritten a short story from a few months ago. It feels good.

Posted in My Thoughts on Writing, Television

long before the idea of a corporation or land ownership or anything else modern, people traded and bartered for goods and services. We use money in our modern society, but humans for most of our very long history, have in one way or another purchased items for life from each other. It may have been that I have a cow and you have an orange tree so I traded milk for oranges.

But beyond this, let’s think about whether we truly want to model human society based on what other species do or do not do: ……

No more central heat, air conditioning or indoor plumbing.
Give up your cars, bicycles and all other forms of transportation other than swimming or walking.
Build your own house with no power tools.
Build that house without tools made with any form of power tools.
Quit your job to do nothing but hunt and garden and fight for resources.

No more flower gardens.
No one will be allowed to keep pets. In fact, you can’t have that cow I mentioned above because what other species keeps cows? Or dogs? Cats? Horses? You’re on your own. PETA will be happy.
No music.
No dancing.

Sun isn’t considered Hemingway’s greatest work and it was a strange book in that there really was no plot. Or maybe a very weak plot that the reader has to kind of search out. It was just a story about a series of events that happened to this group of people. But I still enjoyed it on a certain level. Almost like a course in creative writing without having to sit in a classroom.

I’m discovering how very much I have in common with this man. It’s all very strange. I’ve always said that my time as a journalist was the best thing that ever happened to my creative writing style. The quick, active, short way one must write newspaper articles — getting to the point quickly and using limited space to convey a vast amount of information — translates excellently into short story and novel-writing. One learns to not waste words or over-describe. Turns out, Hemingway learned the exact same lesson in the exact same way I did. He began his writing career in journalism and he learned to write fiction by emulating the journalistic style.

I hate to compare myself to him because he’s considered so widely to be one of the best writers in history and especially of the twentieth century and I haven’t even published a short story. Maybe I’m arrogant in my comparison, but I see so much of my style in his. I see the writer I maybe am not yet but want to be some day.

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Microtubule-Associated Protein Assembled on Polymerized Microtubules

Posted in Bio Instrumentation in Experimental Life Sciences Research, Curation, Proteins, Proteomics, tagged CAP-Gly domain, dynactin's, intermolecular interface determination, magic angle spinning NMR, microtubules, structure determination on November 26, 2015| Leave a Comment »

Microtubule-Associated Protein Assembled on Polymerized Microtubules

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Magic-Angle-Spinning NMR Enables First-Ever Determination of Atomic-Resolution Structure of a Microtubule-Associated Protein Assembled on Polymerized Microtubules

http://www.bioquicknews.com/node/3201

https://upload.wikimedia.org/wikipedia/commons/0/06/Microtubule_structure.png

A latticework of tiny tubes called microtubules gives your cells their shape and also acts like a railroad track that essential proteins travel on. But if there is a glitch in the connection between train and track, diseases can occur. In the November 24, 2015 issue of PNAS, Tatyana Polenova, Ph.D., Professor of Chemistry and Biochemistry, and her team at the University of Delaware (UD), together with John C. Williams, Ph.D., Associate Professor at the Beckman Research Institute of City of Hope in Duarte, California, reveal for the first time — atom by atom — the structure of a protein bound to a microtubule. The protein of focus, CAP-Gly, short for “cytoskeleton-associated protein-glycine-rich domains,” is a component of dynactin, which binds with the motor protein dynein to move cargoes of essential proteins along the microtubule tracks. Mutations in CAP-Gly have been linked to such neurological diseases and disorders as Perry syndrome and distal spinal bulbar muscular dystrophy. The research team used magic-angle-spinning nuclear magnetic resonance spectrometry (NMR) in the Department of Chemistry and Biochemistry at UD to unveil the structure of the CAP-Gly protein assembled on polymerized microtubules. The CAP-Gly protein has 1,329 atoms, and each tubulin dimer, which is a building block for microtubules, has nearly 14,000 atoms. “This is the first time anyone has been able to get an atomic-resolution structure of any microtubule-associated protein assembled on polymerized microtubules,” Dr. Polenova says. “With magic-angle-spinning NMR, we can look into the structure of this and other assemblies of microtubules and their associated proteins and gain critical insights into their function and dynamics, as well as begin to gather clues as to how mutations cause disease.” In magic-angle-spinning NMR, a sample is placed in the NMR’s small, tube-like rotor, which is then spun inside the NMR magnet at an angle of 54.74 degrees — called the “magic angle” because it suppresses the atoms from interacting magnetically. The result is a high-resolution protein fingerprint, a graph of hundreds of peaks representing the frequencies of two or more interacting atoms. These data are then used to calculate the 3-D structures.

The 3-D structures of CAP-Gly, which show the spatial arrangement of atoms in the protein molecule, are different between the free state of the protein and its bound state to the microtubule. These structures reveal how the protein interacts with microtubules, predominantly through its loop regions, which adopt specific conformations upon binding.

However, static structures of CAP-Gly do not tell the whole story about the protein.

“Just as we are always moving our arms and legs about, proteins are very dynamic. They do not stand still,” Dr. Polenova says.

“These motions are essential to their biological function, and NMR spectroscopy is the only technique that can record such movements, with atomic resolution, on a variety of time scales, from picoseconds to arbitrarily long time scales — seconds, days, weeks — to help us understand the protein’s function.”

“We know from our prior studies that CAP-Gly is dynamic on timescales from nano- to milliseconds, and this mobility is essential for the protein’s ability to interact with microtubules and with multiple other binding partners.”

The research, which has been ongoing since 2008 when the first data sets were collected, required the development of new protocols for preparing the samples, new NMR experiments to gather various information on structure and dynamics, and new protocols for data analysis.

In the future, Dr. Polenova and her team envision using NMR in combination with cryo-electron microscopy, in which samples are studied at extremely low temperatures, typically below -200 degrees Fahrenheit, to look at even more complex systems in a highly preserved form.

Dr. Polenova’s research team at UD included Dr. Si Yan, who received her doctorate from the University in 2014, current doctoral student Changmiao Guo, NMR spectroscopist Guangjin Hou, and postdoctoral researchers Dr. Huilan Zhang and Dr. Xingyu Lu. Dr. Williams, at Beckman Research Institute, was also a co-author of the study.

Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

Si Yan^a,¹, Changmiao Guo^a,¹, Guangjin Hou^a, Huilan Zhang^a, Xingyu Lu^a, John Charles Williams^b, and Tatyana Polenova^a,²Author Affiliations

PNAS nov 2015; 112(47):14611–14616 http://dx.doi.org:/10.1073/pnas.1509852112

Fig. 1.

http://www.pnas.org/content/112/47/14611/F1.small.gif

Significance

Microtubules and their associated proteins are central to most cellular functions. They have been extensively studied at multiple levels of resolution; however, significant knowledge gaps remain. Structures of microtubule-associated proteins bound to microtubules are not known at atomic resolution. We used magic angle spinning NMR to solve a structure of dynactin’s cytoskeleton-associated protein glycine-rich (CAP-Gly) domain bound to microtubules and to determine the intermolecular interface, the first example, to our knowledge, of the atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. The results reveal remarkable structural plasticity of CAP-Gly, which enables CAP-Gly’s binding to microtubules and other binding partners. This approach offers atomic-resolution information of microtubule-binding proteins on microtubules and opens up the possibility to study critical parameters such as protonation states, strain, and dynamics on multiple time scales.

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

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Optical neurons

Posted in Biological Networks, Gene Regulation and Evolution, Clinical Genomics, Curation, Developmental biology, Embryology, tagged Adult stem cell, Embryonic stem cell, Neuron, Sympathetic nervous system, Umbilical cord on November 26, 2015| Leave a Comment »

Optical Neurons

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Umbilical Cells Help Eye’s Neurons Connect

Factor released by cells helps connections, not longevity

November 24, 2015 https://today.duke.edu/2015/11/synaptogen

https://today.duke.edu/sites/default/files/stories/SynaptogenDuke_small.jpg?1448396221

Cells isolated from newborns’ umbilical cords help neurons make new connections (shown in yellow) with their neighbors. Image credit: Sehwon Koh, Duke University

Cells isolated from human umbilical cord tissue have been shown to produce molecules that help retinal neurons from the eyes of rats grow, connect and survive, according to Duke University researchers working with Janssen Research & Development, LLC.

The findings, which appear Nov. 25 in the Journal of Neuroscience, implicate one family of molecules in particular — thrombospondins — that may have therapeutic potential for the treatment of degenerative eye diseases.

“By learning more about how these cells work, we are one step closer to understanding the disease states in which these cells should be studied,” said Cagla Eroglu, an assistant professor of cell biology and neurobiology at the Duke University Medical Center, who led the research.

Umbilical cord tissue-derived cells (hUTC) differ from umbilical cord blood cells in that they are isolated from cord tissue itself, rather than the blood. The Duke

team used an established cell culture system to determine whether and how the hUTCs might affect the growth of neurons isolated from the retinas of rat eyes.

In an experimental setup that allowed the two types of cells to bathe in the same fluid without coming into physical contact, retinal neurons in a bath with hUTCs formed new connections between neurons called synapses, and they sprouted new ‘neurites’ — tiny branches that lead to additional connections.

These cells also survived longer than rat neurons placed in a bath lacking the umbilical cord tissue-derived cells.

Something present in the fluid surrounding the neurons in the bath with the hUTCs was apparently affecting the neurons. Through a series of experiments, the researchers determined that relatively large molecules, thrombospondin 1, 2 and 4, were primarily responsible for the effect.

Blocking thrombospondins was found to reduce new connections among neurons. By genetically inhibiting the individual members of the thrombospondin family, the researchers found that TSP1, TSP2, and TSP4 in particular were required to create both neurites and new connections.

“It’s exciting that thrombospondins had a really strong effect on neurite outgrowth,” said Eroglu, who is also a member of the Duke Institute for Brain Sciences (DIBS). She added that making neurites and forming new connections between them are crucial for helping neurons grow when faced with injury and neurodegenerative diseases.

However, blocking TSP1, 2 and 4 did not affect neuron survival, suggesting that there is some other factor in the UTC cells that promotes cell longevity. Her group is now searching for those molecules.

Eroglu’s earlier work has shown that thrombospondins are released by brain cells called astrocytes and boost new synapse formation between neurons in the brain.

Eroglu said there may be deficiencies in thrombospondin signaling in neurodegenerative disease, and the group is actively pursuing this hypothesis in animal studies.

Postdoctoral fellow Sehwon Koh is the lead author of this study and a member of the Eroglu lab. Other authors include Namsoo Kim and Henry H. Yin from Duke’s department of psychology and neuroscience. This research was supported by a research agreement with Janssen Research & Development, LLC.

CITATION: “Human Umbilical Tissue-Derived Cells (hUTC) Promote Synapse Formation and Neurite Outgrowth via Thrombospondin Family Proteins,” Sehwon Koh, Namsoo Kim, Henry H. Yin, Ian R. Harris, Nadine S. Dejneka, and Cagla Eroglu. Journal of Neuroscience, November 25, 2015. http://dx.doi.org:/10.1523/JNEUROSCI.1364-15.2015

ScienceDaily

http://www.sciencedaily.com/releases/2015/11/151124204332.htm

Cells isolated from the human umbilical cord have been shown to produce molecules that help retinal neurons from the eyes of rats grow, connect and survive. The findings implicate one family of molecules in particular — thrombospondins – that may have therapeutic potential for the treatment of degenerative eye diseases.

The findings, which appear Nov. 25 in the Journal of Neuroscience, implicate one family of molecules in particular — thrombospondins — that may have therapeutic potential for the treatment of degenerative eye diseases.

Umbilical cord tissue-derived cells (hUTC) differ from umbilical cord blood cells in that they are isolated from cord tissue itself, rather than the blood. The Duke team used an established cell culture system to determine whether and how the hUTCs might affect the growth of neurons isolated from the retinas of rat eyes.

Golgi Cells Have Active Dendrites

Stephanie Rudolph, Court Hull, and Wade G. Regehr

The Journal of Neuroscience, Nov 25, 2015 • 35(47):i • i (see pages 15492–15504)

http://www.jneurosci.org/content/35/47.abstract.pdf

The cerebellum coordinates multijoint movements and contributes to motor learning. These functions require precise spike timing in Purkinje cells, the cerebellar output neurons. Purkinje cell spiking is driven partly by granule cells, which receive information about ongoing movements from mossy fibers, and the timing and spatial extent of granule cell output is determined largely by inhibitory input from spontaneously active interneurons called Golgi cells.

Golgi cell spiking is modulated by excitatory input from both mossy fibers and granule cells. How these inputs are integrated in Golgi cell dendrites remains poorly understood. Finding no evidence for active conductances in Golgi cell dendrites, Vervaeke et al. (2012, Science 30: 1624) hypothesized that dendritic gap junctions enable granule cell inputs to influence Golgi cell activity. Although gap junctions likely do contribute to dendritic processing in Golgi cells, Rudolph et al. now show that Golgi cell dendrites also express voltage-gated channels.

If dendrites lacked active conductances, one would expect signals to decay with distance from the soma. But calcium imaging in rat cerebellar slices revealed that action potentials caused uniform calcium elevation throughout Golgi cell dendrites. Moreover, applying a voltage-gated sodium channel (VGSC) blocker selectively to dendrites reduced spike-associated calcium elevation in distal dendrites. In addition, blocking T- and R-type voltage-gated calcium channels (VGCCs) attenuated calcium elevation selectivelyin distal dendrites,while blocking N-type channels reduced calcium elevation only in proximal dendrites.

Blocking voltage-gated channels also had functional consequences. Blocking N-type channels decreased the amplitude of the spike afterhyperpolarization and increased the spike rate of Golgi cells. In contrast, T-type channel blockers had little effect on baseline firing frequency. Nonetheless, blocking T-type channels attenuated rebound spiking after hyperpolarization and reduced the amplitude of EPSPs evoked by stimulation of granule cell axons.

These experiments suggest that VGSCs help depolarize distal dendrites to enhance activation of T-type VGCCs, which in turn amplify responses to granule cells and promote rebound bursting. Meanwhile, N-type VGCCs located near the soma appear to be tightly coupled to calcium-activated potassium channels, which regulate the spontaneous spike rate of Golgi cells. Thus, Golgi cell dendrites have multiple types of voltage-sensitive channels that are differently distributed and serve distinct roles in ensuring the precise timing of cerebellar output.

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Naïve Human Pluripotency

Posted in Stem Cells for Regenerative Medicine, tagged naive human pluripotency on November 25, 2015| Leave a Comment »

Naïve Human Pluripotency

Larry H. Bernstein, MD, FCAP, Curator

LPBI

The Current State of Naïve Human Pluripotency
Benjamin T. Dodsworth, Rowan Flynn, Sally A. Cowley

Stem Cells Nov 2015; 33(11): 3181–3186 DOI: http://dx.doi.org:/10.1002/stem.2085

The newly discovered state of ‘naïve’™ human pluripotency is not only an extremely interesting biological phenomenon, but also promises to overcome some of the problems posed by conventional ‘primed’™ human pluripotent stem cells. These problems include variable differentiation capability, difficult single-cell passaging, and low gene editing efficiency – all bottlenecks for applications such as regenerative medicine. A flurry of recent papers describe new ways of accessing naïve human pluripotency. However, there are important differences between these protocols, making this concise yet comprehensive review a timely necessity to navigate the complexities of this emerging field.

Naïve or ground state pluripotency is a cellular state in vitro which resembles cells of the preimplantation epiblast in vivo. This state was first observed in mouse embryonic stem cells and is characterized by high rates of proliferation, the ability to differentiate widely, and global hypomethylation. Human pluripotent stem cells (hPSCs) correspond to a later or “primed” stage of embryonic development. The conversion of hPSCs to a naïve state is desirable as their features should facilitate techniques such as gene editing and more efficient differentiation. Here we review protocols which now allow derivation of naïve human pluripotent stem cells by transgene expression or the use of media formulations containing inhibitors and growth factors and correlate this with pathways involved. Maintenance of these ground state cells is possible using a combination of basic fibroblast growth factor and human leukemia inhibitory factor together with dual inhibition of glycogen synthase kinase 3 beta, and mitogen-activated protein kinase kinase (MEK). Close similarity between the ground state hPSC and the in vivo preimplantation epiblast have been shown both by demonstrating similar upregulation of endogenous retroviruses and correlation of global RNA-seq data. This suggests that the human naïve state is not an in vitro artifact. Stem Cells 2015;33:3181–3186

In mice, two pluripotent states have been captured in vitro. Mouse embryonic stem cells (mESCs) are sourced from the inner cell mass (ICM) of the preimplantation blastocyst [1, 2]. When derived and maintained using a combination of leukemia inhibitory factor (LIF) and 2i (dual inhibition of extracellular signal-regulated protein kinases 1/2 [ERK1/2] pathway and glycogen synthase kinase 3 beta [GSK3β]) they are described as being in a naïve or ground state [3]. When injected back into an early embryo, these cells can contribute to all lineages without tumorigenesis [4]. A more recent discovery has been mouse epiblast stem cells (mEpiSCs—Fig. 1). These are sourced from postimplantation epiblast cells [5, 6] and are termed primed, due to their inability to integrate into a preimplantation blastocyst. They can, however, be differentiated into all three germ layers in vitro. The most striking difference is the very high expression of de novo methyltransferases, which leads to condensing of chromatin [7]. Additionally, these cells require basic fibroblast growth factor (bFGF also known as FGF2) and transforming growth factor beta (TGFβ) for self-renewal, instead of 2i and LIF [3, 5]. mEpiSCs can be converted back to the naïve state by transfection with Klf4 or other reprogramming factors or using small molecules [8, 9].

Naïve pluripotent stem cells have been successfully captured in vitro from primed rhesus monkey induced pluripotent stem cell (iPSC) lines using specialized media containing 2i and LIF [10]. Since naïve pluripotent stem cells can be generated from primates, this suggests that the state of naivety might be conserved across species. Using primate cells also allows dissection of genetic background and species to species differences. Primate naïve iPSCs require bFGF, whereas bFGF causes differentiation in mESCs. Additionally, TGFβ is not required for maintenance of primate naïve iPSCs, indicating that TGFβ might not be essential in the human system [10].

Embryogenesis is inherently different between species, which is reflected by the difficulties in generating truly naïve human pluripotent stem cells (hPSCs) in vitro. For ethical reasons, information on human embryogenesis is lacking and many assumptions are made based on the mouse model [11]. Despite being sourced from the same point in development as mESCs, hESCs resemble mEpiSCs. Both form large, flat, 2D colonies and require bFGF for self-renewal. The ability to convert mEpiSCs to mESCs has led to the prediction that naïve hPSCs might also be accessible by reverting primed hESCs. This has prompted several recent publications of strategies to capture the human naïve state, either relying on transgene overexpression [12-14] or different combinations of small molecule inhibitors [15-20]. Here we review and compare all these published protocols, including a protocol devised by Duggal et al. [16] published in this issue.

The concept of naïve hPSCs has been contentious. Pera [41] argues that since this state was actively searched for in humans, it is highly likely that it is purely an artifact generated in the lab. However, Wang et al. used RNA-seq data which was available from cells taken directly from the ICM of early embryos and showed a tight correlation to naïve cells generated in vitro [27].

This was confirmed when Huang, Maruyama, and Fan took a systems biology approach and compared datasets from many previous publications [42]. Their analysis revealed poor conservation of gene networks between mPSCs and hPSCs but a high resemblance to the ICM of their respective blastocysts. They also found variations in transcriptomes from different naïve conversion protocols, but all established naïve cells showed clear resemblance to human late preimplantation embryos. According to this study, naïve cells generated by Takashima et al. [14] and Theunissen et al. [19] most closely resembled the human preimplantation blastocyst. The protocols by Valamehr et al. [18] and Duggal et al. [16] were not included in the study. In conclusion, the authors propose comparing the combination of transcriptome analysis and epigenetic characterization to in vivo data from embryogenesis as a gold standard for naivety [42].

The description of just two states, naïve and primed, is an oversimplification [11, 27, 43, 44]. Two studies [27, 43] used single-cell RNA-seq and reported a polyclonal spectrum of cell states ranging between these extremes and that naïve PSCs are present as a subpopulation in cultures previously considered entirely primed. Wang et al. [27] used a reporter system based on the endogenous retrovirus HERVH’s LTR7 promoter which is only active in naïve cells. This approach showed a consistent 4% of cells with naïve reporter expression which can be selected for using 2i and LIF and do not need prior conversion. Recently, Wu et al. were able to capture another alternative state designated “region-selective primed” pluripotency in vitro in both mouse and human which are distinct from both naïve and primed states [44].

There remain many challenges in the field of naïve pluripotency. All protocols for generating human naïve PSCs yield slightly different cellular states. It is still unclear which of these is closest to its in vivo counterpart. The in vivo naïve state is inherently transient, so continuous in vitro culture may be detrimental. For example, female cells maintained in the naïve state that do not exhibit X-inactivation might suffer from double dosage effects. With protocols now readily available which allow the generation and maintenance of naïve cells, these questions can be addressed. Meanwhile, their faster rate of growth, single cell survival, and enhanced gene editing efficiency will be used. In the near future, naïve hPSCs may be useful for accessing paths of differentiation which have been previously unreachable.

Siân Gregory, Sushma Swamy, Zoe Hewitt, Andrew Wood, Richard Weightman, Harry Moore, Autophagic response to cell culture stress in pluripotent stem cells, Biochemical and Biophysical Research Communications, 2015

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Protein regulator of HIV replication

Posted in Curation, Proteins, Viral diseases, Virology, tagged CAP-Gly domain, dynactin's, endoplasmic reticulum stress (ER stress), endoplasmic-reticulum-associated protein degradation (ERAD), functional proteomics, glycoprotein, glycoprotein biosynthesis, human immunodeficiency virus (HIV), intermolecular interface determination, magic angle spinning NMR, microtubules, protein degradation, structure determination, Unfolded Protein Response (UPR), viral protein on November 25, 2015| Leave a Comment »

Protein regulator of HIV replication

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Updated 11/26/2015

Closing the loop on an HIV escape mechanism

University of Delaware

http://www.rdmag.com/news/2015/11/closing-loop-hiv-escape-mechanism

Tatyana Polenova, professor of chemistry and biochemistry at UD (background, left), with her UD research team involved in the HIV study. Next to her is Huilan Zhang. In the foreground, from left, are Guangjin Hou and Manman Lu.

http://www.rdmag.com/sites/rdmag.com/files/newsletter-ads/CHEM-Polenova_Research_Groups-111015-015.jpg

Nearly 37 million people worldwide are living with HIV. When the virus destroys so many immune cells that the body can’t fight off infection, AIDS will develop. The disease took the lives of more than a million people last year.

For the past three and a half years, a team of researchers from six universities, led by the University of Delaware and funded by the National Institutes of Health and the National Science Foundation, has been working to uncover new information about a protein that regulates HIV’s capability to hijack a cell and start replicating. Their findings, reported recently in the Proceedings of the National Academy of Sciences, point to a new avenue for developing potential strategies to thwart the virus.

The team included scientists from UD, the University of Pittsburgh School of Medicine, University of Illinois at Urbana-Champaign, Carnegie Mellon University, the National High Magnetic Field Laboratory at Florida State University and Vanderbilt University School of Medicine. They used a combination of high-tech tools and techniques, including magic-angle-spinning nuclear magnetic resonance (NMR) spectroscopy and computer simulations of molecules, to examine the interactions between HIV and the host-cell protein cyclophilin A (CypA), right down to the movement of individual atoms.

“In a nutshell, we found that the infectivity of HIV is regulated by the motions of these proteins,” says Tatyana Polenova, professor of chemistry and biochemistry at the University of Delaware, who led the study. “It’s a subtle regulation strategy that does not involve major structural changes in the virus.”

Sixty times smaller than a red blood cell, HIV contains a cone-shaped shell, or capsid, made of protein, which surrounds two strands of RNA and the enzymes the virus needs for replication. Like any virus, HIV can only produce copies of itself once it has invaded a host organism. Then it will begin directing certain host cells to begin producing the virus.

But how does HIV invade a cell? In humans, the protein CypA can either promote or inhibit viral infection through interactions with the HIV capsid, although the exact mechanism is not yet known. A portion of the HIV capsid protein, called the CypA loop, is responsible for binding to the CypA in the human host cell. Once this occurs, the virus typically becomes infectious.

However, a change of just one amino acid in the CypA loop can cause the virus to operate opposite from how it does normally, allowing the virus to become non-infectious when CypA is present, and to become infectious when there is no CypA present. Such changes are called “escape mutations,” Polenova says, because they allow the virus to “escape” from its dependence on CypA.

To home in on this escape mechanism, the research team examined assemblies of different variants of HIV capsid protein complexed with CypA. Using magic-angle-spinning NMR, they recorded the motions in these assemblies, atom by atom, on time scales ranging from nanoseconds to milliseconds, from a billionth of a second to a thousandth of a second.

The team found that a reduction in the naturally occurring motions in the binding region due to the mutations allowed the virus to escape from CypA dependence. Magic-angle-spinning NMR experiments provided a direct probe of these motions, recording the changes in the magnetic interactions between nuclei. Computer simulations allowed the team to visualize the motions.

Some portions of the capsid protein do not move at all or move only a little while other portions undergo large-amplitude motions distributed over a wide range of time scales, with the most dynamic region being the CypA loop. Polenova says it is rather surprising that such extensive motions are present in the assembled capsid, and that these dynamics could be detected by both NMR and computer simulations.

“It is the first time that quantitative agreement between experiment and computation was achieved in a dynamics study, and it’s particularly exciting that this was attained for such a complex system,” Polenova says. “We hope this work may guide the development of new therapeutic interventions, such as small molecules that would serve as interactors with the HIV capsid and inhibit these dynamics.”

Polenova says the diverse team of researchers, with expertise in HIV virology, structural biology, biophysics and biochemistry, was critical to the study’s success, along with access to national high-field NMR facilities through the National High Magnetic Field Laboratory. The team was assembled through the NIH-funded Pittsburgh Center for HIV Protein Interactions. Led by Prof. Angela Gronenborn, the center brings together high-caliber scientists and facilities to elucidate the interactions of HIV proteins with host cell factors.

Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

Si Yan^a,¹, Changmiao Guo^a,¹, Guangjin Hou^a, Huilan Zhang^a, Xingyu Lu^a, John Charles Williams^b, and Tatyana Polenova^a,²Author Affiliations

PNAS Nov 24, 2015; 112(47): 14611–14616 http://dx.doi.org:/10.1073/pnas.1509852112

Significance

How Viruses Commandeer Human Proteins

http://www.technologynetworks.com/Proteomics/news.aspx?ID=185156

Researchers have produced the first image of an important human protein as it binds with ribonucleic acid (RNA), a discovery that could offer clues to how some viruses, including HIV, control expression of their genetic material.

RNA is one of three macromolecules — along with DNA and proteins — essential to all forms of life. By understanding how hnRNP A1 binds to RNA, the scientists may find ways to jam up components of the replication machinery when the protein is coopted by disease.

The team of scientists reveals the mechanism used by the protein, hnRNP A1 to link to the section of RNA, called the ‘hairpin loop.’

They found that hnRNP A1, a protein essential to cell function and virus replication, has a significantly different structure than its only previously known form: binding to DNA.

“We solved the three-dimensional structure of the protein bound to an RNA hairpin derived from the HIV virus,” said Blanton Tolbert, a chemistry professor at Case Western Reserve. “But because the hairpin loop is found in other viruses and throughout healthy cells, our findings may help explain how the protein connects to the other hairpin targets.”

Tolbert began this research six years ago, frustrated that the only information available was the structure of the protein bound to a synthetic DNA, which isn’t its natural target.

Proteins that bind hairpins sense both the structure and the sequence information presented in the loop. The structure of the DNA complex did not demonstrate the molecular recognition that must take place to bind RNA hairpins.

The process

To discover the structure bound to RNA, the researchers combined three techniques: X-ray crystallography, nuclear magnetic resonance spectroscopy and small angle x-ray scattering. Each technique yielded a piece of the puzzle.

To bind to RNA, hnRNP A1 has two domains, RRM1 and RRM2, which are akin to hands. Scientists already knew both hands are needed to connect to RNA.

But the researchers found that, instead of each domain grabbing a section of the loop, only RRM1 makes contact with the RNA. RRM2 acts as support, helping organize RRM1 into the structure needed to conform to a certain section of the loop.

To confirm that the structures are key to binding, the researchers inserted mutations by changing amino acids on the surface of the domains.

Surprisingly, mutations on the far side of RRM1 — the surface not in contact with the RNA but with the RRM2 — caused decoupling at that site and substantially weakened the affinity for RNA.

Without the normal connection between the two domains, RRM1 fails to adopt the geometric shape that conforms to the RNA hairpin loop.

The researchers are further investigating how the protein transmits the effects of RRM2 to RRM1 and bind. They are also exploring the development of antagonistic agents that would disrupt the interaction of the protein with viruses.

Natural defense protein against HIV discovered

HIV-1, ERManI, antiretroviral, defense protein

Earlier research had shown that it was possible to interfere with HIV spread but the exact molecular mechanisms had not been identified. For the first time, scientists have identified ERManI (Endoplasmic Reticulum Class I α-Mannosidase) as the essential host protein that slows the spread of HIV-1. Scientists investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the HIV-1 envelope (Env) degradation process. Ectopic expression of these four α-mannosidases uncovered that only ERManI inhibited HIV-1 Env expression in a dose-dependent manner. Basically, ERManI is a host enzyme that adds sugars to proteins. The Env glycoprotein is targeted to the endoplasmic reticulum-associated protein degradation pathway for degradation after infecting cells. And ERManI was found to interact with the Env and initiate this degradation pathway.

With this discovery, ERManI has the potential as a new antiretroviral treatment option. Currently there is no cure for HIV-1 and once patients are infected, they have it for life. Current antiretroviral therapies can prolong life but cannot fully cure a patient. ERManI is different from current treatments in the sense that it can help the body protect itself.

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway (Sep 2015)

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

Zhou T¹, Frabutt DA², Moremen KW³, Zheng YH⁴. Author information

J Biol Chem. 2015 Sep 4;290(36):22184-92. http://dx.doi.org:/ 10.1074/jbc.M115.675207. Epub 2015 Jul 23

Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.

T cell editing using CRISPR/Cas9 could revolutionize HIV therapeutics
September 15, 2015

T cell therapy, HIV

Reinforcing the immune system by engineering lymphocytes to target and destroy viruses has the potential to be an effective therapy for many diseases. One potential approach to this strategy is to alter the genome of lymphocytes so that proteins that are typically hijacked by viruses are no longer present. While conceptually feasible, editing T cells has been challenging in practice; however, with the advent of mammalian cell editing using CRISPR/Cas9, T-cell editing is closer to becoming a reality.

How can CRISPR/Cas9 bring us closer to finding a cure for HIV?

In a study recently published in PNAS, scientists have optimized a protocol to introduce nucleotide replacements that would inhibit CXCR4 expression. The authors streamlined the CRISPR/Cas9 editing process by electroporating Cas9 ribonucleoproteins (RNPs) into CD4+ T cells. The RNPs, consisting of both a recombinant Cas9 enzyme and guide RNA, vastly improved editing efficiency, ultimately promoting knock-out of the CXCR4 cell-surface receptor. Taken together, these result suggest the potential of a new cell therapy approach for the fight against HIV.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins
Kathrin Schumann ^a , ^b , ¹ , Steven Lin ^c , ¹ , Eric Boyer ^a , ^b , Dimitre R. Simeonov ^a , ^b , ^d , Meena Subramaniam ^e , ^f , Rachel E. Gate ^e , ^f , et al. PNAS. 2015; 112(33): 10437-10442. http://dx.doi.org:/10.1073/pnas.1512503112

Significance

T-cell genome engineering holds great promise for cancer immunotherapies and cell-based therapies for HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been inefficient. We achieved efficient genome editing by delivering Cas9 protein pre-assembled with guide RNAs. These active Cas9 ribonucleoproteins (RNPs) enabled successful Cas9-mediated homology-directed repair in primary human T cells. Cas9 RNPs provide a programmable tool to replace specific nucleotide sequences in the genome of mature immune cells—a longstanding goal in the field. These studies establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4⁺ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

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