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Audio Podcasts

Reporter: Gail S. Thornton, M.A.

UPDATED on 1/11/2020

In May 2019, Aviva Lev-Ari, Ph.D., R.N., Stephen Williams, Ph.D., and Irina Robu, Ph.D., spoke with Partners in Health and Biz, an half-hour audio podcast that reaches 40,000 listeners, about the topic of 3D Medical BioPrinting Technology: A Revolution in Medicine. The topic is also the title of a recently offered e-book by the LPBI Group on 3D BioPrinting, available on Amazon/Kindle Direct [https://www.amazon.com/Medical-BioPrinting-Technologies-Patient-centered-Patient-Centered-ebook/dp/B078QVDV2W]. https://www.spreaker.com/user/pihandbiz/bioprinting-2019-final

The 3D BioPrinting technology is being used to develop advanced medical practices that will help with previously difficult processes, such as delivering drugs via micro-robots, targeting specific cancer cells and even assisting in difficult eye operations. 

The table of contents in this book includes: Chapter 1: 3D Bioprinting: Latest Innovations in a Forty year-old Technology. Chapter 2: LPBI Initiative on 3D BioPrinting, Chapter 3: Cardiovascular BioPrinting, Chapter 4: Medical and Surgical Repairs – Advances in R&D Research, Chapter 5: Organ on a Chip, Chapter 6: FDA Regulatory Technology Issues, Chapter 7: DNA Origami, Chapter 8: Aptamers and 3D Scaffold Binding, Chapter 9: Advances and Future Prospects, Chapter 10: BioInks and MEMS, Chapter 11: BioMedical MEMS, Chapter 12: 3D Solid Organ Printing and Chapter 13: Medical 3D Printing: Sources and Trade Groups – List of Secondary Material. 

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Heat Shock Proteins (HSP) and Molecular Chaperones

Curator: Larry H. Bernstein, MD, FCAP

 

HSP

 

Report on the VIIth International Symposium on Heat Shock Proteins in Biology & Medicine

The major themes of this meeting were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation.
This symposium was the seventh symposium in a series presided over by Dr Stuart Calderwood aimed at exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes. The biochemistry and ultrastructure of molecular chaperones was not emphasized, as these topics are well represented at other meetings. The major themes were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation. This report gives a thematic overview and does not include all the topics presented.
NEW PROPERTIES OF HSPS AND HSF, AND ROLE IN THE ETIOLOGY OF CANCER 

One of the exciting aspects of the meeting involved advances made in understanding the biology of Hsp90. In recent years, we have understood the molecular chaperone activities of Hsp90 mostly in terms of its biochemistry, cooperative interactions with cochaperones. However, Dr Len Neckers (NCI/NIH), the conference keynote speaker, has opened up new areas in our understanding of this chaperone by characterizing the role of posttranslational modification (PTM) in terms of phosphorylation, acetylation, and sumoylation in Hsp90 biology. One particularly intriguing possibility is that altered signaling mechanisms characteristic of cancer may target such PTMs, and this could contribute to the “addiction to chaperones” observed in malignant cells. (Also discussed later by Dr Mehdi Mollapour, SUNY Upstate Medical University).

In addition, interesting differences in properties of the two Hsp90 isoforms have been detected. Dr Wei Li (University of Southern California) has shown that Hsp90a can be released into the extracellular environment and there take part in cell regulation, mediating for instance wound healing effects. In addition, proteomic studies carried out by Thomas Prince (NCI/NIH) in the Neckers lab indicate that Hsp90β may be more dedicated to “housekeeping” molecular chaperone functions while Hsp90α may play more glamorous roles in cell regulation. These distinctions might not be anticipated based on the rather minimal sequence differences between the Hsp90s but offer keen insights into the biology of this chaperone. Finally, Dr Tim Haystead (Duke University) discussed the approach of targeting ectopically expressed Hsp90 for imaging and treatment.

Another PTM with implications in the stress response is the modification of intracellular proteins by monosaccharides of O-linked β-N- acetylglucosamine (O-GlcNAc). Dr Natasha Zachara (Johns Hopkins University School of Medicine) discussed targets for this modification and roles in cytoprotection.

The poster session was also rich in Hsp90 studies, mostly from the Neckers lab—presentations by Kristin Beebe et al. (NCI/NIH) Posttranslational modification state of Hsp90 differentially affects binding of small molecule inhibitors; Toshiki Kijima et al. (NCI/NIH), Defined interactions between HSF1 and Hsp90; T. Prince et al. (NCI/NIH) Hsp90 and tyrosine kinase inhibitors: A synergistic approach towards combating cancer; Andrew W. Truman (University of Chicago)Quantitive ptoteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperonedependent regulation of ribonucleotide reductase. Inhibition of Hsp90 via Cdomain induces temporally distinct phosphorylation patterns; and Diana M. Dunn (SUNY Upstate Medical University)Phosphorylation of human Hsp90 threonine 115 modulates chaperone function and drug sensitivity.

Hsp70 is also emerging as a factor in cell regulation, exhibiting properties beyond a narrow role in chaperoning. Dr Michael Sherman (Boston University) showed a key role for Hsp72 in mammary cancer, and this property did not seem to depend on alterations in protein folding. Instead, Hsp72 appeared to function through its co-chaperone Bag3, a major regulatory molecule in cell signaling. In addition, a presentation by Stuart Calderwood (Harvard Medical School) that included work by Jianlin Gong showed that Hsp72 is required for tumor initiation and metastasis in murine spontaneous breast cancer. These effects appeared to be partially mediated through regulation of expression of the protoooncogene cMet, a key player in invasion and metastasis in cancer. We anticipate advances in understanding of the roles of individual members of the Hsp70 family, as is currently emerging for Hsp90. The prospect of targeting Hsp70 with small molecule inhibitors was elegantly discussed by Maureen Murphy (The Wistar Institute), who introduced a novel class of drugs that could selectively kill cancer cells by inhibiting Hsp70 function. In a related topic, Dr Mathias P. Mayer (University of Heidelberg) showed a detailed analysis of the activities of inhibitors targeting various domains in Hsp70.

Dr Takanori Eguchi (Harvard Medical School) then described his studies showing an unconventional role for the extracellular protease MMP3 as a nuclear protein that could trigger molecular chaperone synthesis (HspA7) in mammary cancer. Interestingly, a role in cancer for the Hsp70 co-chaperone Hsp40 was also shown by Dr Jane Trepel (NCI/NIH).

One presentation that stood apart was that of Dr Carmen Garrido (INSERM U866) who has shown very impressive studies indicating a key role for Hsp70 in hematopoiesis, acting through the factor GATA1. This role appeared to depend on nuclear localization of Hsp70, and Dr Garrido is attempting to study the role of PTM, particularly phosphorylation in this function/localization of Hsp70. This continued the theme of HSP PTM and regulation in the cell.

MOLECULAR CHAPERONES IN AGING

A symposium on molecular chaperones in aging was organized by Dr Shelley Buffenstein (University of Texas Health Science Center San Antonio). This symposium featured some fascinating studies on the naked mole rat (NMR), a rodent with a remarkable lifespan based on size (32 years compared to 3 years in the comparably sized mouse). This has permitted comparative biology studies that have uncovered important aspects of the aging process in mammals. Dr Buffenstein showed that one aspect of the proteotoxic response was enhanced in NMR—proteasome activity that was resistant to oxidative stress as well as conventional proteasome inhibitors. Such proteasome resistance appeared to be conferred by Hsp70 and Hsp40. Karl Rodriguez (also from the UTHSC San Antonio) stressed the importance of Hsp25 in the longevity of NMR. This small HSP is expressed to very high levels in this organism. Kenneth B. Storey (Carleton University) finally gave an encyclopedic presentation entitled “HeatShock Proteins and Hypometabolism in Nature”, discussing the multiple roles of chaperones in hibernation and other processes involving a step down in metabolism.

PROTEIN AGGREGATION DISORDERS AND HSP EXPRESSION

Michael Sherman (Boston University) chaired a lively and highly diverse session on protein aggregation disorders and HSPs. Gary Jones (Maynooth University) discussed his studies on the roles of Hsp104, Hsp70, and Hsp40 in prion propagation in yeast, concentrating on Hsp70. The Hsp complex was able to dissolve prions in yeast. Daniel Kaganovich (Hebrew University) then continued in a yeast theme, discussing a further strategy for resolving proteotoxic stress involving asymmetric cell division in which damaged proteins and mitochondria remain with the mother cell after mitosis. Nava Zaarur (Boston University) then discussed the role of aggresome particles in resolving aggregated proteins, in this case in eucaryotes. Alberto Macario (University of Maryland School of Medicine) discussed the role of chaperonins in proteotoxic disorders dealing with the effects of a pathogenic mutation of human CCT5 on its intrinsic properties. Dr Elaine C. Lee (University of Connecticut) discussed another type of stress. She showed significant roles for chaperones in osmotic stress responses of Caenorhabditis elegans models of polyglutamine diseases.

EXTRACELLULAR HSPS, INFLAMMATION, AND IMMUNITY

Although it is now generally accepted that HSPs can escape the confines of the cell, many questions still remain regarding their extracellular properties, particularly with regard to their immune effects. These questions include: whether HSPs are mostly immunostimulatory or immunosuppressive, whether they can induce sterile inflammation, and what structures on the immune cells recognize the HSPs. Dr Cristina Bonorino (Pontifícia Universidade Católica do Rio Grande do Sul) chaired a symposium “HSP as modulators of immunity: prokaryotic meets eukaryotic” featuring presentations by Robert Binder (University of Pittsburgh), Eckhart R. Podack (University of Miami), Renata Pasqualini (University of New Mexico Medical School), and Cristina Bonorino. In short, the talks indicated that while the prokaryotic chaperone DNA-PK can be immunosppressive and prolong the lifetime of transplanted tissues and reduce the morbidity of arthritis (Drs Bonorino and Kamal Moudgil (University of Maryland School of Medicine)), HSPs can also be immunostimulatory and act as cancer vaccines when associated with cancer antigens (Drs Binder and Podack). In the discussion, it was stressed that these effects may be related to HSP dose, with low doses of HSP antigen complex favoring immunity while higher doses may lead to immunoregulatory effects (Dr Binder). Most parties agreed that much future study is required to resolve all these issues. It was also suggested, inspired by the presentation of Dr Neckers, that HSP PTMs might also be playing roles in shading the immune effects of HSPs (Dr Bonorino). In the next session, Drs Shawn Wang (Virginia Commonwealth University School of Medicine) and John Subjeck (Roswell Park Cancer Institute) discussed the molecular foundations of their highly effective large HSP vaccines that are now in clinical trial for tumor immunotherapy. They indicated that the high avidity for antigen of the larger HSPs might be key for effectiveness. Although the nature of HSP receptors is still not fully resolved, Ayesha Murshid (Harvard Medical School) made a strong case for the scavenger receptor SREC-I as a key molecule in the effects of HSPs on immune cells. As many of the HSPs are in large families, it has not been clear whether all members of Hsp90 or Hsp70 can function outside the cell. Dr Wei Li (University of Southern California) showed that HSP90 family member Hsp90α is the major secreted factor while Dr John Williams (University of Chester) showed potent extracellular effects for human HSP70 isoform HSPA1A. Extracellular roles are not restricted to Hsp90, and Edward O’Brien (Libin Cardiovascular Institute of Alberta/University of Calgary) discussed the extracellular role of heat shock protein 27 (HSPB1) in inflammatory vascular disease. Another lively issue is whether HSPs are released as free proteins, packaged in exosomes, or whether both forms co-exist. This issue was discussed by Monika Fleshner (University of Colorado) and Antonio De Maio (University of California San Diego). Dr De Maio brought up the interesting scenario of Hsp70 binding directly to lipid membranes and perhaps forming membrane channels (Ryan White, University of Maryland).

HSPs are evidently not the only types of stress proteins that can function in the extracellular milieu, as indicated by Dr Michael A. Lynes (University of Connecticut). In a presentation entitled Therapeutic manipulation of the stress response during inflammatory disease, Dr Lynes showed a significant role for extracellular metallothionen in inflammatory bowel disease. Along those lines, Dr George Perdrizet (University of California San Diego) discussed the use of hyperbaric oxygen for enhanced wound healing in diabetic neuropathy, showing impressive clinical findings.

see more at — doi:  10.1007/s12192-014-0562-z

Lens Intermediate Filaments

 The ocular lens assembles two separate Intermediate Filament systems sequentially with differentiation. Canonical 8–11 nm IFs composed of Vimentin are assembled in lens epithelial cells and younger fiber cells, while the fiber cell-specific Beaded Filaments are switched on as fiber cell elongation initiates. Some of the key features of both filament systems are reviewed. Actin filaments and microtubules are essential to the most elemental functions of eukaryotic cells. These filamentous structures are assembled from proteins derived from small, highly conserved gene families. Though tissue specificity exists in the expression of some actin and tubulin family members, they are generally expressed in a ubiquitous manner, and are required for eukaryotic cell survival and replication. In contrast, the family of proteins that comprise the cytoplasmic Intermediate Filaments (IFs) is one of the largest in the human genome with greater than 60 members. IFs are generally not required for cell survival, and are absent from single cell eukaryotes, suggesting a more recent appearance on the evolutionary stage, and a less-essential role in the biology of the cell.
The IF family also differs sharply from actins and tubulins in that there is great variation in both size and sequence among the IF proteins, with sequence identity falling below 30% between more distant members of the human IF family. However, despite the large number of IF proteins available for the construction of IFs, any given cell typically expresses only 1–3 IF proteins, with expression tightly restricted to cell type and state of differentiation. This suggests a considerable degree of cell-specific specialization.
While IF proteins show considerable sequence and size variation, they are unified into a family on the basis of three major features:
1. Predicted domain structure (see figure 1): Algorithms that predict coiled-coil structure show a common predicted domain structure consisting of a) head and tail domains which are quite variable in both size and sequence, and b) a central rod domain where the size (~310 amino acids) and predicted secondary structure is strongly conserved. The rod domain consists of large regions of alpha helical structure (coil domains) interrupted by short non-helical regions (“linkers”) that connect the coil domains. The size, number, and placement of linkers and coils are well-conserved. Moreover, the coil domains exhibit a heptad repeat pattern where the 1, 4 positions in the heptad are dominated by hydrophobic amino acids. Because the 1,4 positions are aligned on one  side of the helix, they form a largely hydrophobic “stripe” that runs along one side of the alpha helix. This stripe mediates the dimerization of two matched coil domains. The hydrophobic stripe gently twists around the axis of the helix, giving rise to a supercoiling of two alpha helices, hence the “coiled-coil”.
The requisite hydrophobicity at the 1, 4 positions of the heptad can be conferred by any of several amino acids, thus the central rod domain of IF proteins, while showing conserved secondary structure, also exhibits a generally high degree of sequence variation. The exceptions to this are two short motifs found at either end of the central rod domain, commonly called the Helix Initiation Motif (HIM) and the Helix termination Motif (HTM). At these two sites the primary sequence among IF proteins is well conserved. Not surprisingly, the HIM and HTM motifs are intolerant of mutations, with the majority of known IF diseases arising from point mutations in these sites (http://www.interfil.org).
2. Conserved gene structure: Sequence analysis of IF proteins has allowed clustering of IF proteins into several major classes. Sequence conservation in the rod domain is high within a class (typically greater than 70%) but low between classes. Analysis of the IF genes shows that there is conservation of gene structure as well within the IF family, with the number and placement of introns and exons well conserved, especially in the central rod domain. The degree of gene structure conservation correlates well with the degree of primary sequence conservation, and reinforces the grouping of IF proteins into classes on the basis of primary sequence.
The Type I and II IF classes are called cytokeratins, and these comprise the IFs of epithelia. These begin assembly as an obligate heterodimer of one Type I and one Type II cytokeratin. The Type III IF proteins include vimentin, desmin, GFAP, and peripherin, and these are commonly found in tissues of mesenchymal origin. While Type III IF proteins can heterodimerize, they are more commonly found as homomeric filaments. The Type IV IF proteins are the neurofilament proteins Heavy (NFH), Medium (NFM) and Light (NFL), which assemble collectively into the IFs of neurons.
3. IF proteins form 8–11 nm diameter IFs. Ultimately, despite the differences in head/ tail size and sequence, and variation in the rod domain sequence, all cytoplasmic IF proteins typically assemble into 8–11 nm IFs.
The mechanism by which vimentin is removed as the cell transitions to the organelle-free state is unknown. In cells undergoing mitosis, vimentin IFs are routinely dismantled by phosphorlylation (Inagaki, Nishi et al. 1987), a modification that causes the relatively stable IF polymer break up into smaller subunits, thought to be tetramers. These are subsequently reassembled after cell division is complete. However, vimentin in lens fiber cells appears to removed, and not simply dismantled. IFs are known to be among the first targets of calcium activated proteases (calpains) in cells that are damaged, and many investigators have demonstrated the calcium-activated degradation of both vimentin and BFs in lens(Yoshida, Murachi et al. 1984; Truscott, Marcantonio et al. 1990; Marcantonio and Duncan 1991; Bettelheim, Qin et al. 1995; Andersson, Sjostrand et al. 1996; Sanderson, Marcantonio et al. 1996; Sanderson, Marcantonio et al. 2000). The dismantling of organelles implies a potential release of calcium from organelles in which it is otherwise routinely sequestered. Whether this release occurs, and whether it alters cytoplasmic calcium levels to a degree that would activate those calpains present in the fiber cell is not known.
Caspases activated in the apoptotic cascade can target conserved sites in IF proteins(Caulin, Salvesen et al. 1997). The elimination of organelles from the fiber cell represents an incomplete apoptotic event, and thus those enzymes responsible for organelle elimination may also represent a viable mechanism for explanation of vimentin’s suggested disappearance(Oshima 2002; Omary, Coulombe et al. 2004).
The loss or reduction of vimentin levels does not leave the mature lens fiber cell devoid of an IF system, however. In a manner that emulates IF switching seen in stratified epithelia, a second generation IF system is switched on in the lens as vimentin is being switched off. It is here where the story of the lens IF system takes the most unusual turn yet described in the IF field.
The initial recognition that the mature lens fiber cells departed from the IF dogma was made when Maisel and co-workers noted the presence of “Beaded Chain Filaments” or Beaded Filaments (BFs) in an electron microscopic analysis of chick lens homogenates (Maisel and Perry 1972; Maisel 1977; Bradley and Maisel 1978; Bradley, Ireland et al. 1979). These studies noted a clearly filamentous structure that was distinct from thin filaments, microtubules, and IFs, which at that time were emerging as the universal cytoskeletal structures common to essentially all vertebrate cells. Speculation emerged that these structures were thin filaments with bound alpha crystallin particles, or nucleosomes on DNA, but these explanations were ruled out experimentally (Bloemendal, Berbers et al. 1984; Ireland and Maisel 1984).
Consistent with the emerging role of IFAPs in modulating and adapting IF function is the observation that fiber cell vimentin IFs interact with the N Cadherin-gamma catenin complex (Leonard, Chan et al. 2008), lengsin(Wyatt, Gao et al. 2008), MIP(Lindsey Rose, Gourdie et al. 2006), periplakin(Yoon and FitzGerald 2008), tropomodulin(Fischer, Quinlan et al. 2003) and possibly other complexes which are present in lens(Bagchi, Katar et al. 2002; Straub, Boda et al. 2003; Bagchi, Katar et al. 2004). The number of candidate linker proteins demonstrated in lens leads to the expectation that the BF and IF are likely to accomplish multiple functions, and that these may be modulated as differentiation progresses, and as the fiber cell proteome changes, either by expression or proteolysis. Similarly, the small heat shock proteins, whose chaperone function appears essential to IF/BF assembly and maintenance, must be considered as critical parts of the biology of IFs in lens. Mutations in the small heat shock proteins have been shown to precipitate a failure in the IF systems in many tissues, and in lens specifically, and to subsequently emulate IF diseases (FitzGerald and Graham 1991; Nicholl and Quinlan 1994; Carter, Hutcheson et al. 1995; Vicart, Caron et al. 1998; Muchowski, Valdez et al. 1999; Perng, Cairns et al. 1999; Perng, Muchowski et al. 1999; Evgrafov, Mersiyanova et al. 2004; Treweek, Rekas et al. 2005; Song, Hanson et al. 2008). The growing multiplicity of IF interactions underscores the need to expect that failure in “IF function” in the lens can result from failure in a wide spectrum of proteins that affect assembly, phosphorylation, proteolytic modification, stability, removal, or linkage to other cellular structures, and that “IF failure” is likely to show considerable variability in phenotype.

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens    Edited by Harry Maisel

Daniel L BoyleLarry TakemotoJames P Brady and Eric F Wawrousek
BMC Ophthalmology 2003; 3:3   http://dx.doi.org:/10.1186/1471-2415-3-3

Background: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods: Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results: Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion: These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.

Dogfish a-Crystallin Sequences COMPARISON WITH SMALL HEAT SHOCK PROTEINS AND SCHISTOSOMA EGG ANTIGEN*

Wilfried W. de JongS, Jack A. M. Leunissen, Pieter J. M. Leenen, Anneke Zweers, and Marlies Versteeg
J BIOL CHEM  1988; 263(11):5141-5149

The amino acid sequences of the a-crystallin A and B chains of the dogfish, Squalus acanthias, have been determined. Comparison with a-crystallins from other species reveals that charged amino acid replacements have been strongly avoided in the evolution of this lens protein. The homology of a-crystallins with the small heat shock proteins is pronounced throughout the major part of the proteins, starting from the position of the first intron in the a-crystallin genes, but is also detectable in the amino-terminal sequences of human, Xenopus, and Drosophila small heat shock proteins. In addition, a remarkable short sequence similarity is present only in the amino termini of dogfish aB and Drosophila HSP22. The Schistosoma egg antigen p40 turns out to have a tandomly repeated region of homology with the common sequence domain of a-crystallins and small heat shock proteins. Comparison of hydropathy profiles indicates the conservation of conformation of the common domains in these three families of proteins. Construction of phylogenetic trees suggests that the aA and aB genes apparently originated from a single ancestral small heat shock protein gene and indicates that introns have been lost during the evolution of the heat shock protein.

Acknowledgment – Maisel, H. (ed) (1985) The Ocular Lens, Marcel Dekker, New. York

 

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New subgroups of ILC immune cells discovered through single-cell RNA sequencing

Reporter: Stephen J Williams, PhD

 

UPDATED on 8/8/2020

A Hybrid Deep Clustering Approach for Robust Cell Type Profiling Using Single-cell RNA-seq Data

  1. Suhas Srinivasan1,
  2. Anastasia Leshchyk1,
  3. Nathan J Johnson2 and
  4. Dmitry Korkin1,3

+Author Affiliations

  1. 1 Worcester Polytechnic Institute;
  2. 2 Harvard Medical School and Dana Farber Cancer Institute
  1. * Corresponding author; email: korkin@korkinlab.org

Abstract

Single-cell RNA sequencing (scRNA-seq) is a recent technology that enables fine-grained discovery of cellular subtypes and specific cell states. It routinely uses machine learning methods, such as feature learning, clustering, and classification, to assist in uncovering novel information from scRNA-seq data. However, current methods are not well suited to deal with the substantial amounts of noise that is created by the experiments or the variation that occurs due to differences in the cells of the same type. Here, we develop a new hybrid approach, Deep Unsupervised Single-cell Clustering (DUSC), that integrates feature generation based on a deep learning architecture with a model-based clustering algorithm, to find a compact and informative representation of the single-cell transcriptomic data generating robust clusters. We also include a technique to estimate an efficient number of latent features in the deep learning model. Our method outperforms both classical and state-of-the-art feature learning and clustering methods, approaching the accuracy of supervised learning. We applied DUSC to single-cell transcriptomics dataset obtained from a triple-negative breast cancer tumor to identify potential cancer subclones accentuated by copy-number variation and investigate the role of clonal heterogeneity. Our method is freely available to the community and will hopefully facilitate our understanding of the cellular atlas of living organisms as well as provide the means to improve patient diagnostics and treatment.

Keywords

  • Received January 3, 2020.
  • Accepted May 22, 2020.

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

 

New subgroups of ILC immune cells discovered through single-cell RNA sequencing

SOURCE

http://ki.se/en/news/new-subgroups-of-ilc-immune-cells-discovered-through-single-cell-rna-sequencing?elqTrackId=f79885cef36049e281109c02da213910&elq=ac700a4d4374478b9d6e10e301ae6b90&elqaid=14707&elqat=1&elqCampaignId=14

Updated on 2016-02-15. Published on 2016-02-15Denna sida på svenska

Jenny Mjösberg and Rickard Sandberg are principal investigators at Karolinska Institutet’s Department of Medicine, Huddinge and Department of Cell and Molecular Biology, respectively. Credit: Stefan Zimmerman.

A relatively newly discovered group of immune cells known as ILCs have been examined in detail in a new study published in the journal Nature Immunology. By analysing the gene expression in individual tonsil cells, scientists at Karolinska Institutet have found three previously unknown subgroups of ILCs, and revealed more about how these cells function in the human body.

Innate lymphoid cells (ILCs) are a group of immune cells that have only relatively recently been discovered in humans. Most of current knowledge about ILCs stems from animal studies of e.g. inflammation or infection in the gastrointestinal tract. There is therefore an urgent need to learn more about these cells in humans.

Previous studies have shown that ILCs are important for maintaining the barrier function of the mucosa, which serves as a first line of defence against microorganisms in the lungs, intestines and elsewhere. However, while there is growing evidence to suggest that ILCs are involved in diseases such as inflammatory bowel disease, asthma and intestinal cancer, basic research still needs to be done to ascertain exactly what part they play.

Two research groups, led by Rickard Sandberg and Jenny Mjösberg, collaborated on a study of ILCs from human tonsils. To date, three main groups of human ILCs are characterized. In this present study, the teams used a novel approach that enabled them to sort individual tonsil cells and measure their expression across thousands of  genes. This way, the researchers managed to categorise hundreds of cells, one by one, to define the types of ILCs found in the human tonsils.

Unique gene expression profiles

Rickard Sandberg, credit: Stefan Zimmerman,

“We used cluster analyses to demonstrate that ILCs congregate into ILC1, ILC2, ILC3 and NK cells, based on their unique gene expression profiles,” says Professor Sandberg at Karolinska Institutet’sDepartment of Cell and Molecular Biology, and the Stockholm branch of Ludwig Cancer Research. “Our analyses also discovered the expression of numerous genes of previously unknown function in ILCs, highlighting that these cells are likely doing more than what we previously knew.”

By analysing the gene expression profiles (or transcriptome) of individual cells, the researchers found that one of the formerly known main groups could be subdivided.

Jenny Mjösberg, credit: Stefan Zimmerman.

“We’ve identified three new subgroups of ILC3s that evince different gene expression patterns and that differ in how they react to signalling molecules and in their ability to secrete proteins,” says Dr Mjösberg at Karolinska Institutet’s Department of Medicine in Huddinge, South Stockholm. “All in all, our study has taught us a lot about this relatively uncharacterised family of cells and our data will serve as an important resource for other researchers.”

The study was financed by grants from a number of bodies, including the Swedish Research Council, the Swedish Cancer Society, the EU Framework Programme for Research and Innovation, the Swedish Society for Medical Research, the Swedish Foundation for Strategic Research and Karolinska Institutet.

Publication

The heterogeneity of human CD127+ innate lymphoid cells revealed by single-cell RNA sequencing
Åsa K. Björklund, Marianne Forkel, Simone Picelli, Viktoria Konya, Jakob Theorell, Danielle Friberg, Rickard Sandberg, Jenny Mjösberg
Nature Immunology, online 15 February 2016, doi:10.1038/ni.3368

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Two New Drugs for Inflammatory Bowel Syndrome Are Giving Patients Hope

Reporter: Stephen J. Williams, Ph.D.

Actavis Receives FDA Approval for VIBERZI (eluxadoline) for the Treatment of Irritable Bowel Syndrome with Diarrhea (IBS-D) in Adults -First in class treatment for IBS-D treats hallmark symptoms of IBS-D; abdominal pain and diarrhea

DUBLIN, May 27, 2015 /PRNewswire/ — Actavis plc (NYSE: ACT) announced today that VIBERZI™ (eluxadoline) was approved by the Food and Drug Administration (FDA) as a twice-daily, oral treatment for adults suffering from irritable bowel syndrome with diarrhea (IBS-D). VIBERZI (eluxadoline) has mixed opioid receptor activity, it is a mu receptor agonist, a delta receptor antagonist, and a kappa receptor agonist.

Logo – http://photos.prnewswire.com/prnh/20130124/NY47381LOGO

“The FDA’s approval of VIBERZI is the first step to providing physicians with a new, evidence-based, treatment option for their adult patients with IBS-D,” said David Nicholson, Executive Vice President, Actavis Global Brands R&D. “At Actavis, we are dedicated to providing new treatment options, and the development of new agents that help address the most bothersome symptoms of IBS-D. We are very pleased to be working with the FDA to advance this IBS-D treatment and we eagerly await DEA scheduling determination later this year.”

IBS-D is a multifactorial disorder marked by recurrent abdominal pain or discomfort and altered bowel function that affects as many as 15 million adult Americans, impacting about twice as many women as men.i,ii,iii There are few treatment options available for IBS-D, particularly options that relieve both the diarrhea and abdominal pain associated with IBS-D.

“The unpredictable symptoms experienced by patients with IBS-D can have a significant impact on everyday life,” said William D. Chey, MD, Nostrant Professor of Gastroenterology at the University of Michigan Health System. “It’s exciting when physicians are able to add an additional treatment option like VIBERZI to their toolbox for patients with IBS-D.”

The FDA has recommended that VIBERZI be classified as a controlled substance. This recommendation has been submitted to the U.S. Drug Enforcement Administration (DEA).  Once VIBERZI receives final scheduling designation, the updated label will be available. Pending final scheduling designation, product launch is anticipated in Q1 2016.

About VIBERZI

VIBERZI is an orally active compound indicated for the treatment of irritable bowel syndrome with diarrhea (IBS-D) in men and women. VIBERZI (eluxadoline) has mixed opioid receptor activity, it is a mu receptor agonist, a delta receptor antagonist, and a kappa receptor agonist.

Efficacy was established in two Phase III clinical studies, demonstrating significant superiority over placebo on the composite endpoint of simultaneous improvement in both abdominal pain and diarrhea at both 75 mg and 100 mg twice daily doses. The primary efficacy responder endpoint was evaluated over the duration of double-blind, placebo-controlled treatment. Response rates were compared based on patients who met the daily composite response criteria (improvement in both abdominal pain and stool consistency on the same day) for at least 50% of the days from weeks 1 to 12 (FDA endpoint) and weeks 1 to 26 (European Medicines Agency endpoint).

The most common adverse events in the two Phase III clinical trials were constipation (7% and 8% for eluxadoline 75 mg and 100 mg; 2% for placebo) and nausea (8% and 7% for eluxadoline 75 mg and 100 mg; 5% for placebo). Rates of severe constipation were less than 1% in patients receiving 75 mg and 100 mg eluxadoline. Rates of discontinuation due to constipation were low for both eluxadoline and placebo (≤2%) and similar rates of constipation occurred between the active and placebo arms beyond 3 months of treatment. A total of 2,426 subjects were enrolled across the two studies.

For more information including full prescribing information about VIBERZI at http://www.actavis.com/Actavis/media/PDFDocuments/VIBERZI_PI.pdf

About IBS-D

Irritable bowel syndrome with diarrhea (IBS-D) is a functional bowel disorder characterized by chronic abdominal pain and frequent diarrhea, which affects approximately 15 million patients in the U.S.  Although the exact cause of IBS-D is not known, symptoms are thought to result from a disturbance in the way the gastrointestinal tract and nervous system interact.

IBS-D can be debilitating and there are limited therapeutic options for managing the chronic symptoms. IBS-D is associated with economic burden in direct medical costs and indirect social costs such as absenteeism and lost productivity, along with decreased quality of life.

About Actavis
Actavis plc (NYSE: ACT), headquartered in Dublin, Ireland, is a unique, global pharmaceutical company and a leader in a new industry model—Growth Pharma. Actavis is focused on developing, manufacturing and commercializing innovative branded pharmaceuticals, high-quality generic and over-the-counter medicines and biologic products for patients around the world.

Actavis markets a portfolio of best-in-class products that provide valuable treatments for the central nervous system, eye care, medical aesthetics, gastroenterology, women’s health, urology, cardiovascular and anti-infective therapeutic categories, and operates the world’s third-largest global generics business, providing patients around the globe with increased access to affordable, high-quality medicines. Actavis is an industry leader in research and development, with one of the broadest development pipelines in the pharmaceutical industry and a leading position in the submission of generic product applications globally.

With commercial operations in approximately 100 countries, Actavis is committed to working with physicians, healthcare providers and patients to deliver innovative and meaningful treatments that help people around the world live longer, healthier lives.

Actavis intends to adopt a new global name – Allergan – pending shareholder approval in 2015.

For more information, visit Actavis’ website at www.actavis.com.

Actavis Cautionary Statement Regarding Forward-Looking Statements

Statements contained in this communication that refer to Actavis’ estimated or anticipated future results, including estimated synergies, or other non-historical facts are forward-looking statements that reflect Actavis’ current perspective of existing trends and information as of the date of this communication. Actual results may differ materially from Actavis’ current expectations depending upon a number of factors affecting Actavis’ business. These factors include, among others, the timing and success of product launches; the difficulty of predicting the timing or outcome of product development efforts and regulatory agency approvals or actions, if any; market acceptance of and continued demand for Actavis’ products; difficulties or delays in manufacturing; and such other risks and uncertainties detailed in Actavis’ periodic public filings with the Securities and Exchange Commission, including but not limited to Actavis plc’s Quarterly Report on Form 10-Q for the quarter ended March 31, 2015 and from time to time in Actavis’ other investor communications. Except as expressly required by law, Actavis disclaims any intent or obligation to update or revise these forward-looking statements.

i Camilleri M. Current and future pharmacological treatments for diarrhea-predominant irritable bowel syndrome. Expert Opinion on Pharmacotherapy. 2013;14:1151.

ii Grundmann O, Yoon SL. Irritable bowel syndrome: epidemiology, diagnosis, and treatment: an update for health-care practitioners. Journal of Gastroenterology and Hepatology. 2010;25:691–699.

iii Eluxadoline Xifaxin Summary Final. November 2014.

CONTACTS:
Investors:
Lisa DeFrancesco
(862) 261-7152

Media:
David Belian
(862) 261-8141

SOURCE Actavis plc

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Synergy’s Looming FDA Filing Makes It Pharma of the Month

By James Passeri Follow

| Jan 05, 2016 | 8:39 AM EST  | 0

Keep an eye on Synergy Pharmaceuticals (SGYP) this month: Analysts like it, its shares have waned since a big spike this summer, and the official filing of its star product is expected any day.

When the New York-based pharmaceutical company, which specializes in gastrointestinal therapy, announced that it passed clinical trials on its flagship drug plecanatide this summer, shares rocketed 95%.

But today analysts appear mystified at why the stock has receded 45% from its July high, especially with plecanatide’s new drug application with the Food and Drug Administration expected this month. (It’s currently trading below $6, and the consensus price target is over $13, according to data provided by Bloomberg.)

Synergy should be raking in $600 million from plecanatide, a daily tablet that treats patients with irritable bowel syndrome (IBS), within five years of obtaining FDA approval (expected in 2017, according to equity research firm BTIG. Synergy currently has a market capitalization of just $645 million.

BTIG’s $11 price target is also buoyed by roughly $142 million on the balance sheet, as well as newly appointed management including CFO Gary Sender and COO Troy Hamilton, both former executives at pharma success story Shire (SHPG). Though Shire shares are down just under 4% over the past 12 month, they have rocketed 112% over the past two years.

Synergy also stands to benefit from a growing demand for gastrointestinal treatments, feeding the appetite of Big Pharma for potential acquisitions, according to BTIG.

“With about 45 million Americans suffering from chronic constipation and IBS, and major companies like Allergan(AGN) and Valeant (VRX) focusing their marketing efforts on GI treatments, it seems logical to imagine SGYP as a takeover candidate,” BTIG analyst Timothy Chiang wrote in a November report.

Whether or not this leads to a buyout or another stock surge, Synergy certainly can be counted on for a healthy dose of small-cap volatility as its chief product takes the final steps toward reaching its customers.

 

 

Synergy Pharmaceuticals Announces Successful End-of-Phase 2 Meeting with FDA for Plecanatide in Irritable Bowel Syndrome with Constipation

Download PDF

Pivotal Phase 3 IBS-C Program to be Initiated in the Fourth Quarter of 2014

NEW YORK– Synergy Pharmaceuticals Inc. (NASDAQ:SGYP) today announced that it has successfully completed an End-of-Phase 2 meeting with the U.S. Food and Drug Administration (FDA) on its lead drug plecanatide for the treatment of irritable bowel syndrome with constipation (IBS-C). Agreement was reached with the FDA for the plecanatide pivotal phase 3 IBS-C clinical development program that is scheduled to begin in the fourth quarter of this year.

“We are very pleased with the outcome of our meeting with the FDA and have a clear path forward to start the IBS-C registration program with plecanatide this year,” said Dr. Gary S. Jacob, Chairman and CEO of Synergy. “The pivotal phase 3 IBS-C trials will include both 3.0 mg and 6.0 mg plecanatide, which are consistent with the doses currently being evaluated in our phase 3 chronic idiopathic constipation (CIC) program. Plecanatide has demonstrated a clinical dose-response for efficacy with an excellent tolerability profile that is observed across trials. This is an important advantage as we look to bring two doses to market in both indications and provide physicians with options for addressing individual patient needs.”

Synergy’s pivotal phase 3 IBS-C clinical development program will consist of two registration trials, each including 1,050 patients who will receive either placebo, 3.0 mg or 6.0 mg plecanatide. IBS-C patients successfully completing either of the 12-week placebo-controlled registration trials will be offered enrollment into a long-term safety trial in order to complement and support the ongoing long-term safety database for the CIC indication.

About Plecanatide

Plecanatide is Synergy’s lead uroguanylin analog in late-stage clinical development to treat patients with CIC and IBS-C. Uroguanylin is a natural gastrointestinal (GI) hormone produced by humans in the small intestine and plays a key role in regulating the normal functioning of the digestive tract through its activity on the guanylate cyclase-C (GC-C) receptor. The GC-C receptor is known to be a primary source for stimulating a variety of beneficial physiological responses. Orally administered plecanatide mimics uroguanylin’s functions by binding to and activating the GC-C receptor to stimulate fluid and ion transit required for normal bowel function. Synergy has successfully completed a phase 2b trial of plecanatide in 951 patients with CIC and is currently enrolling patients in two pivotal phase 3 CIC trials. The company also recently announced positive top-line data results from a phase 2b dose-ranging study with plecanatide in patients with IBS-C.

About Synergy Pharmaceuticals

Synergy Pharmaceuticals (NASDAQ:SGYP) is a biopharmaceutical company focused on the development of novel therapies based on the natural human hormone, uroguanylin, to treat GI diseases and disorders. Synergy has created two unique analogs of uroguanylin – plecanatide and SP-333 – designed to mimic the natural hormone’s activity on the GC-C receptor and target a variety of GI conditions. SP-333 is currently in phase 2 development for opioid-induced constipation and is also being explored for ulcerative colitis. For more information, please visit www.synergypharma.com.

 

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PD1 Inhibitor atezolizumab may show promise in bladder cancer in patients with high PDL1 expression

Reporter: Stephen J Williams

Updated 4/15/2016

Promising Immunotherapy Agents on Horizon in Bladder Cancer

Reported from OncLive

Virginia Powers, PhD

Published Online: Monday, November 16, 2015 at http://www.onclive.com/web-exclusives/promising-immunotherapy-agents-on-horizon-in-bladder-cancer

 

thompson

Thomas Powles, MD

The dramatic and often practice-changing findings demonstrated by trials of immunotherapies in melanoma and lung cancer may soon be reflected in the treatment of bladder cancer, according to a summary of ongoing studies1 presented at the 7th European Multidisciplinary Meeting on Urological Cancers (EMUC).

“Immune therapy is a promising new treatment in transitional cell carcinoma (TCC) of the bladder,” said Thomas Powles, MD, medical oncologist, director of St Bartholomew’s Cancer Centre, London. “Until recently, bladder cancer research has been somehow left behind.”

Powles underscored that immune checkpoint inhibitors are active in urothelial bladder cancer (UBC) and provided an overview of the emergence of immune therapy in bladder cancer that focused on agents targeting the immune checkpoint axis, especially the programed death receptor (PD1) and its ligand (PD-L1).

“Each drug has a unique companion diagnostic but the strongest data so far are seen with blocking PD-L1 and atezolizumab,” he said.

The confirmed overall response rate (ORR) by RECIST to atezolizumab are associated with PD-L1 expression levels in the tumor. In a phase I trial of second line atezolizumab (MPDL3280A) in TCC, a response was demonstrated in patients that had previously showed only a 10% response rate to chemotherapy. The ORRs were 43% for patients with tumors expressing high levels of PD-L1 (IHC 2/3) compared to 11% in patients with tumors having low expression (IHC 0 or 1).2

PD-L1 expression on the immune cells (IC) infiltrating the tumor has also been shown to be associated with response. The PD-L1 expression on ICs was evaluated as low, medium, and high in approximately one-third each of 311 patients with locally-advanced or metastatic urothelial carcinoma (mUC) participating in the phase II IMvigor 210 trial, which corresponded to an ORR with atezolizumab of 9%, 10%, and 27% in the respective expression groups.

Overall survival (OS) at a median follow-up of 7 months (range, 0-11) also correlated with expression levels and was 6.7 months in low (IC0/1), not reached in high (IC2/3) expressing patients, and 7.9 months in overall population. However, no difference was seen in progression-free survival (PFS) according to expression levels; median PFS was 2.1 months in the overall population and in patients having both low (IC0/1) and high (IV2/3) expression levels, respectively. These data were emphasized as early response data that are expected to mature in further analyses.3

Powles commented that his team is beginning a phase III randomized trial of atezolizumab in 767 patients with locally-advanced UBC who were also chemotherapy-resistant following 1 to 2 prior lines of a platinum-based regimen. Patients have been stratified by chemotherapy regimen, PD-L1 expression, IHC status, risk factors, and the presence of liver metastasis. The primary endpoint is OS and secondary endpoints include ORR, PFS, and duration of response (DoR), safety, and tolerability. Other objectives include disease control rate and potential biomarkers.

“PD-L1 expression appears important but we need to find other biomarkers,” he remarked.

Powles moved on to discuss the KEYNOTE-012 phase Ib trial of pembrolizumab, an anti-PD1 antibody that blocks interaction with both PD-L1 and PD-L2. In KEYNOTE, pembrolizumab demonstrated anti-tumor activity in patients with recurrent or metastatic PD-L1–positive UBC in 64% of patients experiencing a decrease in target lesions from baseline.4

Combination and adjuvant studies are ongoing, according to Powles. A trial of atezolizumab as adjuvant therapy versus placebo is underway in patients with TCC whose tumors express PD-L1. The trial has a primary endpoint of disease-free survival (DFS).

“Next-generation combination therapy with nivolumab plus ipilimumab is a common sense approach that was tested in advanced melanoma and is now being evaluated in the Danube trial,” Powles said.

Nivolumab, a PD-1 blocking antibody, and ipilimumab, which blocks CTLA-4, will be evaluated in Danube, a randomized phase III study that will enroll 800 patients with untreated metastatic TCC. The endpoints are PFS and OS. Patients are required to have available tissue for PD-L1 testing and no contraindications for immune therapy.

The rationale for the combination was demonstrated in melanoma, where confirmed objective responses were seen in 61% of patients receiving nivolumab plus ipilimumab versus 11% in patients receiving ipilimumab and placebo (P <0.001). Complete responses were reported in 16 patients (22%) with combination compared to no patients receiving ipilimumab monotherapy.5

“It looks like checkpoint inhibition works particularly well in node positive patients; in the future we can see treatment with first-line immunotherapeutic agents,” said Powles.

“We hope that immune therapy will identify a subset of patients who get long-term benefits from immune therapy,” Powles said. “The future looks bright for immunotherapy in bladder cancer.”

References

  1. Powles T. Update on systemic treatments in bladder cancer. Presented at: 7th European Multidisciplinary Meeting on Urological Cancers (EMUC), Barcelona, Spain, November 12–15, 2015.
  2. Powles T, Eder JP, Fine GD, et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature. 2014;515(7528):558-562.
  3. Rosenberg J, Petrylak D, Abidoye O, et al. Atezolizumab in patients (pts) with locally-advanced or metastatic urothelial carcinoma (mUC): Results from a pivotal multicenter phase II study (IMvigor 210). Presented at: 2015 European Cancer Congress; September 25-29; Vienna, Austria. Abstract 21LBA.
  4. Plimack ER, Bellmunt J, Gupta S, et al. Pembrolizumab (MK-3475) for advanced urothelial cancer: Updated results and biomarker analysis from KEYNOTE-012. J Clin Oncol 33, 2015 (suppl; abstr 4502).
  5. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015; 373:23-34.

– See more at: http://www.onclive.com/web-exclusives/promising-immunotherapy-agents-on-horizon-in-bladder-cancer#sthash.c63jReGo.dpuf

Speedy review for Merck’s Keytruda in head and neck cancer

DAILY NEWS | APRIL 14, 2016

SELINA MCKEE

Speedy review for Merck's Keytruda in head and neck cancer

US regulators have agreed to undertake a speedy review of Merck & Co’s application to market immunotherapy Keytruda for the treatment of certain patients with head and neck cancer, it third potential indication in the country.

 

The company is targeting the drug towards patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing chemotherapy.

 

“We are encouraged by the data emerging from our program in this type of cancer, and welcome today’s news as this is an important step toward making Keytruda (pembrolizumab) available to these patients,” said Roger Dansey, senior vice president and therapeutic area head, oncology late-stage development, Merck Research Laboratories.

 

The US Food and Drug Administration has set an action date for Keytruda – an anti-PD-1 therapy dosed as a single agent intravenously every three weeks – of August 9.

 

Keytruda is a humanised monoclonal antibody that boosts the ability of the body’s immune system to help detect and fight tumour cells. The drug has already racked up approvals in melanoma and lung cancer in the US.

Read more at: http://www.pharmatimes.com/Article/16-04-14/Speedy_review_for_Merck_s_Keytruda_in_head_and_neck_cancer.aspx#ixzz45uMyaCdc
Follow us: @PharmaTimes on Twitter

 

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Lab Grown Brains and more from Twittersphere on 3D Bio-Printing News

Curator: Stephen J. Williams, Ph.D

How Tiny Lab-Grown Human Brains Are Giving Big Insights Into Autism and more from the Twittershpere

 

https://twitter.com/singularityhub/status/664508353771610112

(more…)

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New Frontiers in Gene Editing
Gene editing is rapidly progressing from being a research/screening tool to one that promises important applications downstream in drug development, cell therapy and bioprocessing. Cambridge Healthtech Institute’s second annual symposium on New Frontiers in Gene Editing will bring together experts from all aspects of basic science and clinical research to talk about the progress being made in gene editing and how it’s being applied. Knowing the strengths and limitations of the different tools, how does one decide when to use the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system, as opposed to Transcription Activator-Like Effector Nucleases (TALENs), zinc finger nucleases (ZFNs) and other systems? What is being done to overcome some of the inherent challenges with design, delivery and off-target effects, associated with each of these techniques? Experts from pharma/biotech, academic and government labs will share their experiences leveraging the utility of gene editing for diverse applications. REGISTER
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HOT TOPICS to be discussed:

  • How to pick the right tools for gene editing
  • How to set-up and run genome-scale CRISPR screens
  • Striving for better design, targeted delivery and performance
  • CRISPR Screening for drug target identification
  • Gene editing in stem cells
  • Gene editing for cell therapy and regenerative medicine
  • Understanding the pitfalls of gene editing
  • Dealing with off-target effects
Interested in Gene Editing? You may also want to attend our focused short course:

A Primer to Gene Editing

Cambridge Healthtech Institute 250 First Avenue, Suite 300 | Needham, MA 02494 | 781-972-5400 | www.healthtech.com

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FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials

Reporter: Stephen J. Williams, PhD

The recent announcement by Editas CEO Katrine Bosley to pursue a CRSPR/Cas9 gene therapy trial to correct defects in an yet to be disclosed gene to treat one form of a rare eye disease called Leber congenital amaurosis (multiple mutant genes have been linked to the disease) have put an interesting emphasis on the need for a regulatory framework to initiate these trials. Indeed at the 2015 EmTechMIT Conference Editas CEO Katrine Bosley had mentioned this particular issue: the need for discourse with FDA and regulatory bodies to establish guidelines for design of clinical trials using the CRSPR gene editing tool.

See the LIVE NOTES from Editas CEO Katrine Bosley on using CRSPR as a gene therapy from the 2015 EmTechMIT Conference at https://pharmaceuticalintelligence.com/2015/11/03/live-1132015-130pm-the-15th-annual-emtech-mit-mit-media-lab-top-10-breakthrough-technologies-2015-innovators-under-35/

To this effect, I have listed below, the multiple FDA Guidance Documents surrounding gene therapy to show that, in the past year, the FDA has shown great commitment to devise a regulatory framework for this therapeutic area.

Cellular & Gene Therapy Guidance Documents

Withdrawn Guidance Documents

Three other posts on this site goes into detail into three of the above-mentioned Guidance Documents

FDA Guidance on Use of Xenotransplanted Products in Human: Implications in 3D Printing

New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

FDA Guidance Documents Update Nov. 2015 on Devices, Animal Studies, Gene Therapy, Liposomes

 

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Early Diagnosis

Reporter: Stephen J. Williams, Ph.D.

This post contains a curation of all Early Diagnosis posts on this site as well as a curation of the Early Detection Research Network.

Early Research Detection Network (EDRN)

Welcome to EDRN

The Early Detection Research Network (EDRN), an initiative of the National Cancer Institute (NCI), brings together dozens of institutions to help accelerate the translation of biomarker information into clinical applications and to evaluate new ways of testing cancer in its earliest stages and for cancer risk.

Getting Started…

Check out the EDRN Highlights — a listing of our accomplishments and milestones.

 

► Scientific Components ► For Public, Patients, Advocates
► Collaborative Opportunities (how to join EDRN) ► For Researchers

Highlights

Highlights of the accomplishments of the Early Detection Research Network.

A brief list of major EDRN-developed assays that have been adapted for clinical use is described in the table below:

Detection/Biomarker Assay Discovery Refine/Adapt for Clin Use Clinical Validation Clinical Translation
Blood proPSA FDA approved
Urine PCA3 FDA approved
OVA1™ for Ovarian Cancer FDA approved
ROMA Algorithm for CA125 and HE4 Tests for Pelvic Mass Malignancies FDA approved
Blood/DCP and AFP-L3 for Hepatocellular Carcinoma FDA approved
Blood GP73 Together with AFP-L3 used  for monitoring cirrhotic patients for HCC in China
MiPS (Mi Prostate Score Urine test), Multiplex analysis of T2-ERG gene fusion, PCA3 and serum PSA In CLIA Lab
FISH to detect T2S:Erg fusion for Prostate Cancer In CLIA Lab
GSTP1 methylation for repeat biopsies in prostate cancer In CLIA Lab
Mitochondrial deletion for detection of prostate cancer In CLIA Lab
Somalogic 12-marker panel for Lung Cancer In CLIA Lab
80-gene panel for Lung Cancer In CLIA Lab
Vimentin Methylation Marker for Colon Cancer In CLIA Lab
Galectin-3 ligand for detection of adenomas and colon cancer In CLIA Lab
8-gene panel for Barrett’s Esophagus In CLIA Lab
SOPs for Blood (Serum, Plasma), Urine, Stool Frequently used by biomarker research community
EDRN Pre/Validation Specimen Reference Sets (specimens from well characterized and matched cases and controls from specific disease spectra) Frequently used by biomarker research community

Since its inception in 1999 EDRN has achieved several key milestones, summarized below:

1998 through 2000: Inception and Inauguration of EDRN

2001 to 2003: Meeting the Challenges to Harness and Share Emerging Scientific Knowledge

  • EDRN Second Report, Translational Research to Identify Early Cancer and Cancer Risk, October 2002, http://edrn.nci.nih.gov/docs.) published.
  • EDRN joined the Gordon Research Conferences to co-host the New Frontiers in Cancer detection and Diagnosis in 2002.

 

  • Guidelines Set for Studies Measuring Biomarker Predictive Power Journal of National Cancer Institute (Vol. 93, No. 14, July 18, 2001).
  • EDRN Associate Membership Program Initiated: This novel approach to make EDRN inclusive has been extremely successful. EDRN has now more than 120 Associate Members who are significantly contributing to EDRN efforts in biomarker discovery, development and validation.

2003 to 2004: Network Surges Ahead in Real-time

  • Collaborative Discovery and Validation Projects:  More than 100 collaborative projects spanned the various organ sites. These projects are monitored through the EDRN’s electronic System Information System (eSIS).
  • EDRN Virtual Specimen Bank and Validation Management System Launched: The EDRN Virtual Specimen Bank, also known as ERNE knowledge system, was deployed to 10 institutions in early 2003, allowing a common web-based query to search for available specimens across the EDRN Clinical Epidemiology and Validation Centers https://ginger.fhcrc.org/edrn/imp/GateServlet?pwd. VSIMS was created to allow multiple studies to be administered efficiently by minimizing development time with standardization of information and data management across multiple activities and research sites. This system encompasses all the security features of Food and Drug Administration (FDA)-required auditing systems.
  • Partnership on the Plasma Proteome Project (PPP) Initiative of the Human Proteome Organization (HUPO): PPP project was initiated to evaluate multiple technology platforms, develop bioinformatic tools and standards for protein identification, and create a database of the plasma proteome. The entire study was published in the August issue of the journal Proteomics August 2005, Volume 4 (4), pp 1045-1450.

2005 to 2008: An Investment in Prevention

  • In late 2006, EDRN’s Program for Rapid, Independent Diagnostic Evaluation (PRIDE), was established (http://grants.nih.gov/grants/guide/notice-files/NOT-CA-07-003.html ) as an administrative means to assist extramural investigators in successfully conducting cross-laboratory validation of biomarkers. Ten applications have been reviewed and five are being supported.
  • EDRN underwent external reviews in 2007 and 2008.
  • The Canary Foundation, Palo Alto, CA signed a Memorandum of Understanding with EDRN, NCI on supporting prostate cancer surveillance network of investigators from seven institutions. The tissue and serum will be collected during a three-year period and will be made available to extramural scientists for discovery and validation research.
  • The Lustgarten Foundation, N.Y., funded 6 institutions to generate monoclonal antibodies and associated hybridoma cell lines for pancreatic cancer antigens (biomarkers) identified by EDRN and non-EDRN investigators. These resources will be stored at the NCI-Frederick Facility for distribution to extramural investigators.

2009 to 2011: Realizing Investment for Clinical Use

  • Two biomarker tests approved by FDA and two IVDs pending FDA review.
  • Six biomarker tests offered by CLIA labs.
  • One biomarker test approved for clinical use outside the USA

A Curation of Posts on Early Detection of Cancer and Other Early Detection Networks is Included Below

 

BRCA 1 and 2 and Early Detection of Cancer

Early Detection of Prostate Cancer: American Urological Association (AUA) Guideline

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

Warning signs may lead to better early detection of ovarian cancer

Cancer Detection

Biomarker tool development for Early Diagnosis of Pancreatic Cancer: Van Andel Institute and Emory University

China, India, and Russia account for 46% of all new cancer cases globally, as well as 52% of cancer-related mortality per 4/2014 Lancet Oncology article

 

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New Generation of Platinated Compounds to Circumvent Resistance

Curator/Writer: Stephen J. Williams, Ph.D.

Resistance to chemotherapeutic drugs continues to be a major hurdle in the treatment of neoplastic disorders, irregardless if the drug is a member of the cytotoxic “older” drugs or the cytostatic “newer” personalized therapies like the tyrosine kinase inhibitors.  For the platinatum compounds such as cisplatin and carboplatin, which are mainstays in therapeutic regimens for ovarian and certain head and neck cancers, development of resistance is often regarded as the final blow, as new options for these diseases have been limited.

Although there are many mechanisms by which resistance to platinated compounds may develop the purpose of this posting is not to do an in-depth review of this area except to refer the reader to the book   Ovarian Cancer and just to summarize the well accepted mechanisms of cisplatin resistance including:

  • Decreased cellular cisplatin influx
  • Increased cellular cisplatin efflux
  • Increased cellular glutathione and subsequent conjugation, inactivation
  • Increased glutathione-S-transferase activity (GST) and subsequent inactivation, conjugation
  • Increased γ-GGT
  • Increased metallothionenes with subsequent conjugation, inactivation
  • Increased DNA repair: increased excision repair
  • DNA damage tolerance: loss of mismatch repair (MMR)
  • altered cell signaling activities and cell cycle protein expression

Williams, S.J., and Hamilton, T.C. Chemotherapeutic resistance in ovarian cancer. In: S.C. Rubin, and G.P. Sutton (eds.), Ovarian Cancer, pp.34-44. Lippincott, Wilkins, and Williams, New York, 2000.

Also for a great review on clinical platinum resistance by Drs. Maritn, Hamilton and Schilder please see the following Clinical Cancer Research link here.

This curation represents the scientific rationale for the development of a new class of platinated compounds which are meant to circumvent mechanisms of resistance, in this case the loss of mismatch repair (MMR) and increased tolerance to DNA damage.

An early step in the production of cytotoxicity by the important anticancer drug cisplatin and its analog carboplatin is the formation of intra- and inter-strand adducts with tumor cell DNA 1-3. This damage triggers a cascade of events, best characterized by activation of damage-sensing kinases (reviewed in 4), p53 stabilization, and induction of p53-related genes involved in apoptosis and cell cycle arrest, such as bax and the cyclin-dependent kinase inhibitor p21waf1/cip1/sdi1 (p21), respectively 5,6. DNA damage significantly induces p21 in various p53 wild-type tumor cell lines, including ovarian carcinoma cells, and this induction is responsible for the cell cycle arrest at G1/S and G2/M borders, allowing time for repair 7,8.  DNA lesions have the ability of  to result in an opening of chromatin structure, allowing for transcription factors to enter 56-58.  Therefore the anti-tumoral ability of cisplatin and other DNA damaging agents is correlated to their ability to bind to DNA and elicit responses, such as DNA breaks or DNA damage responses which ultimately lead to cell cycle arrest and apoptosis.  Therefore either repair of such lesions, the lack of recognition of such lesions, or the cellular tolerance of such lesions can lead to resistance of these agents.

resistmech2

Mechanisms of Cisplatin Sensitivity and Resistance. Red arrows show how a DNA lesion results in chemo-sensitivity while the beige arrow show common mechanisms of resistance including increased repair of the lesion, effects on expression patterns, and increased inactivation of the DNA damaging agent by conjugation reactions

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mechPtresistance

 

 

Increased DNA Repair Mechanisms of Platinated Lesion Lead to ChemoResistance

 

DNA_repair_pathways

Description of Different Types of Cellular DNA Repair Pathways. Nucleotide Excision Repair is commonly up-regulated in highly cisplatin resistant cells

 

 

 

 

 

 

 

 

 

 

 

Loss of Mismatch Repair Can Lead to DNA Damage Tolerance

dnadamage tolerance

 

 

 

 

 

 

 

 

In the following Cancer Research paper Dr. Vaisman in the lab of Dr. Steve Chaney at North Carolina (and in collaboration with Dr. Tom Hamilton) describe how cisplatin resistance may arise from loss of mismatch repair and how oxaliplatin lesions are not recognized by the mismatch repair system.
Cancer Res. 1998 Aug 15;58(16):3579-85.

The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: correlation with replicative bypass of platinum-DNA adducts.

Abstract

Defects in mismatch repair are associated with cisplatin resistance, and several mechanisms have been proposed to explain this correlation. It is hypothesized that futile cycles of translesion synthesis past cisplatin-DNA adducts followed by removal of the newly synthesized DNA by an active mismatch repair system may lead to cell death. Thus, resistance to platinum-DNA adducts could arise through loss of the mismatch repair pathway. However, no direct link between mismatch repair status and replicative bypass ability has been reported. In this study, cytotoxicity and steady-state chain elongation assays indicate that hMLH1 or hMSH6 defects result in 1.5-4.8-fold increased cisplatin resistance and 2.5-6-fold increased replicative bypass of cisplatin adducts. Oxaliplatin adducts are not recognized by the mismatch repair complex, and no significant differences in bypass of oxaliplatin adducts in mismatch repair-proficient and -defective cells were found. Defects in hMSH3 did not alter sensitivity to, or replicative bypass of, either cisplatin or oxaliplatin adducts. These observations support the hypothesis that mismatch repair defects in hMutL alpha and hMutS alpha, but not in hMutS beta, contribute to increased net replicative bypass of cisplatin adducts and therefore to drug resistance by preventing futile cycles of translesion synthesis and mismatch correction.

 

 

The following are slides I had co-prepared with my mentor Dr. Thomas C. Hamilton, Ph.D. of Fox Chase Cancer Center on DNA Mismatch Repair, Oxaliplatin and Ovarina Cancer.

edinborough2mmrtranslesion1

 

 

 

 

 

 

Multiple Platinum Analogs of Cisplatin (like Oxaliplatin )Had Been Designed to be Sensitive in MMR Deficient Tumors

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mmroxaliplatin

 

 

 

 

 

 

edinborough2ptanalogsresist

 

 

 

 

 

 

edinborough2relresistptanalogsdifflines

 

 

 

 

 

 

edinborough2msimlmh2refract

 

 

 

 

 

 

edinborough2gogoxaliplatintrial

 

 

 

 

 

 

 

Please see below video on 2015 Nobel Laureates and their work to elucidate the celluar DNA repair mechanisms.

Clinical genetics expert Kenneth Offit gives an overview of Lynch syndrome, a genetic disorder that can cause colon (HNPCC) and other cancers by defects in the MSH2 DNA mismatch repair gene. (View Video)

 

 

References

  1. Johnson, S. W. et al. Relationship between platinum-DNA adduct formation, removal, and cytotoxicity in cisplatin sensitive and resistant human ovarian cancer cells. Cancer Res 54, 5911-5916 (1994).
  2. Eastman, A. The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology and Therapeutics 34, 155-166 (1987).
  3. Zhen, W. et al. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Molecular and Cellular Biology 12, 3689-3698 (1992).
  4. Durocher, D. & Jackson, S. P. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 13, 225-231 (2001).
  5. el-Deiry, W. S. p21/p53, cellular growth control and genomic integrity. Curr Top Microbiol Immunol 227, 121-37 (1998).
  6. Ewen, M. E. & Miller, S. J. p53 and translational control. Biochim Biophys Acta 1242, 181-4 (1996).
  7. Gartel, A. L., Serfas, M. S. & Tyner, A. L. p21–negative regulator of the cell cycle. Proc Soc Exp Biol Med 213, 138-49 (1996).
  8. Chang, B. D. et al. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells. Oncogene 19, 2165-70 (2000).
  9. Davies, N. P., Hardman, L. C. & Murray, V. The effect of chromatin structure on cisplatin damage in intact human cells. Nucleic Acids Res 28, 2954-2958 (2000).
  10. Vichi, P. et al. Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP. Embo J 16, 7444-7456 (1997).
  11. Xiao, G. et al. A DNA damage signal is required for p53 to activate gadd45. Cancer Res 60, 1711-9 (2000).

Other articles in this Open Access Journal on ChemoResistance Include:

Cancer Stem Cells as a Mechanism of Resistance

An alternative approach to overcoming the apoptotic resistance of pancreatic cancer

Mutation D538G – a novel mechanism conferring acquired Endocrine Resistance causes a change in the Estrogen Receptor and Treatment of Breast Cancer with Tamoxifen

Can IntraTumoral Heterogeneity Be Thought of as a Mechanism of Resistance?

Nitric Oxide Mitigates Sensitivity of Melanoma Cells to Cisplatin

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

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