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Mimicking vaginal cells and microbiome interactions on chip microfluidic culture

Posted in Automated Cell Processing, Bio Instrumentation in Experimental Life Sciences Research, Biological Engineering, Biological Networks, Biotechnology, Cell Level, Clinical & Translational, Clinical Diagnostics, Diagnostics and Lab Tests, Medical Device Therapies for Altzheimer’s Disease, Microbiology, MicroEngineering Cell-Tissue & Systems, Microfuidics, Mobile Healthcare, Organ-on-a-Chip, Patient-centered Medicine, Personal Health Applications: Tech Innovations serves HealhCare, Personalized and Precision Medicine & Genomic Research, Population Health Management, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, Signaling, Tissue Engineering, Women Health, tagged artificial vagina, reproduction, reproductive health, reproductive medicine, reproductive research, reproductive toxicology on February 14, 2023| Leave a Comment »

Mimicking vaginal cells and microbiome interactions on chip microfluidic culture

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Scientists at Harvard University’s Wyss Institute for Biologically Inspired Engineering have developed the world’s first “vagina-on-a-chip,” which uses living cells and bacteria to mimic the microbial environment of the human vagina. It could help to test drugs against bacterial vaginosis, a common microbial imbalance that makes millions of people more susceptible to sexually transmitted diseases and puts them at risk of preterm delivery when pregnant. Vaginal health is difficult to study in a laboratory setting partly because laboratory animals have “totally different microbiomes” than humans. To address this, scientists have created an unique chip, which is an inch-long, rectangular polymer case containing live human vaginal tissue from a donor and a flow of estrogen-carrying material to simulate vaginal mucus.

The organs-on-a-chip mimic real bodily function, making it easier to study diseases and test drugs. Previous examples include models of the lungs and the intestines. In this case, the tissue acts like that of a real vagina in some important ways. It even responds to changes in estrogen by adjusting the expression of certain genes. And it can grow a humanlike microbiome dominated by “good” or “bad” bacteria. The researchers have demonstrated that Lactobacilli growing on the chip’s tissue help to maintain a low pH by producing lactic acid. Conversely, if the researchers introduce Gardnerella, the chip develops a higher pH, cell damage and increased inflammation: classic bacterial vaginosis signs. So, the chip can demonstrate how a healthy / unhealthy microbiome affects the vagina.

The next step is personalization or subject specific culture from individuals. The chip is a real leap forward, it has the prospect of testing how typical antibiotic treatments against bacterial vaginosis affect the different bacterial strains. Critics of organ-on-a-chip technology often raise the point that it models organs in isolation from the rest of the body. There are limitations such as many researchers are interested in vaginal microbiome changes that occur during pregnancy because of the link between bacterial vaginosis and labor complications. Although the chip’s tissue responds to estrogen, but it does not fully mimic pregnancy without feedback loops from other organs. The researchers are already working on connecting the vagina chip to a cervix chip, which could better represent the larger reproductive system.

All these information indicate that the human vagina chip offers a new model to study host-vaginal microbiome interactions in both optimal and non-optimal states, as well as providing a human relevant preclinical model for development and testing of reproductive therapeutics, including live bio-therapeutics products for bacterial vaginosis. This microfluidic human vagina chip that enables flow through an open epithelial lumen also offers a unique advantage for studies on the effect of cervicovaginal mucus on vaginal health as clinical mucus samples or commercially available mucins can be flowed through this channel. The role of resident and circulating immune cells in host-microbiome interactions also can be explored by incorporating these cells into the vagina chip in the future, as this has been successfully done in various other organ chip models.

References:

https://www.scientificamerican.com/article/first-vagina-on-a-chip-will-help-researchers-test-drugs/

https://www.webmd.com/infertility-and-reproduction/news/20230209/scientists-create-vagina-on-chip-what-to-know

https://www.livescience.com/vagina-on-a-chip

https://link.springer.com/article/10.1186/s40168-022-01400-1

https://www.nature.com/articles/s41585-022-00717-8

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Medicine in 2045 – Perspectives by World Thought Leaders in the Life Sciences & Medicine

Posted in Advanced Computing Platform, Artificial Intelligence - General, Big Data, BioBanking, BioIT: BioInformatics, NGS, Clinical & Translational, Pharmaceutical R&D Informatics, Clinical Genomics, Cancer Informatics, Cancer and Current Therapeutics, CAST - Alternative to CRISPR/Cas9, Computational Biology/Systems and Bioinformatics, CRISPR/Cas9 & Gene Editing, Diagnostic Immunology, Digital HealthCare – biotech & internet joint ventures, Disease Biology, DNA repair, Drug Delivery Platform Technology, Gene Editing Impact on Longevity, Genetics & Innovations in Treatment, Genome Biology, Genomic Testing: Methodology for Diagnosis, Health Care System by Country, Health Economics and Outcomes Research, HealthCare IT, Human Immune System in Health and in Disease, Immuno-Oncology & Genomics, Immunodiagnostics, Liquid Biopsy: Circulating Tumor Cells in Urine and Blood, Microfuidics, Single Cell Genomics, Variation in human protein-coding regions on December 9, 2019| Leave a Comment »

Medicine in 2045 – Perspectives by World Thought Leaders in the Life Sciences & Medicine

Reporter: Aviva Lev-Ari, PhD, RN

This report is based on an article in Nature Medicine | VOL 25 | December 2019 | 1800–1809 | http://www.nature.com/naturemedicine

Looking forward 25 years: the future of medicine.

Nat Med 25, 1804–1807 (2019) doi:10.1038/s41591-019-0693-y

Aviv Regev, PhD

Core member and chair of the faculty, Broad Institute of MIT and Harvard; director, Klarman Cell Observatory, Broad Institute of MIT and Harvard; professor of biology, MIT; investigator, Howard Hughes Medical Institute; founding co-chair, Human Cell Atlas.

millions of genome variants, tens of thousands of disease-associated genes, thousands of cell types and an almost unimaginable number of ways they can combine, we had to approximate a best starting point—choose one target, guess the cell, simplify the experiment.
In 2020, advances in polygenic risk scores, in understanding the cell and modules of action of genes through genome-wide association studies (GWAS), and in predicting the impact of combinations of interventions.
we need algorithms to make better computational predictions of experiments we have never performed in the lab or in clinical trials.
Human Cell Atlas and the International Common Disease Alliance—and in new experimental platforms: data platforms and algorithms. But we also need a broader ecosystem of partnerships in medicine that engages interaction between clinical experts and mathematicians, computer scientists and engineers

Feng Zhang, PhD

investigator, Howard Hughes Medical Institute; core member, Broad Institute of MIT and Harvard; James and Patricia Poitras Professor of Neuroscience, McGovern Institute for Brain Research, MIT.

fundamental shift in medicine away from treating symptoms of disease and toward treating disease at its genetic roots.
Gene therapy with clinical feasibility, improved delivery methods and the development of robust molecular technologies for gene editing in human cells, affordable genome sequencing has accelerated our ability to identify the genetic causes of disease.
1,000 clinical trials testing gene therapies are ongoing, and the pace of clinical development is likely to accelerate.
refine molecular technologies for gene editing, to push our understanding of gene function in health and disease forward, and to engage with all members of society

Elizabeth Jaffee, PhD

Dana and Albert “Cubby” Broccoli Professor of Oncology, Johns Hopkins School of Medicine; deputy director, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins.

a single blood test could inform individuals of the diseases they are at risk of (diabetes, cancer, heart disease, etc.) and that safe interventions will be available.
developing cancer vaccines. Vaccines targeting the causative agents of cervical and hepatocellular cancers have already proven to be effective. With these technologies and the wealth of data that will become available as precision medicine becomes more routine, new discoveries identifying the earliest genetic and inflammatory changes occurring within a cell as it transitions into a pre-cancer can be expected. With these discoveries, the opportunities to develop vaccine approaches preventing cancers development will grow.

Jeremy Farrar, OBE FRCP FRS FMedSci

Director, Wellcome Trust.

shape how the culture of research will develop over the next 25 years, a culture that cares more about what is achieved than how it is achieved.
building a creative, inclusive and open research culture will unleash greater discoveries with greater impact.

John Nkengasong, PhD

Director, Africa Centres for Disease Control and Prevention.

To meet its health challenges by 2050, the continent will have to be innovative in order to leapfrog toward solutions in public health.
Precision medicine will need to take center stage in a new public health order— whereby a more precise and targeted approach to screening, diagnosis, treatment and, potentially, cure is based on each patient’s unique genetic and biologic make-up.

Eric Topol, MD

Executive vice-president, Scripps Research Institute; founder and director, Scripps Research Translational Institute.

In 2045, a planetary health infrastructure based on deep, longitudinal, multimodal human data, ideally collected from and accessible to as many as possible of the 9+ billion people projected to then inhabit the Earth.
enhanced capabilities to perform functions that are not feasible now.
AI machines’ ability to ingest and process biomedical text at scale—such as the corpus of the up-to-date medical literature—will be used routinely by physicians and patients.
the concept of a learning health system will be redefined by AI.

Linda Partridge, PhD

Professor, Max Planck Institute for Biology of Ageing.

Geroprotective drugs, which target the underlying molecular mechanisms of ageing, are coming over the scientific and clinical horizons, and may help to prevent the most intractable age-related disease, dementia.

Trevor Mundel, MD

President of Global Health, Bill & Melinda Gates Foundation.

finding new ways to share clinical data that are as open as possible and as closed as necessary.
moving beyond drug donations toward a new era of corporate social responsibility that encourages biotechnology and pharmaceutical companies to offer their best minds and their most promising platforms.
working with governments and multilateral organizations much earlier in the product life cycle to finance the introduction of new interventions and to ensure the sustainable development of the health systems that will deliver them.
deliver on the promise of global health equity.

Josep Tabernero, MD, PhD

Vall d’Hebron Institute of Oncology (VHIO); president, European Society for Medical Oncology (2018–2019).

genomic-driven analysis will continue to broaden the impact of personalized medicine in healthcare globally.
Precision medicine will continue to deliver its new paradigm in cancer care and reach more patients.
Immunotherapy will deliver on its promise to dismantle cancer’s armory across tumor types.
AI will help guide the development of individually matched
genetic patient screenings
the promise of liquid biopsy policing of disease?

Pardis Sabeti, PhD

Professor, Harvard University & Harvard T.H. Chan School of Public Health and Broad Institute of MIT and Harvard; investigator, Howard Hughes Medical Institute.

the development and integration of tools into an early-warning system embedded into healthcare systems around the world could revolutionize infectious disease detection and response.
But this will only happen with a commitment from the global community.

Els Toreele, PhD

Executive director, Médecins Sans Frontières Access Campaign

we need a paradigm shift such that medicines are no longer lucrative market commodities but are global public health goods—available to all those who need them.
This will require members of the scientific community to go beyond their role as researchers and actively engage in R&D policy reform mandating health research in the public interest and ensuring that the results of their work benefit many more people.
The global research community can lead the way toward public-interest driven health innovation, by undertaking collaborative open science and piloting not-for-profit R&D strategies that positively impact people’s lives globally.

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Lab on a chip Enters a New Field

Posted in Bio Instrumentation in Experimental Life Sciences Research, Diagnostics and Lab Tests, Microfuidics, tagged electric field, electrodes, fluidic chamber, lab-on-chip, microfluidics on May 19, 2019| Leave a Comment »

Updated 70/16/2020

In this article, they review the past applications of in vitro models in identifying human hepatotoxins and focus on the use of multiscale experimental models in drug development, including the use of zebrafish and human cell-based, 3-dimensional (3D), microfluidic systems of liver functions as key components in applying Quantitative Systems
Pharmacology (QSP). They have implemented QSP as a platform to improve the rate of success in the process of drug discovery and development of therapeutics.

The stakeholders involved in drug development from academia, industry, and government agencies know the need to improve drug candidate selection by optimizing efficacy while screening out potential toxins so as to concentrate efforts with favorable chances for market approval. A survey of the number of new drugs released between 2000 and 2009 demonstrated a 25-year low in drug approvals despite increases in research and development (R&D) investment.

SOURCE

Click to access nihms-1002432.pdf

Lab on a chip Enters a New Field

Reporter: Irina Robu, PhD

The basis of the lab-on-a-chip is to integrate thousands of biochemical operations onto a single chip that could be done by splitting a single drop of blood collected from the patient in order to get a precise diagnosis of potential diseases. Research on lab-on-a-chip primarily focuses on human diagnostics and DNA analysis. Miniaturization of biochemical operations normally handled in a laboratory has numerous advantages, such as cost efficiency, diagnostic speed and sensitivity. The emergence of the lab-on-a-chip field mainly relies on two core technologies: microfluidics and molecular biology.

The team led by Govind Kaigala at IBM Research-Zurich and the group of Moran Bercovici at Technion-Israel Institute of Technology designed a new device that can effectively control liquids and materials on the micro-scale and have demonstrated that the key to dynamic control of fluid mechanics may be electric. Their research is published on Proceedings of the National Academy of Sciences.

The research team turned to electric field to control the control the motion of fluid in a way that is adjustable. When liquid contacts a surface, it develops a layer of charge; applying an electric field to this layer moves the charges, dragging the liquid with them and creating a net flow.

Using this knowledge, the team calculated a device that uses disk-shaped electrodes implanted in the bottom of a fluidic chamber to produce dipole-like flow patterns in the liquid when an electric field is applied. Placing multiple electrodes together in an array generates “virtual channels” that guide the fluid stream. By altering the voltages on the electrodes, they could then reverse the pattern to create an inner region of flow bounded by an outer region of stagnation, which is useful for selective on-demand mixing. While more applications of these flow patterns have yet to be explored, the control and flexibility the team’s device offers recommend that the lab-on-a-chip dream may finally be within grasp.

SOURCE

https://physicsworld.com/a/microfluidics-enters-a-new-field/

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Print’s Technology Used to Help Produce 3D Printed Glass Molds for Droplet Microfluidic Chips

Posted in 3D Printing for Medical Application, BioPrinting in Regenerative Medicine, Microfuidics, Tissue Engineering, tagged 3D printer, glass, microfluidics, photolithography, polydimethylsiloxane (PDMS) on September 2, 2018| Leave a Comment »

Print’s Technology Used to Help Produce 3D Printed Glass Molds for Droplet Microfluidic Chips

Reporter: Irina Robu, PhD

Scientists from Leibniz HKI, Friedrich Schiller University, the Ilmenau University of Technology, FEMTOprint and the Fraunhofer Institute for Applied Optics and Precision Engineering fabricated 3D polydimethylsiloxane (PDMS) chips for droplet microfluidics by using FEMTOprint’s innovative glass technology to make 3D printed glass molds. The 3D printed glass mold can pack 192 nozzles into a design that’s 25 mm long and 4 mm wide, including all inlets and outlets, which produce monodisperse droplets of 70 µm. It’s also easy to scale this structure so it is capable of holding 1,000 nozzles in a 6.5 cm structure.

FEMTOprint’s direct writing process makes it possible to produce microfluidic designs with diverse levels, continuously changing heights, and complex 3D shapes, along with sub-micrometric resolution. 3D printed glass molds are used to combine the replication and ease of production that soft lithography is capable of with the advantages of high-resolution prototyping. Moreover, it can facilitate fabrication of multilevel structures even ones with gradients of confinement, which can make important droplet microfluidic operations better.

This technique, paired with simple polydimethylsiloxane replica molding, can offer users with a solution for non-specialized and specialized labs in order to customize and expand microfluidic experimentation. In order to leverage the immense potential of droplet microfluidics, the process of chip design and fabrication needs to be simplified. While the PDMS replica molding has significantly transformed the chip-production process, its dependence on 2D-limited photolithography has limited the design possibilities, as well as further dissemination of microfluidics to non-specialized labs. The technique permits new possibilities in the university, meanwhile as of right now, no other methodology exists except this one that allows architectures with structures from 15 µm to hundreds of micrometers in all dimensions to be produced.

According to FEMTOprint, 3D printed glass structures characterize a negative part, and can be used as chips by bonding them to a PDMS slab or glass, which makes it possible to utilize structures, like mirrors, lenses, and filters, that replica molding cannot recreate. Chip fabrication doesn’t have to be the holdup for non-microfluidic labs adopting microfluidic approaches, instead it should be looked at as a way to device novel functionalities, like optical fiber incorporation for fluorescence detection.

SOURCE

https://www.industrial-lasers.com/articles/2018/07/3d-printing-creates-molds-for-droplet-microfluidic-chips.html

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The Role of Exosomes in Metabolic Regulation

Posted in Biological Networks, Chemical Biology and its relations to Metabolic Disease, Exosomes: Natural Carriers for siRNA Delivery, Microfuidics, RNA Biology, tagged alix, annexins, autoimmunity, body fluids, cancer genomics and therapy, caspase-1, cellular immunity, DNA, ESCRT, Exosomes, extracellular vesicles (EVs), flotillin, heat shock proteins, Hsp60, Hsp70, Hsp90, IL18, IL1b, immune cells, immune resistance, Inflammasome, intercellular communication, interleukins, intraluminal vessicles, LRR, metabolic diseases, MicroRNA, microvessicles, miRNA, multiple vesicle bodies (MVBs), myocardial hypertrophy, neurodegenerative diseases, NLRP3, platelets, RNA, Schwann cells, siRNA, tetraspanin on October 8, 2017| Leave a Comment »

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in sciencemission.com

We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.

Background

During the period between 9/2015 and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI) has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group” has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:

QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN

Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD

Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc

SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN

A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD

Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN

Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP

Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP

Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP

Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN

Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN

The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular, neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).

REFERENCES

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Sahasrabudhe P, Rohrberg J, Biebl MM, Rutz DA, Buchner J. The Plasticity of the Hsp90 Co-chaperone System. Molecular Cell 2017 Sept; 67:947–961. http://dx.doi.org/10.1016/j.molcel.2017.08.004

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Genomic Diagnostics: Three Techniques to Perform Single Cell Gene Expression and Genome Sequencing Single Molecule DNA Sequencing

Posted in Analytical Instruments Industry, Bio Instrumentation in Experimental Life Sciences Research, BioIT: BioInformatics, NGS, Clinical & Translational, Pharmaceutical R&D Informatics, Clinical Genomics, Cancer Informatics, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Cell Biology, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, DNA repair, Drug Discovery Chemistry, Gene Regulation and Evolution, Genetics & Innovations in Treatment, Genome Biology, Genomic Testing: Methodology for Diagnosis, Meta-analysis of transcriptome data, Microfuidics, Transcriptomics, tagged ancestor's germline cells, Cell-free nucleic acid sequencing, Immune repertoire sequencing, microfluidic large-scale integration, Single-cell sequencing, Single-neuron genome sequencing on July 4, 2017| Leave a Comment »

Genomic Diagnostics: Three Techniques to Perform Single Cell Gene Expression and Genome Sequencing Single Molecule DNA Sequencing

Curator: Aviva Lev-Ari, PhD, RN

4.2.3

4.2.3 Genomic Diagnostics: Three Techniques to Perform Single Cell Gene Expression and Genome Sequencing Single Molecule DNA Sequencing, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 4: Single Cell Genomics

This article presents Three Techniques to Perform Single Cell Gene Expression and Genome Sequencing Single molecule DNA sequencing

Stephen Quake, Quake Lab @Stanford University
Ido Amit, AmitLab @Department of Immunology at the Weizmann Institute of Science, Rehovot, Israel
Gilad Evrony – EPPENDORF 2016 GRAND PRIZE WINNER

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3D Liver Model in a Droplet

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomedical Measurement Science, Cell Biology, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Diagnostics, Liver & Digestive Diseases Research, Microfuidics, Organ-on-a-Chip & 3D Printing in Life Sciences, tagged 3D Liver Model in a Droplet, liver toxicity, microfluidics on February 14, 2017| Leave a Comment »

3D Liver Model in a Droplet

Curator: Marzan Khan, BSc

Recently, a Harvard University Professor of Physics and Applied Physics, David Weitz and his team of researchers have successfully generated 3D models of liver tissue composed of two different kinds of liver cells, precisely compartmentalized in a core-shell droplet, using the microfluidics approach(1). Compared to alternative in-vitro methods, this approach comes with more advantages – it is cost-effective, can be quickly assembled and produces millions of organ droplets in a second(1). It is the first “organ in a droplet” technology that enables two disparate liver cells to physically co-exist and exchange biochemical information, thus making it a good mimic of the organ in vivo(1).

Liver tissue models are used by researchers to investigate the effect of drugs and other chemical compounds, either alone or in combination on liver toxicity(2). The liver is the primary center of drug metabolism, detoxification and removal and all of these processes need to be carried out systematically in order to maintain a homeostatic environment within the body(2) Any deviation from the steady state will shift the dynamic equilibrium of metabolism, leading to production of reactive oxygen species (ROS)(2). These are harmful because they will exert oxidative stress on the liver, and ultimately cause the organ to malfunction. Drug-induced liver toxicity is a critical problem – 10% of all cases of acute hepatitis, 5% of all hospital admissions, and 50% of all acute liver failures are caused by it(2).

Before any novel drug is launched into the market, it is tested in-vitro, in animal models, and then progresses onto human clinical trials(1). Weitz’s system can produce up to one-thousand organ droplets per second, each of which can be used in an experiment to test for drug toxicity(1). Clarifying further, he asserts that “Each droplet is like a mini experiment. Normally, if we are running experiments, say in test tubes, we need a milliliter of fluid per test tube. If we were to do a million experiments, we would need a thousand liters of fluid. That’s the equivalent of a thousand milk jugs! Here, each droplet is only a nanoliter, so we can do the whole experiment with one milliliter of fluid, meaning we can do a million more experiments with the same amount of fluid.”

Testing hepatocytes alone on a petri dish is a poor indicator of liver-specific functions because the liver is made up of multiple cells systematically arranged on an extracellular matrix and functionally interdependent(3). The primary hepatocytes, hepatic stellate cells, Kupffer cells, endothelial cells and fibroblasts form the basic components of a functioning liver(3). Weitz’s upgraded system contains hepatocytes (that make up the majority of liver cells and carry out most of the important functions) supported by a network of fibroblasts(3). His microfluidic chip is comprised of a network of constricted, circular channels spanning the micrometer range, the inner phase of which contains hepatocytes mixed in a cell culture solution(3). The surrounding middle phase accommodates fibroblasts in an alginate solution and the two liquids remain separated due to differences in their chemical properties as well as the physics of fluids travelling in narrow channels. Addition of a fluorinated carbon oil interferes with the two aqueous layers, forcing them to become individual monodisperse droplets(3). The hydrogel shell is completed when a 0.15% solution of acetic acid facilitates the cross-linking of alginate to form a gelatinous shell, locking the fibroblasts in place(3). Thus, the aqueous core of hepatocytes are encapsulated by fibroblasts confined to a strong hydrogel network, creating a core-shell hydrogel scaffold of 3D liver micro-tissue in a droplet(3). Using empirical analysis, scientists have shown that albumin secretion and urea synthesis (two important markers of liver function) were significantly higher in a co-culture of hepatocytes and fibroblasts 3D core-shell spheroids compared to a monotypic cell-culture of hepatocyte-only spheroids(3). These results validate the theory that homotypic as well as heterotypic communication between cells are important to achieve optimal organ function in vitro(3).

This system of creating micro-tissues in a droplet with enhanced properties is a step-forward in biomedical science(3). It can be used in experiments to test for a myriad of drugs, chemicals and cosmetics on different human tissue samples, as well as to understand the biological connectivity of contrasting cells(3).

Image source: DOI: 10.1039/c6lc00231

A simple demonstration of the microfluidic chip that combines different solutions to create a core-shell droplet consisting of two different kinds of liver cells.

References:

National Institute of Biomedical Imaging and Bioengineering. (2016, December 13). New device creates 3D livers in a droplet.ScienceDaily. Retrieved February 9, 2017 from https://www.sciencedaily.com/releases/2016/12/161213112337.htm
Singh, D., Cho, W. C., & Upadhyay, G. (2015). Drug-Induced Liver Toxicity and Prevention by Herbal Antioxidants: An Overview.Frontiers in Physiology,6, 363. http://doi.org/10.3389/fphys.2015.00363
Qiushui Chen, Stefanie Utech, Dong Chen, Radivoje Prodanovic, Jin-Ming Lin and David A. Weitz; Controlled assembly of heterotypic cells in a core– shell scaffold: organ in a droplet; Lab Chip, 2016, 16, 1346; DOI: 10.1039/c6lc00231