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Archive for the ‘Microbiology’ Category


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

 

The trillions of microbes in the human gut are known to aid the body in synthesizing key vitamins and other nutrients. But this new study suggests that things can sometimes be more adversarial.

 

Choline is a key nutrient in a range of metabolic processes, as well as the production of cell membranes. Researchers identified a strain of choline-metabolizing E. coli that, when transplanted into the guts of germ-free mice, consumed enough of the nutrient to create a choline deficiency in them, even when the animals consumed a choline-rich diet.

 

This new study indicate that choline-utilizing bacteria compete with the host for this nutrient, significantly impacting plasma and hepatic levels of methyl-donor metabolites and recapitulating biochemical signatures of choline deficiency. Mice harboring high levels of choline-consuming bacteria showed increased susceptibility to metabolic disease in the context of a high-fat diet.

 

DNA methylation is essential for normal development and has been linked to everything from aging to carcinogenesis. This study showed changes in DNA methylation across multiple tissues, not just in adult mice with a choline-consuming gut microbiota, but also in the pups of those animals while they developed in utero.

 

Bacterially induced reduction of methyl-donor availability influenced global DNA methylation patterns in both adult mice and their offspring and engendered behavioral alterations. This study reveal an underappreciated effect of bacterial choline metabolism on host metabolism, epigenetics, and behavior.

 

The choline-deficient mice with choline-consuming gut microbes also showed much higher rates of infanticide, and exhibited signs of anxiety, with some mice over-grooming themselves and their cage-mates, sometimes to the point of baldness.

 

Tests have also shown as many as 65 percent of healthy individuals carry genes that encode for the enzyme that metabolizes choline in their gut microbiomes. This work suggests that interpersonal differences in microbial metabolism should be considered when determining optimal nutrient intake requirements.

 

References:

 

https://news.harvard.edu/gazette/story/2017/11/harvard-research-suggests-microbial-menace/

 

http://www.cell.com/cell-host-microbe/fulltext/S1931-3128(17)30304-9

 

https://www.ncbi.nlm.nih.gov/pubmed/23151509

 

https://www.ncbi.nlm.nih.gov/pubmed/25677519

 

http://mbio.asm.org/content/6/2/e02481-14

 

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Medical Scientific Discoveries for the 21st Century & Interviews with Scientific Leaders at https://www.amazon.com/dp/B078313281 – electronic Table of Contents 

Author, Curator and Editor: Larry H Bernstein, MD, FCAP

Available on Kindle Store @ Amazon.com since 12/9/2017

List of Contributors & Contributors’ Biographies

Volume Author, Curator and Editor

Larry H Bernstein, MD, FCAP

Preface, all Introductions, all Summaries and Epilogue

Part One:

1.4, 1.5, 1.6, 2.1.1, 2.1.2, 2.1.3, 2.1.4, 2.2.1, 2.2.2, 2.2.3, 2.3, 2.4, 2.4.1, 2.4.2, 2.5, 2.6.1, 2.6.2, 2.6.3, 2.6.4, 2.7, 2.8, 2.9, 2.10, 3.1, 3.2, 3.3, 3.4, 4.1, 4.2, 4.3

Part Two:

5.2, 5.3, 5.6, 6.1.2, 6.1.4, 6.2.1, 6.2.2, 6.3.2, 6.3.4, 6.3.5, 6.3.6, 6.3.8, 6.3.10, 6.4.1, 6.4.2, 6.5.1.2, 6.5.1.3, 6.5.2.2, 7.1, 7.2, 7.3, 7.4, 7.5, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 8.9.1, 8.9.3, 8.9.4, 8.9.5, 8.9.6, 8.10.1, 8.10.2, 8.10.3, 8.10.4, 9.2, 9.3, 9.5, 9.6, 9.7, 9.8, 9.9, 9.10, 9.11, 9.12, 9.13, 9.14, 9.15, 9.16, 10.2, 10.5, 10.6, 10.7, 10.8, 10.10, 10.11, 11.1, 11.2, 11.3, 11.5, 11.6, 11.7, 12.1, 12.2, 12.3, 12.4, 12.5, 12.7, 12.8, 12.9, 12.10, 12.11, 12.12, 13.1, 13.2, 13.3, 13.6, 13.12, 13.13, 14.1, 14.2

Guest Authors:

Pnina Abir-Am, PhD Part Two: 6.1.1

Stephen J Williams, PhDPart Two: 6.2.6, 6.5.2.2, 10.4, 10.9, 13.4

Aviva Lev-Ari, PhD, RN:

Part One:

1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 2.2.1, 2.3

Part Two:

5.1, 5.4, 5.5, 5.7, 5.8, 5.9, 5.10, 5.11, 6.1.3, 6.2.3, 6.2.4, 6.2.5, 6.3.1, 6.3.3, 6.3.7, 6.3.9, 6.4.3, 6.5.1.1, 6.5.2.1, 6.5.2.2, 6.5.3.1, 6.5.4, 6.5.5, 6,5,6, 8.9.2, 8.10.2, 9.1, 9.4, 10.1, 10.3, 11.4, 12.6, 13.5, 13.7, 13.8, 13.9, 13.10, 13.11

Adam Sonnenberg, BSC, MSc(c)Part Two: 13.9

 

electronic Table of Contents

PART ONE:

Physician as Authors, Writers in Medicine and Educator in Public Health

 

Chapter 1: Physicians as Authors

Introduction

1.1  The Young Surgeon and The Retired Pathologist: On Science, Medicine and HealthCare Policy – Best writers Among the WRITERS

1.2 Atul Gawande: Physician and Writer

1.3 Editorial & Publication of Articles in e-Books by  Leaders in Pharmaceutical Business Intelligence:  Contributions of Larry H Bernstein, MD, FCAP

1.4 Abraham Verghese, MD, Physician and Notable Author

1.5 Eric Topol, M.D.

1.6 Gregory House, MD

1.7 Peter Mueller, MD  Professor of Radiology @MGH & HMS – 2015 Synergy’s Honorary Award Recipient

Summary

Chapter 2: Professional Recognition

Introduction

2.1 Proceedings

2.1.1 Research Presentations

2.1.2 Proceedings of the NYAS

2.1.3 Cold Spring Harbor Conference Meetings

2.1.4 Young Scientist Seminars

2.2 Meet Great Minds

2.2.1 Meet the Laureates

2.2.2 Richard Feynman, Genius and Laureate

2.2.3 Fractals and Heat Energy

2.3 MacArthur Foundation Awards

2.4 Women’s Contributions went beyond Rosie the Riveter

2.4.1 Secret Maoist Chinese Operation Conquered Malaria

2.4.2 Antiparasite Drug Developers Win Nobel

2.5 Impact Factors and Achievement

2.6   RAPsodisiac Medicine

2.6.1 Outstanding-achievements-in-radiology-or-radiotherapy

2.6.2 Outstanding-achievement-in-anesthesiology

2.6.3 Outstanding-achievement-in-pathology

2.6.4 Topics in Pathology – Special Issues from Medscape Pathology

2.7 How to win the Nobel Prize

2.8 Conversations about Medicine

2.9 Current Advances in Medical Technology

2.10 Atul Butte, MD, PhD

Summary

Chapter 3:  Medical and Allied Health Sciences Education

Introduction

3.1 National Outstanding Medical Student Award Winners

3.2 Outstanding Awards in Medical Education

3.3 Promoting Excellence in Physicians and Nurses

3.4 Excellence in mentoring

Summary

Chapter 4: Science Teaching in Math and Technology (STEM)

Introduction

4.1 Science Teaching in Math and Technology

4.2 Television as a Medium for Science Education

4.2.1 Science Discovery TV

4.3 From Turing to Watson

Summary

PART TWO:

Medical Scientific Discoveries Interviews with Scientific Leaders

Chapter 5: Cardiovascular System

Introduction

5.1 Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

5.2 Mitochondrial Dysfunction and Cardiac Disorders

5.3 Notable Contributions to Regenerative Cardiology

5.4 For Accomplishments in Cardiology and Cardiovascular Diseases: The Arrigo Recordati International Prize for Scientific Research

5.5 Becoming a Cardiothoracic Surgeon: An Emerging Profile in the Surgery Theater and through Scientific Publications

5.6 Diagnostics and Biomarkers: Novel Genomics Industry Trends vs Present Market Conditions and Historical Scientific Leaders Memoirs

5.7 CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin

5.8 2013 as A Year of Revolutionizing Medicine and Top 11 Cardiology Stories

5.9 Bridging the Gap in Medical Innovations – Elazer Edelman @ TEDMED 2013

5.10 Development of a Pancreatobiliary Chemotherapy Eluting Stent for Pancreatic Ductal Adenocarcinoma PIs: Jeffrey Clark (MGH), Robert Langer (Koch), Elazer Edelman (Harvard:MIT HST Program)

5.11 Publications on Heart Failure by Prof. William Gregory Stevenson, M.D., BWH

Summary

Chapter 6: Genomics

Introduction
6.1 Genetics before the Human Genome Project

6.1.1 Why did Pauling Lose the “Race” to James Watson and Francis Crick? How Crick Describes his Discovery in a Letter to his Son

6.1.2 John Randall’s MRC Research Unit and Rosalind Franklin’s role at Kings College

6.1.3 Interview with the co-discoverer of the structure of DNA: Watson on The Double Helix and his changing view of Rosalind Franklin

6.1.4 The Initiation and Growth of Molecular Biology and Genomics, Part I

6.2 The Human Genome Project: Articles of Note  @ pharmaceuticalintelligence.com by multiple authors

6.2.1 CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

6.2.2 What comes after finishing the Euchromatic Sequence of the Human Genome?

6.2.3 Human Genome Project – 10th Anniversary: Interview with Kevin Davies, PhD – The $1000 Genome

6.2.4 University of California Santa Cruz’s Genomics Institute will create a Map of Human Genetic Variations

6.2.5 Exceptional Genomes: The Process to find them

6.2.6 Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

6.3 The Impact of Genome Sequencing on Biology and Medicine

6.3.1 Genomics in Medicine – Establishing a Patient-Centric View of Genomic Data

6.3.2 Modification of genes by homologous recombination – Mario Capecchi, Martin Evans, Oliver Smithies

6.3.3 AAAS February 14-18, 2013, Boston: Symposia – The Science of Uncertainty in Genomic Medicine

6.3.4 The Metabolic View of Epigenetic Expression

6.3.5  Pharmacogenomics

6.3.6 Neonatal Pathophysiology

6.3.7 Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

6.3.8 3D mapping of genome in combine FISH and RNAi

6.3.9 Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

6.3.10 DNA mutagenesis and DNA repair

6.4 Scientific Leadership Recognition for Contributions to Genomics

6.4.1 Interview with Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak (44 minutes)

6.4.2 DNA Repair Pioneers Win Nobel – Tomas Lindahl, Paul Modrich, and Aziz Sancar 2015 Nobel Prize in Chemistry for the mechanisms of DNA repair

6.4.3  Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

6.5 Contemporary Field Leaders in Genomics

6.5.1 ROBERT LANGER

6.5.1.1 2014 Breakthrough Prizes Awarded in Fundamental Physics and Life Sciences for a Total of $21 Million – MIT’s Robert Langer gets $3 Million

6.5.1.2 National Medal of Science – 2006 Robert S. Langer

6.5.1.3  Confluence of Chemistry, Physics, and Biology

6.5.2 JENNIFER DOUDNA

6.5.2.1 Jennifer Doudna, cosmology teams named 2015 Breakthrough Prize winners

6.5.2.2 UPDATED – Medical Interpretation of the Genomics Frontier – CRISPR – Cas9: Gene Editing Technology for New Therapeutics

6.5.3 ERIC LANDER

6.5.3.1  2012 Harvey Prize in April 30: at the Technion-Israel Institute of Technology to Eric S. Lander @MIT & Eli Yablonovitch @UC, Berkeley

6.5.4 2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

6.5.5 Recognitions for Contributions in Genomics by Dan David Prize Awards

6.5.6   65 Nobel Laureates meet 650 young scientists covering the fields of physiology and medicine, physics, and chemistry, 28 June – 3 July, 2015, Lindau & Mainau Island, Germany

Summary

Chapter 7: The RNAs

Introduction

7.1 RNA polymerase – molecular basis for DNA transcription – Roger Kornberg, MD

7.2  One gene, one protein – Charles Yanofsky

7.3 Turning genetic information into working proteins – James E. Darnell Jr.

7.4 Small but mighty RNAs – Victor Ambros, David Baulcombe, and Gary Ruvkun, Phillip A. Sharp

7.5 Stress-response gene networks – Nina V. Fedoroff

Summary

Chapter 8: Proteomics, Protein-folding, and Cell Regulation
Introduction.

8.1 The Life and Work of Allan Wilson

8.2 Proteomics

8.3 More Complexity in Protein Evolution

8.4 Proteins: An evolutionary record of diversity and adaptation

8.5 Heroes in Basic Medical Research – Leroy Hood

8.6 Ubiquitin researchers win Nobel – Ciechanover, Hershko, and Rose awarded for discovery of ubiquitin-mediated proteolysis

8.7 Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeostatic regulation

8.8 Dynamic Protein Profiling

8.9 Protein folding

8.9.1 Protein misfolding and prions – Susan L. Lindquist, Stanley B. Prusiner

8.9.2 A Curated Census of Autophagy-Modulating Proteins and Small Molecules Candidate Targets for Cancer Therapy

8.9.3 Voluntary and Involuntary S-Insufficiency

8.9.4 Transthyretin and Lean Body Mass in Stable and Stressed State

8.9.5 The matter of stunting in the Ganges Plains

8.9.6 Proteins, Imaging and Therapeutics

8.10 Protein Folding and Vesicle Cargo

8.10.1 Heat Shock Proteins (HSP) and Molecular Chaperones

8.10.2 Collagen-binding Molecular Chaperone HSP47: Role in Intestinal Fibrosis – colonic epithelial cells and sub epithelial myofibroblasts

8.10.3 Biology, Physiology and Pathophysiology of Heat Shock Proteins

8.10.4 The Role of Exosomes in Metabolic Regulation 


Summary

Chapter 9:  Neuroscience

Introduction

9.1 Nobel Prize in Physiology or Medicine 2013 for Cell Transport: James E. Rothman of Yale University; Randy W. Schekman of the University of California, Berkeley; and Dr. Thomas C. Südhof of Stanford University

9.2 Proteins that control neurotransmitter release – Richard H. Scheller

9.3 Heroes in Basic Medical Research – Robert J. Lefkowitz

9.4 MIND AND MEMORY: BIOLOGICAL AND DIGITAL – 2014 Dan David Prize Symposium

9.5 A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

9.6 Role the basal ganglia

9.7 The Neurogenetics of Language – Patricia Kuhl – 2015 George A. Miller Award

9.8 The structure of our visual system

9.9 Outstanding Achievement in Schizophrenia Research

9.10 George A. Miller, a Pioneer in Cognitive Psychology, Is Dead at 92

9.11 – To understand what happens in the brain to cause mental illness

9.12 Brain and Cognition

9.13 – To reduce symptoms of mental illness and retrain the brain

9.14 Behavior

9.15 Notable Papers in Neurosciences

9.16 Pyrroloquinoline quinone (PQQ) – an unproved supplement

Summary

Chapter 10: Microbiology & Immunology

Introduction

10.1 Reference Genes in the Human Gut Microbiome: The BGI Catalogue

10.2 Malnutrition in India, high newborn death rate and stunting of children age under five years

10.3 In His Own Words: Leonard Herzenberg, The Immunologist Who Revolutionized Research, Dies at 81

10.4 Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

10.5 Tang Prize for 2014: Immunity and Cancer

10.6 Halstedian model of cancer progression

10.7 The History of Hematology and Related Sciences

10.8 Pathology Emergence in the 21st Century

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

10.10  T cell-mediated immune responses & signaling pathways activated by TLRs – Bruce A. Beutler, Jules A. Hoffmann, Ralph M. Steinman

10.11 Roeder – the coactivator OCA-B, the first cell-specific coactivator, discovered by Roeder in 1992, is unique to immune system B cells

Summary

Chapter 11: Endocrine Hormones

Introduction

11.1 Obesity – 2010 Douglas L. ColemanJeffrey M. Friedman

11.2 Lonely Receptors: RXR – Jensen, Chambon, and Evans – Nuclear receptors provoke RNA production in response to steroid hormones

11.3 The Fred Conrad Koch Lifetime Achievement Award—the Society’s highest honor—recognizes the lifetime achievements and exceptional contributions of an individual to the field of endocrinology

11.4 Gerald D Aurbach Award for Outstanding Translational Research

11.5 Roy O. Greep Award for Outstanding Research in Endocrinology – Martin M. Matzuk

11.6 American Physiology Society Awards

11.7 Solomon Berson and Rosalyn Yalow

Summary

Chapter 12. Stem Cells

Introduction

12.1 Mature cells can be reprogrammed to become pluripotent – John Gurdon and Shinya Yamanaka

12.2 Observing the spleen colonies in mice and proving the existence of stem cells – Till and McCulloch

12.3 McEwen Award for Innovation: Irving Weissman, M.D., Stanford School of Medicine, and Hans Clevers, M.D., Ph.D., Hubrecht Institute

12.4 Developmental biology

12.5  CRISPR/Cas-mediated genome engineering – Rudolf Jaenisch

12.6 Ribozymes and RNA Machines –  Work of Jennifer A. Doudna

12.7 Ralph Brinster, ‘Father of Transgenesis’

12.8 Targeted gene modification

12.9 Stem Cells and Cancer

12.10 ALPSP Awards

12.11 Eppendorf Award for Young European Investigators

12.12 Breaking news about genomic engineering, T2DM and cancer treatments

Summary
Chapter 13: 3D Printing and Medical Application

Introduction

13.1 3D Printing

13.2 What is 3D printing?

13.3 The Scientist Who Is Making 3D Printing More Human

13.4 Join These Medical 3D Printing Groups on Twitter and LinkedIn for great up to date news

13.5 Neri Oxman and her Mediated Matter group @MIT Media Lab have developed a technique for 3D-printing Molten Glass

13.6 The ‘chemputer’ that could print out any drug

13.7 3-D-Bioprinting in use to Create Cardiac Living Tissue: Print your Heart out

13.8 LPBI’s Perspective on Medical and Life Sciences Applications – 3D Printing: BioInks, BioMaterials-BioPolymer

13.9 Medical MEMS, Sensors and 3D Printing: Frontier in Process Control of BioMaterials

13.10 NIH and FDA on 3D Printing in Medical Applications: Views for On-demand Drug Printing, in-Situ direct Tissue Repair and Printed Organs for Live Implants

13.11 ‘Pop-up’ fabrication technique trumps 3D printing

13.12 Augmentation of the ONTOLOGY of the 3D Printing Research

13.13 Superresolution Microscopy

Summary

Chapter 14: Synthetic Medicinal Chemistry

Introduction

14.1 Insights in Biological and Synthetic Medicinal Chemistry

14.2 Breakthrough work in cancer

Summary to Part Two

Volume Summary and Conclusions

EPILOGUE

 

 

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

During pregnancy, the baby is mostly protected from harmful microorganisms by the amniotic sac, but recent research suggests the baby could be exposed to small quantities of microbes from the placenta, amniotic fluid, umbilical cord blood and fetal membranes. One theory is that any possible prenatal exposure could ‘pre-seed’ the infant microbiome. In other words, to set the right conditions for the ‘main seeding event’ for founding the infant microbiome.

When a mother gives birth vaginally and if she breastfeeds, she passes on colonies of essential microbes to her baby. This continues a chain of maternal heritage that stretches through female ancestry for thousands of generations, if all have been vaginally born and breastfed. This means a child’s microbiome, that is the trillions of microorganisms that live on and in him or her, will resemble the microbiome of his/her mother, the grandmother, the great-grandmother and so on, if all have been vaginally born and breastfed.

As soon as the mother’s waters break, suddenly the baby is exposed to a wave of the mother’s vaginal microbes that wash over the baby in the birth canal. They coat the baby’s skin, and enter the baby’s eyes, ears, nose and some are swallowed to be sent down into the gut. More microbes form of the mother’s gut microbes join the colonization through contact with the mother’s faecal matter. Many more microbes come from every breath, from every touch including skin-to-skin contact with the mother and of course, from breastfeeding.

With formula feeding, the baby won’t receive the 700 species of microbes found in breast milk. Inside breast milk, there are special sugars called human milk oligosaccharides (HMO’s) that are indigestible by the baby. These sugars are designed to feed the mother’s microbes newly arrived in the baby’s gut. By multiplying quickly, the ‘good’ bacteria crowd out any potentially harmful pathogens. These ‘good’ bacteria help train the baby’s naive immune system, teaching it to identify what is to be tolerated and what is pathogen to be attacked. This leads to the optimal training of the infant immune system resulting in a child’s best possible lifelong health.

With C-section birth and formula feeding, the baby is not likely to acquire the full complement of the mother’s vaginal, gut and breast milk microbes. Therefore, the baby’s microbiome is not likely to closely resemble the mother’s microbiome. A baby born by C-section is likely to have a different microbiome from its mother, its grandmother, its great-grandmother and so on. C-section breaks the chain of maternal heritage and this break can never be restored.

The long term effect of an altered microbiome for a child’s lifelong health is still to be proven, but many studies link C-section with a significantly increased risk for developing asthma, Type 1 diabetes, celiac disease and obesity. Scientists might not yet have all the answers, but the picture that is forming is that C-section and formula feeding could be significantly impacting the health of the next generation. Through the transgenerational aspect to birth, it could even be impacting the health of future generations.

References:

https://blogs.scientificamerican.com/guest-blog/shortchanging-a-babys-microbiome/

https://www.ncbi.nlm.nih.gov/pubmed/23926244

https://www.ncbi.nlm.nih.gov/pubmed/26412384

https://www.ncbi.nlm.nih.gov/pubmed/25290507

https://www.ncbi.nlm.nih.gov/pubmed/25974306

https://www.ncbi.nlm.nih.gov/pubmed/24637604

https://www.ncbi.nlm.nih.gov/pubmed/22911969

https://www.ncbi.nlm.nih.gov/pubmed/25650398

https://www.ncbi.nlm.nih.gov/pubmed/27362264

https://www.ncbi.nlm.nih.gov/pubmed/27306663

http://www.mdpi.com/1099-4300/14/11/2036

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464665/

https://www.ncbi.nlm.nih.gov/pubmed/24848255

https://www.ncbi.nlm.nih.gov/pubmed/26412384

https://www.ncbi.nlm.nih.gov/pubmed/28112736

http://ndnr.com/gastrointestinal/the-infant-microbiome-how-environmental-maternal-factors-influence-its-development/

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Milestones in Physiology & Discoveries in Medicine and Genomics: Request for Book Review Writing on Amazon.com


physiology-cover-seriese-vol-3individualsaddlebrown-page2

Milestones in Physiology

Discoveries in Medicine, Genomics and Therapeutics

Patient-centric Perspective 

http://www.amazon.com/dp/B019VH97LU 

2015

 

 

Author, Curator and Editor

Larry H Bernstein, MD, FCAP

Chief Scientific Officer

Leaders in Pharmaceutical Business Intelligence

Larry.bernstein@gmail.com

Preface

Introduction 

Chapter 1: Evolution of the Foundation for Diagnostics and Pharmaceuticals Industries

1.1  Outline of Medical Discoveries between 1880 and 1980

1.2 The History of Infectious Diseases and Epidemiology in the late 19th and 20th Century

1.3 The Classification of Microbiota

1.4 Selected Contributions to Chemistry from 1880 to 1980

1.5 The Evolution of Clinical Chemistry in the 20th Century

1.6 Milestones in the Evolution of Diagnostics in the US HealthCare System: 1920s to Pre-Genomics

 

Chapter 2. The search for the evolution of function of proteins, enzymes and metal catalysts in life processes

2.1 The life and work of Allan Wilson
2.2  The  evolution of myoglobin and hemoglobin
2.3  More complexity in proteins evolution
2.4  Life on earth is traced to oxygen binding
2.5  The colors of life function
2.6  The colors of respiration and electron transport
2.7  Highlights of a green evolution

 

Chapter 3. Evolution of New Relationships in Neuroendocrine States
3.1 Pituitary endocrine axis
3.2 Thyroid function
3.3 Sex hormones
3.4 Adrenal Cortex
3.5 Pancreatic Islets
3.6 Parathyroids
3.7 Gastointestinal hormones
3.8 Endocrine action on midbrain
3.9 Neural activity regulating endocrine response

3.10 Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious Depression

 

Chapter 4.  Problems of the Circulation, Altitude, and Immunity

4.1 Innervation of Heart and Heart Rate
4.2 Action of hormones on the circulation
4.3 Allogeneic Transfusion Reactions
4.4 Graft-versus Host reaction
4.5 Unique problems of perinatal period
4.6. High altitude sickness
4.7 Deep water adaptation
4.8 Heart-Lung-and Kidney
4.9 Acute Lung Injury

4.10 Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

 

Chapter 5. Problems of Diets and Lifestyle Changes

5.1 Anorexia nervosa
5.2 Voluntary and Involuntary S-insufficiency
5.3 Diarrheas – bacterial and nonbacterial
5.4 Gluten-free diets
5.5 Diet and cholesterol
5.6 Diet and Type 2 diabetes mellitus
5.7 Diet and exercise
5.8 Anxiety and quality of Life
5.9 Nutritional Supplements

 

Chapter 6. Advances in Genomics, Therapeutics and Pharmacogenomics

6.1 Natural Products Chemistry

6.2 The Challenge of Antimicrobial Resistance

6.3 Viruses, Vaccines and immunotherapy

6.4 Genomics and Metabolomics Advances in Cancer

6.5 Proteomics – Protein Interaction

6.6 Pharmacogenomics

6.7 Biomarker Guided Therapy

6.8 The Emergence of a Pharmaceutical Industry in the 20th Century: Diagnostics Industry and Drug Development in the Genomics Era: Mid 80s to Present

6.09 The Union of Biomarkers and Drug Development

6.10 Proteomics and Biomarker Discovery

6.11 Epigenomics and Companion Diagnostics

 

Chapter  7

Integration of Physiology, Genomics and Pharmacotherapy

7.1 Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

7.2 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

7.3 Diagnostics and Biomarkers: Novel Genomics Industry Trends vs Present Market Conditions and Historical Scientific Leaders Memoirs

7.4 Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

7.5 Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling

7.6 Imaging Biomarker for Arterial Stiffness: Pathways in Pharmacotherapy for Hypertension and Hypercholesterolemia Management

7.7 Neuroprotective Therapies: Pharmacogenomics vs Psychotropic drugs and Cholinesterase Inhibitors

7.8 Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

7.9 Preserved vs Reduced Ejection Fraction: Available and Needed Therapies

7.10 Biosimilars: Intellectual Property Creation and Protection by Pioneer and by

7.11 Demonstrate Biosimilarity: New FDA Biosimilar Guidelines

 

Chapter 7.  Biopharma Today

8.1 A Great University engaged in Drug Discovery: University of Pittsburgh

8.2 Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

8.3 Predicting Tumor Response, Progression, and Time to Recurrence

8.4 Targeting Untargetable Proto-Oncogenes

8.5 Innovation: Drug Discovery, Medical Devices and Digital Health

8.6 Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects

8.7 Nanotechnology and Ocular Drug Delivery: Part I

8.8 Transdermal drug delivery (TDD) system and nanotechnology: Part II

8.9 The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

8.10 Natural Drug Target Discovery and Translational Medicine in Human Microbiome

8.11 From Genomics of Microorganisms to Translational Medicine

8.12 Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad

 

Chapter 9. BioPharma – Future Trends

9.1 Artificial Intelligence Versus the Scientist: Who Will Win?

9.2 The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and Potential of Pharmacognosy

9.3 The Vibrant Philly Biotech Scene: Focus on Computer-Aided Drug Design and Gfree Bio, LLC

9.4 Heroes in Medical Research: The Postdoctoral Fellow

9.5 NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

9.6 1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

9.7 Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

9.8 Heroes in Medical Research: Green Fluorescent Protein and the Rough Road in Science

9.9 Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

9.10 The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

Epilogue

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Phosphorylation-dependent interaction between antigenic peptides and MHC class I

Curator: Larry H. Bernstein, MD, FCAP

 

 

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Nat Immunol. 2008 Nov;9(11):1236-43.    http://dx.doi.org:/10.1038/ni.1660.  Epub 2008 Oct 5.
Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of ‘transformed self’. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.
Figure 1
Bioinformatic characterization of the HLA-A2–restricted phosphopeptide repertoire. (a) Distribution of phosphorylated residues among naturally processed (A2 phosphopeptide) and predicted HLA-A2 binding phosphopeptides (Phosphosite, EMBL). The frequency of phosphorylated residues at each position is displayed for naturally processed HLA-A2 associated phosphopeptides, and for peptides in EMBL and Phosphosite datasets that contain phosphorylation sites and are predicted, according to criteria described in Methods, to bind HLA-A2. (b) Representation of positively charged residues (Arg or Lys) at P1 among naturally processed HLA-A2 associated phosphopeptides, phosphopeptides from the EMBL or Phosphosite datasets that are predicted to bind HLA-A2 and contain a p-Ser residue at the P4 position, and datasets of naturally processed non-phosphorylated peptides (B-LCL) and known HLA-A2 binding peptides (Immune Epitope). Selection criteria for the latter two datasets are described in Methods. * = P<0.001, NS= not significant. (c, d) Representation of subdominant residues at the P2 anchor position (c) and the PC (P9) position (d) in naturally processed HLA-A2 associated phosphopeptides and in datasets of naturally processed non-phosphorylated peptides and known HLA-A2 binding peptides.
Changes in protein expression or metabolism due to intracellular infection or cellular transformation modify the repertoire of peptides generated and therefore displayed by class I MHC molecules, resulting in presentation of “altered self” to the immune system. T cell receptor (TCR)-mediated recognition of specific MHC-bound peptides by CD8 T lymphocytes results in cytolytic activity and release of pro-inflammatory cytokines, which are key components of anti-viral and anti-tumor immunity. Evidence suggests that peptides containing post-translational modifications (PTM), including deamidation, cysteinylation, glycosylation, and phosphorylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition2. Indeed, the majority of naturally occurring PTM-bearing peptides defined to date can be discriminated from their unmodified homologs specifically by T cells2-4.  …..
Recent studies have highlighted protein phosphorylation as a process with the capacity to generate unique peptides bound to class I MHC molecules. Significant numbers of different phosphorylated peptides are presented by several HLA-A and HLA-B alleles that are prevalent in humans3,4, demonstrating their widespread potential as antigens. Moreover, CD8+ T lymphocytes recognize these phosphopeptides in a manner that is both peptide sequence-specific and phosphate-dependent3, 4. Thus, phosphopeptides can be immunologically distinguished from their non-phosphorylated counterparts. Consistent with their presentation by class I MHC molecules, most phosphorylated peptides are derived from proteins that function intracellularly, and processing of both model and naturally occurring phosphopeptides is dependent on transport into the endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP)3, 5. Furthermore, rapid degradation by the proteasome, a process that regulates the activity of many transcription factors, cell growth modulators, signal transducers and cell cycle proteins6-8, is frequently dependent on target protein phosphorylation9-11. ….
Phosphopeptide antigens are of significant therapeutic interest because deregulation of protein kinase activity, normally tightly controlled, is one of the hallmarks of malignant transformation and is thought to contribute directly to oncogenic signaling pathways involved in cell growth, differentiation and survival13-15. In addition, mutation-induced deregulation of a limited number of critical kinases can often lead to activation of several signaling cascades and increases in the extent of protein phosphorylation within the cell16-18. These considerations strongly suggest that alterations in protein phosphorylation during malignancy represent a distinctive immunological signature of “transformed self”. Consistent with this notion, the phosphopeptides presented by HLA-A*0201….

Nα-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I–Restricted Nα-Acetylpeptide Presentation

As one of the most common posttranslational modifications (PTMs) of eukaryotic proteins, Nα-terminal acetylation (Nt-acetylation) generates a class of Nα-acetylpeptides that are known to be presented by MHC class I at the cell surface. Although such PTM plays a pivotal role in adjusting proteolysis, the molecular basis for the presentation and T cell recognition of Nα-acetylpeptides remains largely unknown. In this study, we determined a high-resolution crystallographic structure of HLA (HLA)-B*3901 complexed with an Nα-acetylpeptide derived from natural cellular processing, also in comparison with the unmodified-peptide complex. Unlike the α-amino–free P1 residues of unmodified peptide, of which the α-amino group inserts into pocket A of the Ag-binding groove, the Nα-linked acetyl of the acetylated P1-Ser protrudes out of the groove for T cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide but also switches the residues in the α1-helix of HLA-B*3901, which may impact the T cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from different species reveal reduced stability. Our findings provide the insight into the mode of Nα-acetylpeptide–specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.

Produced by Ag processing and proteasomal degradation of intracellular proteins, polypeptides serve as CTL epitopes presented by MHC class I molecules, which play a critical role in cellular immunity (1). Eukaryotic proteins bearing various posttranslational modifications (PTMs) can generate a group of modified Ags, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for TCR-mediated recognition. A modified peptide may become a new Ag because of the distinguished antigenicity compared with its unmodified homolog. A variety of natural peptide Ags containing modification have been observed that can be immunologically discriminated by T cells from their unmodified homologs as “altered self” (2). Thus, the significance of PTMs on epitopes and the application of modified peptides in vaccine development for immunotherapy against cancer and autoimmune diseases have been increasingly appreciated (3, 4).

The molecular bases of the presentation of peptides with several PTMs by MHC class I molecules have been successfully explicated. For instance, the formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of H2-M3 (5); both the glycan and the phosphate moieties of the central region of the glycopeptides (6, 7) and the phosphopeptides (8, 9), respectively, are exposed to enable TCR binding, and the deimination (citrullination) of arginine on a peptide presented by two HLA-B27 subtypes induces distinct peptide conformations (10).

Nα-terminal acetylation (Nt-acetylation) is one of the most common PTMs, occurring on the vast majority of eukaryotic proteins. In humans, >80% of the different varieties of intracellular proteins are irreversibly Nt-acetylated by Nα-acetyltransferases, often after the removal of the initiator methionine. Only a subset of the penultimate residues (Ala, Ser, Thr, Cys, and Val) or the retained initiator methionine can be acetylated at the α-amino (NH2) groups (11). A recent study found that acetylated N-terminal residues of eukaryotic proteins act as specific degradation signals (Ac-N-degrons) that are recognized by specific ubiquitin ligases (12). A subsequent systematic analysis demonstrated that Nt-acetylation can also represent an early determining factor in the cellular sorting for prevention of protein targeting to the secretory pathway (13). These findings suggested that Nt-acetylation–mediated inhibition of secretion could contribute to the retention of proteins in the cytosol where they may subsequently be ubiquitinylated through the specific recognition of their Ac-N-degrons and thereby generating Nt-acetylated proteasomal digestion products (14). Hence, these Nt-acetylated polypeptides in the form of MHC-associated neoantigens stand a good chance to be recognized by T cells. This has indeed been illuminated in an Nt-acetylated MHC class II–restricted peptide derived from myelin basic protein, which stimulates murine T cells to elicit experimental autoimmune encephalomyelitis, whereas the nonacetylated form does not (15). A structural study subsequently suggested that the Nt-acetylation of this peptide is essential for MHC class II binding (16).

For MHC class I, the first Nt-acetylated natural ligand was identified more than a decade ago (17). However, the mode of interaction of this acetylated peptide with class I molecules remained largely enigmatic. To understand this, we determined the crystal structures of a naturally occurring Nt-acetylated self-peptide (NAc-SL9) and two nonmodified variants (SL9 and HL8), respectively, in complex with HLA-B*3901. Taken together with the thermostability analyses of Nα-acetylpeptides complexed with a series of class I molecules of human and murine origin, we elucidated that Nt-acetylation exerts a destabilizing effect on peptide–MHC (pMHC) complex, thereby influencing TCR recognition.

……

Our results here provide the structural and thermodynamic insights into the presentation of Nt-acetylated peptides by MHC class I molecules. The structure of the Nα-acetylpeptide in complex with HLA-B*3901 outlines a molecular interpretation of the reduced stability of MHC class I–bound Nt-acetylated peptides and also highlights a potential influence of Nt-acetylation on antigenic identity and T cell recognition. In addition, the structure elucidation of HLA-B*3901, the predominant B39 subtype, also is valuable in studying immune diseases associated with this MHC allele.

In a previous report, the Nt-formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of the murine MHC class I H2-M3 playing an anchoring role for MHC class I binding (Supplemental Fig. 2A) (5). In our study, the methyl and carbonyl groups of the acetyl are rotated upwards like two arms that push the peptide-binding groove open (Fig. 2G, Supplemental Fig. 2B), thereby altering its immunogenicity at the expense of the pMHC stability. The thermostability we tested from seven human and one murine complexes indicates a general feature of Nα-acetylpeptide in weakening the binding affinity to MHC class I, which could be revealed by the gel-filtration chromatography of pMHC refolding assays as well (Supplemental Fig. 3). Their instability would partially explain why, as yet, such epitopes are rarely found. Within N-terminal residues of eukaryotic proteins, Ser is the most frequently acetylated in vivo (11). The Ala, Thr, Cys, and Val residues can also be Nt-acetylated and have small side chains like Ser. Thus, the rotation of P1 residues observed in the pHLA-B*3901 complex with an acetylated P1-Ser could very well be a general mode in Nα-acetylpeptide binding. In contrast, the long side chain of Met precludes it from being rotated into pocket A, but a certain reorientation is presumed to take place in the acetylated P1-Met based on the thermal instability (Fig. 6H). Besides the accommodation of the acetyl moiety, Nt-acetylation is presumed to decrease the stability of the pHLA-B*3901 complex as a result of the conformational switch of the Arg62. Arg62 in the α1-helix is largely conserved in almost all HLA-B and -C allotypes (Table V). For other HLA class I (Table V, Fig. 8), the long charged side chains of the residues in position 62 (Glu62 of A24 and Gln62 of A11 and so on) also may interact with the acetyl. Hence, the residue in position 62 plays a key role in the interaction between acetyl group and the H chain, which may influence not only the Nα-acetylpeptide binding to HLA molecules but also the TCR docking.

The discoveries that intracellular proteins with Ac-N-degrons are inhibited from being secreted (13) and then are degraded via ubiquitylation (12) raise many questions on the biological significance of acetylation-mediated proteolysis (14). The Nt-acetylated peptides with the size of MHC class I ligands (8–11 aa) as neoepitopes for CD8+ T cells, represent one of the possible roles of the Nt-acetylated digestion products. The vast armory of intracellular proteins that are frequently Nt-acetylated can create a large pool of Nα-acetylpeptides for Ag presentation and T cell surveying. The Nt-acetylation potentially impacts the TCR-MHC interaction in three different aspects: 1) the direct interaction of the solvent-exposed acetyl moiety; 2) the altered conformation of the central region of the peptide main chain; and 3) the conformational switches of the MHC residues. The Nt-acetylation creation of a distinctive pMHC landscape and participation in a potential binding element for TCR engagement described in our results highlights needs for further investigation into the Nα-acetylpeptide–specific TCR repertoires.  ……

see…J Immunol 2014; 192:5509-5519   http://dx.doi.org:/10.4049/jimmunol.1400199   http://www.jimmunol.org/content/192/12/5509

Supplementary http://www.jimmunol.org/content/suppl/2014/05/14/jimmunol.1400199.DCSupplemental.html
References http://www.jimmunol.org/content/192/12/5509.full#ref-list-1

 

The Cellular Redox Environment Alters Antigen Presentation*

Jonathan A. Trujillo,§12Nathan P. Croft,1Nadine L. Dudek,1Rudragouda ChannappanavarAlex TheodossisAndrew I. Webb,…., Jamie Rossjohn,‡‡,§§5Stanley Perlman,§6 and Anthony W. Purcell,7
The Journal of Biological Chemistry 289; 27979-27991.
http://dx.doi.org:/10.1074/jbc.M114.573402

Capsule

Background: Modification of cysteine residues, including glutathionylation, commonly occurs in peptides bound to and presented by MHC molecules.

Results: Glutathionylation of a coronavirus-specific T cell epitope results in diminished CD8 T cell recognition.

Conclusion: Cysteine modification of a T cell epitope negatively impacts the host immune response.

Significance: Cross-talk between virus-induced oxidative stress and the T cell response probably occurs, diminishing host cell recognition of infected cells.

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5–10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.

Antigen Presentation     Antigen Processing     Glutathionylation     Mass Spectrometry (MS)     Oxidation-Reduction (Redox)     Redox Regulation     T-cell     Viral Immunology

Small fragments of proteins (peptides) derived from both intracellular and extracellular sources are displayed on the surface of cells by molecules encoded within the major histocompatibility complex (MHC). These peptides are recognized by T lymphocytes and provide the immune system with a surveillance mechanism for the detection of pathogens and cancer cells. The fidelity with which antigen presentation communicates changes in the intracellular proteome is critical for immune surveillance. Not only do antigens expressed at vastly different abundances need to be represented within the array of peptides selected and presented at the cell surface (collectively termed the immunopeptidome (1, 2)), but changes in their post-translational state also need to be conveyed within this complex mixture of peptides. For example, changes in antigen phosphorylation have been linked to cancer, and the presentation of phosphorylated peptides has been shown to communicate the cancerous state of cells to the immune system (36). Other types of post-translational modification play a central role in the pathogenesis of autoimmune diseases (7), such as arginine citrullination in arthritis (810), deamidation of glutamine residues in wheat proteins in celiac disease (1115), and cysteine oxidation in type 1 diabetes (16, 17). Cysteine is predicted to be present in up to 14% of potential T cell epitopes based on its prevalence in various pathogen and host proteomes (18). However, reports of cysteine-containing epitopes are much less frequent due to technical difficulties associated with synthesis and handling of cysteine-containing peptides and their subsequent avoidance in many epitope mapping studies (19). Cysteine can be modified in numerous ways, including cysteinylation (the disulfide linkage of free cysteine to peptide or protein cysteine residues), oxidation to cysteine sulfenic (oxidation), sulfinic (dioxidation) and sulfonic acids (trioxidation), S-nitrosylation, and S-glutathionylation. Such modifications may occur prior to or during antigen processing; however, the role of cysteine modification in T-cell-mediated immunity has not been systematically addressed.

In addition to constitutive presentation of a subset of oxidatively modified peptides, it is anticipated that changes in the proportion of these ligands will occur upon infection because oxidative stress, triggering of the unfolded protein response, and modulation of host cell synthesis by the virus are hallmarks of this process (2027). For example, host cell stress responses modulate expression, localization, and function of Toll-like receptors, a key event in the initiation of the immune response (28). Oxidative stress would also be predicted to affect protein function through post-translational modification of amino acids, such as cysteine. Indeed, because of the reactive nature of cysteine and the requirements for cells to regulate the redox state of proteins to maintain function, a number of scavenging systems for redox-reactive intermediates exist. The tripeptide glutathione (GSH) is one of the key intracellular antioxidants, acting as a scavenger for reactive oxygen species. Reduced GSH is equilibrated with its oxidized form, GSSG, with normal cytosolic conditions being that of the reduced state in a ratio of ∼50:1 (GSH/GSSG) (29). Modification of proteins and peptides with GSH (termed S-glutathionylation) occurs following reaction of GSSG with the thiol group of cysteine in a reaction catalyzed by the detoxifying enzyme, glutathione S-transferase (GST). A variety of cellular processes and signaling pathways, such as the induction of innate immunity, apoptosis, redox homeostasis, and cytokine production, are modulated by this GST-catalyzed post-translational modification (3032). S-Glutathionylation can eventuate via oxidative stress, whereby the intracellular levels of GSSG increase.

Given that viruses are known to induce oxidative stress (3335), the intracellular environment of viral infection may lead to an increase inS-glutathionylated cellular proteins and viral antigens. For instance, HSV infection induces an early burst of reactive oxygen species, resulting in S-glutathionylation of TRAF family members, which in turn is linked to downstream signaling and interferon production (36). The potential for modification of viral antigens subsequent to reactive oxygen species production is highlighted by S-glutathionylation of several retroviral proteases, leading to host modulation of protease function (37). Indeed large scale changes in protein S-glutathionylation are observed in HIV-infected T cell blasts (38), suggesting that functional modulation of both host and viral proteins occurs via this mechanism. Whether these S-glutathionylated proteins inhibit or enhance immune responses to the unmodified epitope or generate novel T-cell epitopes that are subsequently recognized by the adaptive immune system is unclear.

Here, we investigate the frequency of modification of cysteine-containing MHC-bound peptides by interrogating a large database of naturally processed self-peptides derived from B-lymphoblastoid cells, murine tissues, and cytokine-treated cells. In addition, the functional consequences of Cys modification of T cell epitopes was investigated using an established model of infection that involves an immunodominant cysteine-containing epitope derived from a neurotropic strain of mouse hepatitis virus, strain JHM (JHMV)8(3941). We describe S-glutathionylation of this viral T cell epitope and the functional and structural implications of redox-modulated antigen presentation. Collectively our studies suggest that S-glutathionylation plays a key, previously unappreciated role in adaptive immune recognition.

PDF(Free)        RESULTS      DISCUSSION       REFERENCES

 

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Bacterial synthetic factories

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Bacteria seeded with synthetic pathways

http://www.rsc.org/chemistryworld/2016/04/bacteria-living-chemical-synthesis

 

Chinese scientists have taken a biosynthetic pathway from plants and introduced it into bacteria to create potentially health-boosting chemicals. Their route provides an alternative to complicated chemical syntheses or farming hectares of crops.

Shared photosynthetic components between plant chloroplasts and cyanobacteria make these microbes ideal hosts for expressing foreign plant enzymes. Ping Xu and colleagues at the Shanghai Jiao Tong University have genetically engineered the cyanobacterium Synechococcus elongatusPC7942 with plant-derived enzymes. In total, the team created 18 bacterial strains expressing different combinations of enzymes. The different strains generate a variety of compounds with a six-carbon, phenyl group and three-carbon propene tail, called phenylpropanoids.

Phenylpropanoids perform diverse functions in plants, ranging from ultraviolet light protection to pathogen defence. One such compound, resveratrol, is made when the bacteria express the plant enzyme stilbene synthase downstream of enzymes tyrosine ammonia lyase and 4-coumarate:coenzyme A-ligase. Found in the skin of grapes and other berries, resveratrol reduces the risk of heart disease and is a valuable pharmaceutical commodity. Different versions of the engineered bacteria can also churn out the phenylpropanoid antioxidants caffeic acid, naringenin and coumaric acid.

The Shanghai Jia Tong University team genetically engineered cyanobacteria to produce compounds like flavonoids, stilbenes and curcuminoids usually only found in plants

http://www.rsc.org/chemistryworld/sites/default/files/upload/Photoautotrophic-platform_c6gc00317f-f1_630m.jpg

What’s more, the team added feedback-inhibition resistant enzymes to the bacteria so that the chemical yields would surpass physiological levels. Photosynthesis within the cyanobacteria generates the chemicals from just water, carbon dioxide and a few mineral nutrients.

The bacterial growth medium houses the products, but isolating them at an industrially relevant yield is currently the biggest challenge. However, by not needing to harvest crops, generating the compounds from bacteria is potentially more sustainable. Xu stresses the potential of this point: ‘For the production of 1 tonne of natural resveratrol, our method may save about 485 hectare of farmland at its current production level.’

‘The approach deftly sidesteps major economic challenges by targeting chemicals with high intrinsic value,’ comments Paul Fowler, executive director of the Wisconsin Institute for Sustainable Technology in the US.  A world-scale production plant under these circumstances is not a pre-requisite for commercialising this research.’

 REFERENCES

This article is free until 06 June 2016

J Ni et al, Green Chem., 2016, DOI: 10.1039/c6gc00317f

A photoautotrophic platform for the sustainable production of valuable plant natural products from CO2

Jun Ni,ab   Fei Tao,ab   Yu Wang,ab   Feng Yaoab and   Ping Xu
Many plant natural products have remarkable pharmacological activities. They are mainly produced directly by extraction from higher plants, which can hardly keep up with the surging global demand. Furthermore, the over-felling of many medicinal plants has undesirable effects on the ecological balance. In this study, we constructed a photoautotrophic platform with the unicellular cyanobacterium Synechococcus elongatus PCC7942 to directly convert the greenhouse gas CO2 into an array of valuable healthcare products, including resveratrol, naringenin, bisdemethoxycurcumin, p-coumaric acid, caffeic acid, and ferulic acid. These six compounds can be further branched to many other precious and useful natural products. Various strategies including introducing a feedback-inhibition-resistant enzyme, creating functional fusion proteins, and increasing malonyl-CoA supply have been systematically investigated to increase the production. The highest titers of these natural products reached 4.1–128.2 mg L−1 from the photoautotrophic system, which are highly comparable with those obtained by many other heterotrophic microorganisms using carbohydrates. Several advantages such as independence from carbohydrate feedstocks, functionally assembling P450s, and availability of plentiful NADPH and ATP support that this photosynthetic platform is uniquely suited for producing plant natural products. This platform also provides a green route for direct conversion of CO2 to many aromatic building blocks, a promising alternative to petrochemical-based production of bulk aromatic compounds.
Graphical abstract: A photoautotrophic platform for the sustainable production of valuable plant natural products from CO2

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CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease


CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease

Curator: Larry H. Bernstein, MD, FCAP

 

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_Fig1_Cas9InVivo.png

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_GenomeEditingGlossary.png

One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool in Molecular Biology

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Figure 2. CRISPR/Cas9 System Applications

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_Fig2_Cas9forGenomeEditing.png?device=modal

  1. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.
    B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
    C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Targeting Efficiency and Off-target Mutations

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Figure 3. T7 Endonuclease I Targeting Efficiency Assay

https://www.neb.com/~/media/NebUs/Files/Feature%20Articles/Images/FA_Cas9_Fig3_T7Assay_TargetEfficiency.png

Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editing and Genome Targeting Tool

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila (45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The Future of CRISPR/Cas9

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.

 

Scientists urge caution in using new CRISPR technology to treat human genetic disease

By Robert Sanders, Media relations | MARCH 19, 2015
http://news.berkeley.edu/2015/03/19/scientists-urge-caution-in-using-new-crispr-technology-to-treat-human-genetic-disease/

http://news.berkeley.edu/wp-content/uploads/2015/03/crispr350.jpg

The bacterial enzyme Cas9 is the engine of RNA-programmed genome engineering in human cells. (Graphic by Jennifer Doudna/UC Berkeley)

A group of 18 scientists and ethicists today warned that a revolutionary new tool to cut and splice DNA should be used cautiously when attempting to fix human genetic disease, and strongly discouraged any attempts at making changes to the human genome that could be passed on to offspring.

Among the authors of this warning is Jennifer Doudna, the co-inventor of the technology, called CRISPR-Cas9, which is driving a new interest in gene therapy, or “genome engineering.” She and colleagues co-authored a perspective piece that appears in the March 20 issue of Science, based on discussions at a meeting that took place in Napa on Jan. 24. The same issue of Science features a collection of recent research papers, commentary and news articles on CRISPR and its implications.    …..

A prudent path forward for genomic engineering and germline gene modification

David Baltimore1,  Paul Berg2, …., Jennifer A. Doudna4,10,*, et al.
http://science.sciencemag.org/content/early/2015/03/18/science.aab1028.full
Science  19 Mar 2015.  http://dx.doi.org:/10.1126/science.aab1028

 

Correcting genetic defects

Scientists today are changing DNA sequences to correct genetic defects in animals as well as cultured tissues generated from stem cells, strategies that could eventually be used to treat human disease. The technology can also be used to engineer animals with genetic diseases mimicking human disease, which could lead to new insights into previously enigmatic disorders.

The CRISPR-Cas9 tool is still being refined to ensure that genetic changes are precisely targeted, Doudna said. Nevertheless, the authors met “… to initiate an informed discussion of the uses of genome engineering technology, and to identify proactively those areas where current action is essential to prepare for future developments. We recommend taking immediate steps toward ensuring that the application of genome engineering technology is performed safely and ethically.”

 

Amyloid CRISPR Plasmids and si/shRNA Gene Silencers

http://www.scbt.com/crispr/table-amyloid.html

Santa Cruz Biotechnology, Inc. offers a broad range of gene silencers in the form of siRNAs, shRNA Plasmids and shRNA Lentiviral Particles as well as CRISPR/Cas9 Knockout and CRISPR Double Nickase plasmids. Amyloid gene silencers are available as Amyloid siRNA, Amyloid shRNA Plasmid, Amyloid shRNA Lentiviral Particles and Amyloid CRISPR/Cas9 Knockout plasmids. Amyloid CRISPR/dCas9 Activation Plasmids and CRISPR Lenti Activation Systems for gene activation are also available. Gene silencers and activators are useful for gene studies in combination with antibodies used for protein detection.    Amyloid CRISPR Knockout, HDR and Nickase Knockout Plasmids

 

CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome


Mehrabian M, Brethour D, MacIsaac S, Kim JK, Gunawardana C.G, Wang H, et al.
PLoS ONE 2014; 9(12): e114594. http://dx.doi.org/10.1371/journal.pone.0114594

The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer’s disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.

http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0114594.g001

http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0114594.g003

 

Development and Applications of CRISPR-Cas9 for Genome Engineering

Patrick D. Hsu,1,2,3 Eric S. Lander,1 and Feng Zhang1,2,*
Cell. 2014 Jun 5; 157(6): 1262–1278.   doi:  10.1016/j.cell.2014.05.010

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

The development of recombinant DNA technology in the 1970s marked the beginning of a new era for biology. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and harness them to develop novel medicine and biotechnology. Recent advances in genome engineering technologies are sparking a new revolution in biological research. Rather than studying DNA taken out of the context of the genome, researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level, as well as identify causal genetic variations.

Broadly speaking, genome engineering refers to the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). The ability to do so easily and efficiently in eukaryotic and especially mammalian cells holds immense promise to transform basic science, biotechnology, and medicine (Figure 1).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f1.jpg

For life sciences research, technologies that can delete, insert, and modify the DNA sequences of cells or organisms enable dissecting the function of specific genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale. Similarly, manipulating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell, illuminating relationships between the architecture of the genome and its functions. In biotechnology, precise manipulation of genetic building blocks and regulatory machinery also facilitates the reverse engineering or reconstruction of useful biological systems, for example, by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops. Additionally, genome engineering is stimulating a new generation of drug development processes and medical therapeutics. Perturbation of multiple genes simultaneously could model the additive effects that underlie complex polygenic disorders, leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy (Tebas et al., 2014).

Eukaryotic genomes contain billions of DNA bases and are difficult to manipulate. One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination (HR), which integrates exogenous repair templates that contain sequence homology to the donor site (Figure 2A) (Capecchi, 1989). HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research. However, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur extremely infrequently (1 in 106–109 cells) (Capecchi, 1989), presenting enormous challenges for large-scale applications of gene-targeting experiments.

Genome Editing Technologies Exploit Endogenous DNA Repair Machinery

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f2.gif

To overcome these challenges, a series of programmable nuclease-based genome editing technologies have been developed in recent years, enabling targeted and efficient modification of a variety of eukaryotic and particularly mammalian species. Of the current generation of genome editing technologies, the most rapidly developing is the class of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats), which can be easily targeted to virtually any genomic location of choice by a short RNA guide. Here, we review the development and applications of the CRISPR-associated endonuclease Cas9 as a platform technology for achieving targeted perturbation of endogenous genomic elements and also discuss challenges and future avenues for innovation.   ……

Figure 4   Natural Mechanisms of Microbial CRISPR Systems in Adaptive Immunity

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f4.gif

……  A key turning point came in 2005, when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated origins (Mojica et al., 2005Pourcel et al., 2005Bolotin et al., 2005). This insight was tremendously exciting, especially given previous studies showing that CRISPR loci are transcribed (Tang et al., 2002) and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes (Mojica et al., 2005). Together, these findings led to the speculation that CRISPR arrays serve as an immune memory and defense mechanism, and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids (Mojica et al., 2005Pourcel et al., 2005). Despite these compelling realizations that CRISPR loci might be involved in microbial immunity, the specific mechanism of how the spacers act to mediate viral defense remained a challenging puzzle. Several hypotheses were raised, including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism (Makarova et al., 2006) or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions (Bolotin et al., 2005).   …..

As the pace of CRISPR research accelerated, researchers quickly unraveled many details of each type of CRISPR system (Figure 4). Building on an earlier speculation that protospacer adjacent motifs (PAMs) may direct the type II Cas9 nuclease to cleave DNA (Bolotin et al., 2005), Moineau and colleagues highlighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR interference (Deveau et al., 2008). Additionally, for types I and II, the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system. In type III systems, however, mismatches between the 5′ end of the crRNA and the DNA target are required for plasmid interference (Marraffini and Sontheimer, 2010).  …..

In 2013, a pair of studies simultaneously showed how to successfully engineer type II CRISPR systems from Streptococcus thermophilus (Cong et al., 2013) andStreptococcus pyogenes (Cong et al., 2013Mali et al., 2013a) to accomplish genome editing in mammalian cells. Heterologous expression of mature crRNA-tracrRNA hybrids (Cong et al., 2013) as well as sgRNAs (Cong et al., 2013Mali et al., 2013a) directs Cas9 cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing. Multiple guide RNAs can also be used to target several genes at once. Since these initial studies, Cas9 has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems (Sander and Joung, 2014). ……

The majority of CRISPR-based technology development has focused on the signature Cas9 nuclease from type II CRISPR systems. However, there remains a wide diversity of CRISPR types and functions. Cas RAMP module (Cmr) proteins identified in Pyrococcus furiosus and Sulfolobus solfataricus (Hale et al., 2012) constitute an RNA-targeting CRISPR immune system, forming a complex guided by small CRISPR RNAs that target and cleave complementary RNA instead of DNA. Cmr protein homologs can be found throughout bacteria and archaea, typically relying on a 5 site tag sequence on the target-matching crRNA for Cmr-directed cleavage.

Unlike RNAi, which is targeted largely by a 6 nt seed region and to a lesser extent 13 other bases, Cmr crRNAs contain 30–40 nt of target complementarity. Cmr-CRISPR technologies for RNA targeting are thus a promising target for orthogonal engineering and minimal off-target modification. Although the modularity of Cmr systems for RNA-targeting in mammalian cells remains to be investigated, Cmr complexes native to P. furiosus have already been engineered to target novel RNA substrates (Hale et al., 20092012).   ……

Although Cas9 has already been widely used as a research tool, a particularly exciting future direction is the development of Cas9 as a therapeutic technology for treating genetic disorders. For a monogenic recessive disorder due to loss-of-function mutations (such as cystic fibrosis, sickle-cell anemia, or Duchenne muscular dystrophy), Cas9 may be used to correct the causative mutation. This has many advantages over traditional methods of gene augmentation that deliver functional genetic copies via viral vector-mediated overexpression—particularly that the newly functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient (such as transthyretin-related hereditary amyloidosis or dominant forms of retinitis pigmentosum), it may also be possible to use NHEJ to inactivate the mutated allele to achieve therapeutic benefit. For allele-specific targeting, one could design guide RNAs capable of distinguishing between single-nucleotide polymorphism (SNP) variations in the target gene, such as when the SNP falls within the PAM sequence.

 

 

CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases

Zhuchi Tu, Weili Yang, Sen Yan, Xiangyu Guo and Xiao-Jiang Li

Molecular Neurodegeneration 2015; 10:35  http://dx.doi.org:/10.1186/s13024-015-0031-x

Animal models are extremely valuable to help us understand the pathogenesis of neurodegenerative disorders and to find treatments for them. Since large animals are more like humans than rodents, they make good models to identify the important pathological events that may be seen in humans but not in small animals; large animals are also very important for validating effective treatments or confirming therapeutic targets. Due to the lack of embryonic stem cell lines from large animals, it has been difficult to use traditional gene targeting technology to establish large animal models of neurodegenerative diseases. Recently, CRISPR/Cas9 was used successfully to genetically modify genomes in various species. Here we discuss the use of CRISPR/Cas9 technology to establish large animal models that can more faithfully mimic human neurodegenerative diseases.

Neurodegenerative diseases — Alzheimer’s disease(AD),Parkinson’s disease(PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and frontotemporal dementia (FTD) — are characterized by age-dependent and selective neurodegeneration. As the life expectancy of humans lengthens, there is a greater prevalence of these neurodegenerative diseases; however, the pathogenesis of most of these neurodegenerative diseases remain unclear, and we lack effective treatments for these important brain disorders.

CRISPR/Cas9,  Non-human primates,  Neurodegenerative diseases,  Animal model

There are a number of excellent reviews covering different types of neurodegenerative diseases and their genetic mouse models [812]. Investigations of different mouse models of neurodegenerative diseases have revealed a common pathology shared by these diseases. First, the development of neuropathology and neurological symptoms in genetic mouse models of neurodegenerative diseases is age dependent and progressive. Second, all the mouse models show an accumulation of misfolded or aggregated proteins resulting from the expression of mutant genes. Third, despite the widespread expression of mutant proteins throughout the body and brain, neuronal function appears to be selectively or preferentially affected. All these facts indicate that mouse models of neurodegenerative diseases recapitulate important pathologic features also seen in patients with neurodegenerative diseases.

However, it seems that mouse models can not recapitulate the full range of neuropathology seen in patients with neurodegenerative diseases. Overt neurodegeneration, which is the most important pathological feature in patient brains, is absent in genetic rodent models of AD, PD, and HD. Many rodent models that express transgenic mutant proteins under the control of different promoters do not replicate overt neurodegeneration, which is likely due to their short life spans and the different aging processes of small animals. Also important are the remarkable differences in brain development between rodents and primates. For example, the mouse brain takes 21 days to fully develop, whereas the formation of primate brains requires more than 150 days [13]. The rapid development of the brain in rodents may render neuronal cells resistant to misfolded protein-mediated neurodegeneration. Another difficulty in using rodent models is how to analyze cognitive and emotional abnormalities, which are the early symptoms of most neurodegenerative diseases in humans. Differences in neuronal circuitry, anatomy, and physiology between rodent and primate brains may also account for the behavioral differences between rodent and primate models.

 

Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases

Hsiuchen Chen and David C. Chan
Human Molec Gen 2009; 18, Review Issue 2 R169–R176
http://dx.doi.org:/10.1093/hmg/ddp326

Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. We review the general features of mitochondrial dynamics, incorporating recent findings on mitochondrial fusion, fission, transport and mitophagy. Defects in these key features are associated with neurodegenerative disease. Charcot-Marie-Tooth type 2A, a peripheral neuropathy, and dominant optic atrophy, an inherited optic neuropathy, result from a primary deficiency of mitochondrial fusion. Moreover, several major neurodegenerative diseases—including Parkinson’s, Alzheimer’s and Huntington’s disease—involve disruption of mitochondrial dynamics. Remarkably, in several disease models, the manipulation of mitochondrial fusion or fission can partially rescue disease phenotypes. We review how mitochondrial dynamics is altered in these neurodegenerative diseases and discuss the reciprocal interactions between mitochondrial fusion, fission, transport and mitophagy.

 

Applications of CRISPR–Cas systems in Neuroscience

Matthias Heidenreich  & Feng Zhang
Nature Rev Neurosci 2016; 17:36–44   http://dx.doi.org:/10.1038/nrn.2015.2

Genome-editing tools, and in particular those based on CRISPR–Cas (clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR–Cas systems has the potential to advance both basic and translational neuroscience research.
Cellular neuroscience
, DNA recombination, Genetic engineering, Molecular neuroscience

Figure 3: In vitro applications of Cas9 in human iPSCs.close

http://www.nature.com/nrn/journal/v17/n1/carousel/nrn.2015.2-f3.jpg

a | Evaluation of disease candidate genes from large-population genome-wide association studies (GWASs). Human primary cells, such as neurons, are not easily available and are difficult to expand in culture. By contrast, induced pluripo…

  1. Genome-editing Technologies for Gene and Cell Therapy

Molecular Therapy 12 Jan 2016

  1. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

Scientific Reports 31 Mar 2016

  1. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

Scientific Reports 12 Nov 2015

 

Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century

https://www.physicsforums.com/insights/can-gene-editing-eliminate-alzheimers-disease/

The development of the CRISPR/Cas9 system has made gene editing a relatively simple task.  While CRISPR and other gene editing technologies stand to revolutionize biomedical research and offers many promising therapeutic avenues (such as in the treatment of HIV), a great deal of debate exists over whether CRISPR should be used to modify human embryos. As I discussed in my previous Insight article, we lack enough fundamental biological knowledge to enhance many traits like height or intelligence, so we are not near a future with genetically-enhanced super babies. However, scientists have identified a few rare genetic variants that protect against disease.  One such protective variant is a mutation in the APP gene that protects against Alzheimer’s disease and cognitive decline in old age. If we can perfect gene editing technologies, is this mutation one that we should be regularly introducing into embryos? In this article, I explore the potential for using gene editing as a way to prevent Alzheimer’s disease in future generations. Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century Can gene editing be the missing piece in the battle against Alzheimer’s? (Source: bostonbiotech.org) I chose to assess the benefit of germline gene editing in the context of Alzheimer’s disease because this disease is one of the biggest challenges medicine faces in the 21st century. Alzheimer’s disease is a chronic neurodegenerative disease responsible for the majority of the cases of dementia in the elderly. The disease symptoms begins with short term memory loss and causes more severe symptoms – problems with language, disorientation, mood swings, behavioral issues – as it progresses, eventually leading to the loss of bodily functions and death. Because of the dementia the disease causes, Alzheimer’s patients require a great deal of care, and the world spends ~1% of its total GDP on caring for those with Alzheimer’s and related disorders. Because the prevalence of the disease increases with age, the situation will worsen as life expectancies around the globe increase: worldwide cases of Alzheimer’s are expected to grow from 35 million today to over 115 million by 2050.

Despite much research, the exact causes of Alzheimer’s disease remains poorly understood. The disease seems to be related to the accumulation of plaques made of amyloid-β peptides that form on the outside of neurons, as well as the formation of tangles of the protein tau inside of neurons. Although many efforts have been made to target amyloid-β or the enzymes involved in its formation, we have so far been unsuccessful at finding any treatment that stops the disease or reverses its progress. Some researchers believe that most attempts at treating Alzheimer’s have failed because, by the time a patient shows symptoms, the disease has already progressed past the point of no return.

While research towards a cure continues, researchers have sought effective ways to prevent Alzheimer’s disease. Although some studies show that mental and physical exercise may lower ones risk of Alzheimer’s disease, approximately 60-80% of the risk for Alzheimer’s disease appears to be genetic. Thus, if we’re serious about prevention, we may have to act at the genetic level. And because the brain is difficult to access surgically for gene therapy in adults, this means using gene editing on embryos.

Reference https://www.physicsforums.com/insights/can-gene-editing-eliminate-alzheimers-disease/

 

Utilising CRISPR to Generate Predictive Disease Models: a Case Study in Neurodegenerative Disorders


Dr. Bhuvaneish.T. Selvaraj  – Scottish Centre for Regenerative Medicine

http://www.crisprsummit.com/utilising-crispr-to-generate-predictive-disease-models-a-case-study-in-neurodegenerative-disorders

  • Introducing the latest developments in predictive model generation
  • Discover how CRISPR is being used to develop disease models to study and treat neurodegenerative disorders
  • In depth Q&A session to answer your most pressing questions

 

Turning On Genes, Systematically, with CRISPR/Cas9

http://www.genengnews.com/gen-news-highlights/turning-on-genes-systematically-with-crispr-cas9/81250697/

 

Scientists based at MIT assert that they can reliably turn on any gene of their choosing in living cells. [Feng Zhang and Steve Dixon]  http://www.genengnews.com/media/images/GENHighlight/Dec12_2014_CRISPRCas9GeneActivationSystem7838101231.jpg

With the latest CRISPR/Cas9 advance, the exhortation “turn on, tune in, drop out” comes to mind. The CRISPR/Cas9 gene-editing system was already a well-known means of “tuning in” (inserting new genes) and “dropping out” (knocking out genes). But when it came to “turning on” genes, CRISPR/Cas9 had little potency. That is, it had demonstrated only limited success as a way to activate specific genes.

A new CRISPR/Cas9 approach, however, appears capable of activating genes more effectively than older approaches. The new approach may allow scientists to more easily determine the function of individual genes, according to Feng Zhang, Ph.D., a researcher at MIT and the Broad Institute. Dr. Zhang and colleagues report that the new approach permits multiplexed gene activation and rapid, large-scale studies of gene function.

The new technique was introduced in the December 10 online edition of Nature, in an article entitled, “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.” The article describes how Dr. Zhang, along with the University of Tokyo’s Osamu Nureki, Ph.D., and Hiroshi Nishimasu, Ph.D., overhauled the CRISPR/Cas9 system. The research team based their work on their analysis (published earlier this year) of the structure formed when Cas9 binds to the guide RNA and its target DNA. Specifically, the team used the structure’s 3D shape to rationally improve the system.

In previous efforts to revamp CRISPR/Cas9 for gene activation purposes, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

After investigating single-guide RNA targeting rules for effective transcriptional activation, demonstrating multiplexed activation of 10 genes simultaneously, and upregulating long intergenic noncoding RNA transcripts, the research team decided to undertake a large-scale screen. This screen was designed to identify genes that confer resistance to a melanoma drug called PLX-4720.

“We … synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor,” wrote the authors of the Nature paper. “The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual [single-guide RNA] and complementary DNA overexpression.”

A gene signature based on the top screening hits, the authors added, correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patient-derived samples. It was also suggested that large-scale screens such as the one demonstrated in the current study could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

More at –  http://www.genengnews.com/gen-news-highlights/turning-on-genes-systematically-with-crispr-cas9/81250697/

 

Susceptibility and modifier genes in Portuguese transthyretin V30M amyloid polyneuropathy: complexity in a single-gene disease
Miguel L. Soares1,2, Teresa Coelho3,6, Alda Sousa4,5, …, Maria Joa˜o Saraiva2,5 and Joel N. Buxbaum1
Human Molec Gen 2005; 14(4): 543–553   http://dx.doi.org:/10.1093/hmg/ddi051
https://www.researchgate.net/profile/Isabel_Conceicao/publication/8081351_Susceptibility_and_modifier_genes_in_Portuguese_transthyretin_V30M_amyloid_polyneuropathy_complexity_in_a_single-gene_disease/links/53e123d70cf2235f352733b3.pdf

Familial amyloid polyneuropathy type I is an autosomal dominant disorder caused by mutations in the transthyretin (TTR ) gene; however, carriers of the same mutation exhibit variability in penetrance and clinical expression. We analyzed alleles of candidate genes encoding non-fibrillar components of TTR amyloid deposits and a molecule metabolically interacting with TTR [retinol-binding protein (RBP)], for possible associations with age of disease onset and/or susceptibility in a Portuguese population sample with the TTR V30M mutation and unrelated controls. We show that the V30M carriers represent a distinct subset of the Portuguese population. Estimates of genetic distance indicated that the controls and the classical onset group were furthest apart, whereas the late-onset group appeared to differ from both. Importantly, the data also indicate that genetic interactions among the multiple loci evaluated, rather than single-locus effects, are more likely to determine differences in the age of disease onset. Multifactor dimensionality reduction indicated that the best genetic model for classical onset group versus controls involved the APCS gene, whereas for late-onset cases, one APCS variant (APCSv1) and two RBP variants (RBPv1 and RBPv2) are involved. Thus, although the TTR V30M mutation is required for the disease in Portuguese patients, different genetic factors may govern the age of onset, as well as the occurrence of anticipation.

Autosomal dominant disorders may vary in expression even within a given kindred. The basis of this variability is uncertain and can be attributed to epigenetic factors, environment or epistasis. We have studied familial amyloid polyneuropathy (FAP), an autosomal dominant disorder characterized by peripheral sensorimotor and autonomic neuropathy. It exhibits variation in cardiac, renal, gastrointestinal and ocular involvement, as well as age of onset. Over 80 missense mutations in the transthyretin gene (TTR ) result in autosomal dominant disease http://www.ibmc.up.pt/~mjsaraiv/ttrmut.html). The presence of deposits consisting entirely of wild-type TTR molecules in the hearts of 10– 25% of individuals over age 80 reveals its inherent in vivo amyloidogenic potential (1).

FAP was initially described in Portuguese (2) where, until recently, the TTR V30M has been the only pathogenic mutation associated with the disease (3,4). Later reports identified the same mutation in Swedish and Japanese families (5,6). The disorder has since been recognized in other European countries and in North American kindreds in association with V30M, as well as other mutations (7).

TTR V30M produces disease in only 5–10% of Swedish carriers of the allele (8), a much lower degree of penetrance than that seen in Portuguese (80%) (9) or in Japanese with the same mutation. The actual penetrance in Japanese carriers has not been formally established, but appears to resemble that seen in Portuguese. Portuguese and Japanese carriers show considerable variation in the age of clinical onset (10,11). In both populations, the first symptoms had originally been described as typically occurring before age 40 (so-called ‘classical’ or early-onset); however, in recent years, more individuals developing symptoms late in life have been identified (11,12). Hence, present data indicate that the distribution of the age of onset in Portuguese is continuous, but asymmetric with a mean around age 35 and a long tail into the older age group (Fig. 1) (9,13). Further, DNA testing in Portugal has identified asymptomatic carriers over age 70 belonging to a subset of very late-onset kindreds in whose descendants genetic anticipation is frequent. The molecular basis of anticipation in FAP, which is not mediated by trinucleotide repeat expansions in the TTR or any other gene (14), remains elusive.

Variation in penetrance, age of onset and clinical features are hallmarks of many autosomal dominant disorders including the human TTR amyloidoses (7). Some of these clearly reflect specific biological effects of a particular mutation or a class of mutants. However, when such phenotypic variability is seen with a single mutation in the gene encoding the same protein, it suggests an effect of modifying genetic loci and/or environmental factors contributing differentially to the course of disease. We have chosen to examine age of onset as an example of a discrete phenotypic variation in the presence of the particular autosomal dominant disease-associated mutation TTR V30M. Although the role of environmental factors cannot be excluded, the existence of modifier genes involved in TTR amyloidogenesis is an attractive hypothesis to explain the phenotypic variability in FAP. ….

ATTR (TTR amyloid), like all amyloid deposits, contains several molecular components, in addition to the quantitatively dominant fibril-forming amyloid protein, including heparan sulfate proteoglycan 2 (HSPG2 or perlecan), SAP, a plasma glycoprotein of the pentraxin family (encoded by the APCS gene) that undergoes specific calcium-dependent binding to all types of amyloid fibrils, and apolipoprotein E (ApoE), also found in all amyloid deposits (15). The ApoE4 isoform is associated with an increased frequency and earlier onset of Alzheimer’s disease (Ab), the most common form of brain amyloid, whereas the ApoE2 isoform appears to be protective (16). ApoE variants could exert a similar modulatory effect in the onset of FAP, although early studies on a limited number of patients suggested this was not the case (17).

In at least one instance of senile systemic amyloidosis, small amounts of AA-related material were found in TTR deposits (18). These could reflect either a passive co-aggregation or a contributory involvement of protein AA, encoded by the serum amyloid A (SAA ) genes and the main component of secondary (reactive) amyloid fibrils, in the formation of ATTR.

Retinol-binding protein (RBP), the serum carrier of vitamin A, circulates in plasma bound to TTR. Vitamin A-loaded RBP and L-thyroxine, the two natural ligands of TTR, can act alone or synergistically to inhibit the rate and extent of TTR fibrillogenesis in vitro, suggesting that RBP may influence the course of FAP pathology in vivo (19). We have analyzed coding and non-coding sequence polymorphisms in the RBP4 (serum RBP, 10q24), HSPG2 (1p36.1), APCS (1q22), APOE (19q13.2), SAA1 and SAA2 (11p15.1) genes with the goal of identifying chromosomes carrying common and functionally significant variants. At the time these studies were performed, the full human genome sequence was not completed and systematic singlenucleotide polymorphism (SNP) analyses were not available for any of the suspected candidate genes. We identified new SNPs in APCS and RBP4 and utilized polymorphisms in SAA, HSPG2 and APOE that had already been characterized and shown to have potential pathophysiologic significance in other disorders (16,20–22). The genotyping data were analyzed for association with the presence of the V30M amyloidogenic allele (FAP patients versus controls) and with the age of onset (classical- versus late-onset patients). Multilocus analyses were also performed to examine the effects of simultaneous contributions of the six loci for determining the onset of the first symptoms.  …..

The potential for different underlying models for classical and late onset is supported by the MDR analysis, which produces two distinct models when comparing each class with the controls. One could view the two onset classes as unique diseases. If this is the case, then the failure to detect a single predictive genetic model is consistent with two related, but different, diseases. This is exactly what would be expected in such a case of genetic heterogeneity (28). Using this approach, a major gene effect can be viewed as a necessary, but not sufficient, condition to explain the course of the disease. Analyzing the cases but omitting from the analysis of phenotype the necessary allele, in this case TTR V30M, can then reveal a variety of important modifiers that are distinct between the phenotypes.

The significant comparisons obtained in our study cohort indicate that the combined effects mainly result from two and three-locus interactions involving all loci except SAA1 and SAA2 for susceptibility to disease. A considerable number of four-site combinations modulate the age of onset with SAA1 appearing in a majority of significant combinations in late-onset disease, perhaps indicating a greater role of the SAA variants in the age of onset of FAP.

The correlation between genotype and phenotype in socalled simple Mendelian disorders is often incomplete, as only a subset of all mutations can reliably predict specific phenotypes (34). This is because non-allelic genetic variations and/or environmental influences underlie these disorders whose phenotypes behave as complex traits. A few examples include the identification of the role of homozygozity for the SAA1.1 allele in conferring the genetic susceptibility to renal amyloidosis in FMF (20) and the association of an insertion/deletion polymorphism in the ACE gene with disease severity in familial hypertrophic cardiomyopathy (35). In these disorders, the phenotypes arise from mutations in MEFV and b-MHC, but are modulated by independently inherited genetic variation. In this report, we show that interactions among multiple genes, whose products are confirmed or putative constituents of ATTR deposits, or metabolically interact with TTR, modulate the onset of the first symptoms and predispose individuals to disease in the presence of the V30M mutation in TTR. The exact nature of the effects identified here requires further study with potential application in the development of genetic screening with prognostic value pertaining to the onset of disease in the TTR V30M carriers.

If the effects of additional single or interacting genes dictate the heterogeneity of phenotype, as reflected in variability of onset and clinical expression (with the same TTR mutation), the products encoded by alleles at such loci could contribute to the process of wild-type TTR deposition in elderly individuals without a mutation (senile systemic amyloidosis), a phenomenon not readily recognized as having a genetic basis because of the insensitivity of family history in the elderly.

 

Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis

Coelho T, Adams D, Silva A, et al.
N Engl J Med 2013;369:819-29.    http://dx.doi.org:/10.1056/NEJMoa1208760

Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin.

Methods We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers.

Results Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively.

ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene.

 

Alnylam May Seek Approval for TTR Amyloidosis Rx in 2017 as Other Programs Advance


https://www.genomeweb.com/rnai/alnylam-may-seek-approval-ttr-amyloidosis-rx-2017-other-programs-advance

Officials from Alnylam Pharmaceuticals last week provided updates on the two drug candidates from the company’s flagship transthyretin-mediated amyloidosis program, stating that the intravenously delivered agent patisiran is proceeding toward a possible market approval in three years, while a subcutaneously administered version called ALN-TTRsc is poised to enter Phase III testing before the end of the year.

Meanwhile, Alnylam is set to advance a handful of preclinical therapies into human studies in short order, including ones for complement-mediated diseases, hypercholesterolemia, and porphyria.

The officials made their comments during a conference call held to discuss Alnylam’s second-quarter financial results.

ATTR is caused by a mutation in the TTR gene, which normally produces a protein that acts as a carrier for retinol binding protein and is characterized by the accumulation of amyloid deposits in various tissues. Alnylam’s drugs are designed to silence both the mutant and wild-type forms of TTR.

Patisiran, which is delivered using lipid nanoparticles developed by Tekmira Pharmaceuticals, is currently in a Phase III study in patients with a form of ATTR called familial amyloid polyneuropathy (FAP) affecting the peripheral nervous system. Running at over 20 sites in nine countries, that study is set to enroll up to 200 patients and compare treatment to placebo based on improvements in neuropathy symptoms.

According to Alnylam Chief Medical Officer Akshay Vaishnaw, Alnylam expects to have final data from the study in two to three years, which would put patisiran on track for a new drug application filing in 2017.

Meanwhile, ALN-TTRsc, which is under development for a version of ATTR that affects cardiac tissue called familial amyloidotic cardiomyopathy (FAC) and uses Alnylam’s proprietary GalNAc conjugate delivery technology, is set to enter Phase III by year-end as Alnylam holds “active discussions” with US and European regulators on the design of that study, CEO John Maraganore noted during the call.

In the interim, Alnylam continues to enroll patients in a pilot Phase II study of ALN-TTRsc, which is designed to test the drug’s efficacy for FAC or senile systemic amyloidosis (SSA), a condition caused by the idiopathic accumulation of wild-type TTR protein in the heart.

Based on “encouraging” data thus far, Vaishnaw said that Alnylam has upped the expected enrollment in this study to 25 patients from 15. Available data from the trial is slated for release in November, he noted, stressing that “any clinical endpoint result needs to be considered exploratory given the small sample size and the very limited duration of treatment of only six weeks” in the trial.

Vaishnaw added that an open-label extension (OLE) study for patients in the ALN-TTRsc study will kick off in the coming weeks, allowing the company to gather long-term dosing tolerability and clinical activity data on the drug.

Enrollment in an OLE study of patisiran has been completed with 27 patients, he said, and, “as of today, with up to nine months of therapy … there have been no study drug discontinuations.” Clinical endpoint data from approximately 20 patients in this study will be presented at the American Neurological Association meeting in October.

As part of its ATTR efforts, Alnylam has also been conducting natural history of disease studies in both FAP and FAC patients. Data from the 283-patient FAP study was presented earlier this year and showed a rapid progression in neuropathy impairment scores and a high correlation of this measurement with disease severity.

During last week’s conference call, Vaishnaw said that clinical endpoint and biomarker data on about 400 patients with either FAC or SSA have already been collected in a nature history study on cardiac ATTR. Maraganore said that these findings would likely be released sometime next year.

Alnylam Presents New Phase II, Preclinical Data from TTR Amyloidosis Programs
https://www.genomeweb.com/rnai/alnylam-presents-new-phase-ii-preclinical-data-ttr-amyloidosis-programs

 

Amyloid disease drug approved

Nature Biotechnology 2012; (3http://dx.doi.org:/10.1038/nbt0212-121b

The first medication for a rare and often fatal protein misfolding disorder has been approved in Europe. On November 16, the E gave a green light to Pfizer’s Vyndaqel (tafamidis) for treating transthyretin amyloidosis in adult patients with stage 1 polyneuropathy symptoms. [Jeffery Kelly, La Jolla]

 

Safety and Efficacy of RNAi Therapy for Transthyretin …

http://www.nejm.org/…/NEJMoa1208760?&#8230;

The New England Journal of Medicine

Aug 29, 2013 – Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart.

 

Alnylam’s RNAi therapy targets amyloid disease

Ken Garber
Nature Biotechnology 2015; 33(577)    http://dx.doi.org:/10.1038/nbt0615-577a

RNA interference’s silencing of target genes could result in potent therapeutics.

http://www.nature.com/nbt/journal/v33/n6/images/nbt0615-577a-I1.jpg

The most clinically advanced RNA interference (RNAi) therapeutic achieved a milestone in April when Alnylam Pharmaceuticals in Cambridge, Massachusetts, reported positive results for patisiran, a small interfering RNA (siRNA) oligonucleotide targeting transthyretin for treating familial amyloidotic polyneuropathy (FAP).  …

  1. Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases

Nature Biotechnology 11 April 2016

  1. CRISPR-Cas systems for editing, regulating and targeting genomes

Nature Biotechnology 02 March 2014

  1. Near-optimal probabilistic RNA-seq quantification

Nature Biotechnology 04 April 2016

 

Translational Neuroscience: Toward New Therapies

https://books.google.com/books?isbn=0262029863

Karoly Nikolich, ‎Steven E. Hyman – 2015 – ‎Medical

Tafamidis for Transthyretin Familial Amyloid Polyneuropathy: A Randomized, Controlled Trial. … Multiplex Genome Engineering Using CRISPR/Cas Systems.

 

Is CRISPR a Solution to Familial Amyloid Polyneuropathy?

Author and Curator: Larry H. Bernstein, MD, FCAP

Originally published as

https://pharmaceuticalintelligence.com/2016/04/13/is-crispr-a-solution-to-familial-amyloid-polyneuropathy/

 

http://scholar.aci.info/view/1492518a054469f0388/15411079e5a00014c3d

FAP is characterized by the systemic deposition of amyloidogenic variants of the transthyretin protein, especially in the peripheral nervous system, causing a progressive sensory and motor polyneuropathy.

FAP is caused by a mutation of the TTR gene, located on human chromosome 18q12.1-11.2.[5] A replacement of valine by methionine at position 30 (TTR V30M) is the mutation most commonly found in FAP.[1] The variant TTR is mostly produced by the liver.[citation needed] The transthyretin protein is a tetramer.    ….

 

 

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