Category Archives: Molecular Medicine

In the compelling and unstoppable research in favor of cellular rejuvenation, Sentcell LTD is looking for PhD or Postdoc Researchers to investigate molecular mechanisms and innovative therapies at basis of T cell and systemic rejuvenation. The studies will be held at our new laboratory located at the Toscana Life Sciences, within the GSK Campus, in Siena, Italy.

Expertises required will span across:

a) Flow cytometry b) In vivo studies c) Immunoblotting/Immunoprecipitation d) Elisa assays e) PCR f) Primary culturesBioinformatic skills are strongly appreciated (docking predictions).

The ideal candidate will possess: Degree in Biology, or Biotechnology, or Medicine or other Biological Sciences Completed or about to complete PhD in Cellular/Molecular Biology or Molecular Medicine or Immunology Excellent level of spoken and written English Team working skills

The candidate, preferably with work experience abroad, will have in depth understanding in the field of Immunology and/or Biochemistry with particular regard to signal transduction.

The candidate(s) will be offered: A competitive three-year contract commensurate to track record Excellent career progression prospects with important salary adjustments and bonuses Opportunities for professional growth of the highest level

Sentcell LTD was founded by Prof. Dr Alessio Lanna in the UK back in 2019, and has rapidly been recognised at the top of all biotech companies operating in the country.

Sentcell first of its kind work started from the understanding that T lymphocytes, particularly CD4+ cells, are at the pinnacle of the aging process in all organs. Despite so, no T cell rejuvenation therapies presently exist and Sentcell is committed to fill in the gap.We were the first to discover the "sMAC" macromolecular complex, responsible for the cellular aging program of T lymphocytes (Lanna et al., Nature Immunology, 2014 and Lanna et al., Nature Immunology 2017), and a new anti-aging mechanism based on transfer of telomeres in vesicles between immune cells (Lanna et al., Nature Cell Biology, 2022).

On this basis, we introduced the "DOS", which- to date- represents the first drug in the world capable of reversing T cell aging resulting in immune remodelling as well systemic rejuvenation as a whole, via both sMAC disruption and telomere transfer induction (Lanna et al., under consideration) In the near future, these T cell rejuvenating approaches will span to fight variety of diseases across the organism.

A first in human clinical trial of DOS will be held in the UK in 2025; as such candidates will be exposed to an extraordinary work flow spanning from target identification to drug discovery and in human medical translation to treat human diseases.

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Postdoc to investigate molecular mechanisms in the field immune-rejuvenation for drug discovery job with Sentcell LTD ... - Nature.com

4 June 2024 - Beth Amato - Faculty Communications

The Division of Molecular Medicine and Haematology in the School of Pathology

The Division of Molecular Medicine and Haematology in the School of Pathology spearheads vital clinical and diagnostic work in both the private and public sectors. A joint initiative of Wits University and the National Health Laboratory Service, the division comprises specialised units focusing on science and research, teaching and learning and clinical work. It is represented in the main teaching hospitals, Charlotte Maxeke, Chris Hani Baragwanath, Helen Joseph, Rahima Moosa Childrens Hospital, and the Wits Donald Gordon Medical Centre.

The division plays a critical role in providing undergraduate teaching, as well as facilitating rigorous training for a large number of postgraduate students. The division provides specialist training to registrars preparing for College of Medicine examinations.

Division head, Professor Johnny Mahlangu

Figure 1: Professor Johnny Mahlangu, the division head at the Department of Molecular Medicine and Haematology in the School of Pathology

Professor Johnny Mahlangu heads the Division of Molecular Medicine and Haematology in the Faculty of Health Sciences of the University of the Witwatersrand and the National Health Laboratory Service. He is also a consultant clinical haematologist at the Charlotte Maxeke Johannesburg Academic Hospital. Professor Mahlangu received his undergraduate and postgraduate training in science and medicine at the University of the Witwatersrand with haematology specialist and clinical haematology sub-specialist qualifications through the Colleges of Medicine of South Africa. His main area of research is novel therapies in bleeding disorders, in which he has served as Principal Investigator for many international multicentre studies. He has published peer-reviewed journal articles and presented over 500 oral talks and posters at national and international scientific meetings. His academic citizenship includes membership in a wealth of national and international scientific committees.

Once-off gene therapy could resolve the many challenges of living with a bleeding disorder

A once-off adeno-associated gene therapy (AAV) for haemophilia types A and B will revolutionise the treatment of inherited bleeding disorders. There are promising results from four clinical trials which show that 80% of patients can live without replacement therapy and that the side effects of gene therapy are minimal.

Patients born with mutant F8 or F9 genes have impaired thrombin generation (the final and crucial step in clotting), resulting in spontaneous or trauma-induced bleeding. The hallmark of haemophilia bleeding is bleeding into the joints (known as haemarthroses).

The global standard of care for these patients is replacement therapy with plasma-derived or recombinant FVIII or FIX proteins. Replacement therapy often results in suboptimal treatment. Moreover, the cost of treating haemophilia is about R400000 to R600000 per patient yearly. The development of alternative therapies such as gene therapy may be able to mitigate this, explains Professor Johnny Mahlangu, head of the Department of Molecular Medicine and Haematology in the Wits School of Pathology.

Now in its fifth year post-gene therapy, Mahlangu's research reveals that giving a single infusion of AAV-mediated gene therapy carrying the FVIII or FIX transgene results in. The patients quality of life drastically improves.

In AAV-mediated gene therapy, the liver cells assist the transgene in finding its way to what is known as the hepatocyte nuclear. It then uses the hepatocyte protein synthesis machinery to transcribe the AAV-delivered FVIII or FIX gene into an mRNA. The vector isnt integrated, so we arent changing anyones genetic makeup.

Figure 2: AAV mediated gene therapy

Figure 3: The lab has carefully controlled refrigeration, with remote sensing abilities

This is the ultimate treatment that haemophilia patients require to remain bleed-free while not taking replacement therapy. Significantly, both the FDA and EMA have approved two gene therapies for haemophilia A and B, which are ommercially available. The major challenge in South Africa is that it is R60 million per treatment. Obviously, this isnt affordable, but we are looking at models to see how we can make it viable, says Professor Mahlangu.

Figure 4: Professor Mahlangu's world-class haematology clinical set-up

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2024 - Welcome to Molecular Medicine - Wits University

An advanced algorithm that has been developed by Google DeepMind has gone some way to cracking one of the biggest unsolved mysteries in biology. AlphaFold aims to predict the 3D structures of proteins from the instruction code in their building blocks. The latest upgrade has recently been released. The latest upgrade has recently been released.

Proteins are essential parts of living organisms and take part in virtually every process in cells. But their shapes are often complex, and they are difficult to visualise. So being able to predict their 3D structures offers windows into the processes inside living things, including humans.

This provides new opportunities for creating drugs to treat disease. This in turn opens up new possibilities in what is called molecular medicine. This is where scientists strive to identify the causes of disease at the molecular scale and also develop treatments to correct them at the molecular level.

The first version of DeepMinds AI tool was unveiled in 2018. The latest iteration, released this year, is AlphaFold3. A worldwide competition to evaluate new ways of predicting the structures of proteins, the Critical Assessment of Structure Prediction (Casp) has been held biannually since 1994 In 2020, the Casp competition got to test AlphaFold2 and was very impressed. Since then, researchers eagerly anticipate each new incarnation of the algorithm.

However, as a masters student I was once reprimanded for using AlphaFold2 in some of my coursework. This was because it was deemed only a predictive tool. In other words, how could anyone know whether what was predicted matched the real-life protein without experimental verification?

This is a legitimate point. The area of experimental molecular biology has undergone its own revolution in the past decade with strong advances in a microscope technique called cryo-electron microscopy (cryo-EM), which uses frozen samples and gentle electron beams to capture the structures of biomolecules in high resolution.

The advantage of AI tools such as AlphaFold is that it can elucidate protein structures much faster (in a matter of minutes) at almost no cost. Results are more readily available and accessible globally online. They can also predict the structure of proteins that are notoriously difficult to experimentally verify, such as membrane proteins.

However, AlphaFold2 was not designed to address something called the quaternary structure of proteins, where multiple protein subunits form a larger protein. This involves a dynamic visualisation of how different units of the protein molecule are folded. And some researchers reported that it sometimes appeared to have difficulty predicting structural elements of proteins known as coils.

When my professor contacted me in May to relay the news that AlphaFold3 had been released, my first question was about its ability to predict quaternary structures. Had it succeeded? Were we now able to take the massive leap towards predicting a complete structure? Early reports suggest the answers to those questions are positive.

Experimental methods are slower. And when they are able to capture the 3D structure of molecules, it is more akin to looking at a statue - a snapshot of the protein rather than seeing how it moves and interacts to carry out actions in the body. In other words, we want a movie, rather than a photo.

Experimental methods have also traditionally struggled with membrane proteins key molecules that are attached to or are associated with the membranes of cells. These are often crucial in understanding and treating many of the worst diseases.

Here is where AlphaFold3 could truly change the landscape. If it is successful at predicting quaternary structures at a level equal to or greater than experimental methods such as crystallography, cryo-EM and others, and it can visualise membrane proteins better than the competition, then we will indeed have a gigantic leap forwards in our race towards true molecular medicine.

AlphaFold3 can only be accessed from a DeepMind server, but it is easy to use. Researchers can get their results in minutes simply from the sequence. The other promise of AlphaFold3 is further disruption. DeepMind is not alone in its ambitions to master the problem of protein folding. As the next Casp competition approaches there are others looking to win the race. For example, Liam McGuffin and his team at the University of Reading are making gains in quality assessment and predicting the stoichiometry of protein complexes. Stoichiometry refers to the proportions in which elements or chemical compounds react with one another.

Not all scientists in this area are chasing the goal in the same way. Others are trying to solve similar challenges in terms of the quality of the 3D models or specific barriers such as those presented by membrane proteins. The competition has been marvellous for progress in this field.

However, experimental methods are not going away anytime soon, and nor should they. The progress of cryo-EM is laudable, and X-ray crystallography still gives us the finest resolution on biomolecules. The European XFEL laser in Germany could be the next breakthrough. These technologies will only continue to improve.

My biggest question as we survey this new field is whether our human instinct to relent until we have absolute proof will fold with AlphaFold. If this new technology is able to give results comparable to, or greater than, experimental verification, will we be prepared to accept it? If we can, its speed and accuracy could have a major effect on areas such as drug development.

For the first time, with AlphaFold3, we may have cleared the most significant hurdle in the protein prediction revolution. What will we make of this new world? And what medicine can we make with it?

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An AI tool for predicting protein shapes could be transformative for medicine, but it challenges science's need for proof - The Conversation

About the job

We have vacancy for one PhD candidate at theOtterlei groupat the Department of Clinical and Molecular Medicine. The project addresses the need for new antibiotics with novel mechanisms of action and new treatment regimens to handle the emerging antimicrobial resistance (AMR).

Based on knowledge of peptides with antibacterial activities and conserved mechanisms for mutagenesis, we have designed synthetic peptides with strong anti-mutagenic, antibacterial and anti-biofilm activities. These peptides interfere with DNA translesion synthesis (i.e. mutagenesis), bacterial replication, and cellular signalling, and is targeting the bacterial DNA sliding clamp, the b-clamp. The project specifically focuses on development of second-generation antibacterial peptides. This project is part of a multidisciplinary collaboration project (TAMiR - NTNU) between two faculties at NTNU, University of Oslo / Oslo University Hospitaland University of Copenhagen and cover competences from basic molecular microbiology, chemical synthesis and structural biology/modelling, bioinformatics, cell biology and immunology/infectious diseases.

The PhD candidate will study antimicrobial activities of the peptides using several different methods used in medical microbiology and evaluate the toxicity of the peptides in cell line based assays as well as initial toxicity studies in animals.

Required selection criteria

The PhD-position's main objective is to qualify for work in research positions. The qualification requirement is that you have completed a masters degree or second degree (equivalent to 120 credits) with a strong academic background in biochemistry, microbiology, molecular and cell biology or equivalent education with a grade of B or better in terms ofNTNUs grading scale. If you do not have letter grades from previous studies, you must have an equally good academic foundation. If you are unable to meet these criteria you may be considered only if you can document that you are particularly suitable for education leading to a PhD degree.

Master's students can apply, but the master's degree must be obtained and documented by the end of june 2024.

The appointment is to be made in accordance withRegulations on terms of employment for positions such as postdoctoral fellow, Phd candidate, research assistant and specialist candidateandRegulations concerning the degrees ofPhilosophiaeDoctor (PhD)andPhilosodophiaeDoctor (PhD) in artistic researchnational guidelines for appointment as PhD, post doctor and research assistant

Preferred selection criteria

Personal characteristics

In the evaluation of which candidate is best qualified, emphasis will be placed on education, experience and personal suitability.

We offer

Salary and conditions

As a PhD candidate (code 1017) you are normally paid from gross NOK 532200 per annum before tax, depending on qualifications and seniority. From the salary, 2% is deducted as a contribution to the Norwegian Public Service Pension Fund.

The period of employment is 3years.

Appointment to a PhD position requires that you are admitted to thePhD programme in Medicine and Health Scienceswithin three months of employment, and that you participate in an organized PhD programme during the employment period.

The engagement is to be made in accordance with the regulations in force concerningState Employees and Civil Servants, and the acts relating to Control of the Export of Strategic Goods, Services and Technology. Candidates who by assessment of the application and attachment are seen to conflict with the criteria in the latter law will be prohibited from recruitment to NTNU. After the appointment you must assume that there may be changes in the area of work.

It is a prerequisite you can be present at and accessible to the institution on a daily basis.

About the application

The application and supporting documentation to be used as the basis for the assessment must be in English.

Publications and other scientific work must be attached to the application. Please note that your application will be considered based solely on information submitted by the application deadline. You must therefore ensure that your application clearly demonstrates how your skills and experience fulfil the criteria specified above.

The application must include:

If all,or parts,of your education has been taken abroad, we also ask you to attach documentation of the scope and quality of your entire education, both bachelor's and master's education, in addition to other higher education. Description of the documentation required can befoundhere. If you already have a statement fromNOKUT,pleaseattachthisas well.

We will take joint work into account. If it is difficult to identify your efforts in the joint work, you must enclose a short description of your participation.

NTNU is committed to following evaluation criteria for research quality according toThe San Francisco Declaration on Research Assessment - DORA.

General information

Working at NTNU

NTNU believes that inclusion and diversity is our strength. We want to recruit people with different competencies, educational backgrounds, life experiences and perspectives to contribute to solving our social responsibilities within education and research. We will facilitate for our employees needs.

The city of Trondheimis a modern European city with a rich cultural scene. Trondheim is the innovation capital of Norway with a population of 200,000. The Norwegian welfare state, including healthcare, schools, kindergartens and overall equality, is probably the best of its kind in the world. Professional subsidized day-care for children is easily available. Furthermore, Trondheim offers great opportunities for education (including international schools) and possibilities to enjoy nature, culture and family life and has low crime rates and clean air quality.

As an employeeatNTNU, you must at all times adhere to the changes that the development in the subject entails and the organizational changes that are adopted.

A public list of applicants with name, age, job title and municipality of residence is prepared after the application deadline. If you want to reserve yourself from entry on the public applicant list, this must be justified. Assessment will be made in accordance withcurrent legislation. You will be notified if the reservation is not accepted.

If you have any questions about the position, please contact Professor Marit Otterlei, telephone +47 72573075, emailmarit.otterlei@ntnu.no. If you have any questions about the recruitment process, please contact Vebjrn F. Andreassen, e-mail: vebjorn.andreassen@ntnu.no

If you think this looks interesting and in line with your qualifications, please submit your application electronically via jobbnorge.no with your CV, diplomas and certificates attached. Applications submitted elsewhere will not be considered. Upon request, you must be able to obtain certified copies of your documentation.

Application deadline: 10.06.2024

NTNU - knowledge for a better world

The Norwegian University of Science and Technology (NTNU) creates knowledge for a better world and solutions that can change everyday life.

The Department of Clinical and Molecular Medicine (IKOM):

The Department of Clinical and Molecular Medicine (IKOM) is NTNUs largest department, with 450 employees. Our research and teaching help to improve treatment and health.

IKOM has expertise in basic, clinical and translational research within broad disciplinary areas. We study childrens and womens health, cancers, blood disorders and infectious diseases, gastroenterology, inflammation, metabolic disorders, laboratory sciences and medical ethics. The Department offers teaching in medicine at masters and PhD level. We also offer continuing education for employees in the health services.

Deadline10th June 2024 EmployerNTNU - Norwegian University of Science and Technology MunicipalityTrondheim ScopeFulltime Duration Project Place of serviceErling Skjalgssons gate 1, 7030 Trondheim

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PhD Candidate within Molecular Medicine job with NORWEGIAN UNIVERSITY OF SCIENCE & TECHNOLOGY - NTNU ... - Times Higher Education

Professor Eugenia Piddini has been elected to the Academy of Medical Sciences respected and influential Fellowship. She joins 58 exceptional biomedical and health scientists selected for their exceptional contributions to the advancement of medical science.

The newFellows, announced on Tuesday 21 May, have been recognised for their remarkable contributions to advancing biomedical and health sciences, groundbreaking research discoveries and translating developments into benefits for patients and wider society.

Awardees join an esteemed Fellowship of over 1,400 researchers who are at the heart of the Academy's work, which includes nurturing the next generation of researchers and shaping research and health policy in the UK and worldwide. The expertise of Fellows elected this year spans a wide range of clinical and non-clinical disciplines, from midwifery to cancer stem cell biology.

Eugenia Piddini,Professor of Cell Biology in theSchool of Cellular and Molecular Medicine, is conducting innovative work to identify cell competition-based strategies to gain control over tissue colonisation, its impact in tissue colonisation in regenerative medicine and to prevent tumour expansion in cancer.

A cell and developmental biologist,Eugenia is known for her seminal work in the field of cell competition the mechanism of tissue quality control that removes damaged cells from tissues. Eugenias discoveries have helped widen the scope of cell competition in terms of physiological relevance and potential therapeutic impact. Recently, Eugenias group demonstrated that cell competition acts in adult tissues. There it can potentially slow down the onset of disease/ageing by eliminating damaged cells.

Eugenias team has also shown that tumour cells kill surrounding normal cells via cell competition to free space for their own growth. Their work has identified many mechanisms and signals that cells use to compete. By explaining the mechanisms that cells use to compete the Piddini group aims to identify cell competition-based strategies to gain control over tissue colonisation.

In recognition of her work Eugenia, who is also School Research Director, was awarded theBritish Society for Cell Biology Hooke Medalin 2019 and in 2023, was elected as aMember of the European Molecular Biology Organisation.

Alongside Professor Piddini, Professor Gene Feder OBE, has also been elected from the University. Gene Feder,is a GP and Professor of Primary Care at BristolsCentre for Academic Primary Care, Bristol Medical School and Director of VISION, aUK Prevention Research Partnership(UKPRP) consortium.

Professor Andrew Morris PMedSci, President of theAcademy of Medical Sciences, said: It is an honour to welcome these brilliant minds to our Fellowship. Our new Fellows lead pioneering work in biomedical research and are driving remarkable improvements in healthcare. We look forward to working with them, and learning from them, in our quest to foster an open and progressive research environment that improves the health of people everywhere through excellence in medical science.

This year's cohort marks a significant milestone in the Academy's efforts to promote equality, diversity and inclusion (EDI) within its Fellowship election. Among the new Fellows, 41 per cent are women, the highest percentage ever elected. Additionally, Black, Asian and minority ethnic representation is 29 per cent, an 11 per cent increase from the previous year. The new Fellows hold positions at institutions across the UK, including in Edinburgh, Birmingham, Liverpool, Manchester, Sheffield, Nottingham and York.

Professor Morris added: It is also welcoming to note that this year's cohort is our most diverse yet, in terms of gender, ethnicity and geography. While this progress is encouraging, we recognise that there is still much work to be done to truly diversify our Fellowship. We remain committed to our EDI goals and will continue to take meaningful steps to ensure our Fellowship reflects the rich diversity of the society we serve."

The new Fellows will be formally admitted to the Academy at a ceremony on Wednesday 18 September 2024.

The Academy of Medical Sciences is the independent, expert body representing the diversity of medical science in the UK. Its mission is to advance biomedical and health research and its translation into benefits for society. The Academy's elected Fellows are the most influential scientists in the UK and worldwide, drawn from the NHS, academia, industry and the public service.

About theAcademy of Medical SciencesTheAcademy of Medical Sciencesis the independent, expert voice of biomedical and health research in the UK. Our Fellowship comprises the most influential scientists in the UK and worldwide, drawn from the NHS, academia, industry, and the public service. Our mission is to improve the health of people everywhere by creating an open and progressive research sector. We do this by working with patients and the public to influence policy and biomedical practice, strengthening UK biomedical and health research, supporting the next generation of researchers through funding and career development opportunities, and working with partners globally.

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2024: Prof Eugenia Piddini Medical Sciences Fellow | School of Cellular and Molecular Medicine - University of Bristol

A recent paper published in the Journal of Physiology deepened the case for the youthfulness-promoting effects of exercise on ageing organisms, building on previous work done with lab mice nearing the end of their natural lifespan that had access to a weighted exercise wheel.

The densely detailed paper, "A molecular signature defining exercise adaptation with ageing and in vivo partial reprogramming in skeletal muscle," lists a whopping 16 co-authors, six of whom are affiliated with the University of Arkansas. The corresponding author is Kevin Murach, an assistant professor in the University's Department of Health, Human Performance and Recreation, and the first author is Ronald G. Jones III, a Ph.D. student in Murach's Molecular Muscle Mass Regulation Laboratory.

ALSO READ: From skipping meals to crash diets: Debunking the top weight loss myths

For this paper, the researchers compared aging mice that had access to a weighted exercise wheel with mice that had undergone epigenetic reprogramming via the expression of Yamanaka factors.

The Yamanaka factors are four protein transcription factors (identified as Oct3/4, Sox2, Klf4 and c-Myc, often abbreviated to OKSM) that can revert highly specified cells (such as a skin cell) back to a stem cell, which is a younger and more adaptable state. The Nobel Prize in Physiology or Medicine was awarded to Dr. Shinya Yamanaka for this discovery in 2012. In the correct dosages, inducing the Yamanaka factors throughout the body in rodents can ameliorate the hallmarks of aging by mimicking the adaptability that is common to more youthful cells.

that have been reprogrammed through exercise -- "reprogramming" in the latter case reflecting how an environmental stimulus can alter the accessibility and expression of genes.

The researchers compared the skeletal muscle of mice who had been allowed to exercise late in life to the skeletal muscle of mice that overexpressed OKSM in their muscles, as well as to genetically modified mice limited to the overexpression of just Myc in their muscles.

Ultimately, the team determined that exercise promotes a molecular profile consistent with epigenetic partial programming. That is to say: exercise can mimic aspects of the molecular profile of muscles that have been exposed to Yamanaka factors (thus displaying molecular characteristics of more youthful cells). This beneficial effect of exercise may in part be attributed to the specific actions of Myc in muscle.

While it would be easy to hypothesize that someday we might be able to manipulate Myc in muscle to achieve the effects of exercise, thus sparing us the actual hard work, Murach cautions that would be the wrong conclusion to draw.

First, Myc would never be able to replicate all the downstream effects exercise has throughout the body. It is also the cause of tumors and cancers, so there are inherent dangers to manipulating its expression. Instead, Murach thinks manipulating Myc might best be employed as an experimental strategy to understand how to restore exercise adaptation to old muscles showing declining responsiveness. Possibly it could also be a means of supercharging the exercise response of astronauts in zero gravity or people confined to bed rest who only have a limited capacity for exercise. Myc has many effects, both good and bad, so defining the beneficial ones could lead to a safe therapeutic that could be effective for humans down the road.

Murach sees their research as further validation of exercise as a polypill. "Exercise is the most powerful drug we have," he says, and should be considered a health-enhancing -- and potentially life-extending -- treatment along with medications and a healthy diet.

Murach and Jones' co-authors at the U of A included exercise science professor Nicholas Greene, as well as contributing researchers Francielly Morena Da Silva, Seongkyun Lim and Sabin Khadgi.

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Exercise promotes a molecular profile in muscle: Research

The Molecular Diagnostics Laboratory is responsible for the development and performance of molecular diagnostic tests for nucleic acid targets found in a variety of settings in medicine. We offer a wide array of tests at the forefront of molecular diagnostics and precision medicine. The three broad areas of testing we provide are:

Hematopathology is a main focus of our lab with testing performed in genetic analysis of hematologic malignancies for diagnosis and therapeutic decision-making, coagulation genetics, and evaluation of stem-cell transplant patients. We also perform several additional genetic tests including hemochromatosis and cystic fibrosis screens for adults and in conjunction with the prenatal laboratory for newborns.

A listing of our testing can be found here.

Other department laboratories perform molecular testing for microorganisms. The Clinical Virology Laboratory tests for multiple viral pathogens, whereas the Clinical Microbiology Laboratory tests for M. tuberculosis, C. trachomatis, and N. gonorrhoeae. The Molecular Diagnostics Laboratory complements these tests by performing in situ hybridization and quantitative PCR to assay for Epstein-Barr virus and supports the 16S ribosomal RNA DNA sequencing test to identify bacteria.

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Molecular Diagnostics Laboratory < Laboratory Medicine

Set of methods inmolecular biology

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.[1] The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.[2]

In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMO).[3] This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.[4]

Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. There is also a lower chance of success when inserting large-sized DNA sequences. Inserts larger than 10kbp have very limited success, but bacteriophages such as bacteriophage can be modified to successfully insert a sequence up to 40 kbp.[5]

Prior to the 1970s, the understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleases, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered.[6] They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation. Using a second enzyme, DNA ligase, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972.[7][8]

Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the transgenic organism.

Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.

Before actual cloning experiments are performed in the lab, most cloning experiments are planned in a computer, using specialized software. Although the detailed planning of the cloning can be done in any text editor, together with online utilities for e.g. PCR primer design, dedicated software exist for the purpose. Software for the purpose include for example ApE [1] (open source), DNAStrider [2] (open source), Serial Cloner [3] (gratis), Collagene [4] (open source), and SnapGene (commercial). These programs allow to simulate PCR reactions, restriction digests, ligations, etc., that is, all the steps described below.

In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts and biological properties.

Notably, the growing capacity and fidelity of DNA synthesis platforms allows for increasingly intricate designs in molecular engineering. These projects may include very long strands of novel DNA sequence and/or test entire libraries simultaneously, as opposed to of individual sequences. These shifts introduce complexity that require design to move away from the flat nucleotide-based representation and towards a higher level of abstraction. Examples of such tools are GenoCAD, Teselagen [5] (free for academia) or GeneticConstructor [6] (free for academics).

Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.[3] If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome[10] or yeast artificial chromosome vector is often chosen.

Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.

Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility:[3]

The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA (see DNA end). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are located within a gene (frequently beta-galactosidase) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.[11]

For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from extinct animals),[12] as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography). Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning.

DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene synthesis). cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by the designer. Such a designed sequence may be required when moving genes across genetic codes (for example, from the mitochrondria to the nucleus)[13] or simply for increasing expression via codon optimization.[14]

The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA (linkers) containing desired restriction sites may be added to create end structures that are compatible with the vector.[3][11]

The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.

DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting in a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present. This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells.[3][11]

The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g. transformation, transduction, transfection, electroporation).[3][11]

When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA are said to be competent.[15] In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.

Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present).[16] In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.

Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.[3][11]

When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the plasmid will survive when exposed to the antibiotic, while those that have failed to take up plasmid sequences will die. When mammalian cells (e.g. human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin.

Modern bacterial cloning vectors (e.g. pUC19 and later derivatives including the pGEM vectors) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted). In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme so that colonies containing transformed DNA remain colorless (white). Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.

The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library. Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously cloned DNA fragment into a different plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained. This may be accomplished through a very wide range of experimental methods, including the use of nucleic acid hybridizations, antibody probes, polymerase chain reaction, restriction fragment analysis and/or DNA sequencing.[3][11]

Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from any genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science. A few of the more important applications are summarized here.

Molecular cloning has led directly to the elucidation of the complete DNA sequence of the genomes of a very large number of species and to an exploration of genetic diversity within individual species, work that has been done mostly by determining the DNA sequence of large numbers of randomly cloned fragments of the genome, and assembling the overlapping sequences. Further, cloning can be used to produce gene therapies for the treatment of serious disease indications, such as cystic fibrosis, cancer, AIDS and others. It is interesting to note that gene cloning can be a potential solution to organ scarcity. It also plays an important role in synthesis of antibiotics, vitamins and hormones.[17]

At the level of individual genes, molecular clones are used to generate probes that are used for examining how genes are expressed, and how that expression is related to other processes in biology, including the metabolic environment, extracellular signals, development, learning, senescence and cell death. Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to inactivate the genes, or make more subtle mutations using regional mutagenesis or site-directed mutagenesis. Genes cloned into expression vectors for functional cloning provide a means to screen for genes on the basis of the expressed protein's function.

Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein. In practice, it is frequently more difficult to develop an organism that produces an active form of the recombinant protein in desirable quantities than it is to clone the gene. This is because the molecular signals for gene expression are complex and variable, and because protein folding, stability and transport can be very challenging.

Many useful proteins are currently available as recombinant products. These include--(1) medically useful proteins whose administration can correct a defective or poorly expressed gene (e.g. recombinant factor VIII, a blood-clotting factor deficient in some forms of hemophilia,[18] and recombinant insulin, used to treat some forms of diabetes[19]), (2) proteins that can be administered to assist in a life-threatening emergency (e.g. tissue plasminogen activator, used to treat strokes[20]), (3) recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g. hepatitis B vaccine[21]), and (4) recombinant proteins as standard material for diagnostic laboratory tests.

Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, generating transgenic organisms, also termed genetically modified organisms (GMOs). Although most GMOs are generated for purposes of basic biological research (see for example, transgenic mouse), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds (pharming), herbicide-resistant crop plants, and fluorescent tropical fish (GloFish) for home entertainment.[1]

Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This germ line gene therapy is considered by many to be unethical in human beings.[22] The second type of gene therapy, somatic cell gene therapy, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.[23]

Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success.[23] The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.

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Trends in Molecular Medicine objective is to provide concise and contextualized views on the latest research moving biomedical science closer to improved diagnosis, treatment, and prevention of human diseases. As such, TMM is dedicated to research disciplines at the interface between basic biology and clinical research. Articles cover new concepts in mechanisms of human biology and pathology with clear implications for diagnostics and therapy. Bridging bench and bedside, reviews published in TMM have clear implications for human health and disease and discuss not only preclinical studies but also research conducted on patient samples, first-in-man studies, and patient-enrolled trials. The major themes covered in TMM in include:Disease mechanismsTools and technologiesDiagnosticsTherapeutics

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Trends in Molecular Medicine objective is to provide concise and contextualized views on the latest research moving biomedical science closer to improved diagnosis, treatment, and prevention of human diseases. As such, TMM is dedicated to research disciplines at the interface between basic biology

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Researchers from Insilico Medicine, University of Copenhagen, and University of Chicago unravel molecular secrets hidden in premature aging diseases and cancer using AI  GlobeNewswire

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Researchers from Insilico Medicine, University of Copenhagen, and University of Chicago unravel molecular secrets hidden in premature aging diseases...