Translational Research Studies Research Articles

Abstract 2779: NGS characterization of multiple immune receptors from a single multiplex PCR reaction

Abstract Background: Immune cell analysis by next-generation sequencing (NGS) has shown utility in the fields of hematology-oncology and immuno-oncology research. In hemato-oncology research, advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, as well as eliminate sample-specific primer design often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR immune cell assays require separate reactions to survey each receptor (TCRB, TCRG, IGH, IGK, IGL,⋯), depleting samples and leading to a longer time-to-answer. Two assays for immune receptor analysis based on Ion AmpliSeq technology have been developed to circumvent these issues, allowing for the effective use of up to thousands of primers in a single reaction. These two highly multiplexed NGS assays provide for efficient single-reaction library preparation for detection of IGH, IGK, and IGL chains and the TCRB and TCRG chains, respectively. Methods: Primer panels target the variable gene and joining gene regions of the individual loci (IGH, IGK, IGL and TCRB/TCRG) for all alleles found within the IMGT database, which allows readout of the complementary-determining region 3 (CDR3) sequence of each receptor. The B cell assay includes primers to amplify IGK loci rearrangements involving the kappa deletion and constant region intronic elements. To evaluate the performance of each assay, clonality detection assessment and limit-of-detection testing used gDNA from a total of 65 research samples representing common B and T cell malignancies. Sequencing was performed using the Ion GeneStudio S5 System and analysis was completed using Ion Reporter 5.16. Results: Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, DLBCL, and T-ALL) show a >90% overall positive detection rate. Measurements of linearity-of-response and limit-of-detection were carried out using T and B cell lines and clinical research samples diluted in PBL gDNA to between 10-2 and 10-6 by mass. Both the TCR and BCR multi-receptor assays perform as expected, with linear response of detection to the sample dilution, including the ability to detect clones of interest at 10-6. Conclusions: These results demonstrate the robustness of newly developed Ion AmpliSeq assays for multi-receptor B and T cell analysis. These assays will cut the time-to-answer for translational research studies as well as simplify clonality assessment and rare clone detection in hematology research. For research use only. Citation Format: Geoffrey Marc Lowman, Michelle Toro, Loni Pickle, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang. NGS characterization of multiple immune receptors from a single multiplex PCR reaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2779.

Brain organoids: A promising model to assess oxidative stress-induced central nervous system damage.

Oxidative stress (OS) is one of the most significant propagators of systemic damage with implications for widespread pathologies such as vascular disease, accelerated aging, degenerative disease, inflammation, and traumatic injury. OS can be induced by numerous factors such as environmental conditions, lifestyle choices, disease states, and genetic susceptibility. It is tied to the accumulation of free radicals, mitochondrial dysfunction, and insufficient antioxidant protection, which leads to cell aging and tissue degeneration over time. Unregulated systemic increase in reactive species, which contain harmful free radicals, can lead to diverse tissue-specific OS responses and disease. Studies of OS in the brain, for example, have demonstrated how this state contributes to neurodegeneration and altered neural plasticity. As the worldwide life expectancy has increased over the last few decades, the prevalence of OS-related diseases resulting from age-associated progressive tissue degeneration. Unfortunately, vital translational research studies designed to identify and target disease biomarkers in human patients have been impeded by many factors (e.g., limited access to human brain tissue for research purposes and poor translation of experimental models). In recent years, stem cell-derived three-dimensional tissue cultures known as "brain organoids" have taken the spotlight as a novel model for studying central nervous system (CNS) diseases. In this review, we discuss the potential of brain organoids to model the responses of human neural cells to OS, noting current and prospective limitations. Overall, brain organoids show promise as an innovative translational model to study CNS susceptibility to OS and elucidate the pathophysiology of the aging brain.

David Katzenstein and James Hakim-In Memoriam.

The world lost two wonderful doctors who devoted their lives to the fight against HIV/AIDS in Africa. Ironically, they both died of COVID-19 within hours of each other in late January in Zimbabwe. David Katzenstein, MD, began his career in the early 1980 s working with underserved populations at a Native Tribal clinic in New Mexico and with persons suffering from AIDS at the Haight Ashbury Clinic in San Francisco, conducted CMV research at the University of Minnesota, and then took a position working as a microbiologist at the University of Zimbabwe in Harare between 1985 and 1987. In 1989, David joined the faculty at Stanford University where he remained until his retirement in 2017. Dr Katzenstein, led many important clinical and translational research studies related to HIV. With his colleagues in the AIDS Clinical Trials Group (ACTG), he led studies that resulted in the approval of life-saving antiretroviral medications. David dedicated a major portion of his academic endeavors to study HIV drug resistance, serving as director of one of the Virology Specialty Laboratories for the ACTG for many years. He was a protocol team member and leader on several ACTG studies and served on numerous ACTG committees, including the Performance Evaluation Committee and the original Virology Committee. He was co-chair of ACTG 384 that dealt with protease and/or non-nucleoside reverse transcriptase inhibitors, and was one of the first investigators to call attention to the issues of drug resistance in Africa. Recognizing the disparate impact of HIV in resource limited settings, David was a pioneer in Global HIV Research, in the fields of perinatal HIV transmission prevention, and studying different approaches to optimize antiretroviral treatment and laboratory monitoring in various settings around the world. He worked diligently to bring laboratory technology to Zimbabwe. Following his retirement from Stanford in 2017, David became the director of the Biomedical Research Training Institute in Zimbabwe where he led the molecular diagnostics laboratory to support laboratory monitoring of community-based treatment programs. In all his work, David engaged researchers and trainees from different parts of the world. Generous with his time, effort and ideas, David was a cherished mentor for many young and mid-career physicians and scientists, now scattered throughout different academic institutions and agencies in the U.S. He nurtured careers of clinicians, basic and social scientists, while encouraging multidisciplinary efforts to tackle the global HIV pandemic. He worked tirelessly and with great joy, giving of himself without reserve. James Gita Hakim, MRCP-UK, Professor of Medicine, University of Zimbabwe College of Health Sciences, to COVID-19. Dr Hakim was an accomplished investigator who made seminal contributions in HIV treatment and prevention research. He was known internationally for his accomplishments as a research leader, a builder of African research capacity, a leader of modernizing medical education in Africa, and as a mentor of young African investigators. Dr Hakim was at the forefront of clinical research on HIV/AIDS in Africa for over 20 years. He founded the University of Zimbabwe Clinical Research Centre (UZ-CRC) in 2001 as a site for international clinical trials on HIV treatment and prevention. He was a leader in pivotal international clinical trials including the landmark HIV ‘treatment as prevention’ trial HPTN 052, the MRC-funded “Development of Anti-Retroviral Therapy in Africa” (DART) trial, and the ACTG “Prospective Evaluation of Antiretroviral Therapy in Resource Limited Settings” (PEARLS) study. These studies contributed to the knowledge needed for successful implementation of antiretroviral therapy in southern Africa. Dr Hakim was as Principal Investigator of the University of Zimbabwe's program, the Medical Education Partnership Initiative (MEPI) which serves as a model for medical education in Africa. He was as a representative on the International AIDS Society (IAS) Governing Council from 2016 to 2020 and on the World Health Organization Guidelines Review Committee, the UNAIDS Advisory Committee, and on program committees for IAS Conference on HIV Science, the Conference on Retroviruses and Opportunistic Infections (CROI). Dr Hakim was Co-chair of the World AIDS Conference, Amsterdam, Netherlands in 2018. James Hakim's life demonstrated how investment in biomedical research and modern medical education can pay large dividends and be transformative for Africa. The world is better for the lives you lived, David and James. May light perpetual shine upon you.

A human ACTH-secreting corticotroph tumoroid model: Novel Human ACTH-Secreting Tumor Cell in vitro Model

BackgroundCushing disease (CD), although rare, is a life-threatening disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, which leads to excess adrenal-derived cortisol. Efficacious and safe medical therapies that control both hormonal hypersecretion and pituitary corticotroph tumor growth remain an unmet need in the management of CD. Translational research in pituitary tumors has been significantly hampered by limited quantities of surgically resected tissue for ex vivo studies, and unavailability of human pituitary tumor cell models. MethodsTo characterize human corticotroph tumors at the cellular level, we employed single cell RNA-sequencing (scRNA-seq) to study 4 surgically resected tumors. We also used microarrays to compare individualized paired consecutive culture passages to understand transcriptional shifts as in vitro cultures lost ACTH secretion. Based on these findings, we then modified our in vitro culture methods to develop sustained ACTH-secreting human corticotroph tumoroid cultures. FindingsscRNA-seq identified 4 major cell populations, namely corticotroph tumor (73.6%), stromal (11.2%), progenitor (8.3%), and immune cells (6.8%). Microarray analysis revealed striking changes in extracellular matrix, cell adhesion and motility-related genes concordant with loss of ACTH secretion during conventional 2D culture. Based on these findings, we subsequently defined a series of crucial culture nutrients and scaffold modifications that provided a more favorable trophic and structural environment that could maintain ACTH secretion in in vitro human corticotroph tumor cultures for up to 4 months. InterpretationOur human corticotroph tumoroid model is a significant advance in the field of pituitary tumors and will further enable translational research studies to identify critically needed therapies for CD. FundingThis work was partly funded by NCI P50-CA211015 and the Warley Trust Foundation.

American Journal of Geriatric Psychiatry: Translating Research Evidence into Clinical Geriatric Mental Health Practice

This symposium will describe the role of the American Journal of Geriatric Psychiatry in translating research evidence into clinical practice. The publishing team will provide an overview of defining impact and the methodology for measuring impact. The members of the AJGP editorial board will describe metrics used to evaluate the impact of research evidence on the readership of the journal and the field of geriatric mental health. The Editor-in-Chief and three Associate Editors will describe the top three papers in the areas of translational research, clinical interventions, and epidemiological studies with the highest impact in the last year.

46213 Florida Community-Engaged Research Alliance Against COVID-19 in Disproportionately Affected Communities (FL-CEAL): addressing education, awareness, access, and inclusion of underserved communities in COVID-19 research

ABSTRACT IMPACT: Understanding the needs and barriers or facilitators to participation in research, especially among minority communities is critical not only for COVID-19 research but also for future clinical and translational research and health disparities studies. OBJECTIVES/GOALS: The overall goal of this project is to enhance education, awareness, access, and inclusion of underserved communities across Florida in COVID-19 research, especially among Black and Hispanic minority groups that are disproportionately affected by COVID-19. METHODS/STUDY POPULATION: Through strategic partnership among five academic institutions and community-based organizations across the state of Florida, the FL-CEAL team will implement focus groups and surveys in minority communities in Florida to gauge the awareness and understanding of COVID-19, and the barriers and facilitators for participation in COVID-19 research studies. These communities include but are not limited to Latinx and Black populations in South and Central Florida, and Black communities in North Florida. The outcomes will help shape strategies for outreach and dissemination activities and minority recruitment plans to promote participation of minorities into vaccine and therapeutic trials. RESULTS/ANTICIPATED RESULTS: An estimated 75-125 participants will be recruited for focus groups. Four focus groups with minority communities have been conducted and the results are being analyzed. A common Community-Based Needs Assessment survey is being finalized and will be deployed across the 11 states that are part of the national CEAL consortium. Community Health Workers are being engaged to support outreach and dissemination to educate targeted communities on COVID-19 research and the importance of participation in COVID trials. To date, 243 CHWs and 880 community members have been engaged. Minority participation in COVID-19 vaccine trials at University of Miami has been higher than the national average. DISCUSSION/SIGNIFICANCE OF FINDINGS: The FL-CEAL Alliance has successfully demonstrated a coordinated effort to engage minority communities affected by COVID. Through strategic geographic partnerships, FL-CEAL will positively impact minority communities throughout the state that has one of the most diverse populations in the nation.

Omics in immunology

The term “Omics” broadly refers to a set of genomics technologies that are utilized in cellular and molecular biology to investigate molecular (DNA, RNA), protein and epigenetics signatures. Omics are utilized to study both host and pathogens, and across many disciplines such as immunology, oncology and cell biology. With the high-throughput technologies and the development of new bioinformatics tools, these approaches have significantly contributed to our understanding of how molecular mechanisms determine the structure, function and dynamics of organisms. With the large and growing volume of data generated with these approaches, bioinformatics has become an integral part of research in biology and notably immunology and microbiology. Large omics data sets are now also important components in clinical and translational research. For instance, databases of gene expression, somatic mutations and protein networks can be used to predict disease outcome or to develop new molecular diagnostic tools. In this Special Feature, the four reviews describe some of the most prominent applications of omics and bioinformatics in immunology research and clinical translational applications. These articles exemplify the vast research work that has occurred in the last two decades on the application of omics in medical and basic science research, with the objective to provide the reader with an introductory view of the current state of the art. T-cell receptors are formed from a complex set of genes undergoing homologous recombination, and it has been the result of very long evolutionary processes. T-cell receptor drives one of the most sophisticated weapons that the immune system has against pathogens. Understanding the biology of T-cell receptors is an ongoing subject of investigation since their discovery and sequence in the early 1980s.1 The review from Watkins and Miles 2 brings the reader up to speed with sequencing technologies and bioinformatics that have enabled diagnostic applications and predictive analyses in basic and clinical immunology. Autoimmunity: High-throughput sequencing of genomics has significantly impacted research in autoimmune disorders, with the possibility to screen large sample size and identify genetic variants across the full genome. The review from Field 3 reports the bioinformatics challenges that arose with the high-throughput detection of pathogenic variants and discusses the future applications toward personalized medicine. Single-cell multi-omics: In the last decade, single-cell genomics have permitted unprecedented detection of biological complexity and heterogeneity, notably in immunology.4 Advances in microfluidics and other technologies have allowed investigation of individual cells, thus removing the unwanted noise arising from the analysis of heterogenous cell populations. More recently, new technologies and bioinformatics analyses have enabled the simultaneous investigation of multiple modalities within the same cells utilizing more than one omic technology. The review from Louie and Luciani 5 discusses the recent advances in multi-omics to study immune cells, and also how the near future may be shaped by these technologies. Omics technology in the clinic: In the clinical setting, the application of omics technology has shown considerable promise. From patient stratification and therapeutic selection to the realization of personalized medicine, these technologies are beginning to fundamentally alter the way we approach molecular studies in translational research. The review from Giles et al.6 review current applications of omics technologies in immunology and oncology, along with ethical and practical challenges currently constraining adoption. Omics technologies and bioinformatics are increasingly becoming essential components in biology and specifically immunology. The development of high-throughput technologies has permitted the generation of numerous data sets across human and pathogens, introducing the need for large, centralized repositories, such as the Human Cell Atlas for single-cell genomics.7 Bioinformatics analysis and integration of very large omics data are providing the foundation to address unanswered questions including heterogeneity of biological processes and cell types, and how these can be translated into new treatment and interventions. These reviews represent key examples of the questions that are currently being addressed and a view of the future directions that omics and bioinformatics will shape the next decade of research. Samuel Foster: Conceptualization. Fabio Luciani: Conceptualization; Writing-original draft; Writing-review & editing. The authors declare no conflict of interest for this article.

Long-term inflammatory pain does not impact exploratory behavior and stress coping strategies in mice.

Pain puts patients at risk for developing psychiatric conditions such as anxiety and depression. Preclinical mouse models of pain-induced affective behavior vary widely in methodology and results, impairing progress towards improved therapeutics. To systematically investigate the effect of long-term inflammatory pain on exploratory behavior and stress coping strategy, we assessed male C57BL/6J mice in the forced swim test (FST), elevated zero maze, and open field test at 4 and 6 weeks postinjection of Complete Freund's Adjuvant, while controlling for testing order and combination. Inflammatory pain did not induce a passive stress coping strategy in the FST and did not reduce exploratory behavior in the elevated zero maze or the open field test. Using systematic correlational analysis and composite behavioral scores, we found no consistent association among measures for mice with or without inflammatory pain. A meta-analysis of similar studies indicated a modest, significant effect of Complete Freund's Adjuvant on exploratory behavior, but not immobility in the FST, and high heterogeneity among effect sizes in all 3 paradigms. Given the urgency for understanding the mechanisms of pain comorbidities and identifying novel therapies, these findings support the reallocation of our limited resources away from such unreliable assays and toward motivated and naturalistic behaviors. Future studies in pain and psychiatric translational research may benefit by considering outcomes beyond binary categorization, quantifying the associations between multiple measured behaviors, and agnostically identifying subtle yet meaningful patterns in behaviors.

Spontaneous In Vitro and In Vivo Interaction of (-)-Oleocanthal with Glycine in Biological Fluids: Novel Pharmacokinetic Markers.

Since the first discovery of its ibuprofen-like anti-inflammatory activity in 2005, the olive phenolic (-)-oleocanthal gained great scientific interest and popularity due to its reported health benefits. (-)-Oleocanthal is a monophenolic secoiridoid exclusively occurring in extra-virgin olive oil (EVOO). While several groups have investigated oleocanthal pharmacokinetics (PK) and disposition, none was able to detect oleocanthal in biological fluids or identify its PK profile that is essential for translational research studies. Besides, oleocanthal could not be detected following its addition to any fluid containing amino acids or proteins such as plasma or culture media, which could be attributed to its unique structure with two highly reactive aldehyde groups. Here, we demonstrate that oleocanthal spontaneously reacts with amino acids, with high preferential reactivity to glycine compared to other amino acids or proteins, affording two products: an unusual glycine derivative with a tetrahydropyridinium skeleton that is named oleoglycine, and our collective data supported the plausible formation of tyrosol acetate as the second product. Extensive studies were performed to validate and confirm oleocanthal reactivity, which were followed by PK disposition studies in mice, as well as cell culture transport studies to determine the ability of the formed derivatives to cross physiological barriers such as the blood-brain barrier. To the best of our knowledge, we are showing for the first time that (-)-oleocanthal is biochemically transformed to novel products in amino acids/glycine-containing fluids, which were successfully monitored in vitro and in vivo, creating a completely new perspective to understand the well-documented bioactivities of oleocanthal in humans.

Clinical Research Center for Neuromodulation in Psychiatry: A Multi-Center Initiative to Advance Interventional Psychiatry in India.

This manuscript introduces a unique program titled “Clinical Research Center (CRC) for Neuromodulation in Psychiatry” supported by the prestigious CRC/Public Health Research Center Grant of the DBT Wellcome Trust India Alliance. This multi-institutional research program will be led by NIMHANS (Bengaluru) in collaboration with the Central Institute of Psychiatry (Ranchi), and Kasturba Medical College (Manipal). The goal of this CRC is in alignment with the editorial titled “Need to Develop “Interventional Psychiatry” as a subspecialty in India” published in the January 2020 issue of the Indian Journal of Psychiatry. The translational research studies and the training programs envisaged through this center will facilitate the development of cost-effective, advanced interventional psychiatry tailored to resource-limited Indian clinical settings and similar other countries as well.

Immunotherapy in Breast Cancer Patients: A Focus on the Use of the CurrentlyAvailable Biomarkers in Oncology

Immune checkpoint inhibitors (ICIs) have remarkably modified the way solid tumors are managed, including breast cancer. Unfortunately, only a relatively small number of breast cancer patients significantly respond to these treatments. To maximize the immunotherapy benefit in breast cancer, several efforts are currently being put forward for the identification of i) the best therapeutic strategy (i.e. ICI monotherapy or in association with chemotherapy, radiotherapy, or other drugs); ii) the optimal timing for administration (e.g. early/advanced stage of disease; adjuvant/neoadjuvant setting); iii) the most effective and reliable predictive biomarkers of response (e.g. tumor-infiltrating lymphocytes, programmed death-ligand 1, microsatellite instability associated with mismatch repair deficiency, and tumor mutational burden). In this article, we review the impacts and gaps in the characterization of immune-related biomarkers raised by clinical and translational research studies with immunotherapy treatments. Particular emphasis has been put on the documented evidence of significant clinical benefits of ICI in different randomized clinical trials, along with preanalytical and analytical issues in predictive biomarkers pathological assessment.

A Structured Evaluation of Cryopreservation in Generating Single Cell Transcriptomes From Cerebrospinal Fluid

Abstract Importance A robust cerebrospinal fluid (CSF) cell cryopreservation protocol using high resolution single-cell (sc) transcriptomic data would enable the deployment of this important modality in multi-center translational research studies and clinical trials in which many sites do not have the expertise or resources to produce data from fresh samples. It would also serve to reduce technical variability in larger projects. Objective To test a reliable cryopreservation protocol adapted for CSF cells, facilitating the characterization of these rare, fragile cells in moderate to large scale studies. Design Diagnostic lumbar punctures were performed on twenty-one patients at two independent sites. Excess CSF was collected and cells were isolated. Each cell sample was split into two fractions for single cell analysis using one of two possible chemistries: 3’ sc-RNA-Sequencing or 5’sc-RNA-Sequencing. One cell fraction was processed fresh while the second sample was cryopreserved and profiled at a later time after thawing. Setting The research protocol was deployed at two academic medical centers taking care of multiple sclerosis and other neurological conditions. Participants 21 subjects (age 24 – 72) were recruited from individuals undergoing a diagnostic lumbar puncture for suspected neuroinflammatory disease or another neurologic illness; they donated excess CSF. Findings Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. The proportion of all cell types was similar between the fresh and the cryopreserved cells processed, and RNA expression was not significantly different. Results were comparable at both performance sites, and with different single cell sequencing chemistries. Cryopreservation also did not affect recovery of T and B cell clonotype diversity. Conclusion and relevance Our cryopreservation protocol for CSF-cells provides an important alternative to fresh processing of fragile CSF cells: cryopreservation enables the involvement of sites with limited capacity for experimental manipulation and reduces technical variation by enabling batch processing and pooling of samples. Key points Question How efficient is CSF cryopreservation for single-cell transcriptome analysis and can it be implemented in large multi-center translational and clinical trial settings? Findings We compared single-cell transcriptomes of paired fresh and cryopreserved CSF from 21 patients at two independent sites. We validate the efficacy of a simple and cost effective CSF cryopreservation method that preserves the composition and the transcriptomes of CSF cells stored for weeks-months. The protocol is deployed in a large multicenter Phase 4 MS clinical trial. Meaning A validated CSF cryopreservation method that would significantly advance basic science and biomarker research in neurological disorders by implementing single-cell transcriptome analyses in multi-center research and clinical trials.

Annotation and extraction of age and temporally-related events from clinical histories

BackgroundAge and time information stored within the histories of clinical notes can provide valuable insights for assessing a patient’s disease risk, understanding disease progression, and studying therapeutic outcomes. However, details of age and temporally-specified clinical events are not well captured, consistently codified, and readily available to research databases for study.MethodsWe expanded upon existing annotation schemes to capture additional age and temporal information, conducted an annotation study to validate our expanded schema, and developed a prototypical, rule-based Named Entity Recognizer to extract our novel clinical named entities (NE). The annotation study was conducted on 138 discharge summaries from the pre-annotated 2014 ShARe/CLEF eHealth Challenge corpus. In addition to existing NE classes (TIMEX3, SUBJECT_CLASS, DISEASE_DISORDER), our schema proposes 3 additional NEs (AGE, PROCEDURE, OTHER_EVENTS). We also propose new attributes, e.g., “degree_relation” which captures the degree of biological relation for subjects annotated under SUBJECT_CLASS. As a proof of concept, we applied the schema to 49 H&P notes to encode pertinent history information for a lung cancer cohort study.ResultsAn abundance of information was captured under the new OTHER_EVENTS, PROCEDURE and AGE classes, with 23%, 10% and 8% of all annotated NEs belonging to the above classes, respectively. We observed high inter-annotator agreement of >80% for AGE and TIMEX3; the automated NLP system achieved F1 scores of 86% (AGE) and 86% (TIMEX3). Age and temporally-specified mentions within past medical, family, surgical, and social histories were common in our lung cancer data set; annotation is ongoing to support this translational research study.ConclusionsOur annotation schema and NLP system can encode historical events from clinical notes to support clinical and translational research studies.

How selecting best upfront therapy for metastatic disease?-Focus on ROS1-rearranged disease.

ROS proto-oncogene 1 (ROS1) rearrangements defines a distinct group of non-small cell lung cancer (NSCLC), mainly represented by younger subjects, never smokers and with adenocarcinoma histology. Fusions involving ROS1 gene are present in 1-2% of lung adenocarcinomas and other solid tumors. Identification of patients harboring ROS1 rearrangements is a critical issue and current guidelines recommend screening of all advanced non-squamous NSCLC and certain squamous lung cancer patients. A number of trials have supported crizotinib as the best option for NSCLC patients with ROS1 translocations, irrespective of line of therapy. Unfortunately, the majority of patients become insensitive to crizotinib, due to the occurrence of secondary ROS1 mutations or failure within the central nervous system (CNS). Several highly potent and CNS penetrant ROS1 inhibitors have been developed and recent data highlight their potential role in the front-line treatment of this disease. Among them entrectinib, also known as RXDX-101, is a potent second-generation, multitarget oral inhibitor against the neurotrophin receptors TRKA, TRKB, TRKC ALK, and ROS1 with the ability to cross the blood-brain barrier. In the next few years, results of ongoing trials with novel ROS1 inhibitors and dedicated translational research studies might help to define the optimal sequence of treatment for ROS1-positive NSCLC patients.

Optimization of Biodiesel (Rubber Seed Oil) Process Parameters using Taguchi Method Based on Experimental Procedure in A Batch Stirred Tank Reactor

Due to the increase in the demand for an alternate fuel the research studies towards searching for a low cost source and optimum conditions with new reactors were gaining momentum. Hence the present investigation is focused on the design of a batch stirred tank reactor with two different impellers to produce biodiesel with the use of rubber seed oil. Further an experiment was conducted in optimizing the process parameters using Taguchi methodology. The Taguchi methodology is mainly focused on the enhancement of the product produced and this is the most precise method used. The best combinations were obtained through the L9 orthogonal array. The reactor specifications were fixed to be 7.5cm. height, 7.5cm diameter, 2.5 cm agitator diameter, 0.625cm baffle thickness with 2.5cm as distance between bottom of the tank and impeller. Two impellers with 0.5cm. Width, 0.625cm length and others with 1.1cm length were used to test the individual performance towards yield. The optimum conditions obtained were found to be Impeller type-Straight blade impeller, Temperature- 65ºC, Time - 45min, Reactant ratio - 1:6, Catalyst amount- 22.46% (mol% of Oil), and Impeller Speed- 500rpm with the yield of 88 % . Hence BSTR reactors could be scaled up for translational research studies too.

Major Scientific Hurdles in HIV Vaccine Development: Historical Perspective and Future Directions.

Following the discovery of HIV as a causative agent of AIDS, the expectation was to rapidly develop a vaccine; but thirty years later, we still do not have a licensed vaccine. Progress has been hindered by the extensive genetic variability of HIV and our limited understanding of immune responses required to protect against HIV acquisition. Nonetheless, valuable knowledge accrued from numerous basic and translational science research studies and vaccine trials has provided insight into the structural biology of the virus, immunogen design and novel vaccine delivery systems that will likely constitute an effective vaccine. Furthermore, stakeholders now appreciate the daunting scientific challenges of developing an effective HIV vaccine, hence the increased advocacy for collaborative efforts among academic research scientists, governments, pharmaceutical industry, philanthropy, and regulatory entities. In this review, we highlight the history of HIV vaccine development efforts, highlighting major challenges and future directions.

Clinical nurses' perspectives on discharge practice changes from participating in a translational research study.

To describe clinical nurses' experiences with practice change associated with participation in a multi-site nursing translational research study implementing new protocols for hospital discharge readiness assessment. Nurses' participation in translational research studies provides an opportunity to evaluate how implementation of new nursing interventions affects care processes within a local context. These insights can provide information that leads to successful adoption and sustainability of the intervention. Semi-structured focus groups from 30 of 33 participating study hospitals lead by team nurse researchers. Nurses reported improved and earlier awareness of patients' discharge needs, changes in discharge practices, greater patient/family involvement in discharge, synergy and enhanced discharge processes, and implementation challenges. Participating nurses related the benefits of participation in nursing research. Participation in a unit-level translational research project was a successful strategy for engaging nurses in practice change to improve hospital discharge. Leading unit-based implementation of a structured discharge readiness assessment including nurse assessment and patient self-assessment encourages earlier awareness of patients' discharge needs, improved patient assessment and greater patient/family involvement in discharge preparation. Integrating discharge readiness assessments into existing discharge care promotes communication between health team members that facilitates a timely, coordinated discharge.

Sexual dimorphism of detrusor function demonstrated by urodynamic studies in rhesus macaques

The lower urinary tract (LUT) and micturition reflexes are sexually dimorphic across mammals. Sex as a biological variable is also of critical importance for the development and translation of new medical treatments and therapeutics interventions affecting pelvic organs, including the LUT. However, studies of LUT function with comparisons between the sexes have remained sparse, especially for larger mammals. Detrusor function was investigated by filling cystometry and pressure flow studies in 16 male and 22 female rhesus macaques. By filling cystometry, male subjects exhibited a significantly larger bladder capacity and compliance compared to females. Pressure flow studies showed a significantly higher bladder pressure at voiding onset, peak pressure, and elevation in detrusor-activated bladder pressure from the end of bladder filling to peak pressure in the male subjects. The activation of reflex micturition, with associated detrusor contractions, resulted in voiding in a significantly larger proportion of female compared to male subjects. A higher urethral outlet resistance is suggested in the male subjects. We conclude that sexual dimorphism of detrusor function is prominent in rhesus macaques, shares many features with the human, and merits consideration in translational and pre-clinical research studies of micturition and LUT function in non-human primates.

Melatonin and Autophagy in Aging-Related Neurodegenerative Diseases.

With aging, the nervous system gradually undergoes degeneration. Increased oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, and cell death are considered to be common pathophysiological mechanisms of various neurodegenerative diseases (NDDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), organophosphate-induced delayed neuropathy (OPIDN), and amyotrophic lateral sclerosis (ALS). Autophagy is a cellular basic metabolic process that degrades the aggregated or misfolded proteins and abnormal organelles in cells. The abnormal regulation of neuronal autophagy is accompanied by the accumulation and deposition of irregular proteins, leading to changes in neuron homeostasis and neurodegeneration. Autophagy exhibits both a protective mechanism and a damage pathway related to programmed cell death. Because of its “double-edged sword”, autophagy plays an important role in neurological damage and NDDs including AD, PD, HD, OPIDN, and ALS. Melatonin is a neuroendocrine hormone mainly synthesized in the pineal gland and exhibits a wide range of biological functions, such as sleep control, regulating circadian rhythm, immune enhancement, metabolism regulation, antioxidant, anti-aging, and anti-tumor effects. It can prevent cell death, reduce inflammation, block calcium channels, etc. In this review, we briefly discuss the neuroprotective role of melatonin against various NDDs via regulating autophagy, which could be a new field for future translational research and clinical studies to discover preventive or therapeutic agents for many NDDs.

Ketamine-induced neuromuscular reactivity is associated with aging in female rhesus macaques.

Rhesus macaques represent an important species for translational and pre-clinical research studies across a multitude of disease and injury models, including aging. Ketamine anesthesia is used in humans and non-human primates but may be associated with adverse effects, including neuromuscular reactions. The effects of aging on ketamine adverse effects is not well characterized. Urodynamic recordings and electromyography (EMG) studies were performed in aged (>20 years old) and adult (3.9–14.9 years old) female rhesus macaques under an equal and light plane of sedation by constant rate infusion (CRI) of ketamine. A total of 4 of 41 adult subjects (9.7%) showed clinical signs of ketamine-induced abnormal neuromuscular reactivity, whereas a larger portion of 14 of 26 aged subjects showed similar ketamine-induced neuromuscular reactivity (53.8%; P< 0.001). The ketamine CRI rate was 19.8±0.9 mg/kg/h in adults and lower in aged subjects at 16.5±1.4 mg/kg/h (P<0.05). The ketamine CRI rate was negatively correlated with age (r = -0.30, P<0.05, n = 64). The incidence of ketamine reactivity or CRI rate was not different between aged pre-and post-menopausal females. EMG recordings during neuromuscular reactivity showed coordinated activation of multiple muscles, suggesting a central nervous system (CNS) mechanism for ketamine-associated neuromuscular reactivity. The incidence of ketamine-induced neuromuscular reactivity is age related but not affected by the estrous cycle in female rhesus macaques. A coordinated activation of multiple muscles, innervated by different peripheral nerves, suggests that ketamine-induced neuromuscular reactivity originates in the CNS.