Modeling mRNA-based vaccine YFV.E1988 against yellow fever virus E-protein using immuno-informatics and reverse vaccinology approach.

To surmount constraints of live-attenuated vaccines we have in silico designed mRNA vaccine using envelope protein as a target antigen. From the alignment of 216 envelope proteins, a consensus sequence was obtained which was used for codon optimization. The secondary structure was predicted using Mfold and RNAfold tool. IEDB server was used to predict T-cell and B-cell epitopes, epitope conservancy, immunogenicity, and population coverage. Antigenicity, allergenicity, and toxicity were predicted using Vaxijen, AllerTOP, and ToxinPred tools, respectively. Interactions between MHC and identified epitopes were confirmed by docking and molecular dynamics simulation. In silico immune simulation was done using the C-ImmSim server. Vaccine peptide 3D structure was predicted and validated based on the Ramachandran plot. Finally, we designed the vaccine construct for simulating restriction cloning using the SnapGene tool. Our optimization of consensus E protein is highly immunogenic, conserved, has immune-dominance characteristics, and suggests high translational efficiency in the host cell. We validated the presence of T and B cell epitopes and interestingly we found one CD4+ and four CD8+ T-cell epitopes that satisfied all the criteria of an effective vaccine candidate. We found high-affinity interactions between epitope and HLA alleles that can stimulate the T-cell response. The immune simulation verified the immune cell response to eliminate the antigen. To ensure effective expression of the vaccine, a circular plasmid has been designed using in silico cloning approach for the in vitro transcription process. Obtained results suggest that the vaccine YFV.E1988 will elicit specific immune responses against YFV and it is a potential model ready for laboratory testing. HighlightsThe envelope (E) protein was found to be highly conserved and it has the potential to protect individuals against YFV infection.YFV.E1988 vaccine has been capable to stimulate both the CD8+ and CD4+ T cell, solving the major limitations of the current live-attenuated vaccines against YFV.Presence of T- and B-cell epitopes across the antigen have been validated using several computational tools.Molecular docking ensured the epitope-allele binding and protein-TLR/MR interaction. The vaccine was found to be immune-stimulatory, safe, and stable.The codons were optimized for efficient translation and increased stability into the human host. The UTR regions and poly (A) tail used for the development of YFV.E1988 showed immune stimulatory potential in several experiments.Communicated by Ramaswamy H. Sarma.

A synthetic lethality screen reveals ING5 as a genetic dependency of catalytically dead Set1A/COMPASS in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/complex of proteins associated with SET1 histone methyltransferase (Set1A/COMPASS) in mouse ESCs does not impair their viability or ability to self-renew; however, it leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF (monocytic leukaemia zinc finger protein/monocytic leukaemia zinc finger protein-related factor) and HBO1 (HAT bound to ORC1) acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mouse ESCs decreases the fitness of these cells, and the simultaneous loss of ING5 and in Set1AΔSET leads to up-regulation of differentiation-associated genes. Taken together, our results point toward Set1A/COMPASS and ING5 as potential coregulators of the self-renewal and differentiation status of ESCs.

BMAL1 drives muscle repair through control of hypoxic NAD+ regeneration in satellite cells.

The process of tissue regeneration occurs in a developmentally timed manner, yet the role of circadian timing is not understood. Here, we identify a role for the adult muscle stem cell (MuSC)-autonomous clock in the control of muscle regeneration following acute ischemic injury. We observed greater muscle repair capacity following injury during the active/wake period as compared with the inactive/rest period in mice, and loss of Bmal1 within MuSCs leads to impaired muscle regeneration. We demonstrate that Bmal1 loss in MuSCs leads to reduced activated MuSC number at day 3 postinjury, indicating a failure to properly expand the myogenic precursor pool. In cultured primary myoblasts, we observed that loss of Bmal1 impairs cell proliferation in hypoxia (a condition that occurs in the first 1-3 d following tissue injury in vivo), as well as subsequent myofiber differentiation. Loss of Bmal1 in both cultured myoblasts and in vivo activated MuSCs leads to reduced glycolysis and premature activation of prodifferentiation gene transcription and epigenetic remodeling. Finally, hypoxic cell proliferation and myofiber formation in Bmal1-deficient myoblasts are restored by increasing cytosolic NAD+ Together, we identify the MuSC clock as a pivotal regulator of oxygen-dependent myoblast cell fate and muscle repair through the control of the NAD+-driven response to injury.

SPT5 stabilization of promoter-proximal RNA polymerase II.

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression.

The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

Identifying research obstacles; a cross sectional comparative study of perceptions of postgraduate medical and dental residents in three public sector medical colleges.

OBJECTIVE: To explore the attitude of postgraduate medical and dental residents about research work and to identify barriers in the way of research activities. METHODOLOGY: The cross-sectional study was conducted from May to July, 2018, at three public-sector medical and dental institutions in Quetta, the capital of Pakistan province of Balochistan, and comprised postgraduate residents. Data was collected using a pre-validated 16 item Likert scale questionnaire. Data was analysed using SPSS 23. RESULTS: Of the 72 subjects, 44(61.1%) were males. The overall mean age was 23.3±4.6 years (range: 28-30 years. The residents demonstrated willingness to conduct clinical research with a mean score of 4.36±0.65. There was no association of attitudes and barriers with socio-demographic and personal characteristics (p>0.05). Deficient lab facilities (p-<0.001), and inaccessibility to electronic and hospital data (p=0.003) were the major factors identified as research barriers. CONCLUSIONS: Barriers to research need to be addressed in designing research curricula to encourage and facilitate meaningful research.

Factors contributing to the lack of interest in research activities among postgraduate medical students.

OBJECTIVE: To determine the factors contributing to lack of interest in research activities among postgraduate residents in pediatric medicine. METHODS: This cross-sectional survey was conducted at The Children's Hospital Lahore, Pakistan in August 2017. The questionnaire was distributed to 105 postgraduate residents working in pediatric medicine department and 90 of them returned the completed proforma (response rate; 86%). Data was analyzed by SPSS version 22. RESULTS: Out of 90 residents' females were predominant (n=58/90; 64.4%). Mean age of the participants was 28.22 ± 2.092 years. Majority were unmarried (n=57; 63.3%). The highest mean score (2.31±0.697) was regarding "Lack of proper training for research", followed by "lack of previous exposure" (2.26±0.728) and "over loaded curriculum" (2.13±0.753). "Uncooperative faculty" and "funding issues" as a barrier towards research (p=0.016 and 0.014 respectively) was mentioned by males more than females. "Social and family commitment" was a significant perceived barrier in married residents as compared to unmarried residents (p=0.001). The residents in the younger age group were more reluctant to do research due to "over loaded curriculum" (p=0.038). CONCLUSION: Lack of proper training of research, lack of previous exposure and time management are the major factors affecting resident's research work, whereas married residents face more social/family constraints as compare to unmarried residents.

Oligonucleotides Targeting Telomeres and Telomerase in Cancer.

Telomeres and telomerase have become attractive targets for the development of anticancer therapeutics due to their involvement in cancer cell immortality. Currently, several therapeutics have been developed that directly target telomerase and telomeres, such as telomerase inhibitors and G-quadruplex stabilizing ligands. Telomere-specific oligonucleotides that reduce telomerase activity and disrupt telomere architecture are also in development as novel anticancer therapeutics. Specifically, GRN163L and T-oligos have demonstrated promising anticancer activity in multiple cancers types via induction of potent DNA damage responses. Currently, several miRNAs have been implicated in the regulation of telomerase activity and may prove to be valuable targets in the development of novel therapies by reducing expression of telomerase subunits. Targeting miRNAs that are known to increase expression of telomerase subunits may be another strategy to reduce carcinogenesis. This review aims to provide a comprehensive understanding of current oligonucleotide-based anticancer therapies that target telomeres and telomerase. These studies may help design novel therapeutic approaches to overcome the challenges of oligonucleotide therapy in a clinical setting.

Current Molecular-Targeted Therapies in NSCLC and Their Mechanism of Resistance.

Lung cancer is treated with many conventional therapies, such as surgery, radiation, and chemotherapy. However, these therapies have multiple undesirable side effects. To bypass the side effects elicited by these conventional treatments, molecularly-targeted therapies are currently in use or under development. Current molecularly-targeted therapies effectively target specific biomarkers, which are commonly overexpressed in lung cancers and can cause increased tumorigenicity. Unfortunately, several molecularly-targeted therapies are associated with initial dramatic responses followed by acquired resistance due to spontaneous mutations or activation of signaling pathways. Acquired resistance to molecularly targeted therapies presents a major clinical challenge in the treatment of lung cancer. Therefore, to address this clinical challenge and to improve lung cancer patient prognosis, we need to understand the mechanism of acquired resistance to current therapies and develop additional novel therapies. This review concentrates on various lung cancer biomarkers, including EGFR, ALK, and BRAF, as well as their potential mechanisms of drug resistance.

Haematological parameters and recurrent aphthous stomatitis.

OBJECTIVE: To find out the relationship between recurrent aphthous stomatitis (RAS) with deficiencies of haemoglobin, haematocrit, serum vitamin B12, serum Ferritin and red blood cells (RBC) Folate level. STUDY DESIGN: An analytical cross-sectional study. PLACE AND DURATION OF STUDY: Department of Oral Health Sciences, Shaikh Zayed Federal Postgraduate Medical Complex, Lahore, from February to July 2008. METHODOLOGY: Sixty consecutive subjects with active RAS were taken as the aphthous group; 60 age and gender matched subjects without RAS were as the Non-Aphthous group. Five milliliter blood was taken from both groups to evaluate the levels of serum B12, and RBC Folate through radio immuno assay and serum ferritin with enzyme linked immuno-sorbent assay tests. Complete blood count was carried out to determine the level of haemoglobin and haematocrit in both groups. Proportion of subjects with lower values was compared using 2 text of proportions with significance at p < 0.05. RESULTS: Serum Ferritin (p = 0.001), haematocrit (p < 0.001), RBC Folate (p < 0.001) and serum B12 (p < 0.001) were significantly lower in the RAS group. Combined deficiency state (haemoglobin, serum Ferritin, haematocrit, RBC Folate and serum B12) was identified in 13% (n = 8) RAS patients. CONCLUSION: Frequency of haematinic deficiencies was high in RAS patients. Serum B12 and RBC Folate were significantly low in aphthous group.

Effect on blood pressure and pulse rate after administration of an epinephrine containing dental local anaesthetic in hypertensive patients.

OBJECTIVE: To document safety for use, through recording changes in blood pressure and pulse rate, after administering 3.6 ml of a 2% lignocaine with 1:100,000 epinephrine dental local anaesthetic in a group of hypertensive patients. METHODOLOGY: The present study was conducted at Shaikh Zayed Medical Complex, Lahore, from May to December 2008, using a convenience sampling technique. The first 60 walk-in patients in the dental outpatient department planned for tooth extraction were enrolled in the study. Out of these 60 patients, 10 had pre-hypertension (BP = 130/90), 10 had stage 1 hypertension (BP =140-159/90-99) and 10 study subjects were suffering from stage 2 hypertension (BP = 160-179/100-109). Thirty age and sex matched normo-tensive patients requiring tooth extraction acted as the control group. All patients were administered two cartridges each of 1.8 ml of dental local anaesthesia containing 2% Lignocaine with 1:100,000 epinephrine. Blood pressure and pulse rate (PR) were the risk indicators that were measured thrice; pre-injection, 2 minutes and 5 minutes after injection. RESULTS: A decrease in systolic in stage 2 hypertension patients after 2 and 5 minutes of injections was noted. The diastolic BP (DBP) fell in all the groups after injections. Mean pulse rate increased from three to four beats per minute in all groups except in stage 2 hypertension patients where it slightly decreased. CONCLUSION: Epinephrine containing dental local anaesthesia decreased systolic blood pressure in stage 2 hypertension patients included in this study. There was an observed decrease of 21 mm Hg in systolic blood pressure hypertension patients but with no adverse effects.