Two-way pharmacodynamic modeling of drug combinations and its application to pairs of repurposed Ebola and SARS-CoV-2 agents.

Existing pharmacodynamic (PD) mathematical models for drug combinations discriminate antagonistic, additive, multiplicative, and synergistic effects, but fail to consider how concentration-dependent drug interaction effects may vary across an entire dose-response matrix. We developed a two-way pharmacodynamic (TWPD) model to capture the PD of two-drug combinations. TWPD captures interactions between upstream and downstream drugs that act on different stages of viral replication, by quantifying upstream drug efficacy and concentration-dependent effects on downstream drug pharmacodynamic parameters. We applied TWPD to previously published in vitro drug matrixes for repurposed potential anti-Ebola and anti-SARS-CoV-2 drug pairs. Depending on the drug pairing, the model recapitulated combined efficacies as or more accurately than existing models and can be used to infer efficacy at untested drug concentrations. TWPD fits the data slightly better in one direction for all drug pairs, meaning that we can tentatively infer the upstream drug. Based on its high accuracy, TWPD could be used in concert with PK models to estimate the therapeutic effects of drug pairs in vivo.

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.

Harnessing the MYB-dependent TAL1 5'super-enhancer for targeted therapy in T-ALL.

The acquisition of genetic abnormalities engendering oncogene dysregulation underpins cancer development. Certain proto-oncogenes possess several dysregulation mechanisms, yet how each mechanism impacts clinical outcome is unclear. Using T-cell acute lymphoblastic leukemia (T-ALL) as an example, we show that patients harboring 5'super-enhancer (5'SE) mutations of the TAL1 oncogene identifies a specific patient subgroup with poor prognosis irrespective of the level of oncogene dysregulation. Remarkably, the MYB dependent oncogenic 5'SE can be targeted using Mebendazole to induce MYB protein degradation and T-ALL cell death. Of note Mebendazole treatment demonstrated efficacy in vivo in T-ALL preclinical models. Our work provides proof of concept that within a specific oncogene driven cancer, the mechanism of oncogene dysregulation rather than the oncogene itself can identify clinically distinct patient subgroups and pave the way for future super-enhancer targeting therapy.

TAL1 activation in T-cell acute lymphoblastic leukemia: a novel oncogenic 3' neo-enhancer.

T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.

Clinico-epidemiological study of an outbreak of typhoid in North India.

Introduction: Typhoid fever is endemic in India. There have been various outbreaks of typhoid fever reported from different parts of India. Considering the outbreak potential of typhoid, the present study was undertaken wherein an outbreak of typhoid occurred in a city in northern India. Methodology: The study design used was a cross-sectional descriptive study. Detailed information was obtained from each reported case. Active case finding surveys were conducted. Case definitions for suspect, probable, and confirmed case taken for our study, have already been validated by the World health organisation (WHO). All patients were subjected to requisite investigations. A sanitary survey was carried out to locate defects in water supply and sewage disposal. Record of bacteriological survey of water samples from the area was scrutinized. Descriptive epidemiology of cases was carried out. Results: Attack rate was 16.1% and 17.4% among men and women, respectively. Fever was present in 52 (69.3%) cases. The epidemic curve displayed a sudden peak and an abrupt fall of cases. This is suggestive of a common point source outbreak. There were no fatalities. Sanitary survey teams found a sewage leak in the locality where maximum cases were reported. Lab reports tested positive for salmonella species in water. The outbreak was promptly controlled after repair of the leaking sewage pipeline; and provision of alternative source of water supply. Conclusion: This study reemphasises the requirement of a basic public health measure and infrastructure, that is, water quality monitoring by concerned authorities to break the chain of transmission of typhoid fever.

Drug abuse in North Kashmir - An experience from a drug de-addiction center.

Background: The history of substance abuse is as old as mankind itself. Easy transit of drugs across the state of Jammu and Kashmir is facilitated due to its geographical location. Materials and Methods: A descriptive study was carried out among individuals with substance use disorders in a de-addiction center in Northern Kashmir from August 2017 to July 2018. The study sample included all patients with substance use disorders attending the de-addiction center. Inclusion criteria: Individuals using substances attending the de-addiction center where the study was carried out were included in the study. Exclusion criteria: Individuals using substances attending the de-addiction center and not willing to participate were excluded from the study. A semi-structured pretested questionnaire was utilized as the study tool. Excel sheet and SPSS version 23 were used for data analysis. Ethical approval was obtained from the institutional ethical committee. Results: The mean (SD) age of patients was 29.58 years (8.8) ranging from 10 to 62 years and all were males. The commonest age of onset for substance use was between 11 and 20 years (56%). Nicotine abuse was the most common substance in our study. Peer pressure (52.8%) was the most common reason for drug dependence, followed by relief from negative symptoms on abstinence (29.6%). Conclusion: There is a worrying trend of the early age of initiation into substance use disorders. Since more youth are becoming engaged in substance use, it is important to evolve and apply preventive, curative, and rehabilitative strategies before it is too late.

Challenges and Road Map towards Starting a New COVID Hospital.

Background and Aim: With advent of the Coronavirus Disease 2019 (covid-19) pandemic, need for a dedicated government hospital was felt. Following directions after a cabinet decision, a dedicated Covid hospital was made functional within a month in Central district of Delhi. This manuscript briefs the journey and challenges experienced during this mission. Method: As per decision of the state health ministry, bed allocation was planned along with provision for diagnosis, treatment and prevention of Covid -19. Various trainings were simultaneously conducted, licences were obtained and manpower and material were arranged starting with procurement to service provision and waste management. Result: Concerted efforts resulted in initiation of clinical and diagnostic services within one month of initiation of teamwork. Government supported in all the licencing requirements and material management. The hospital became functional during the first wave; and by the start of second wave, 20-bedded fully equipped Intensive Care Unit (ICU) was ready with pressure swing adsorber (PSA) oxygen generator in premises. Conclusion: A well-coordinated action in the right direction with administrative support can help in achieving difficult targets. Opening a new hospital amidst lockdown and resource constraints in an emergency situation was a rewarding achievement.

Modeling explains prolonged SARS-CoV-2 nasal shedding relative to lung shedding in remdesivir-treated rhesus macaques.

In clinical trials, remdesivir decreased recovery time in hospitalized patients with SARS- CoV-2 and prevented hospitalization when given early during infection, despite not reducing nasal viral loads. In rhesus macaques, early remdesivir prevented pneumonia and lowered lung viral loads, but viral loads increased in nasal passages after five days. We developed mathematical models to explain these results. Our model raises the following hypotheses: 1) in contrast to nasal passages, viral load monotonically decreases in lungs during therapy because of infection-dependent generation of refractory cells, 2) slight reduction in lung viral loads with an imperfect agent may result in a substantial decrease in lung damage, and 3) increases in nasal viral load may occur because of a blunting of peak viral load that decreases the intensity of the innate immune response. We demonstrate that a higher potency drug could lower viral loads in nasal passages and lungs.

Modeling-Based Response-Guided DAA Therapy for Chronic Hepatitis C to Identify Individuals for Shortening Treatment Duration.

Shortening duration of direct-acting antiviral therapy for chronic hepatitis C could provide cost savings, reduce medication exposure, and foster adherence and treatment completion in special populations. The current analysis indicates that measuring hepatitis C virus at baseline and on days 7 and 14 of therapy can identify patients for shortening therapy duration.

Multi-scale modelling reveals that early super-spreader events are a likely contributor to novel variant predominance.

The emergence of new SARS-CoV-2 variants of concern (VOC) has hampered international efforts to contain the COVID-19 pandemic. VOCs have been characterized to varying degrees by higher transmissibility, worse infection outcomes and evasion of vaccine and infection-induced immunologic memory. VOCs are hypothesized to have originated from animal reservoirs, communities in regions with low surveillance and/or single individuals with poor immunologic control of the virus. Yet, the factors dictating which variants ultimately predominate remain incompletely characterized. Here we present a multi-scale model of SARS-CoV-2 dynamics that describes population spread through individuals whose viral loads and numbers of contacts (drawn from an over-dispersed distribution) are both time-varying. This framework allows us to explore how super-spreader events (SSE) (defined as greater than five secondary infections per day) contribute to variant emergence. We find stochasticity remains a powerful determinant of predominance. Variants that predominate are more likely to be associated with higher infectiousness, an SSE early after variant emergence and ongoing decline of the current dominant variant. Additionally, our simulations reveal that most new highly infectious variants that infect one or a few individuals do not achieve permanence in the population. Consequently, interventions that reduce super-spreading may delay or mitigate emergence of VOCs.

Estimation of the in vivo neutralization potency of eCD4Ig and conditions for AAV-mediated production for SHIV long-term remission.

The engineered protein eCD4Ig has emerged as a promising approach to achieve HIV remission in the absence of antiviral therapy. eCD4Ig neutralizes nearly all HIV-1 isolates and induces antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. To characterize the in vivo antiviral neutralization and possible ADCC effects of eCD4Ig, we fit mathematical models to eCD4Ig, anti­eCD4Ig-drug antibody (ADA), and viral load kinetics from healthy and simian-human immunodeficiency virus AD8 (SHIV-AD8) infected nonhuman primates that were treated with single or sequentially dosed eCD4Ig passive administrations. Our model predicts that eCD4Ig transiently decreases SHIV viral loads due to neutralization only with an in vivo IC50 of ~25 µg/ml but with limited effect due to ADA. Simulations suggest that endogenous, continuous expression of eCD4Ig at levels greater than 105 µg/day, as is possible with Adeno-associated virus (AAV) vector-based production, could overcome the diminishing effects of ADA and allow for long-term remission of SHIV viremia in nonhuman primates.

Epigenomic technologies for precision oncology.

Epigenetic patterns in a cell control the expression of genes and consequently determine the phenotype of a cell. Cancer cells possess altered epigenomes which include aberrant patterns of DNA methylation, histone tail modifications, nucleosome positioning and of the three-dimensional chromatin organization within a nucleus. These altered epigenetic patterns are potential useful biomarkers to detect cancer cells and to classify tumor types. In addition, the cancer epigenome dictates the response of a cancer cell to therapeutic intervention and, therefore its knowledge, will allow to predict response to different therapeutic approaches. Here we review the current state-of-the-art technologies that have been developed to decipher epigenetic patterns on the genomic level and discuss how these methods are potentially useful for precision oncology.

Antiretroviral therapy for HIV and intrahepatic hepatitis C virus replication.

OBJECTIVE: HIV alters host responses to hepatitis C virus (HCV). However, the impact of antiretroviral therapy (ART) on HCV is rarely understood in relevant tissues and never before within individual hepatocytes. DESIGN: HIV and HCV kinetics were studied before and after ART initiation among 19 HIV/HCV co-infected persons. From five persons with the largest decline in plasma HCV RNA, liver tissues collected before and during ART, when plasma HIV RNA was undetectable, were studied. METHODS: We used single-cell laser capture microdissection and quantitative PCR to assess intrahepatic HCV. Immunohistochemistry was performed to characterize intrahepatic immune cell populations. RESULTS: Plasma HCV RNA declined by 0.81 (0.52-1.60) log10 IU/ml from a median (range) 7.26 (6.05-7.29) log10 IU/ml and correlated with proportions of HCV-infected hepatocytes (r = 0.89, P = 2 × 10-5), which declined from median (range) of 37% (6-49%) to 23% (0.5-52%) after plasma HIV clearance. Median (range) HCV RNA abundance within cells was unchanged in four of five participants. Liver T-cell abundance unexpectedly decreased, whereas natural killer (NK) and NK T-cell infiltration increased, correlating with changes in proportions of HCV-infected hepatocytes (r = -0.82 and r = -0.73, respectively). Hepatocyte expression of HLA-E, an NK cell restriction marker, correlated with proportions of HCV-infected hepatocytes (r = 0.79). CONCLUSION: These are the first data to show that ART control of HIV reduces the intrahepatic burden of HCV. Furthermore, our data suggest that HIV affects the pathogenesis of HCV infection by an NK/NK T-cell-mediated mechanism that may involve HLA-E and can be rescued, at least in part, by ART.

Machine learning for mathematical models of HCV kinetics during antiviral therapy.

Mathematical models for hepatitis C virus (HCV) dynamics have provided a means for evaluating the antiviral effectiveness of therapy and estimating treatment outcomes such as the time to cure. Recently, a mathematical modeling approach was used in the first proof-of-concept clinical trial assessing in real-time the utility of response-guided therapy with direct-acting antivirals (DAAs) in chronic HCV-infected patients. Several retrospective studies have shown that mathematical modeling of viral kinetics predicts time to cure of less than 12 weeks in the majority of individuals treated with sofosbuvir-based as well as other DAA regimens. A database of these studies was built, and machine learning methods were evaluated for their ability to estimate the time to cure for each patient to facilitate real-time modeling studies. Data from these studies exploring mathematical modeling of HCV kinetics under DAAs in 266 chronic HCV-infected patients were gathered. Different learning methods were applied and trained on part of the dataset ('train' set), to predict time to cure on the untrained part ('test' set). Our results show that this machine learning approach provides a means for establishing an accurate time to cure prediction that will support the implementation of individualized treatment.

ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

Mathematical Modeling of Vaccines That Prevent SARS-CoV-2 Transmission.

SARS-CoV-2 vaccine clinical trials assess efficacy against disease (VEDIS), the ability to block symptomatic COVID-19. They only partially discriminate whether VEDIS is mediated by preventing infection completely, which is defined as detection of virus in the airways (VESUSC), or by preventing symptoms despite infection (VESYMP). Vaccine efficacy against transmissibility given infection (VEINF), the decrease in secondary transmissions from infected vaccine recipients, is also not measured. Using mathematical modeling of data from King County Washington, we demonstrate that if the Moderna (mRNA-1273QS) and Pfizer-BioNTech (BNT162b2) vaccines, which demonstrated VEDIS > 90% in clinical trials, mediate VEDIS by VESUSC, then a limited fourth epidemic wave of infections with the highly infectious B.1.1.7 variant would have been predicted in spring 2021 assuming rapid vaccine roll out. If high VEDIS is explained by VESYMP, then high VEINF would have also been necessary to limit the extent of this fourth wave. Vaccines which completely protect against infection or secondary transmission also substantially lower the number of people who must be vaccinated before the herd immunity threshold is reached. The limited extent of the fourth wave suggests that the vaccines have either high VESUSC or both high VESYMP and high VEINF against B.1.1.7. Finally, using a separate intra-host mathematical model of viral kinetics, we demonstrate that a 0.6 log vaccine-mediated reduction in average peak viral load might be sufficient to achieve 50% VEINF, which suggests that human challenge studies with a relatively low number of infected participants could be employed to estimate all three vaccine efficacy metrics.

Modeling reveals no direct role of the extent of HBV DNA integrations on the outcome of infection.

Hepatitis B virus (HBV) with its high prevalence and death toll is one of the most important infectious diseases to study. Yet, there is very little progress in the development of within-host models for HBV, which has subsequently hindered our understanding of this virus. The uncertainty around the proliferation of infected hepatocytes has been studied but never in association with other important biological continuous events such as integrations and superinfections. This is despite the fact that these processes affect the diversity and composition of infected cell population in the liver and an improved understanding of the cellular composition will undoubtedly assist in strategizing against this viral infection. Here, we developed novel mathematical models that incorporate these key biological processes and analyzed them both analytically and numerically. Unaffected by the extent of integrated DNA (IDNA), the outcome of HBV infection was primarily dictated by the balance between processes generating and killing infected hepatocytes containing covalent closed circular DNA (cccDNA). The superinfection was found to be a key process in the spread of HBV infection as its exclusion could not reproduce experimentally observed composition of infected hepatocytes at peak of acute HBV infection, a stage where our model predicts that infected hepatocytes most likely carry both cccDNA and IDNA. Our analysis further suggested the existence of some form of selective advantage of infected hepatocytes containing only IDNA to explain the viral dynamics observed during antiviral treatment and the transition from peak to acute infection. Finally, the fine line between liver destruction and resolution of acute HBV infection was found to be highly influenced by the fate of cccDNA during cellular proliferation.

Efficacy of Autologous Conditioned Serum (ACS), Platelet-Rich Plasma (PRP), Hyaluronic Acid (HA) and Steroid for Early Osteoarthritis Knee: A Comparative Analysis.

BACKGROUND: Intra-articular injection therapy constituting corticosteroids, viscosupplements and blood-derived products are considered to have a key role in non-operative management of osteoarthritis knee. While corticosteroids and viscosupplements have proven short-term efficacy in early osteoarthritis; orthobiologics are gaining increased attention in osteoarthritis management. The aim of present study was thus to compare two commonly used biologics (platelet-rich plasma/PRP and autologous conditioned serum/ACS) to each other and to established therapies. METHODS: After required institutional clearances, all patients presenting with early primary osteoarthritis knee who had failed initial conservative management and received only unilateral knee injection were included. Patients in the PRP group were compared to the other groups (comprising the HA/hyaluronic acid group, steroid group, and a matched cohort who had been administered ACS for the same indication earlier). Clinical outcome was evaluated using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire and Visual Analogue scale (VAS) pre-injection and at 6 months. RESULTS: ACS and PRP did not have any significant difference in terms of either WOMAC score (p = 0.154) or VAS score at 6 months (p = 0.850). The scores for both these orthobiologics were better than the control groups (HA group and Steroid group). Between the two control groups, HA group had better VAS scores as compared to the Steroid group (p = 0.008). CONCLUSION: The clinical outcomes following intra-articular injection of ACS and PRP are better than controls (HA and steroid), but a difference between the two orthobiologics could not be demonstrated. LEVEL OF EVIDENCE: 3b.