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bioRxiv2017

The effect of a lifestyle intervention in obese pregnant women on change in gestational metabolic profiles: findings form the UK Pregnancies Better Eating and Activity Trial (UPBEAT) RCT.

Background: Pregnancy metabolic disruption is believed to be enhanced in obese women and lead to adverse outcomes in them and their offspring. The UK Pregnancies Better Eating and Activity Trial (UPBEAT), a randomised controlled trial of a lifestyle intervention in obese pregnant women, has already been shown to improve diet and physical activity. We used UPBEAT to determine (a) the magnitude of change in metabolic profiles in obese pregnant women and (b) the impact of a lifestyle intervention that successfully improved diet and physical activity on these profiles. Methods: Detailed targeted metabolic profiling, with quantification of 158 metabolic features (129 lipid measures, 9 glycerides and phospholipids, and 20 low-molecular weight metabolites) was completed on three occasions (~17-, 28- and 35-weeks of gestation) in the UPBEAT participants using NMR. Random intercept and random slope models were used to quantify metabolite changes in obese women using the control (usual care) group only (N = 577). The effect of the intervention was determined by comparing rates of metabolite change between those randomised to intervention and usual care, using intention to treat analyses (N = 1158). Results: There were adverse changes across pregnancy in most lipoprotein subclasses, lipids, glycerides, phospholipids, several fatty acids and glucose. All extremely large, very large, large, medium, small and very small VLDL particles increased by 2 to 3 standard deviation units (SD), with IDL, and large, medium and small LDL particles increasing by 1 to 2SD, between 16- and 36-weeks. Triglycerides increased by 3 to 4SD and glucose increased by 2SD, with more modest changes in other metabolites. The intervention reduced the rate of increase in extremely large, very large, large and medium VLDL, in particular those containing triglycerides. Triglyceride to phosphoglyceride ratio was reduced and there were improvements in fatty acid profiles (increases in the proportion of all fatty acids that were linoleic, omega-6 and polyunsaturated and decreases in the proportion of saturated). Conclusion: Systemic metabolism is markedly disrupted in obese pregnant women, but a lifestyle intervention that improved their diet and physical activity has beneficial effects on some of these profiles; these effects might have long-term benefit. Clinical trial registration ID # ISRCTN89971375.

clinical trials↗

bioRxiv2017

Topical Silver Diamine Fluoride For Dental Caries Arrest In Preschool Children: A Randomized Controlled Trial

ObjectivesThe Stopping Cavities Trial investigated effectiveness and safety of 38% silver diamine fluoride in arresting caries lesions.\n\nMaterials and MethodsDouble-blind randomized placebo-controlled superiority trial with 2 parallel groups. Oregon preschools. 66 preschool children with [≥]1 lesion. 38% silver diamine fluoride or placebo (blue-tinted water), applied topically to the lesion. The primary endpoint was caries arrest (lesion inactivity, Nyvad criteria) 14-21 days post intervention. Dental plaque was collected from all children, and microbial composition was assessed by RNA sequencing from 2 lesions and 1 unaffected surface before treatment and at follow-up for 3 children from each group.\n\nResults and ConclusionMean fraction of arrested caries lesions in the silver diamine fluoride group was higher (0.72; 95% CI; 0.55, 0.84) than in the placebo group (0.05; 95% CI; 0.00, 0.16). Confirmatory analysis using generalized estimating equation log-linear regression, accounting for the number of treated surfaces and length of follow-up, indicated the fraction of arrested caries was significantly higher in the treatment group (relative risk, 17.3; 95% CI: 4.3 to 69.4). No harms were observed. RNA sequencing analysis identified no consistent changes in relative abundance of caries-associated microbes, nor emergence of antibiotic or metal resistance gene expression. Topical 38% silver diamine fluoride was effective and safe in arresting cavities in preschool children. The treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.\n\nTrial RegistrationClinicalTrials.gov NCT02536040.\n\nClinical SignificanceIn this clinical trial, 72% of caries lesions were arrested by silver diamine fluoride, with no harms. Contrary to the presumed antibacterial mechanism, lesion bacterial composition changed negligibly. This simple topical treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.

clinical trials↗

bioRxiv2018

Characterization of intact proviruses in blood and lymph node from HIV-infected individuals undergoing analytical treatment interruption

The role of lymphoid tissue as a potential source of HIV-1 rebound following interruption of antiretroviral therapy is uncertain. To address this issue, we compared the latent viruses obtained from CD4+ T cells in peripheral blood and lymph nodes to viruses emerging during treatment interruption. Latent viruses were characterized by sequencing near full-length (NFL) proviral DNA, and env from viral outgrowth cultures (VOAs). 5 HIV-1 infected individuals on antiretroviral therapy (ART) were studied, 4 of whom participated in a clinical trial that included an analytical treatment interruption. Intact or replication competent clonal sequences from blood and lymph node overlapped. In contrast, there was no overlap between 205 latent reservoir and 125 rebound sequences in the 4 individuals who underwent treatment interruption. However, rebound viruses could be accounted for by recombination. The data suggests that CD4+ T cells carrying latent viruses circulate between blood and lymphoid tissues in individuals on ART and support the idea that recombination may play a role in the emergence of rebound viremia.

clinical trials↗

bioRxiv2017

TORC1 inhibition as an immunotherapy to reduce infections in the elderly

mTOR inhibition extends lifespan and ameliorates aging-related pathologies including declining immune function in model organisms. The objective of this Phase 2a clinical trial was to determine if low dose mTOR inhibitor therapy enhanced immune function and thereby decreased infection rates in elderly subjects. The results indicate that 6 weeks of treatment with a low dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor (that selectively inhibits TORC1 downstream of mTOR) was safe, significantly decreased the rate of infections reported by elderly subjects for a year following study drug initiation, upregulated antiviral gene expression, and significantly improved influenza vaccination response. Thus selective TORC1 inhibition with a combination of BEZ235 and RAD001 may be efficacious as immunotherapy to reduce infections, a leading cause of death in the elderly.\n\nOne Sentence SummaryTreatment of elderly subjects with a low dose mTOR inhibitor regimen that selectively inhibits TORC1 significantly decreased infection rates

clinical trials↗

bioRxiv2018

Effect of fluoxetine on adult amblyopia: a placebo-controlled study combining neuroplasticity-enhancing pharmacological intervention and perceptual training

Amblyopia is a common visual disorder that is treatable in childhood. However, therapies have limited efficacy in adult patients with amblyopia. Fluoxetine can reinstate early-life critical period-like neuronal plasticity and has been used to recover functional vision in adult rats with amblyopia. This phase 2, randomized, double-blind (fluoxetine vs. placebo), multicenter clinical trial examined whether or not fluoxetine can improve visual acuity in amblyopic adults. This interventional trial included 42 participants diagnosed with moderate to severe amblyopia. Subjects were randomized to receive either 20 mg fluoxetine (n=22) or placebo (n=20). During the 10-week treatment period, all subjects performed daily computerized perceptual training and eye patching. There was no significant difference in treatment efficacy between the groups. Visual acuity at the primary endpoint had significantly improved over baseline in both the fluoxetine (-0.167 logMAR) and placebo (-0.194 logMAR) groups (both p < 0.001). Because patching alone is not effective in adults, the visual acuity improvement likely resulted from perceptual training. There was a positive correlation between visual acuity improvement and the perceptual training time. While this study failed to provide evidence that fluoxetine enhances neuroplasticity, our data support the usefulness of perceptual training for vision improvement in adults with amblyopia.

clinical trials↗

bioRxiv2022

MRI-based computational model generation for cerebral perfusion simulations in health and ischaemic stroke

Cerebral perfusion models were found to be promising research tools to predict the impact of acute ischaemic stroke and related treatments on cerebral blood flow (CBF) linked to patients functional outcome. To provide insights relevant to clinical trials, perfusion simulations need to become suitable for group-level investigations, but computational studies to date have been limited to a few patient-specific cases. This study set out to overcome issues related to automated parameter inference, that restrict the sample size of perfusion simulations, by integrating neuroimaging data. Seventy-five brain models were generated using measurements from a cohort of 75 healthy elderly individuals to model resting-state CBF distributions. Computational perfusion model geometries were adjusted using healthy reference subjects T1-weighted MRI. Haemodynamic model parameters were determined from CBF measurements corresponding to arterial spin labelling perfusion MRI. Thereafter, perfusion simulations were conducted for 150 acute ischaemic stroke cases by simulating an occlusion and cessation of blood flow in the left and right middle cerebral arteries. The anatomical (geometrical) fitness of the brain models was evaluated by comparing the simulated grey and white matter (GM and WM) volumes to measurements in healthy reference subjects. Statistically significant, strong positive correlations were found in both cases (GM: Pearsons r 0.74, P-value< 0.001; WM: Pearsons r 0.84, P-value< 0.001). Haemodynamic parameter tuning was verified by comparing total volumetric blood flow rate to the brain in reference subjects and simulations resulting in Pearsons r 0.89, and P-value< 0.001. In acute ischaemic stroke cases, the simulated infarct volume using a perfusion-based proxy was 197{+/-}25 ml. Computational results showed excellent agreement with anatomical and haemodynamic literature data corresponding to T1-weighted, T2-weighted, and phase-contrast MRI measurements both in healthy scenarios and in acute ischaemic stroke cases. Simulation results represented solely worst-case stroke scenarios with large infarcts because compensatory mechanisms, e.g. collaterals, were neglected. The established computational brain model generation framework provides a foundation for population-level cerebral perfusion simulations and for in silico clinical stroke trials which could assist in medical device and drug development.

bioengineering↗

bioRxiv2019

Within Visit Test-Retest Reliability of EEG Profiles in Children with Autism Spectrum Disorder and Typical Development

Biomarker development is currently a high priority in neurodevelopmental disorder research. For many types of biomarkers (particularly biomarkers of diagnosis), reliability over short time periods is critically important. In the field of autism spectrum disorder (ASD), resting electroencephalography (EEG) power spectral densities (PSD) are well-studied for their potential as biomarkers. Classically, such data have been decomposed into pre-specified frequency bands (e.g., delta, theta, alpha, beta, and gamma). Recent technical advances, such as the Fitting Oscillations and One-Over-F (FOOOF) algorithm, allow for targeted characterization of the features that naturally emerge within an EEG PSD, permitting a more detailed characterization of the frequency band-agnostic shape of each individuals EEG PSD. Here, using two resting EEGs collected a median of 6 days apart from 22 children with ASD and 25 typically developing (TD) controls during the Feasibility Visit of the Autism Biomarkers Consortium for Clinical Trials, we estimate within visit test-retest reliability based on characterization of the PSD shape in two ways: (1) Using the FOOOF algorithm we estimate six parameters (offset, slope, number of peaks, and amplitude, center frequency and bandwidth of the largest alpha peak) that characterize the shape of the EEG PSD; and (2) using nonparametric functional data analyses, we decompose the shape of the EEG PSD into a reduced set of basis functions that characterize individual power spectrum shapes. We show that individuals exhibit idiosyncratic PSD signatures that are stable over recording sessions using both characterizations. Our data show that EEG activity from a brief two-minute recording provides an efficient window into understanding brain activity at the single-subject level with desirable psychometric characteristics that persist across different analytical decomposition methods. This is a necessary step towards analytical validation of biomarkers based on the EEG PSD, and provides insights into parameters of the PSD that offer short-term reliability (and thus promise as potential biomarkers of trait or diagnosis) versus those that are more variable over the short term (and thus may index state or other rapidly dynamic measures of brain function). Future research should address longer-term stability of the PSD, for purposes such as monitoring development or response to treatment.

neuroscience↗

bioRxiv2022

Licensing Microgels Prolong the Immunomodulatory Phenotype of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) are sensors of inflammation, and they exert immunomodulatory properties through the secretion of cytokines and exosomes and direct cell-cell interactions. MSC are routinely used in clinical trials and effectively resolve inflammatory conditions. Nevertheless, inconsistent clinical outcomes necessitate the need for more robust therapeutic phenotypes. The immunomodulatory properties of MSC can be enhanced and protracted by priming (aka licensing) them with IFN{gamma} and TNF. Yet these enhanced properties rapidly diminish, and prolonged stimulation could tolerize their response. Hence a balanced approach is needed to enhance the therapeutic potential of the MSC for consistent clinical performance. Here, we investigated the concentration-dependent effects of IFN{gamma} and TNF and developed gelatin-based microgels to sustain a licensed MSC phenotype. We show that IFN{gamma} treatment is more beneficial than TNF in promoting an immunomodulatory MSC phenotype. We also show that the microgels possess integrin-binding sites to support MSC attachment and a net positive charge to sequester the licensing cytokines electrostatically. Microgels are enzymatically degradable, and the rate is dependent on the enzyme concentration and matrix density. Our studies show that one milligram of microgels by dry mass can sequester up to 641 {+/-} 81 ng of IFN{gamma}. Upon enzymatic degradation, microgels exhibited a sustained release of IFN{gamma} that linearly correlated with their degradation rate. The MSC cultured on the IFN{gamma} sequestered microgels displayed efficient licensing potential comparable to or exceeding the effects of bolus IFN{gamma} treatment. When cultured with proinflammatory M1-like macrophages, the MSC-seeded on licensing microgel showed an enhanced immunomodulatory potential compared to untreated MSC and MSC treated with bolus IFN{gamma} treatment. Specifically, the MSC seeded on licensing microgels significantly upregulated Arg1, Mrc1, and Igf1, and downregulated Tnfa in M1-like macrophages compared to other treatment conditions. These licensing microgels are a potent immunomodulatory approach that shows substantial promise in elevating the efficacy of current MSC therapies and may find utility in treating chronic inflammatory conditions.

bioengineering↗

bioRxiv2022

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy Against 2D and 3D Breast Cancer Models

Sophorolipids are biosurfactants derived from the non-pathogenic yeast Starmerella bombicola, with potential efficacy in anti-cancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drugscreening. Drug screening typically utilizes 2D cell monolayers due to their simplicity and potential for high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment, and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. We screened two sophorolipid candidates and clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing Optical Coherence Tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

pharmacology and toxicology↗

bioRxiv2023

Intratumoral injection of melatonin enhances tumor regression in cell line-derived and patient-derived xenografts of head and neck cancer by increasing mitochondrial oxidative stress

Head and neck squamous cell carcinoma present a high mortality rate. Melatonin has been shown to have oncostatic effects in different types of cancers. However, inconsistent results have been reported for in vivo applications. Consequently, an alternative administration route is needed to improve bioavailability and establish the optimal dosage of melatonin for cancer treatment. On the other hand, the use of patient-derived tumor models has transformed the field of drug research because they reflect the heterogeneity of patient tumor tissues. In the present study, we explore mechanisms for increasing melatonin bioavailability in tumors and investigate its potential as an adjuvant to improve the therapeutic efficacy of cisplatin in the setting of both xenotransplanted cell lines and primary human HNSCC. We analyzed the effect of two different formulations of melatonin administered subcutaneously or intratumorally in Cal-27 and SCC-9 xenografts and in patient-derived xenografts. Melatonin effects on tumor mitochondrial metabolism was also evaluated as well as melatonin actions on tumor cell migration. In contrast to the results obtained with the subcutaneous melatonin, intratumoral injection of melatonin drastically inhibited tumor progression in HNSCC-derived xenografts, as well as in patient-derived xenografts. Interestingly, intratumoral injection of melatonin potentiated CDDP effects, decreasing Cal-27 tumor growth. We demonstrated that melatonin increases ROS production and apoptosis in tumors, targeting mitochondria. Melatonin also reduces migration capacities and metastasis markers. These results illustrate the great clinical potential of intratumoral melatonin treatment and encourage a future clinical trial in cancer patients to establish a proper clinical melatonin treatment.

cancer biology↗

bioRxiv2023

Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity

Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five-to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/563088v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a21f94org.highwire.dtl.DTLVardef@1c76751org.highwire.dtl.DTLVardef@500930org.highwire.dtl.DTLVardef@8b6c05_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

pharmacology and toxicology↗

bioRxiv2024

Novel derivatives of BCV and (S)-HPMPA inhibit orthopoxviruses and human adenoviruses more potently than BCV

BCV and tecovirimat are the only two chemical drugs that have been approved to treat smallpox and can be requested for Mpox treatment through a single-patient Emergency Investigational New Drug (EIND) application. Disappointedly, the efficacy of tecovirimat manifested in a recent clinical trial is far from being satisfactory, while the clinical efficacy of BCV is still inconclusive. Given that MPXV, variola and other emerging orthopoxviruses are posing serious threats to global health, it is urgent to develop better therapeutics. In this study, we tested the antiviral effects of three novel prodrugs, which were designed based on previously reported parent drugs, either (S)-HPMPC (cidofovir) or (S)-HPMPA. We found that one of the (S)-HPMPA-based prodrugs, ODE-(S)-HPMPA formate, exhibited significantly better anti-orthopoxvirus activity than BCV both in vitro and in vivo, which also inhibited human adenovirus type 2 and type 21 more efficiently than BCV. Most strikingly, the EC50 and EC90 of ODE-(S)-HPMPA formate against MPXV were more than 40-fold lower than those of BCV. In contrast, we observed that the anti-HSV-1 activities of the (S)-HPMPA-based prodrugs were less effective than those of the cidofovir-based prodrugs (BCV and BCV formate), especially in vivo. Moreover, we showed for the first time that cytidine and adenine analog combined therapies could provide mice with complete protection against lethal challenges of both vaccinia and HSV-1. Collectively, we propose that both the ODE-(S)-HPMPA formate and the BCV/ODE-(S)-HPMPA formate combination are worth further investigations for their potential clinical applications.

microbiology↗

bioRxiv2025

Beyond the Cut: Long-read sequencing reveals complex genomic and transcriptomic changes in AAV-CRISPR therapy for Duchenne Muscular Dystrophy

Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5 or 3 ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV-Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

bioengineering↗

bioRxiv2025

The effect of vitamin D and calcium supplementation on gut microbiota composition in adolescents living with HIV in Southern Africa

Adolescents living with HIV are at risk of delayed growth and of increased susceptibility to infections, potentially linked to vitamin D insufficiency, which is common in people living with HIV. We analyzed rectal swab microbiota compositions before and after 48 weeks supplementation with high-dose vitamin D and calcium, or placebo, embedded in a randomized controlled trial conducted in Zambia and Zimbabwe (Pan African Clinical Trials Registry PACTR20200989766029). In a sub-cohort of 244 participants, we first assessed baseline associations of the rectal swab microbiota with vitamin D metabolites, parathyroid hormone (PTH), and bone mineral density (BMD). We then analyzed longitudinal changes in rectal swab microbiota and intestinal barrier integrity during supplementation and examined whether the baseline microbiota influenced the treatment response. At baseline, anaerobic taxa within the class Clostridia were positively associated with PTH, 1,25(OH)2D, and BMD, and negatively associated with 24,25(OH)2D. After supplementation, Shannon diversity was decreased ({beta}Std=-0.495, CI [-0.802, -0.188], p=0.0018), median Bray-Curtis dissimilarity from baseline samples was increased ({beta}Std=0.399, CI [0.086, 0.712], p=0.013), and abundance of Clostridia_vadinBB60_group was decreased (log2fc=-1.715, CI [-2.596, -0.835], q=0.043) in the intervention arm. The intervention did not affect the intestinal barrier integrity, measured by intestinal fatty acid binding protein (iFABP) concentration in blood plasma. On average, PTH concentrations decreased and BMD increased during supplementation. However, higher baseline abundance of Clostridia-member UCG-009 predicted a smaller reduction in PTH concentrations during supplementation (log2fcstd=1.200 CI [0.656, 1.744], q=0.012), while higher baseline abundances of Enterococcus (log2fcstd=-1.220 CI [-1.807, -0.632], q=0.022) and Fournierella (log2fcstd=-1.122 CI [-1.694, -0.551], q=0.025) predicted a smaller increase in BMD. In conclusion, there was a reduction of an anaerobic taxon of the class Clostridia following vitamin D supplementation. Further, the microbiota appeared to moderate the effect of the intervention on PTH and bone density.

microbiology↗

bioRxiv2019

CIViCpy: a Python software development and analysis toolkit for the CIViC knowledgebase

PurposePrecision oncology is dependent upon the matching of tumor variants to relevant knowledge describing the clinical significance of those variants. We recently developed the Clinical Interpretations for Variants in Cancer (CIViC; civicdb.org) crowd-sourced, expert-moderated, open-access knowledgebase, providing a structured framework for evaluating genomic variants of various types (e.g., fusions, SNVs) for their therapeutic, prognostic, predisposing, diagnostic, or functional utility. CIViC has a documented API for accessing CIViC records: Assertions, Evidence, Variants, and Genes. Third-party tools that analyze or access the contents of this knowledgebase programmatically must leverage this API, often reimplementing redundant functionality in the pursuit of common analysis tasks that are beyond the scope of the CIViC web application.\n\nMethodsTo address this limitation, we developed CIViCpy (civicpy.org), a software development kit (SDK) for extracting and analyzing the contents of the CIViC knowledgebase. CIViCpy enables users to query CIViC content as dynamic objects in Python. We assess the viability of CIViCpy as a tool for advancing individualized patient care by using it to systematically match CIViC evidence to observed variants in patient cancer samples.\n\nResultsWe used CIViCpy to evaluate variants from 59,437 sequenced tumors of the AACR Project GENIE dataset. We demonstrate that CIViCpy enables annotation of >1,200 variants per second, resulting in precise variant matches to CIViC level A (professional guideline) or B (clinical trial) evidence for 38.6% of tumors.\n\nConclusionsThe clinical interpretation of genomic variants in cancers requires high-throughput tools for interoperability and analysis of variant interpretation knowledge. These needs are met by CIViCpy, an SDK for downstream applications and rapid analysis. CIViCpy (civicpy.org) is fully documented, open-source, and freely available online.

bioinformatics↗

bioRxiv2026

PRECISION EVOLUTIONARY MEDICINE: A COMPUTATIONAL GRAPH-THEORETICAL FRAMEWORK FOR PATHOGEN-SPECIFIC ANTIBIOTIC CYCLING IN MULTI-DRUG-RESISTANT GRAM-NEGATIVE INFECTIONS

BackgroundThe Proliferation of Multi-drug resistant (MDP) ESKAPE pathogens threatens to compromise the efficacy of standard antibiotic pharmacopoeia. Current antimicrobial stewardship strategies predominantly rely on reactive antibiograms selecting therapeutic agents based on immediate phenotypic susceptibility. This approach, while clinically expedient, often inadvertently selects for cross-resistance, driving the evolutionary trajectory toward pan-drug resistance. A paradigm shift is required toward predictive strategies that exploit evolutionary trade-offs, specifically Collateral Sensitivity (CS), where the acquisition of resistance to one agent induces hypersensitivity to another. MethodsWe developed a computational graph-theory framework to map the evolutionary trajectories of three critical Gram-negative pathogens: Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Drawing upon validated CS interaction matrices from experimental evolution literature, we constructed directed weighted graphs where nodes represent antibiotics and edges represent evolutionary sensitivity trade-offs. A closed-loop cycle optimization algorithm was deployed to identify pathogen-specific "Trap Loops" sequences of three or more antibiotics that force the pathogen into a state of high sensitivity. These loops were validated via stochastic in-silico clinical trials simulating 18 months of treatment, explicitly modeling clinical error and biological noise. ResultsThe model identified distinct, optimal cycling protocols for each pathogen. For E. coli, an Aminoglycoside-Beta Lactam loop (Gentamicin to Cefuroxime to Fosfomycin) demonstrated sustained suppression of resistance accumulation in silico. For K. pneumoniae, a novel Rifampicin to Doxycycline to Colistin loop was identified. For P. aeruginosa, a Tobramycin to Ciprofloxacin to Piperacillin sequence proved optimal. Stochastic simulations demonstrated that while standard reactive care resulted in progressive resistance accumulation (Normalized Resistance > 2.5), the graph-optimized protocols suppressed resistance within the therapeutic window (Normalized Resistance < 0.2) for the duration of the simulation. ConclusionWe demonstrate, through computational modeling, that antibiotic resistance trajectories can be strategically constrained by optimizing the temporal sequence of existing agents. This study provides a computational framework to inform the transition from reactive prescribing toward precision evolutionary steering. These protocols are intended to complement, not replace, clinical judgment and local antibiograms, particularly regarding pharmacokinetic constraints.

microbiology↗

bioRxiv2021

The HIV latency reversal agent HODHBt enhances NK Cell effector and memory-like functions by increasing IL-15 mediated-STAT activation

Elimination of latent HIV reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is shock and kill. This strategy is based on the transcriptional activation of latent HIV with a Latency-Reversing Agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) act as an LRA by increasing Signal Transducers and Activators of Transcription (STAT) activation mediated by IL-15 in cells isolated from aviremic participants. The IL-15 superagonist (N-803) is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanism of action by promoting the activation STATs and have shown some promise in pre-clinical models directed towards HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in NK cells and the biological consequences associated with increased STAT activation in NK effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increased secretion of CXCL10 and IFN-{gamma} and expression of cytotoxic proteins including Granzyme B, Granzyme A, Perforin, Granulysin, FASL and TRAIL. This increased cytotoxic profile results in an increase cytotoxicity against different tumor cell lines and HIV-infected cells. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and targeting this pathway could be further explored for HIV cure interventions. Author SummarySeveral clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit of these trials, they have been crucial to elucidate hurdles that shock and kill strategies have to overcome to promote an effective reduction of the latent reservoir leading. These hurdles include low reactivation potential mediated by LRAs; the negative influence of some LRAs on the activity of Natural Killer and CD8T effector cells; an increased resistance to apoptosis of latently infected cells; and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed towards HIV eradication. In here, we showed that the LRA HODHBt also improves IL-15 mediated NK effector and memory-like functions. As such, pharmacological enhancement of IL-15 mediated STAT activation can open new therapeutic venues towards an HIV cure.

cell biology↗

bioRxiv2019

Metronomic capecitabine as an immune modulator in glioblastoma patients reduces myeloid-derived suppressor cells

BackgroundMyeloid-derived suppressor cells (MDSCs) are elevated in glioblastoma (GBM) patient circulation, present in tumor tissue, and associated with poor prognosis. While low-dose chemotherapy reduces MDSCs in preclinical models, the use of this strategy to reduce MDSCs in GBM patients has yet to be evaluated. MethodsA phase 0/1 dose-escalation clinical trial was conducted in recurrent glioblastoma patients treated 5-7 days prior to surgery with low-dose chemotherapy via capecitabine followed by concomitant low-dose capecitabine and bevacizumab. Clinical outcomes, including progression-free and overall survival, were measured, along with safety and toxicity profiles. Over the treatment time course, circulating MDSC levels were measured by multi-parameter flow cytometry, and tumor tissue immune profiles were assessed via mass cytometry time-of-flight. ResultsA total of 11 patients were enrolled across escalating dose cohorts of 150, 300, and 450 mg bid, with a progression-free survival of 5.8 months (range of 1.8-27.8 months) and an overall survival of 11.5 months (range of 3->28.0 months) from trial enrollment. No serious adverse events related to the drug combination were observed. Compared to pre-treatment baseline, circulating MDSCs were found to be higher after surgery in the 150 mg treatment arm and lower in the 300 mg and 450 mg treatment arms. Increased cytotoxic immune infiltration was observed after low-dose capecitabine compared to untreated GBM patients in the 300 mg and 450 mg treatment arms. ConclusionsLow-dose, metronomic capecitabine in combination with bevacizumab is well tolerated in GBM patients and was associated with a reduction in circulating MDSC levels and an increase in cytotoxic immune infiltration into the tumor microenvironment. Trial registrationNCT02669173 FundingThis research was funded by the Cleveland Clinic, Case Comprehensive Cancer Center, Musella Foundation, and B*CURED. Capecitabine was provided in kind by Mylan Pharmaceuticals.

clinical trials↗