Dual role of signaling pathways in myeloma requires cell-type specific targeting of ligand-receptor interactions.

Although most patients with multiple myeloma respond to treatment initially, therapy resistance develops almost invariably and only a subset of patients show durable responses to immunomodulatory (IMiD) therapies. While the immune microenvironment has been extensively studied in myeloma patients, its composition is currently not used as prognostic markers in clinical routine. We hypothesized that the outcome of immune signaling pathway engagement can be highly variable, depending on which two cellular populations participate in this interaction. This would have important prognostic and therapeutic implications, suggesting that it is crucial for immune pathways to be targeted in a specific cellular context. To test this hypothesis, we investigated a cohort of 27 patients with newly diagnosed multiple myeloma. We examined the complex regulatory networks within the immune compartment and their impact on disease progression. Analysis of immune cell composition and expression profiles revealed significant differences in the B cell compartment associated with treatment response. Transcriptional states in patients with short time to progression demonstrated an enrichment of pathways promoting B cell differentiation and inflammatory responses, which may indicate immune dysfunction. Importantly, the analysis of molecular interactions within the immune microenvironment highlights the dual role of signaling pathways, which can either be associated with good or poor prognosis depending on the cell types involved. Our findings therefore argue that therapeutic strategies targeting ligand-receptor interactions should take into consideration the composition of the microenvironment and the specific cell types involved in molecular interactions.

Terpenes in Cannabis sativa Inhibit Capsaicin Responses in Rat DRG Neurons via Na+/K+ ATPase Activation.

Terpenes in Cannabis sativa exert analgesic effects, but the mechanisms are uncertain. We examined the effects of 10 terpenes on capsaicin responses in an established model of neuronal hypersensitivity. Adult rat DRG neurons cultured with neurotrophic factors NGF and GDNF were loaded with Fura2AM for calcium imaging, and treated with individual terpenes or vehicle for 5 min, followed by 1 µMol capsaicin. In vehicle treated control experiments, capsaicin elicited immediate and sustained calcium influx. Most neurons treated with terpenes responded to capsaicin after 6-8 min. Few neurons showed immediate capsaicin responses that were transient or normal. The delayed responses were found to be due to calcium released from the endoplasmic reticulum, as they were maintained in calcium/magnesium free media, but not after thapsigargin pre-treatment. Terpene inhibition of calcium influx was reversed after washout of medium, in the absence of terpenes, and in the presence of the Na+/K+ ATPase inhibitor ouabain, but not CB1 or CB2 receptor antagonists. Thus, terpenes inhibit capsaicin evoked calcium influx by Na+/K+ ATPase activation. Immunofluorescence showed TRPV1 co-expression with α1ß1 Na+/K+ ATPase in most neurons while others were either TRPV1 or α1ß1 Na+/K+ ATPase positive.

Development of Electronic Health Record-Based Machine Learning Models to Predict Barrett's Esophagus and Esophageal Adenocarcinoma Risk.

INTRODUCTION: Screening for Barrett's esophagus (BE) is suggested in those with risk factors, but remains underutilized. BE/esophageal adenocarcinoma (EAC) risk prediction tools integrating multiple risk factors have been described. However, accuracy remains modest (area under the receiver-operating curve [AUROC] ≤0.7), and clinical implementation has been challenging. We aimed to develop machine learning (ML) BE/EAC risk prediction models from an electronic health record (EHR) database. METHODS: The Clinical Data Analytics Platform, a deidentified EHR database of 6 million Mayo Clinic patients, was used to predict BE and EAC risk. BE and EAC cases and controls were identified using International Classification of Diseases codes and augmented curation (natural language processing) techniques applied to clinical, endoscopy, laboratory, and pathology notes. Cases were propensity score matched to 5 independent randomly selected control groups. An ensemble transformer-based ML model architecture was used to develop predictive models. RESULTS: We identified 8,476 BE cases, 1,539 EAC cases, and 252,276 controls. The BE ML transformer model had an overall sensitivity, specificity, and AUROC of 76%, 76%, and 0.84, respectively. The EAC ML transformer model had an overall sensitivity, specificity, and AUROC of 84%, 70%, and 0.84, respectively. Predictors of BE and EAC included conventional risk factors and additional novel factors, such as coronary artery disease, serum triglycerides, and electrolytes. DISCUSSION: ML models developed on an EHR database can predict incident BE and EAC risk with improved accuracy compared with conventional risk factor-based risk scores. Such a model may enable effective implementation of a minimally invasive screening technology.

Pulmonary Tuberculosis in Severely Malnourished Children Admitted to Nutrition Rehabilitation Centers: A Multicenter Study.

OBJECTIVES: To identify prevalence of pulmonary tuberculosis (TB) in severely malnourished children admitted to nutritional rehabilitation centers. METHODS: A multicenter cross-sectional study involving 41 nutrition rehabilitation centres (NRCs) across India was carried out to document prevalence of pulmonary tuberculosis in acute severe malnourished children admitted in NRCs. After training of the NRC staff to follow algorithm provided by national tuberculosis elimination program, children admitted to NRCs were screened for pulmonary tuberculosis. RESULTS: A total of 4356 children were enrolled across all the sites. Gastric aspirate for Cartridge based nucleic acid amplification test (CBNAAT), tuberculin skin test (TST) and X-ray film of chest were done in more than 99% of enrolled subjects. A total of 189 children (4.3%) had pulmonary tuberculosis. Eighty-seven (1.99%) were microbiologically confirmed by positive CBNAAT. On multivariate analysis, only significant association was with history of contact with TB patient in family. CONCLUSIONS: The present results suggest that a significant proportion (>4%) of children admitted in NRCs suffer from pulmonary tuberculosis. It is feasible to improve diagnosis of tuberculosis as a whole and microbiologically confirmed TB.

Expanding repertoire of SARS-CoV-2 deletion mutations contributes to evolution of highly transmissible variants.

The emergence of highly transmissible SARS-CoV-2 variants and vaccine breakthrough infections globally mandated the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 2.13 million SARS-CoV-2 genomes from 188 countries/territories (up to June 2021) and performed whole-genome viral sequencing from 102 COVID-19 patients, including 43 vaccine breakthrough infections. We identified 92 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a 3-month window. Deletions in the Spike protein N-terminal domain were highly enriched for these 'surge-associated mutations' (Odds Ratio = 14.19, 95% CI 6.15-32.75, p value = 3.41 × 10-10). Based on a longitudinal analysis of mutational prevalence globally, we found an expanding repertoire of Spike protein deletions proximal to an antigenic supersite in the N-terminal domain that may be one of the key contributors to the evolution of highly transmissible variants. Finally, we generated clinically annotated SARS-CoV-2 whole genome sequences from 102 patients and identified 107 unique mutations, including 78 substitutions and 29 deletions. In five patients, we identified distinct deletions between residues 85-90, which reside within a linear B cell epitope. Deletions in this region arose contemporaneously on a diverse background of variants across the globe since December 2020. Overall, our findings based on genomic-epidemiology and clinical surveillance suggest that the genomic deletion of dispensable antigenic regions in SARS-CoV-2 may contribute to the evasion of immune responses and the evolution of highly transmissible variants.

ResidualBind: Uncovering Sequence-Structure Preferences of RNA-Binding Proteins with Deep Neural Networks.

Deep neural networks have demonstrated improved performance at predicting sequence specificities of DNA- and RNA-binding proteins. However, it remains unclear why they perform better than previous methods that rely on k-mers and position weight matrices. Here, we highlight a recent deep learning-based software package, called ResidualBind, that analyzes RNA-protein interactions using only RNA sequence as an input feature and performs global importance analysis for model interpretability. We discuss practical considerations for model interpretability to uncover learned sequence motifs and their secondary structure preferences.

Reversing painful and non-painful diabetic neuropathy with the capsaicin 8% patch: Clinical evidence for pain relief and restoration of function via nerve fiber regeneration.

Introduction: Current oral treatments for pain in diabetic peripheral neuropathy (DPN) do not affect the progression of DPN i.e., "disease modification." We assessed whether Capsaicin 8% patch treatment can provide pain relief and also restore nerve density and function via nerve regeneration, in both painful (PDPN) and non-painful (NPDPN) diabetic peripheral neuropathy. Methods: 50 participants with PDPN were randomized to receive Capsaicin 8% patch Qutenza with Standard of Care (SOC) (PDPN Q+SOC group), or SOC alone (PDPN SOC group). Pain symptoms were assessed with a diary (Numerical Pain Rating Scale, NRPS) and questionnaires. Investigations included quantitative sensory testing (QST) and distal calf skin biopsies, at baseline and 3 months after baseline visit; subsequent options were 3-monthly visits over 1 year. 25 participants with NPDPN had tests at baseline, and 3 months after all received Capsaicin 8% patch treatment. Results: At 3 months after baseline, PDPN Q+SOC group had reduction in NPRS score (p = 0.0001), but not PDPN SOC group. Short-Form McGill Pain Questionnaire (SF-MPQ) showed significant reductions in scores for overall and other pain descriptors only in the PDPN Q+SOC group. Warm perception thresholds were significantly improved only in the PDPN Q+SOC group (p = 0.02), and correlated with reduction in SF-MPQ overall pain score (p = 0.04). NPDPN Q+SOC group did not report pain during the entire study. Density of intra-epidermal nerve fibers (IENF) with PGP9.5 was increased at 3 months in PDPN Q+SOC (p = 0.0002) and NPDPN Q+SOC (p = 0.002) groups, but not in the PDPN SOC group. Increased sub-epidermal nerve fibers (SENF) were observed with GAP43 (marker of regenerating nerve fibers) only in PDPN Q+SOC (p = 0.003) and NPDPN Q+SOC (p = 0.0005) groups. Pain relief in the PDPN Q+SOC group was correlated with the increased PGP9.5 IENF (p = 0.0008) and GAP43 (p = 0.004), whereas those with lack of pain relief showed no such increase; in some subjects pain relief and increased nerve fibers persisted over months. PGP9.5 IENF increase correlated with axon-reflex vasodilatation in a NPDPN Q+SOC subset (p = 0.006). Conclusions: Capsaicin 8% patch can provide pain relief via nerve regeneration and restoration of function in DPN (disease modification). It may thereby potentially prevent diabetic foot complications, including ulcers.

The Endocannabinoid Analgesic Entourage Effect: Investigations in Cultured DRG Neurons.

Background: The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the "entourage effect". We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors. Methods: Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 µM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 µMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3-7 rats, controls N=16), and analysed. Results: In controls, 1 µMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10-100 µMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001-100 µMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P<0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 µMol (P<0.001), in 60% neurons, as did 2-LG at 0.1-100 µMol (P<0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 µMol 2-AG, 5 µMol 2-PG and 10 µMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P<0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist. Conclusion: 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are required to identify the underlying mechanisms.

On the Origins of Omicron's Unique Spike Gene Insertion.

The emergence of a heavily mutated SARS-CoV-2 variant (Omicron; Pango lineage B.1.1.529 and BA sublineages) and its rapid spread to over 75 countries raised a global public health alarm. Characterizing the mutational profile of Omicron is necessary to interpret its clinical phenotypes which are shared with or distinctive from those of other SARS-CoV-2 variants. We compared the mutations of the initially circulating Omicron variant (now known as BA.1) with prior variants of concern (Alpha, Beta, Gamma, and Delta), variants of interest (Lambda, Mu, Eta, Iota, and Kappa), and ~1500 SARS-CoV-2 lineages constituting ~5.8 million SARS-CoV-2 genomes. Omicron's Spike protein harbors 26 amino acid mutations (23 substitutions, 2 deletions, and 1 insertion) that are distinct compared to other variants of concern. While the substitution and deletion mutations appeared in previous SARS-CoV-2 lineages, the insertion mutation (ins214EPE) was not previously observed in any other SARS-CoV-2 lineage. Here, we consider and discuss various mechanisms through which the nucleotide sequence encoding for ins214EPE could have been acquired, including local duplication, polymerase slippage, and template switching. Although we are not able to definitively determine the mechanism, we highlight the plausibility of template switching. Analysis of the homology of the inserted nucleotide sequence and flanking regions suggests that this template-switching event could have involved the genomes of SARS-CoV-2 variants (e.g., the B.1.1 strain), other human coronaviruses that infect the same host cells as SARS-CoV-2 (e.g., HCoV-OC43 or HCoV-229E), or a human transcript expressed in a host cell that was infected by the Omicron precursor.

Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T cell-leukemia cell interface followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing, we demonstrated that a subset of refractory CD19low cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the Bruton's tyrosine kinase inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity. See related Spotlight by Zhao and Melenhorst, p. 1040.

Genetic alteration of human MYH6 is mimicked by SARS-CoV-2 polyprotein: mapping viral variants of cardiac interest.

Acute cardiac injury has been observed in a subset of COVID-19 patients, but the molecular basis for this clinical phenotype is unknown. It has been hypothesized that molecular mimicry may play a role in triggering an autoimmune inflammatory reaction in some individuals after SARS-CoV-2 infection. Here we investigate if linear peptides contained in proteins that are primarily expressed in the heart also occur in the SARS-CoV-2 proteome. Specifically, we compared the library of 136,704 8-mer peptides from 144 human proteins (including splicing variants) to 9926 8-mers from all the viral proteins in the reference SARS-CoV-2 proteome. No 8-mers were exactly identical between the reference human proteome and the reference SARS-CoV-2 proteome. However, there were 45 8-mers that differed by only one amino acid when compared to the reference SARS-CoV-2 proteome. Interestingly, analysis of protein-coding mutations from 141,456 individuals showed that one of these 8-mers from the SARS-CoV-2 Replicase polyprotein 1a/1ab (KIALKGGK) is identical to an MYH6 peptide encoded by the c.5410 C > A (Q1804K) genetic variation, which has been observed at low prevalence in Africans/African Americans (0.08%), East Asians (0.3%), South Asians (0.06%), and Latino/Admixed Americans (0.003%). Furthermore, analysis of 4.85 million SARS-CoV-2 genomes from over 200 countries shows that viral evolution has already resulted in 20 additional 8-mer peptides that are identical to human heart-enriched proteins encoded by reference sequences or genetic variants. Whether such mimicry contributes to cardiac inflammation during or after COVID-19 illness warrants further experimental evaluation. We suggest that SARS-CoV-2 variants harboring peptides identical to human cardiac proteins should be investigated as "viral variants of cardiac interest".

High diversity in Delta variant across countries revealed by genome-wide analysis of SARS-CoV-2 beyond the Spike protein.

The highly contagious Delta variant of SARS-CoV-2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant's Spike protein, the mutational landscape of the rest of the SARS-CoV-2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS-CoV-2 proteins from nearly 2 million SARS-CoV-2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle-associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant's proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.

Cell-free DNA for the detection of emerging treatment failure in relapsed/ refractory multiple myeloma.

Interrogation of cell-free DNA (cfDNA) represents an emerging approach to non-invasively estimate disease burden in multiple myeloma (MM). Here, we examined low-pass whole genome sequencing (LPWGS) of cfDNA for its predictive value in relapsed/ refractory MM (RRMM). We observed that cfDNA positivity, defined as ≥10% tumor fraction by LPWGS, was associated with significantly shorter progression-free survival (PFS) in an exploratory test cohort of 16 patients who were actively treated on diverse regimens. We prospectively determined the predictive value of cfDNA in 86 samples from 45 RRMM patients treated with elotuzumab, pomalidomide, bortezomib, and dexamethasone in a phase II clinical trial (NCT02718833). PFS in patients with tumor-positive and -negative cfDNA after two cycles of treatment was 1.6 and 17.6 months, respectively (HR 7.6, P < 0.0001). Multivariate hazard modelling confirmed cfDNA as independent risk factor (HR 96.6, P = 6.92e-05). While correlating with serum-free light chains and bone marrow, cfDNA additionally discriminated patients with poor PFS among those with the same response by IMWG criteria. In summary, detectability of MM-derived cfDNA, as a measure of substantial tumor burden with therapy, independently predicts poor PFS and may provide refinement for standard-of-care response parameters to identify patients with poor response to treatment earlier than is currently feasible.

Continuous genomic diversification of long polynucleotide fragments drives the emergence of new SARS-CoV-2 variants of concern.

Highly transmissible or immuno-evasive SARS-CoV-2 variants have intermittently emerged, resulting in repeated COVID-19 surges. With over 6 million SARS-CoV-2 genomes sequenced, there is unprecedented data to decipher the evolution of fitter SARS-CoV-2 variants. Much attention has been directed to studying the functional importance of specific mutations in the Spike protein, but there is limited knowledge of genomic signatures shared by dominant variants. Here, we introduce a method to quantify the genome-wide distinctiveness of polynucleotide fragments (3- to 240-mers) that constitute SARS-CoV-2 sequences. Compared to standard phylogenetic metrics and mutational load, the new metric provides improved separation between Variants of Concern (VOCs; Reference = 89, IQR: 65-108; Alpha = 166, IQR: 149-181; Beta 131, IQR: 114-149; Gamma = 164, IQR: 150-178; Delta = 235, IQR: 217-255; and Omicron = 459, IQR: 395-521). Omicron's high genomic distinctiveness may confer an advantage over prior VOCs and the recently emerged and highly mutated B.1.640.2 (IHU) lineage. Evaluation of 883 lineages highlights that genomic distinctiveness has increased over time (R 2 = 0.37) and that VOCs score significantly higher than contemporary non-VOC lineages, with Omicron among the most distinctive lineages observed. This study demonstrates the value of characterizing SARS-CoV-2 variants by genome-wide polynucleotide distinctiveness and emphasizes the need to go beyond a narrow set of mutations at known sites on the Spike protein. The consistently higher distinctiveness of each emerging VOC compared to prior VOCs suggests that monitoring of genomic distinctiveness would facilitate rapid assessment of viral fitness.

Dose-Related Inhibition of Capsaicin Responses by Cannabinoids CBG, CBD, THC and their Combination in Cultured Sensory Neurons.

BACKGROUND: The analgesic effects of Cannabis sativa are mediated by ∆9 tetrahydrocannabinol (THC), but the contributions of other bioactive complex components, including cannabigerol (CBG) and cannabidiol (CBD), are unclear. We describe the individual and combined effects of CBG, CBD and THC, on blocking capsaicin responses in dorsal root ganglion (DRG) neurons, in an in vitro model of nociceptor hypersensitivity. MATERIALS AND METHODS: Adult rat DRG were dissected and enzyme digested to obtain a neuronal suspension in BSF2 medium containing 2% fetal calf serum, and the neurotrophic factors NGF and GDNF. After 48 h, cultured neurons were loaded with Fura-2 AM, to determine the effects of cannabinoids on capsaicin responses using calcium imaging. In control experiments, neurons were treated with vehicle, followed by 1 µM capsaicin. In cannabinoid treated cultures, CBG, CBD or THC were applied individually, or combined (1:1:1 ratio), followed by 1 µM capsaicin. Data from n = 6 experiments were analysed with Student's t-test and Pearson's correlation coefficient. RESULTS: CBG, CBD and THC, applied individually, elicited dose-related calcium influx in a subset of DRG neurons, and a corresponding dose-related reduction of subsequent responses to capsaicin. Maximum inhibition of capsaicin responses was observed at 30 µM CBG, 100 µM CBD, and 100 µM THC individually, and with combined CBD+CBG+THC (1:1:1) at 90 µM. THC+CBD+CBG combined in a 1:1:1 proportion has the potential to enhance the potency of these compounds applied individually. There was a high correlation between cannabinoid-mediated calcium influx and reduction of capsaicin responses: CBG = -0.88, THC = -0.97, CBD = -0.99 and combined CBG + THC + CBD = -1.00. CONCLUSION: CBG, CBD and THC demonstrated potent dose-related inhibition of capsaicin responses in DRG neurons when applied individually in vitro, and enhanced when applied in combination, being most effective at 90 µM. Thus, efficacy and tolerability of THC could be improved in combination with CBG and CBD at optimal concentrations, which deserve further studies in vivo.

Dynamic transcriptional reprogramming leads to immunotherapeutic vulnerabilities in myeloma.

While there is extensive evidence for genetic variation as a basis for treatment resistance, other sources of variation result from cellular plasticity. Using multiple myeloma as an example of an incurable lymphoid malignancy, we show how cancer cells modulate lineage restriction, adapt their enhancer usage and employ cell-intrinsic diversity for survival and treatment escape. By using single-cell transcriptome and chromatin accessibility profiling, we show that distinct transcriptional states co-exist in individual cancer cells and that differential transcriptional regulon usage and enhancer rewiring underlie these alternative transcriptional states. We demonstrate that exposure to standard treatment further promotes transcriptional reprogramming and differential enhancer recruitment while simultaneously reducing developmental potential. Importantly, treatment generates a distinct complement of actionable immunotherapy targets, such as CXCR4, which can be exploited to overcome treatment resistance. Our studies therefore delineate how to transform the cellular plasticity that underlies drug resistance into immuno-oncologic therapeutic opportunities.

Mycobacterium tuberculosis strain lineage in mixed tribal population across India and Andaman Nicobar Island.

In India, the tribal population constitutes almost 8.6% of the nation's total population. Despite their large presence, there are only a few reports available on Mycobacterium tuberculosis (M. tb) strain prevalence in Indian tribal communities considering the mobile nature of this population and also the influence of the mainstream populations they coexist within many areas for their livelihood. This study attempts to provide critical information pertaining to the TB strain diversity, its public health implications, and distribution among the tribal population in eleven Indian states and Andaman & Nicobar (A&N) Island. The study employed a population-based molecular approach. Clinical isolates were received from 66 villages (10 states and Island) and these villages were selected by implying situation analysis. A total of 78 M. tb clinical isolates were received from 10 different states and A&N Island. Among these, 16 different strains were observed by spoligotyping technique. The major M. tb strains spoligotype belong to the Beijing, CAS1_DELHI, and EAI5 family of M. tb strains followed by EAI1_SOM, EAI6_BGD1, LAM3, LAM6, LAM9, T1, T2, U strains. Drug-susceptibility testing (DST) results showed almost 15.4% of clinical isolates found to be resistant to isoniazid (INH) or rifampicin (RMP) + INH. Predominant multidrug-resistant (MDR-TB) isolates seem to be Beijing strain. Beijing, CAS1_DELHI, EAI3_IND, and EAI5 were the principal strains infecting mixed tribal populations across India. Despite the small sample size, this study has demonstrated higher diversity among the TB strains with significant MDR-TB findings. Prevalence of Beijing MDR-TB strains in Central, Southern, Eastern India and A&N Island indicates the transmission of the TB strains.

Capsaicin 8% Patch Treatment in Non-Freezing Cold Injury: Evidence for Pain Relief and Nerve Regeneration.

Introduction: Neuropathic pain associated with Non-freezing Cold Injury (NFCI) is a major burden to military service personnel. A key feature of NFCI is reduction of the intra-epidermal nerve fibre density in skin biopsies, in keeping with painful neuropathy. Current oral treatments are generally ineffective and have undesirable side effects. Capsaicin 8% patch (Qutenza) has been shown to be well-tolerated and effective for reducing neuropathic pain, for up to 3 months after a single 30-minute application. Methods: In this single-centre open label study, 16 military participants with NFCI (mean duration 49 months) received 30-minute Capsaicin 8% patch treatment to the feet and distal calf. Pain symptoms were assessed using a pain diary (with the 11-point Numerical Pain Rating Scale, NPRS) and questionnaires, the investigations included skin biopsies, performed before and three months after treatment. Results: Participants showed significant decrease in spontaneous pain (mean NPRS: -1.1, 95% CI: 0.37 to 1.90; p = 0.006), and cold-evoked pain (-1.2, 95% CI: 0.40 to 2.04; p = 0.006). The time-course of pain relief over 3 months was similar to other painful neuropathies. Patient Global Impression of Change showed improvement (p = 0.0001). Skin punch biopsies performed 3 months after the patch application showed significant increase of nerve fibres with structural marker PGP9.5 (intra-epidermal nerve fibres [IENFs], p < 0.0001; sub-epidermal nerve fibres [SENFs]; p =< 0.0001), and of regenerating nerve fibres with their selective marker GAP43 (p = 0.0001). The increase of IENFs correlated with reduction of spontaneous (p = 0.027) and cold-evoked pain (p = 0.019). Conclusions: Capsaicin 8% patch provides an exciting new prospect for treatment of NFCI, with regeneration and restoration of nerve fibres, for the first time, in addition to pain relief.

Single-Cell Profiling Reveals Metabolic Reprogramming as a Resistance Mechanism in BRAF-Mutated Multiple Myeloma.

PURPOSE: Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment. EXPERIMENTAL DESIGN: Full-length single-cell RNA (scRNA) sequencing (scRNA-seq) was conducted on 3 patients with BRAF-mutated myeloma and 1 healthy donor. We sequenced 1,495 cells before, after 1 week, and at clinical relapse to BRAF/MEK inhibitor treatment. We developed an in vitro model of dabrafenib resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). Mitochondrial metabolism was characterized by metabolic flux analysis. RESULTS: Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in patients with myeloma and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells. CONCLUSIONS: This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation, and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.