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IntroductionThe COVID 19 pandemic was highlighted by a rise in hospital admissions secondary to respiratory decompensation. This was accompanied by an increase in ICU admissions, endotracheal intubation and mechanical ventilation. As a consequence, tracheostomies became essential in preventing complications of prolonged intubation and to facilitate weaning from sedation and mechanical ventilation. With the lack of international consensus on tracheostomy technique and optimal timing, we present our experience with 377 percutaneous tracheostomies performed on critically ill COVID 19 patients.ObjectiveTo report the outcomes of critically ill patients with COVID-19 who underwent percutaneous tracheostomy during a period of 24 months.MethodsA retrospective single-center electronic chart review was performed on all ICU patients who underwent percutaneous tracheostomy after respiratory failure secondary to COVID-19 between March 2020 to March 2022.ResultsA total of 377 percutaneous tracheostomies were performed. The mean duration between intubation and percutaneous tracheostomy was 17.4 days (3-61). The study included 222 males (59%) and 155 females (41%).The mean age of patients was 56.2 years (17-94), with a mean BMI was 31.3 (14-68).The commonest comorbidities among patients were diabetes mellitus (50%) and hypertension (48%).Complications were encountered in 85 cases (23%), with the commonest overall complication being minor bleeding.203 patients (54%) were weaned from sedation. The mean duration between tracheostomy and weaning from sedation was 7.5 days (1 – 47 days). 156 patients (41%) were weaned from MV. The mean duration between tracheostomy and weaning from MV was 12.9 days (1 – 58 days). There was a total of 236 (63%) deaths reported during the period of this study.No deaths were attributable to the surgical procedure.ConclusionPercutaneous tracheostomy can be safely performed in patients with COVID-19. With lack of conclusive objective data regarding the optimal timing for tracheostomy, we recommend that tracheostomy be performed as soon as possible after the 7thday endotracheal intubation.

The pathogenesis of SARS-CoV-2 in the context of a specific immunological niche is not fully understood. Here, we used a golden Syrian hamster model to systematically evaluate the kinetics of host response to SARS-CoV-2 infection, following disease pathology, viral loads, antibody responses, and inflammatory cytokine expression in multiple organs. The kinetics of SARS-CoV-2 pathogenesis and genomewide lung transcriptome was also compared between immunocompetent and immunocompromised hamsters. We observed that the body weight loss was proportional to the SARS-CoV-2 infectious dose and lasted for a short time only in immunocompetent hamsters. Body weight loss was more prominent and prolonged in infected immunocompromised hamsters. While the kinetics of viral replication and peak live viral loads were not significantly different at low and high infectious doses (LD and HD), the HD-infected immunocompetent animals developed severe lung disease pathology. The immunocompetent animals cleared the live virus in all tested tissues by 12 days post-infection and generated a robust serum antibody response. In contrast, immunocompromised hamsters mounted an inadequate SARS-CoV-2 neutralizing antibody response, and the virus was detected in the pulmonary and multiple extrapulmonary organs until 16 days post-infection. These hamsters also had prolonged moderate inflammation with severe bronchiolar-alveolar hyperplasia/metaplasia. Consistent with the difference in disease presentation, distinct changes in the expression of inflammation and immune cell response pathways and network genes were seen in the lungs of infected immunocompetent and immunocompromised animals. This study highlights the interplay between the kinetics of viral replication and the dynamics of SARS-CoV-2 pathogenesis at organ-level niches and maps how COVID-19 symptoms vary in different immune contexts. Together, our data suggest that the histopathological manifestations caused by progressive SARS-CoV-2 infection may be a better predictor of COVID-19 severity than individual measures of viral load, antibody response, and cytokine storm at the systemic or local (lungs) levels in the immunocompetent and immunocompromised hosts.

OBJECTIVES Thoracic endovascular techniques for aneurysm repair offer less invasive alternatives to open strategies. Both approaches, however, are associated with the risk for neurological complications. Despite adjuncts to maintain spinal cord perfusion, ischaemia and paraplegia continue to occur during thoracoabdominal aortic aneurysm (TAAA) repair. Staging of such extensive procedures has been proven to decrease the risk for spinal cord injury. Archived biopsy specimens may offer insight into the molecular signature of the reorganization and expansion of the spinal collateral network during staged endovascular interventions in the setting of TAAA. METHODS Biological replicates of total RNA were isolated from existing paraspinous muscle samples from 22 Yorkshire pigs randomized to 1 of 3 simulated TAAA repair strategies as part of a previous study employing coil embolization of spinal segmental arteries within the thoracic and lumbar spine. Gene expression profiling was performed using the Affymetrix GeneChip Porcine array. RESULTS Microarray analysis identified 649 differentially expressed porcine genes (≥1.3-fold change, P ≤ 0.05) when comparing paralysed and non-paralysed subjects. Of these, 355 were available for further analysis. When mapped to the human genome, 169 Homo sapiens orthologues were identified. Integrated interpretation of gene expression profiles indicated the significant regulation of transcriptional regulators (such as nuclear factor кB), cytokine (including CXCL12) elements contributing to hypoxia signalling in the cardiovascular system (vascular endothelial growth factor and UBE2) and cytoskeletal elements (like dystrophin (DMD) and matrix metallopeptidase (MMP)). CONCLUSIONS This study demonstrates the ability of microarray-based platforms to detect the differential expression of genes in paraspinous muscle during staged TAAA repair. Pathway enrichment analysis detected subcellular actors accompanying the neuroprotective effects of staged endovascular coiling. These observations provide new insight into the potential prognostic and therapeutic value of gene expression profiling in monitoring and modulating the arteriolar remodelling in the collateral network.

Funding Information: The work was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health grants U19AI111276 and U01AI065663 to R.R.R., R.D., J.J.E. and P.S., and NIAID training grant T32AI125185 to A.L. The study sponsors were not involved in the study design, in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. Publisher Copyright: © 2022, The Author(s). Mechanisms underlying variability in transmission of Mycobacterium tuberculosis strains remain undefined. By characterizing high and low transmission strains of M.tuberculosis in mice, we show here that high transmission M.tuberculosis strain induce rapid IL-1R-dependent alveolar macrophage migration from the alveolar space into the interstitium and that this action is key to subsequent temporal events of early dissemination of bacteria to the lymph nodes, Th1 priming, granulomatous response and bacterial control. In contrast, IL-1R-dependent alveolar macrophage migration and early dissemination of bacteria to lymph nodes is significantly impeded in infection with low transmission M.tuberculosis strain; these events promote the development of Th17 immunity, fostering neutrophilic inflammation and increased bacterial replication. Our results suggest that by inducing granulomas with the potential to develop into cavitary lesions that aids bacterial escape into the airways, high transmission M.tuberculosis strain is poised for greater transmissibility. These findings implicate bacterial heterogeneity as an important modifier of TB disease manifestations and transmission. publishersversion published

RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long arrays of unrelated genes. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective slender bloodstream form of African trypanosome, and we show that RBP42 is a key regulator of parasite’s central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Using global quantitative transcriptomic and proteomic analyses, we also show that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a plausible role of RBP42 as a positive regulator of target mRNA translation. Analysis reveals significant changes in central carbon metabolic intermediates following loss of RBP42, further supporting its critical role in cellular energy metabolism.

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated genes in the proximal intestine associated with active calcium transport (Trpv6, S100g, and Atp2b1) are also induced by 1,25(OH)2D3 in the distal intestine of KO/TG mice. In addition, Slc30a10, encoding a manganese efflux transporter, was one of the genes most induced by 1,25(OH)2D3 in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA sequence (RNA-seq) analysis of human enteroids indicated that the effects of 1,25(OH)2D3 observed in mice are conserved in humans. Using Slc30a10-/- mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action. ispartof: MOLECULAR AND CELLULAR BIOLOGY vol:41 issue:1 ispartof: location:United States status: published

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

We discuss the procedure to build an interactive workbench environment for single cell genomics applications with the Open OnDemand (OOD) science gateway. In our approach, an end-user submits a complex single cell RNA sequencing (scRNA) pipeline, checks the status of the job, and visualizes the output results. All of these tasks are accomplished through a web browser, relieving the users from the complexities involved in developing and handling a large-scale workflow. Our approach helped researchers in processing several input data sets of scRNA in the campus HPC cluster. Although the current work is focused on scRNA analysis, the same approach can be extended for any workflow.

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]-activated genes in the proximal intestine associated with active calcium transport (Trpv6, S100g, and Atp2b1) are also induced by 1,25(OH)(2)D(3) in the distal intestine of KO/TG mice. In addition, Slc30a10, encoding a manganese efflux transporter, was one of the genes most induced by 1,25(OH)(2)D(3) in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA sequence (RNA-seq) analysis of human enteroids indicated that the effects of 1,25(OH)(2)D(3) observed in mice are conserved in humans. Using Slc30a10(−/−) mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

Background The mechanisms underlying sex-based differences in pain and analgesia are poorly understood. In this study, we investigated gene expression changes in trigeminal ganglia (TG) of male and female rats exposed to infraorbital nerve chronic constriction injury (IoN-CCI). Methods Somatosensory assessments were performed prior to IoN-CCI and at selected time points postsurgery. Selected gene expression changes were examined with real-time quantitative polymerase chain reaction (RT-PCR) in ipsilateral TG at 21 days postsurgery. Results Rats exposed to IoN-CCI developed significant mechanical allodynia and hyperalgesia on days 19 and 21 postsurgery. During this period, females developed significantly more allodynia but not hyperalgesia compared to males. At 21 days postsurgery, expression levels of 44 of the 84 investigated pain-related genes in ipsilateral TG were significantly regulated relative to naive rats in either sex. Csf1 and Cx3cr1 were up-regulated in both sexes, but the magnitude of regulation was significantly higher in females (p = 0.02 and p = 0.001, respectively). Htr1a and Scn9a were down-regulated in both sexes, but the down-regulation was significantly more pronounced in males (p = 0.04 and p = 0.02, respectively). Additionally, Cck, Il1a, Pla2g1b and Tnf genes were significantly regulated in females but not in males, and Chrna4 gene was significantly down-regulated in males but not in females. Conclusions Our findings suggest sex-dependent gene regulation in response to nerve injury, which may contribute to sex dimorphism of trigeminal neuropathic pain. Further studies are needed to establish gene expression changes over time and correlate these with hormonal and other physiological parameters in male and female. Significance We present novel sex-specific transcriptional regulation in trigeminal ganglia that may contribute to male-/female-based differences in trigeminal neuropathic pain. These findings are expected to open new research horizons, particularly in male versus female targeted therapeutic regimens.

Objectives: USC and its ovarian counterpart (OSC), the most lethal gynecologic malignancies, are resistant to current therapies. We noted that the highly annotated USC-derived cell line ARK1 lacks expression of collagen type III (COL3), the master organizer of VWC deposition during angiogenesis as evidenced by the COL3-deficient vascular Ehlers-Danlos Syndrome (OMIM 130050). In USC, vascular COL3 may therefore originate from interstitial cells via MRMC. We hypothesized ARK1 to express mediators of MRMC. Our objective was to test this hypothesis in standard culture (SC) and in angiogenic overdrive (AO) condition, using both gene expression profiles for anti-cancer drug discovery. Our AO protocol employs FDA-approved medicines that maximally push and maximally brake angiogenesis by inhibiting ketoglutarate-utilizing protein hydroxylases (KPHs), e.g. the enzymes forming peptide-bound hydroxylproline. We reported that such medicines advance angiogenesis via activated molecular oxygen sensing yet at the same time inhibit deposition of the stable collagenous matrix required for angiogenesis, thus trapping cancer cells between ‘hammer and anvil’. Methods: Cell culture, repeat RNA-seq, antigen-specific protein detection. Results: ARK1-SC expressed an extraordinary array of angiogenesis-relevant genes that encode a) the mediators of MRMC (connective tissue growth factor [CTGF], the suite of fibroblast growth factors [FGFs, FGFR1-4, FGFRL1, FRS2-3, FGFBP1] and ligands signaling over a few cell diameters [e.g. WNT5B]; b) all elemental VWCs except COL3 (49 collagen/laminin/fibrillin chains; the entire elastin suite [ELN, LOX, LOXL1-4, EMILIN1-3, MMRN2, FBLN5] and proteoglycans [e.g. HSPG2, VCAN]; c) angiogenic factors, from capillary tube formation to branching morphology (e.g. all 6 subunits of the hypoxia inducible factors [HIF1-3]; the suite of vascular endothelial growth factors [VEGFA-C, FIGF, FLT1, KDR, FLT4]; NRP1; HBEGF; even the beta-2 adrenergic receptor ADRB2]; d) the complete set of 41 KPHs required for physiological function in a-c. After 48 hours at clinical concentrations of the KPH-inhibitory medicine deferiprone, ARK1-AO accumulated underhydroxylated collagens intracellularly, indicating blockade of collagen secretion due to triple helix collapse and mis-folding. Simultaneously, expression of CTGF increased 16.1-fold, FGF2 2.5-fold, FGF1 14.5-fold, and FGF5 23.8-fold over control ARK1-SC (RPKM-based FDR p: 10-2 to 10-6) whereas expression of their corresponding receptors remained unchanged and COL3 expression defective. VEGFA/VEGFB expression fell by almost 50%, with likewise unchanged receptor expression. HBEGF expression rose 9-fold (FDR p Conclusions: ARK1 is a genetically defined USC model to interrogate the targeted disruption of MRMC. The pioneer KPH inhibitory medicine deferiprone exploits the asymmetry between KPH-stabilized matrix proteins (e.g. collagens) and KPH-destabilized oxygen sensors (e.g. HIFs) to disrupt both and in this process, reveal the genetic circuitry for matrix recruitment and angiogenesis. We identify CTGF, FGFs, and HBEGF as key factors that enroll interstitial cells for procurement of tumor perfusion. We anticipate therapeutic value of current CTGF/FGFs/HBEGF antagonists in USC (and OSC).

Background The dorsal root (DRG) and trigeminal (TG) ganglia contain cell bodies of sensory neurons of spinal and trigeminal systems, respectively. They are homologs of each other; however, differences in how the two systems respond to injury exist. Trigeminal nerve injuries rarely result in chronic neuropathic pain (NP). To date, no genes involved in the differential response to nerve injury between the two systems have been identified. We examined transcriptional changes involved in the development of trigeminal and spinal NP. Methods Trigeminal and spinal mononueropathies were induced by chronic constriction injury to the infraorbital or sciatic nerve. Expression levels of 84 genes in the TG and DRG at 4, 8 and 21 days post-injury were measured using real-time PCR. Results We found time-dependent and ganglion-specific transcriptional regulation that may contribute to the development of corresponding neuropathies. Among genes significantly regulated in both ganglia Cnr2, Grm5, Htr1a, Il10, Oprd1, Pdyn, Prok2 and Tacr1 were up-regulated in the TG but down-regulated in the DRG at 4 days post-injury; at 21 days post-injury, Adora1, Cd200, Comt, Maob, Mapk3, P2rx4, Ptger1, Tnf and Slc6a2 were significantly up-regulated in the TG but down-regulated in the DRG. Conclusions Our findings suggest that spinal and trigeminal neuropathies due to trauma are differentially regulated. Subtle but important differences between the two ganglia may affect NP development. Significance We present distinct transcriptional alterations in the TG and DRG that may contribute to differences observed in the corresponding mononeuropathies. Since the trigeminal system seems more resistant to developing NP following trauma our findings lay ground for future research to detect genes and pathways that may act in a protective or facilitatory manner. These may be novel and important therapeutic targets.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity ofMycobacterium tuberculosisto develop drug resistance, are major barriers to global TB control efforts.M. tuberculosisis known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a novel mechanism of genetically encoded but rapidly reversible drug-tolerance inM. tuberculosiscaused by transient frameshift mutations in a homopolymeric tract (HT) of seven cytosines (7C) in theglpKgene. Inactivating frameshift mutations associated with the 7C HT inglpKproduce small colonies that exhibit heritable multi-drug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the sameglpKHT region. These reversible frameshift mutations in the 7C HT ofM. tuberculosis glpKoccur in clinical isolates, accumulate inM. tuberculosisinfected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction ofdosRandsigHand repression ofkstRregulons, similar to that observed in otherin vitromodels ofM. tuberculosistolerance. These results suggest that GlpK phase variation may contribute to drug-tolerance, treatment failure and relapse in human TB. Drugs effective against phase-variantM. tuberculosismay hasten TB treatment and improve cure rates.SIGNIFICANCEThe ability ofM. tuberculosisto survive during prolonged treatment has been attributed to either transient stress responses or fixed heritable drug-resistance producing mutations. We show that phase-variation in theM. tuberculosis glpKgene represents a third type of resistance mechanism. The ability of theseglpKmutants to grow slowly and then rapidly revert suggests that these transiently-heritable changes may also explain how a hidden population of drug-tolerant bacteria develops during TB treatment. As a genetically trackable cause of drug-tolerance,M. tuberculosis glpKmutants provides a unique opportunity to study these phenomena at a cellular and mechanistic level. These mutants could also be used for developing drugs that target tolerant populations, leading to more rapid and effective TB treatments.

Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA—a key component for biosynthesis of the mycolic acid layer of the bacterium’s cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars., We report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis. Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).

Active tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy treatments to achieve durable cures. This problem has partly been attributable to the existence of nonreplicating M. tuberculosis “persisters” that are difficult to kill using conventional anti-TB treatments. Compounds that target the respiratory pathway have the potential to kill both replicating and persistent M. tuberculosis and shorten TB treatment, as this pathway is essential in both metabolic states. We developed a novel respiratory pathway-specific whole-cell screen to identify new respiration inhibitors. This screen identified the biphenyl amide GSK1733953A (DG70) as a likely respiration inhibitor. DG70 inhibited both clinical drug-susceptible and drug-resistant M. tuberculosis strains. Whole-genome sequencing of DG70-resistant colonies identified mutations in menG (rv0558), which is responsible for the final step in menaquinone biosynthesis and required for respiration. Overexpression of menG from wild-type and DG70-resistant isolates increased the DG70 MIC by 4× and 8× to 30×, respectively. Radiolabeling and high-resolution mass spectrometry studies confirmed that DG70 inhibited the final step in menaquinone biosynthesis. DG70 also inhibited oxygen utilization and ATP biosynthesis, which was reversed by external menaquinone supplementation. DG70 was bactericidal in actively replicating cultures and in a nutritionally deprived persistence model. DG70 was synergistic with the first-line TB drugs isoniazid, rifampin, and the respiratory inhibitor bedaquiline. The combination of DG70 and isoniazid completely sterilized cultures in the persistence model by day 10. These results suggest that MenG is a good therapeutic target and that compounds targeting MenG along with standard TB therapy have the potential to shorten TB treatment duration., IMPORTANCE This study shows that MenG, which is responsible for the last enzymatic step in menaquinone biosynthesis, may be a good drug target for improving TB treatments. We describe the first small-molecule inhibitor (DG70) of Mycobacterium tuberculosis MenG and show that DG70 has characteristics that are highly desirable for a new antitubercular agent, including bactericidality against both actively growing and nonreplicating mycobacteria and synergy with several first-line drugs that are currently used to treat TB.

Active tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy treatments to achieve durable cures. This problem has partly been attributable to the existence of nonreplicating M. tuberculosis “persisters” that are difficult to kill using conventional anti-TB treatments. Compounds that target the respiratory pathway have the potential to kill both replicating and persistent M. tuberculosis and shorten TB treatment, as this pathway is essential in both metabolic states. We developed a novel respiratory pathway-specific whole-cell screen to identify new respiration inhibitors. This screen identified the biphenyl amide GSK1733953A (DG70) as a likely respiration inhibitor. DG70 inhibited both clinical drug-susceptible and drug-resistant M. tuberculosis strains. Whole-genome sequencing of DG70-resistant colonies identified mutations in menG ( rv0558 ), which is responsible for the final step in menaquinone biosynthesis and required for respiration. Overexpression of menG from wild-type and DG70-resistant isolates increased the DG70 MIC by 4× and 8× to 30×, respectively. Radiolabeling and high-resolution mass spectrometry studies confirmed that DG70 inhibited the final step in menaquinone biosynthesis. DG70 also inhibited oxygen utilization and ATP biosynthesis, which was reversed by external menaquinone supplementation. DG70 was bactericidal in actively replicating cultures and in a nutritionally deprived persistence model. DG70 was synergistic with the first-line TB drugs isoniazid, rifampin, and the respiratory inhibitor bedaquiline. The combination of DG70 and isoniazid completely sterilized cultures in the persistence model by day 10. These results suggest that MenG is a good therapeutic target and that compounds targeting MenG along with standard TB therapy have the potential to shorten TB treatment duration. IMPORTANCE This study shows that MenG, which is responsible for the last enzymatic step in menaquinone biosynthesis, may be a good drug target for improving TB treatments. We describe the first small-molecule inhibitor (DG70) of Mycobacterium tuberculosis MenG and show that DG70 has characteristics that are highly desirable for a new antitubercular agent, including bactericidality against both actively growing and nonreplicating mycobacteria and synergy with several first-line drugs that are currently used to treat TB.

Cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma overexpressed gene (CCN) comprise a family of matricellular proteins that have multiple physiologic functions including development, tissue repair, cell adhesion, migration, and proliferation. The expression of CCN1, cyclin D1, β-catenin, and p53 was explored by immunohistochemistry in different grades of ductal carcinoma in situ (DCIS) cases. These cases did not contain any infiltrating carcinoma components. In addition, all cysteine-rich protein 61 gene exons (encoding the CCN1 protein) were sequenced in 30 samples. Allred and H-scores were calculated for expression in both DCIS and the surrounding benign breast tissue. All cases of DCIS showed degrees of cytoplasmic CCN1 staining with median H-scores of 170, 160, and 60 in grades 3, 2, and 1, respectively (P = .043). Twelve of 28 DCIS 3, 1 of 15 DCIS 2, and 0 of 18 DCIS 1 also showed nuclear staining for CCN1. The cytoplasmic staining difference was preserved when the cases were divided into estrogen receptor (ER)+/DCIS grade 1, ER+/DCIS 2 and 3, and ER-/DCIS 2 and 3 by the H-score (P = .037). Cyclin D1 expression was positively correlated with the CCN1 cytoplasmic H-score in all DCIS samples (P = .038). Membranous β-catenin expression correlated with the grade of intraepithelial carcinoma by both H-score (P = .047) and Allred score (P = .026). Our results suggest that CCN1 has a role in the development of intraepithelial carcinoma. CCN1 expression correlates with grade of DCIS independent of ER status. It can induce cell cycle progression through cyclin D1. It is warranted to study high expression of CCN1 in DCIS as an independent risk factor in a larger cohort.

Summary Obesity induces lipotoxic cardiomyopathy, a condition in which lipid accumulation in cardiomyocytes causes cardiac dysfunction. Here, we show that glycogen synthase kinase-3α (GSK-3α) mediates lipid accumulation in the heart. Fatty acids (FAs) upregulate GSK-3α, which phosphorylates PPARα at Ser280 in the ligand-binding domain (LBD). This modification ligand independently enhances transcription of a subset of PPARα targets, selectively stimulating FA uptake and storage, but not oxidation, thereby promoting lipid accumulation. Constitutively active GSK-3α, but not GSK-3β, was sufficient to drive PPARα signaling, while cardiac-specific knockdown of GSK-3α, but not GSK-3β, or replacement of PPARα Ser280 with Ala conferred resistance to lipotoxicity in the heart. Fibrates, PPARα ligands, inhibited phosphorylation of PPARα at Ser280 by inhibiting the interaction of GSK-3α with the LBD of PPARα, thereby reversing lipotoxic cardiomyopathy. These results suggest that GSK-3α promotes lipid anabolism through PPARα-Ser280 phosphorylation, which underlies the development of lipotoxic cardiomyopathy in the context of obesity.

Uterine carcinosarcoma (UCS) accounts for approximately 15% of uterine cancer-associated deaths in the United States. With lack of effective treatment modalities, identification of underlying molecular defects may allow the introduction of targeted treatments. The expression of AKT, epithelial growth factor receptor, C-Kit, and C-ErbB-2 were studied by immunohistochemistry and exons 9 and 20 of PIK3CA gene were sequences in a cohort of 37 UCS, including 23 early-stage (I and II) and 14 late-stage (III and IV) tumors. Twenty-three (62%) of the UCS were homologous; the reminder contained heterologous elements. The carcinomatous component was pure serous carcinoma in 13 (35%), endometrioid in 12 (32%) cases. An immunostaining score ranging from 0 to (6+) was calculated for AKT, epithelial growth factor receptor, and C-Kit. C-ErbB-2 staining was scored by American Society of Clinical Oncology/College of American Pathologists criteria. AKT staining was seen in 35/37 cases with an immunostaining score ranging from (2+) to (5+). AKT was expressed significantly more in the early-stage than late-stage disease (P=0.016). The expression of AKT in the epithelial component was associated with the survival (P=0.026). Epithelial growth factor receptor was positive in 21/37 cases. Only 8 cases showed (≤3+) immunostaining score with C-Kit. C-ErbB-2 immunostain was (3+) in only 1 case. An H1047R mutation on PIK3CA gene was detected in both carcinomatous and sarcomatous components in a single case. These results indicate that AKT pathway may be important in pathogenesis of UCS. Further studies with larger cohorts are warranted to confirm the observed associations in this study.

Uric acid is present in human plasma in relatively high concentrations and is considered to be a natural physiological antioxidant. We have earlier shown that in the presence of Cu(II) and molecular oxygen, uric acid causes strand breakage in DNA. In this article, we show that uric acid fluorescence is quenched by addition of DNA, indicating the formation of uric acid-DNA complex. Uric acid-Cu(II)-mediated DNA strand scission is capable of bacteriophage inactivation and such inactivation is mediated through reduction of Cu(II) to Cu(I) and the generation of oxygen-derived radicals. It is indicated that the DNA breakage is repaired in E. coli and involves the repair of DNA polymerase.

In this study we analysed the regulation of gene expression by arvanil and anandamide in human peripheral blood mononuclear cells (PBMCs) to clarify their immunosuppressive properties. PBMCs were activated, leading to CD36 down regulation, that was normalized by arvanil and anandamide. We used microarray technology to identify a regulatory pattern associated with cell proliferation in the presence of both substances. CD3-CD28 stimulated PBMCs showed a pattern of up-regulated and down-regulated genes after treatment with these substances. We selected and analysed several genes chosen by their function in the regulation of cell proliferation. We showed a transcriptional control of the CD36 gene by arvanil and anandamide associated with an increased protein expression, thus suggesting a possible role of CD36 in anandamide and arvanil anti-inflammatory pattern.

We have previously shown that L-DOPA in the presence of Cu(II) caused DNA cleavage through the generation of reactive oxygen species such as the hydroxyl radical. Since L-DOPA is the precursor for the synthesis of melanin, we have studied the action of melanin on DNA in a similar reaction. In this paper, we show that melanin in the presence of Cu(II) also causes DNA strand breakage. However, the rate of such strand breakage is considerably less than l-DOPA. Melanin and L-DOPA are both capable of generating superoxide anion. Furthermore, the action of L-DOPA and Cu(II) on bacteriophage lambda reduces its viability. The results are discussed in relation to the putative role of L-DOPA-Cu(II) system as a source of endogenous generation of reactive oxygen species.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios ( affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.

It has been recently reported that l-DOPA and Cu(II) in the presence of H2O2 leads to extensive DNA damage. In this paper we show that l-DOPA in the presence of Cu(II) alone can cause DNA cleavage through the generation of reactive oxygen species such as the hydroxyl radical. Fluorescence quenching studies indicate that l-DOPA is capable of binding to DNA.