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Abstract Background The cuticle is an important adaptive structure whose origin played a crucial role in the transition of plants from aqueous to terrestrial conditions. HvABCG31/Eibi1 is an ABCG transporter gene, involved in cuticle formation that was recently identified in wild barley (Hordeum vulgare ssp. spontaneum). To study the genetic variation of HvABCG31 in different habitats, its 2 kb promoter region was sequenced from 112 wild barley accessions collected from five natural populations from southern and northern Israel. The sites included three mesic and two xeric habitats, and differed in annual rainfall, soil type, and soil water capacity. Results Phylogenetic analysis of the aligned HvABCG31 promoter sequences clustered the majority of accessions (69 out of 71) from the three northern mesic populations into one cluster, while all 21 accessions from the Dead Sea area, a xeric southern population, and two isolated accessions (one from a xeric population at Mitzpe Ramon and one from the xeric ‘African Slope’ of “Evolution Canyon”) formed the second cluster. The southern arid populations included six haplotypes, but they differed from the consensus sequence at a large number of positions, while the northern mesic populations included 15 haplotypes that were, on average, more similar to the consensus sequence. Most of the haplotypes (20 of 22) were unique to a population. Interestingly, higher genetic variation occurred within populations (54.2%) than among populations (45.8%). Analysis of the promoter region detected a large number of transcription factor binding sites: 121–128 and 121–134 sites in the two southern arid populations, and 123–128,125–128, and 123–125 sites in the three northern mesic populations. Three types of TFBSs were significantly enriched: those related to GA (gibberellin), Dof (DNA binding with one finger), and light. Conclusions Drought stress and adaptive natural selection may have been important determinants in the observed sequence variation of HvABCG31 promoter. Abiotic stresses may be involved in the HvABCG31 gene transcription regulations, generating more protective cuticles in plants under stresses.

Objective: Determine the effects of pimavanserin tartrate [ACP-103; N-(4-flurophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide], a selective serotonin 5-HT(2A) receptor inverse agonist, on slow wave sleep (SWS), other sleep parameters, and attention/vigilance.Methods: Forty-five healthy adults were randomized to pimavanserin (1, 2.5, 5, or 20 mg) or placebo in a double-blind fashion (n=9/group). Pimavanserin or placebo was administered once daily in the morning for 13 consecutive days. The effects of pimavanserin were measured after the first dose and again after 13 days. Sleep parameters were measured by polysomnography. Effects on attention/vigilance were measured by a continuous performance task.Results: Compared to placebo, pimavanserin significantly increased SWS following single and multiple dose administration. Pimavanserin also decreased number of awakenings. PSG variables not affected by pimavanserin included sleep period time, total sleep time, sleep onset latency, number of stage shifts, total time awake, early morning wake, and microarousal index. Changes in sleep architecture parameters, sleep profile parameters, and spectral power density parameters were consistent with a selective increase in SWS. Pimavanserin did not adversely affect performance on the continuous performance test measured in the evening before or morning after polysomnography.Conclusions: These data suggest that pimavanserin selectively increases slow wave sleep and decreases awakenings, an effect that does not diminish with repeated administration. [ABSTRACT FROM AUTHOR]

Cutaneous T-cell lymphoma is a group of non-Hodgkin T-cell lymphomas that develop in and affect the skin but can potentially spread to other organs. There are many subtypes, the most common of which are mycosis fungoides, Sezary syndrome, lymphomatoid papulosis, and primary cutaneous anaplastic large cell lymphoma. Cutaneous lymphoma is a common cause of recalcitrant chronic skin rash and notoriously mimics other dermatologic and hematologic conditions, often resulting in diagnostic delays of months to years. This review provides an introduction to cutaneous T-cell lymphoma, with a primary focus on the clinical presentation, diagnosis, immunopathogenesis, and management of the condition., Competing Interests: Disclosure A.H. Rook is a consultant for TLR Biosciences and speaker for Mallinckrodt. D. M. Weiner has no disclosures., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Prolonged indwelling catheter use is a known risk factor for catheter-associated UTIs (CAUTIs). We sought to reduce catheter use by creating and implementing a trial of void (TOV) algorithm to standardize indwelling Foley catheter removal in surgical patients., Methods: We partnered with the Departments of General Surgery and Nursing to develop an evidence-based TOV algorithm for a step-down unit at a large urban teaching hospital. Our cohort included patients treated with intra-abdominal, thoracic, vascular, urologic, and gynecologic surgeries. The primary outcome was mean cumulative indwelling urethral catheter patient-days. For example, if 2 patients had catheters for 3 and 7 days, respectively, then cumulative catheter days would be 10. We analyzed changes in catheter use 90 days before and after algorithm implementation., Results: The mean number of hospitalized patient-days before and after algorithm introduction did not differ (32.2 vs 32.0, P = .60). After implementation, mean cumulative catheter patient-days decreased (14.8 vs 9.9, P < .01), as did mean daily number of patients with catheters on the unit (3.7 vs 3.1, P = .02). There was 1 CAUTI before and after algorithm implementation, the latter deemed associated with algorithm nonadherence. Catheter use in a surgical floor control group where the algorithm was not implemented did not differ for any outcome over the same time period ( P > .05)., Conclusions: A multidisciplinary approach to standardize catheter care with a TOV algorithm is feasible and effective in reducing catheter use. Further research is needed to determine its impact on CAUTI rate.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [ 18 F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[ 18 F]fluoro-2-deoxytrehalose ([ 18 F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [ 18 F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [ 18 F]FDT from the most globally-abundant organic 18 F-containing molecule, [ 18 F]FDG. The full, pre-clinical validation of both production method and [ 18 F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [ 18 F]FDT directly from the widely-available clinical reagent [ 18 F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer., (© 2024. The Author(s).)

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis . Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these ( 9 ) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients., Competing Interests: Competing interests statement:R.K.J. received consultant fees from Cur, DynamiCure, Elpis, Merck, SPARC, and SynDevRx; owns equity in Accurius, Enlight, and SynDevRx; served on the Board of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund; and received research grants from Boehringer Ingelheim and Sanofi. No funding or reagents from these organizations were used in the study.

Tuberculosis (TB) is a life-threatening infectious disease. The standard treatment is up to 90% effective; however, it requires the administration of four antibiotics (isoniazid, rifampicin, pyrazinamide, and ethambutol [HRZE]) over long time periods. This harsh treatment process causes adherence issues for patients because of the long treatment times and a myriad of adverse effects. Therefore, the World Health Organization has focused goals of shortening standard treatment regimens for TB in their End TB Strategy efforts, which aim to reduce TB-related deaths by 95% by 2035. For this purpose, many novel and promising combination antibiotics are being explored that have recently been discovered, such as the bedaquiline, pretomanid, and linezolid (BPaL) regimen. As a result, testing the number of possible combinations with all possible novel regimens is beyond the limit of experimental resources. In this study, we present a unique framework that uses a primate granuloma modeling approach to screen many combination regimens that are currently under clinical and experimental exploration and assesses their efficacies to inform future studies. We tested well-studied regimens such as HRZE and BPaL to evaluate the validity and accuracy of our framework. We also simulated additional promising combination regimens that have not been sufficiently studied clinically or experimentally, and we provide a pipeline for regimen ranking based on their efficacies in granulomas. Furthermore, we showed a correlation between simulation rankings and new marmoset data rankings, providing evidence for the credibility of our framework. This framework can be adapted to any TB regimen and can rank any number of single or combination regimens., (© 2024 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis . To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Background: The utility of prophylactic endovascular internal iliac balloon placement in the surgical management of placenta accreta spectrum is debated., Objective: In this study, we review outcomes of surgical management of placenta accreta spectrum with and without prophylactic endovascular internal iliac balloon catheter use at a single institution., Study Design: This is a retrospective cohort study of consecutive viable singleton pregnancies with a confirmed pathologic diagnosis of placenta accreta spectrum undergoing scheduled delivery from October 2018 through November 2020. In the T1 period (October 2018-August 2019), prophylactic endovascular internal iliac balloon catheters were placed in the operating room before the start of surgery. Balloons were inflated after neonatal delivery and deflated after hysterectomy completion. In the T2 period (September 2019-November 2020), endovascular catheters were not used. In both time periods, all surgeries were performed by a dedicated multidisciplinary team using a standardized surgical approach. The outcomes compared included the estimated blood loss, anesthesia duration, operating room time, surgical duration, and a composite of surgical complications. Comparisons were made using the Wilcoxon rank-sum test and the Fisher exact test., Results: A total of 30 patients were included in the study (T1=10; T2=20). The proportion of patients with placenta increta or percreta was 80% in both groups, as defined by surgical pathology. The median estimated blood loss was 875 mL in T1 and 1000 mL in T2 (P=.84). The proportion of patients requiring any packed red blood cell transfusion was 60% in T1 and 40% in T2 (P=.44). The proportion of patients requiring >4 units of packed red blood cells was 20% in T1 and 5% in T2 (P=.25). Surgical complications were observed in 1 patient in each group. Median operative anesthesia duration was 497 minutes in T1 and 296 minutes in T2 (P<.001). Median duration of operating room time was 498 minutes in T1 and 205 minutes in T2 (P<.001). Median surgical duration was 227 minutes in T1 and 182 minutes in T2 (P<.05). The median duration of time for prophylactic balloon catheter placement was 74 minutes (range, 46-109 minutes). The median postoperative length of stay was similar in both groups (6 days in T1 and 5.5 days in T2; P=.36)., Conclusion: The use of prophylactic endovascular internal iliac balloon catheters was not associated with decreased blood loss, packed red blood cell transfusion, or surgical complications. Catheter use was associated with increased duration of anesthesia, operating room time, and surgical time., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Competing Interests: None.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with refractory cutaneous T-cell lymphoma (CTCL) through replacement of the bone marrow responsible for lymphoma cells and possibly induction of a graft-versus-lymphoma effect. However, allo-HSCT is not always curative; relapse of CTCL occurs in about half of patients post-transplant. Treatment of relapsed CTCL after allo-HSCT is challenging because post-transplant patients are at high risk of graft-versus-host disease, and this condition may be precipitated or exacerbated by standard CTCL therapies. The benefit of each potential therapy must therefore be weighed against its risk of graft versus host disease (GVHD). In this article, we review the management of relapsed CTCL after allo-HSCT. We begin with an exemplative patient whose relapsed Sezary syndrome was successfully treated without development of GVHD. We also report high-throughput T-cell receptor sequencing data obtained during the patient's disease relapse and remission. We then review general guidelines for management of relapsed CTCL and summarize all reported cases and outcomes of relapsed CTCL after transplant. We conclude by reviewing the current CTCL therapies and their risk of GVHD., (© 2022 Wiley Periodicals LLC.)

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a potentially fatal infectious disease requiring long treatment duration with multiple antibiotics and against which there is no reliable cure. Among the factors that have hampered the development of adequate drug regimens is the lack of an animal model that reproduces the NTM lung pathology required for studying antibiotic penetration and efficacy. Given the documented similarities between tuberculosis and NTM immunopathology in patients, we first determined that the rabbit model of active tuberculosis reproduces key features of human NTM-PD and provides an acceptable surrogate model to study lesion penetration. We focused on clarithromycin, a macrolide and pillar of NTM-PD treatment, and explored the underlying causes of the disconnect between its favorable potency and pharmacokinetics and inconsistent clinical outcome. To quantify pharmacokinetic-pharmacodynamic target attainment at the site of disease, we developed a translational model describing clarithromycin distribution from plasma to lung lesions, including the spatial quantitation of clarithromycin and azithromycin in mycobacterial lesions of two patients on long-term macrolide therapy. Through clinical simulations, we visualized the coverage of clarithromycin in plasma and four disease compartments, revealing heterogeneous bacteriostatic and bactericidal target attainment depending on the compartment and the corresponding potency against nontuberculous mycobacteria in clinically relevant assays. Overall, clarithromycin's favorable tissue penetration and lack of bactericidal activity indicated that its clinical activity is limited by pharmacodynamic, rather than pharmacokinetic, factors. Our results pave the way toward the simulation of lesion pharmacokinetic-pharmacodynamic coverage by multidrug combinations to enable the prioritization of promising regimens for clinical trials.

Background: The acute care surgery model has led to improved outcomes for emergent surgical conditions, but similar models of care have not been implemented in urology. Our department implemented an acute care urology (ACU) service in 2015, and the service evolved in 2018. We aimed to evaluate the impact of the ACU model on the management of nephrolithiasis. Materials and Methods: We conducted a retrospective review of all patients with urology consults in the emergency department for nephrolithiasis, who required surgical intervention from 2013 to 2019. Patients were divided into three cohorts based on date of consultation: Pre-ACU (2013-2014), Phase 1 (2015-2017), and Phase 2 (2018-2019). Results: We identified 733 patients with nephrolithiasis requiring intervention (162 pre-ACU, 334 Phase 1, and 237 Phase 2). Before ACU implementation, median time from consult to definitive intervention was 36 days. After ACU implementation, median time to intervention decreased to 22 days in Phase 1 ( p  < 0.001) and 15 days in Phase 2 ( p  < 0.001). On multivariable Cox regression, the hazard of definitive intervention improved in Phase 1 (hazard ratio [HR] 1.90, p  < 0.001) and in Phase 2 (HR 1.80, p  < 0.001). Rates of primary definitive intervention without initial decompression and loss to follow-up were also significantly improved, compared to the pre-ACU cohort. Conclusions: Implementation of a structured ACU service was associated with improved time to treatment for patients with acute nephrolithiasis, as well as increased primary definitive intervention and improved follow-up care. This model of care has potential to improve patient outcomes for nephrolithiasis and other acute urological conditions.

Objective: To construct a risk prediction model to identify cases of difficult urethral catheterizations (DUC) in order to prevent complications from improper placement., Materials and Methods: Using a single-institution database of urologic consults for Foley catheterizations from June 2016 to January 2020, a model to predict DUC in male patients was constructed. DUC was defined as requiring the use of a guidewire, cystoscopy, urethral dilation, and/or suprapubic tube (SPT) placement, while a simple Foley was defined as an uncomplicated placement of a regular or coudé catheter. A final model to predict DUC was constructed using multivariable logistic regression and internally validated using bootstrap statistics., Results: A total of 841 consults were identified, with 181 (21.5%) classified as a DUC. On multivariable regression, patient-specific factors as overweight BMI (OR: 1.71; P = .014), urethral stricture disease (OR: 7.38; P < .001), BPH surgery (OR: 2.47; P < .001), radical prostatectomy (OR: 4.32; P = .001), and genitourinary (GU) prosthetic implants (OR: 3.44; P = .046) were associated with DUC. Situational factors such as blood at the meatus (OR: 2.40; P < .001), and consulting team (eg, surgery OR: 4.82; P < .001) were also significant. Bootstrap analysis of the final model demonstrated good overall accuracy (predictive accuracy: 75%)., Conclusion: This model is a promising tool to help providers identify patients who likely require catheterization by a urologist and potentially reduce catheterization-related complications. The high rate of uncomplicated catheterizations also highlights the need for continuing education amongst healthcare professionals. External validation and application to the initial Foley encounter will shed light on its overall utility., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tuberculosis (TB) remains a global health problem despite almost universal efforts to provide patients with highly effective chemotherapy, in part, because many infected individuals are not diagnosed and treated, others do not complete treatment, and a small proportion harbor Mycobacterium tuberculosis (Mtb ) strains that have become resistant to drugs in the standard regimen. Development and approval of new drugs for TB have accelerated in the last 10 years, but more drugs are needed due to both Mtb 's development of resistance and the desire to shorten therapy to 4 months or less. The drug development process needs predictive animal models that recapitulate the complex pathology and bacterial burden distribution of human disease. The human host response to pulmonary infection with Mtb is granulomatous inflammation usually resulting in contained lesions and limited bacterial replication. In those who develop progressive or active disease, regions of necrosis and cavitation can develop leading to lasting lung damage and possible death. This review describes the major vertebrate animal models used in evaluating compound activity against Mtb and the disease presentation that develops. Each of the models, including the zebrafish, various mice, guinea pigs, rabbits, and non-human primates provides data on number of Mtb bacteria and pathology resolution. The models where individual lesions can be dissected from the tissue or sampled can also provide data on lesion-specific bacterial loads and lesion-specific drug concentrations. With the inclusion of medical imaging, a compound's effect on resolution of pathology within individual lesions and animals can also be determined over time. Incorporation of measurement of drug exposure and drug distribution within animals and their tissues is important for choosing the best compounds to push toward the clinic and to the development of better regimens. We review the practical aspects of each model and the advantages and limitations of each in order to promote choosing a rational combination of them for a compound's development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Wang, Wen, Weiner and Via.)

In the past few decades, immunotherapy has emerged as an effective therapeutic option for patients with cutaneous T cell lymphoma (CTCL). CTCL is characterized by progressive impairment of multiple arms of the immune system. Immunotherapy targets these deficits to stimulate a more robust antitumor response, thereby both clearing the malignant T cells and repairing the immune dysfunction. By potentiating rather than suppressing the immune system, immunotherapy can result in longer treatment responses than alternatives such as chemotherapy. In recent years, advances in our understanding of the pathogenesis of CTCL have led to the development of several new agents with promising efficacy profiles. The second article in this continuing medical education series describes the current immunotherapeutic options for treatment of CTCL, with a focus on how they interact with the immune system and their treatment outcomes in case studies and clinical trials. We will discuss established CTCL immunotherapies, such as interferons, photopheresis, and retinoids; emerging therapies, such as interleukin-12 and Toll-like receptor agonists; and new approaches to targeting tumor antigens and checkpoint molecules, such as mogamulizumab, anti-programmed cell death protein 1, anti-CD47, and chimeric antigen receptor T cell therapy. We also describe the principles of multimodality immunotherapy and the use of total skin electron beam therapy in such regimens., (Copyright © 2020 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Cutaneous T cell lymphomas (CTCLs) are malignancies of skin-trafficking T cells. Patients with advanced CTCL manifest immune dysfunction that predisposes to infection and suppresses the antitumor immune response. Therapies that stimulate immunity have produced superior progression-free survival compared with conventional chemotherapy, reinforcing the importance of addressing the immune deficient state in the care of patients with CTCL. Recent research has better defined the pathogenesis of these immune deficits, explaining the mechanisms of disease progression and revealing potential therapeutic targets. The features of the malignant cell in mycosis fungoides and Sézary syndrome are now significantly better understood, including the T helper 2 cell phenotype, regulatory T cell cytokine production, immune checkpoint molecule expression, chemokine receptors, and interactions with the microenvironment. The updated model of CTCL immunopathogenesis provides understanding into clinical progression and therapeutic response., (Copyright © 2020 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb's respiratory chain, and highlight the challenges associated with targeting the pathogen's respiratory enzymes for tuberculosis drug development.

Objective: To investigate the rate of bladder cancer in patients undergoing cystoscopic evaluation for asymptomatic microscopic hematuria (AMH) in order to identify groups at sufficiently low-risk for bladder cancer in whom invasive testing may be avoided., Methods: We performed a retrospective review of patients who underwent cystoscopic evaluation for AMH between 2010 and 2018. Age, gender, smoking status, history of pelvic radiation, and number of red blood cells per high-power field on urine microscopy were recorded. We used logistic regression to explore the association between specific risk factors and a diagnosis of bladder cancer on cystoscopy., Results: Among the 2118 patients who underwent cystoscopy for AMH, 25 patients (1.2%) were diagnosed with a bladder cancer, all of which were nonmuscle invasive urothelial carcinoma. There were no bladder cancers detected in patients under the age of 50. Older age and positive smoking history were significantly associated with bladder cancer., Conclusion: Bladder cancer was an uncommon finding on cystoscopy among patients being evaluated for AMH, especially in younger patients. We confirmed several known risk factors for bladder cancer, including older age and smoking history. Further studies are required to evaluate the utility of cystoscopy for identifying latent bladder cancers in low-risk patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The convergence of human molecular genetics and Lewy pathology of Parkinson's disease (PD) have led to a robust, clinical-stage pipeline of alpha-synuclein (α-syn)-targeted therapies that have the potential to slow or stop the progression of PD and other synucleinopathies. To facilitate the development of these and earlier stage investigational molecules, the Michael J. Fox Foundation for Parkinson's Research convened a group of leaders in the field of PD research from academia and industry, the Alpha-Synuclein Clinical Path Working Group. This group set out to develop recommendations on preclinical and clinical research that can de-risk the development of α-syn targeting therapies. This consensus white paper provides a translational framework, from the selection of animal models and associated end-points to decision-driving biomarkers as well as considerations for the design of clinical proof-of-concept studies. It also identifies current gaps in our biomarker toolkit and the status of the discovery and validation of α-syn-associated biomarkers that could help fill these gaps. Further, it highlights the importance of the emerging digital technology to supplement the capture and monitoring of clinical outcomes. Although the development of disease-modifying therapies targeting α-syn face profound challenges, we remain optimistic that meaningful strides will be made soon toward the identification and approval of disease-modifying therapeutics targeting α-syn.

Magnesium plays an important role in infection with Mycobacterium tuberculosis ( Mtb) as a signal of the extracellular environment, as a cofactor for many enzymes, and as a structural element in important macromolecules. Raltegravir, an antiretroviral drug that inhibits HIV-1 integrase is known to derive its potency from selective sequestration of active-site magnesium ions in addition to binding to a hydrophobic pocket. In order to determine if essential Mtb-related phosphoryl transfers could be disrupted in a similar manner, a directed screen of known molecules with integrase inhibitor-like pharmacophores ( N-alkyl-5-hydroxypyrimidinone carboxamides) was performed. Initial hits afforded compounds with low-micromolar potency against Mtb, acceptable cytotoxicity and PK characteristics, and robust SAR. Elucidation of the target of these compounds revealed that they lacked magnesium dependence and instead disappointingly inhibited a known promiscuous target in Mtb, decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1, Rv3790).

Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. In contrast, moxifloxacin, which is active in vitro against a subpopulation of Mycobacterium tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug-resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration may contribute to treatment outcome has wide implications for TB.

Shortening the lengthy treatment duration for tuberculosis patients is a major goal of current drug development efforts. The common marmoset develops human-like disease pathology and offers an attractive model to better understand the basis for relapse and test regimens for effective shorter duration therapy. We treated Mycobacterium tuberculosis-infected marmosets with two drug regimens known to differ in their relapse rates in human clinical trials: the standard four-drug combination of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) that has very low relapse rates and the combination of isoniazid and streptomycin that is associated with higher relapse rates. As early as 2 weeks, the more sterilizing regimen significantly reduced the volume of lung disease by computed tomography (P = 0.035) and also significantly reduced uptake of [(18)F]-2-fluoro-2-deoxyglucose by positron emission tomography (P = 0.049). After 6 weeks of therapy, both treatments caused similar reductions in granuloma bacterial load, but the more sterilizing, four-drug regimen caused greater reduction in bacterial load in cavitary lesions (P = 0.009). These findings, combined with the association in humans between cavitary disease and relapse, suggest that the basis for improved sterilizing activity of the four-drug combination is both its faster disease volume resolution and its stronger sterilizing effect on cavitary lesions. Definitive data from relapse experiments are needed to support this observation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing the apparent discrepancy between in vitro and in vivo activity. However, the results also raise the possibility that subinhibitory concentrations of POA generated by the host could fuel the emergence of resistance to both PZA and POA. In contrast to widespread expectations, we demonstrate good oral bioavailability and exposure in preclinical species in pharmacokinetic studies of oral POA. Baseline exposure of oral POA can be further increased by the xanthine oxidase inhibitor and approved gout drug allopurinol. These promising results pave the way for clinical investigations of oral POA as a therapeutic alternative or an add-on to overcome PZA resistance and salvage this essential TB drug.

Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. Patients who have TB require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here, we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, antiangiogenic, specifically anti-VEGF, treatments can "normalize" their vasculature, reducing hypoxia and creating a window of opportunity for concurrent chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential avenue to improve delivery and efficacy of current treatment regimens.

Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades.

Background: Functional cross-talk between seven transmembrane (7TM) receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing fewer adverse events. Although several studies convincingly have established the existence of 7TM receptor cross-talk, little is known about the frequencey and biological significance of this phenomenon., Methodology/principal Findings: To evaluate the extent of synergism in 7TM receptor signaling, we took a comprehensive approach and co-expressed 123 different 7TM receptors together with the angiotensin II type 1 receptor (AT1R) and analyzed how each receptor affected the angiotensin II (AngII) response. To monitor the effect we used integrative receptor activation/signaling assay called Receptor Selection and Amplification Technology (R-SAT). In this screen the thromboxane A2α receptor (TPαR) was the only receptor which significantly enhanced the AngII-mediated response. The TPαR-mediated enhancement of AngII signaling was significantly reduced when a signaling deficient receptor mutant (TPαR R130V) was co-expressed instead of the wild-type TPαR, and was completely blocked both by TPαR antagonists and COX inhibitors inhibiting formation of thromboxane A2 (TXA2)., Conclusions/significance: We found a functional enhancement of AT1R only when co-expressed with TPαR, but not with 122 other 7TM receptors. In addition, the TPαR must be functionally active, indicating the AT1R enhancement is mediated by a paracrine mechanism. Since we only found one receptor enhancing AT1R potency, our results suggest that functional augmentation through 7TM receptor cross-talk is a rare event that may require specific conditions to occur.

Most atypical antipsychotic drugs (APDs), e.g. risperidone (RIS), produce more extensive blockade of brain serotonin (5-HT)(2A) than dopamine (DA) D(2) receptors. This distinguishes them from typical APDs, e.g. haloperidol (HAL). Our objective was to test the hypothesis that augmentation of low doses of RIS or HAL (2mg/day) with pimavanserin (PIM), a selective 5-HT(2A) inverse agonist, to enhance 5-HT(2A) receptor blockade, can achieve efficacy comparable to RIS, 6mg/day, but with lesser side effects. In a multi-center, randomized, double-blind, 6week trial, 423 patients with chronic schizophrenia experiencing a recent exacerbation of psychotic symptoms were randomized to RIS2mg+placebo (RIS2PBO), RIS2mg+PIM20mg (RIS2PIM), RIS6mg+PBO (RIS6PBO), HAL2mg+PBO (HAL2PBO), or HAL2mg+PIM20mg (HAL2PIM). Improvement in psychopathology was measured by the PANSS and CGI-S. The reduction in PANSS Total Score with RIS2PIM at endpoint was significantly greater than RIS2PBO: -23.0 vs. -16.3 (p=0.007), and not significantly different from the RIS6PBO group: -23.2 points. The percentage of patients with ≥20% improvement at day 15 in the RIS2PIM group was 62.3%, significantly greater than the RIS6PBO (42.1%; p=0.01) and the RIS2PBO groups (37.7%; p=0.002). Weight gain and hyperprolactinemia were greater in the RIS6PBO group than the RIS2PIM group but there was no difference in extrapyramidal side effects (EPS). HAL2PBO and HAL2PIM were not significantly different from each other in efficacy but HAL2PIM had less EPS at end point. Both HAL groups and RIS6PBO showed equal improvement in psychopathology at endpoint, indicating HAL 2mg/day is effective to treat an acute exacerbation in chronic schizophrenia patients. In conclusion, a sub-effective RIS dose combined with PIM to enhance 5-HT(2A) receptor blockade provided faster onset of action, and at endpoint, equal efficacy and better safety, compared to standard dose RIS. These results support the conclusion that 5-HT(2A) receptor blockade is a key component of the action of some atypical APDs and can reduce EPS due to a typical APD., (Copyright © 2012 Elsevier B.V. All rights reserved.)

With a host of new antitubercular chemotherapeutics in development, methods to assess the activity of these agents beyond mouse efficacy are needed to prioritize combinations for clinical trials. Lesions in Mycobacterium tuberculosis-infected rabbits are hypoxic, with histopathologic features that closely resemble those of human tuberculous lesions. Using [(18)F]2-fluoro-deoxy-d-glucose ([(18)F]FDG) positron emission tomography-computed tomography (PET-CT) imaging, we studied the dynamics of tuberculosis infection in rabbits, revealing an initial inflammatory response followed by a consolidative chronic disease. Five weeks after infection, as much as 23% of total lung volume was abnormal, but this was contained and to some extent reversed naturally by 9 weeks. During development of this chronic state, individual lesions in the same animal had very different fates, ranging from complete resolution to significant progression. Lesions that remained through the initial stage showed an increase in volume and tissue density over time by CT. Initiation of chemotherapy using either isoniazid (INH) or rifampin (RIF) during chronic infection reduced bacterial load with quantitative changes in [(18)F]FDG uptake, lesion density and total lesion volume measured by CT. The [(18)F]FDG PET uptake in lesions was significantly reduced with as little as 1 week of treatment, while the volume and density of lesions changed more slowly. The results from this study suggest that rabbits may be a useful surrogate species for evaluating novel chemotherapies and understanding changes in both PET and CT scans in human clinical trials.

Borderline Personality Disorder (BPD) is associated with behavioral and emotional dysregulation, particularly in social contexts; however, the underlying pathophysiology at the level of brain function is not well understood. Previous studies found abnormalities in frontal cortical and limbic areas suggestive of poor frontal regulation of downstream brain regions. However, the striatum, which is closely connected with the medial frontal cortices and plays an important role in motivated behaviors and processing of rewarding stimuli, has been understudied in BPD. Here we hypothesized that, in addition to frontal dysfunction, BPD patients may show abnormal striatal function. In this study, 38 BPD patients with intermittent explosive disorder (BPD-IED) and 36 healthy controls (HC) participated in the Point Subtraction Aggression Paradigm (PSAP), a computer game played with a fictitious other player. (18)Fluoro-deoxyglucose positron emission tomography (FDG-PET) measured relative glucose metabolism (rGMR) within caudate and putamen in response to aggression-provoking and non-provoking versions of the PSAP. Male BPD-IED patients had significantly lower striatal rGMR than all other groups during both conditions, although male and female BPD-IED patients did not differ in clinical or behavioral measures. These sex differences suggest differential involvement of frontal-striatal circuits in BPD-IED, and are discussed in relation to striatal involvement in affective learning and social decision-making., (Published by Elsevier Ltd.)

The loss of dopaminergic neurons in the substantia nigra pars compacta leads to the characteristic motor symptoms of Parkinson's disease: bradykinesia, rigidity and resting tremors. Although these symptoms can be improved using currently available dopamine replacement strategies, there is still a need to improve current strategies of treating these symptoms, together with a need to alleviate non-motor symptoms of the disease. Moreover, treatments that provide neuroprotection and/or disease-modifying effects remain an urgent unmet clinical need. This Review describes the most promising biological targets and therapeutic agents that are currently being assessed to address these treatment goals. Progress will rely on understanding genetic mutations or susceptibility factors that lead to Parkinson's disease, better translation between preclinical animal models and clinical research, and improving the design of future clinical trials.

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.

It has been suggested previously ( AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98 ) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, beta-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling.

A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist and antagonist, ACP-103 [N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1, active:salt)], was evaluated for its ability to reduce the primary motor symptom of tremor using tacrine-induced tremulous jaw movements in rats, which is an animal model of parkinsonian tremor. Furthermore, ACP-103 was evaluated for its ability to reduce levodopa-induced dyskinesias in monkeys rendered parkinsonian with MPTP [1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine]. ACP-103 reduced tacrine-induced tremulous jaw movements in rats. In addition, ACP-103 administered in combination with levodopa caused a dose-related reduction in dyskinesias in monkeys. These data suggest that ACP-103 may have the potential to reduce tremor and levodopa-induced dyskinesias in Parkinson's disease.

The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.

The mechanisms underlying the clinical properties of atypical antipsychotics have been postulated to be mediated, in part, by interactions with the 5-HT2A receptor. Recently, it has been recognized that clinically effective antipsychotic drugs are 5-HT2A receptor inverse agonists rather than neutral antagonists. In the present study, which is part of the clinical development of the novel, selective 5-HT2A receptor inverse agonist ACP-103, we applied positron emission tomography (PET) with the radioligand [11C]N-methylspiperone ([11C]NMSP) to study the relationship between oral dose, plasma level, and uptake of ACP-103 in living human brain. The safety of drug administration was also assessed. Four healthy volunteers were examined by PET at baseline, and after the oral administration of various single doses of ACP-103. Two subjects each received 1, 5, and 20 mg doses, and two subjects each received 2, 10, and 100 mg doses, respectively. ACP-103 was well tolerated. Detectable receptor binding was observed at very low ACP-103 serum levels. Cortical [11C]NMSP binding was found to be dose-dependent and fitted well to the law of mass action. A reduction in binding was detectable after an oral dose of ACP-103 as low as 1 mg, and reached near maximal displacement following the 10-20 mg dose. In conclusion, administration of ACP-103 to healthy volunteers was found to be safe and well tolerated, and single oral doses as low as 10 mg were found to fully saturate 5-HT2A receptors in human brain as determined by PET.