Orally available nucleoside analog UMM-766 provides protection in a murine model of orthopox disease.

Although smallpox has been eradicated, other orthopoxviruses continue to be a public health concern as exemplified by the ongoing Mpox (formerly monkeypox) global outbreak. While medical countermeasures (MCMs) previously approved by the Food and Drug Administration for the treatment of smallpox have been adopted for Mpox, previously described vulnerabilities coupled with the questionable benefit of at least one of the therapeutics during the 2022 Mpox outbreak reinforce the need for identifying and developing other MCMs against orthopoxviruses. Here, we screened a panel of Merck proprietary small molecules and identified a novel nucleoside inhibitor with potent broad-spectrum antiviral activity against multiple orthopoxviruses. Efficacy testing of a 7-day dosing regimen of the orally administered nucleoside in a murine model of severe orthopoxvirus infection yielded a dose-dependent increase in survival. Treated animals had greatly reduced lesions in the lung and nasal cavity, particularly in the 10 µg/mL dosing group. Viral levels were also markedly lower in the UMM-766-treated animals. This work demonstrates that this nucleoside analog has anti-orthopoxvirus efficacy and can protect against severe disease in a murine orthopox model.IMPORTANCEThe recent monkeypox virus pandemic demonstrates that members of the orthopoxvirus, which also includes variola virus, which causes smallpox, remain a public health issue. While currently FDA-approved treatment options exist, risks that resistant strains of orthopoxviruses may arise are a great concern. Thus, continued exploration of anti-poxvirus treatments is warranted. Here, we developed a template for a high-throughput screening assay to identify anti-poxvirus small-molecule drugs. By screening available drug libraries, we identified a compound that inhibited orthopoxvirus replication in cell culture. We then showed that this drug can protect animals against severe disease. Our findings here support the use of existing drug libraries to identify orthopoxvirus-targeting drugs that may serve as human-safe products to thwart future outbreaks.

Carbodiimide and Isocyanate Hydroboration by a Cyclic Carbodiphosphorane Catalyst.

We report hydroboration of carbodiimide and isocyanate substrates catalyzed by a cyclic carbodiphosphorane catalyst. The cyclic carbodiphosphorane outperformed the other Lewis basic carbon species tested, including other zerovalent carbon compounds, phosphorus ylides, an N-heterocyclic carbene, and an N-heterocyclic olefin. Hydroborations of seven carbodiimides and nine isocyanates were performed at room temperature to form N-boryl formamidine and N-boryl formamide products. Intermolecular competition experiments demonstrated the selective hydroboration of alkyl isocyanates over carbodiimide and ketone substrates. DFT calculations support a proposed mechanism involving activation of pinacolborane by the carbodiphosphorane catalyst, followed by hydride transfer and B-N bond formation.

Individualizing busulfan dose in specific populations and evaluating the risk of pharmacokinetic drug-drug interactions.

INTRODUCTION: Busulfan is an alkylating agent widely used in the conditioning of hematopoietic stem cell transplantation possessing a complex metabolism and a large interindividual and intra-individual variability, especially in children. Combined with the strong rationale of busulfan PK/PD relationships, factors altering its clearance (e.g. weight, age, and GST-A genetic polymorphism mainly) can also affect clinical outcomes. AREAS COVERED: This review aims to provide an overview of the current knowledge on busulfan pharmacokinetics, its pharmacokinetics variabilities in pediatric populations, drug-drug interactions (DDI), and their consequences regarding dose individualization. This review was based on medical literature up until October 2021. EXPERT OPINION: To ensure effective busulfan exposure in pediatrics, different weight-based nomograms have been established to determine busulfan dosage and provided improved results (65-80% of patients correctly exposed). In addition to nomograms, therapeutic drug monitoring (TDM) of busulfan measuring plasmatic concentrations to estimate busulfan pharmacokinetic parameters can be used. TDM is now widely carried out in routine practices and aims to ensure the targeting of the reported therapeutic windows by individualizing busulfan dosing based on the clearance estimations from a previous dose.

Evaluating the Abnormality of Bilateral Motor Cortex Activity in Subacute Stroke Patients Executing a Unimanual Motor Task With Increasing Demand on Precision.

Abnormal contralesional M1 activity is consistently reported in patients with compromised upper limb and hand function after stroke. The underlying mechanisms and functional implications of this activity are not clear, which hampers the development of treatment strategies targeting this brain area. The goal of the present study was to determine the extent to which contralesional M1 activity can be explained by the demand of a motor task, given recent evidence for increasing ipsilateral M1 activity with increasing demand in healthy age-matched controls. We hypothesized that higher activity in contralesional M1 is related to greater demand on precision in a hand motor task. fMRI data were collected from 19 patients with ischemic stroke affecting hand function in the subacute recovery phase and 31 healthy, right-handed, age-matched controls. The hand motor task was designed to parametrically modulate the demand on movement precision. Electromyography data confirmed strictly unilateral task performance by all participants. Patients showed significant impairment relative to controls in their ability to perform the task in the fMRI scanner. However, patients and controls responded similarly to an increase in demand for precision, with better performance for larger targets and poorer performance for smaller targets. Patients did not show evidence of elevated ipsilesional or contralesional M1 blood oxygenation level-dependent (BOLD) activation relative to healthy controls and mean BOLD activation levels were not elevated for patients with poorer performance relative to patients with better task performance. While both patients and healthy controls showed demand-dependent increases in BOLD activation in both ipsilesional/contralateral and contralesional/ipsilateral hemispheres, patients with stroke were less likely to show evidence of a linear relationship between the demand on precision and BOLD activation in contralesional M1 than healthy controls. Taken together, the findings suggest that task demand affects the BOLD response in contralesional M1 in patients with stroke, though perhaps less strongly than in healthy controls. This has implications for the interpretation of reported abnormal bilateral M1 activation in patients with stroke because in addition to contralesional M1 reorganization processes it could be partially related to a response to the relatively higher demand of a motor task when completed by patients rather than by healthy controls.

Transcutaneous Tibial Nerve Stimulation in the Management of Overactive Bladder: A Scoping Review.

OBJECTIVE: Overactive bladder (OAB) is a condition that has physical, social, psychosocial, and financial impacts. Transcutaneous tibial nerve stimulation (TTNS) is a modality that stimulates the nerve root fibers of L5-S3, the same spinal segments of the parasympathetic nervous system as the bladder. This scoping review aims to identify current literature available on the feasibility and outcomes of TTNS as a first-line treatment option for OAB. MATERIALS AND METHODS: A scoping review of six electronic data bases was performed to identify full-text articles from 2015 that explored the impact of TTNS on OAB and bladder dysfunction in people aged >18 years. RESULTS: A total of 15 articles met the inclusion criteria. TTNS was compared with sham treatment, parasacral stimulation, pelvic floor muscle training (PFMT), anticholinergic medication, and percutaneous tibial nerve stimulation (PTNS). Heterogeneity in treatment application and parameters existed, with variations in treatment duration, frequency of use, and treatment settings such as pulse width (µs) and frequency (Hz). Results indicated that TTNS has efficacy equal to PFMT and PTNS in the management of OAB; however, it is not as efficacious as anticholinergic medication. CONCLUSIONS: TTNS is a promising first-line management option for people with OAB, particularly in the older population and for those with neurogenic bladder. It can provide symptomatic relief from urinary incontinence, frequency, urgency, and nocturia, while avoiding the bothersome side effects of more invasive or pharmaceutical therapies. Heterogeneity in treatment parameters limits generalizability and translation of the most appropriate clinical application and should be considered in future trials.

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants.

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2.

Junin Virus Activates p38 MAPK and HSP27 Upon Entry.

Junín virus (JUNV), a New World arenavirus, is a rodent-borne virus and the causative agent of Argentine hemorrhagic fever. Humans become infected through exposure to rodent host secreta and excreta and the resulting infection can lead to an acute inflammatory disease with significant morbidity and mortality. Little is understood about the molecular pathogenesis of arenavirus hemorrhagic fever infections. We utilized Reverse Phase Protein Microarrays (RPPA) to compare global alterations in the host proteome following infection with an attenuated vaccine strain, Candid#1 (CD1), and the most parental virulent strain, XJ13, of JUNV in a human cell culture line. Human small airway epithelial cells were infected with CD1 or XJ13 at an MOI of 10, or mock infected. To determine proteomic changes at early timepoints (T = 1, 3, 8 and 24 h), the JUNV infected or mock infected cells were lysed in compatible buffers for RPPA. Out of 113 proteins that were examined by RPPA, 14 proteins were significantly altered following JUNV infection. Several proteins were commonly phosphorylated between the two strains and these correspond to entry and early replication events, to include p38 mitogen-activated protein kinase (MAPK), heat shock protein 27 (HSP27), and nuclear factor kappa B (NFκB). We qualitatively confirmed the alterations of these three proteins following infection by western blot analysis. We also determined that the inhibition of either p38 MAPK, with the small molecule inhibitor SB 203580 or siRNA knockdown, or HSP27, by siRNA knockdown, significantly decreases JUNV replication. Our data suggests that HSP27 phosphorylation at S82 upon virus infection is dependent on p38 MAPK activity. This work sheds light on the nuances of arenavirus replication.

Advances in the Treatment of Hairy Cell Leukemia Variant.

OPINION STATEMENT: Hairy cell leukemia variant (HCL-V) is a rare B cell lymphoproliferative disorder with a clinical-pathological distinction from the classic form of hairy cell leukemia (HCL-C). HCL-V is more aggressive in nature, has a higher tendency to be refractory to conventional purine analog pharmacotherapies, and leads to a poorer prognosis. Hence, these differing features bring paramount importance to the diagnosis and management of HCL-V. While there is no genetic mutation diagnostic of HCL-V, genetic profiling efforts have identified potential therapeutic targets (i.e., MAP2K1, KDM6A, CREBBP, ARID1A, CCND3, U2AF1, KMT2C) and yielded prognostic markers (i.e., IGHV4-34 rearrangements). To date, combination chemoimmunotherapies, such as cladribine and rituximab, have shown the best results in HCL-V. Future directions include targeted therapies such as moxetumomab pasudotox, ibrutinib, trametinib, and binimetinib and potentially anti-CD22 chimeric antigen receptor T cell therapy. The purpose of this review is to provide an outline of the diagnostic approach and an update on the therapeutic advancements in HCL-V.

Engineered Human Cathelicidin Antimicrobial Peptides Inhibit Ebola Virus Infection.

The 2014-2016 West Africa Ebola virus (EBOV) outbreak coupled with the most recent outbreaks in Central Africa underscore the need to develop effective treatment strategies against EBOV. Although several therapeutic options have shown great potential, developing a wider breadth of countermeasures would increase our efforts to combat the highly lethal EBOV. Here we show that human cathelicidin antimicrobial peptide (AMP) LL-37 and engineered LL-37 AMPs inhibit the infection of recombinant virus pseudotyped with EBOV glycoprotein (GP) and the wild-type EBOV. These AMPs target EBOV infection at the endosomal cell-entry step by impairing cathepsin B-mediated processing of EBOV GP. Furthermore, two engineered AMPs containing D-amino acids are particularly potent in blocking EBOV infection in comparison with other AMPs, most likely owing to their resistance to intracellular enzymatic degradation. Our results identify AMPs as a novel class of anti-EBOV therapeutics and demonstrate the feasibility of engineering AMPs for improved therapeutic efficacy.

Stabilization of ß-catenin upon B-cell receptor signaling promotes NF-kB target genes transcription in mantle cell lymphoma.

B-cell receptor (BCR) signaling pathways and interactions with the tumor microenvironment account for mantle cell lymphoma (MCL) cells survival in lymphoid organs. In several MCL cases, the WNT/ß-catenin canonical pathway is activated and ß-catenin accumulates into the nucleus. As both BCR and ß-catenin are important mediators of cell survival and interaction with the microenvironment, we investigated the crosstalk between BCR and WNT/ß-catenin signaling and analyzed their impact on cellular homeostasis as well as their targeting by specific inhibitors. ß-catenin was detected in all leukemic MCL samples and its level of expression rapidly increased upon BCR stimulation. This stabilization was hampered by the BCR-pathway inhibitor Ibrutinib, supporting ß-catenin as an effector of the BCR signaling. In parallel, MCL cells as compared with normal B cells expressed elevated levels of WNT16, a NF-κB target gene. Its expression increased further upon BCR stimulation to participate to the stabilization of ß-catenin. Upon BCR stimulation, ß-catenin translocated into the nucleus but did not induce a Wnt-like transcriptional response, i.e., TCF/LEF dependent. ß-catenin rather participated to the regulation of NF-κB transcriptional targets, such as IL6, IL8, and IL1. Oligo pull down and chromatin immunoprecipitation experiments demonstrated that ß-catenin is part of a protein complex that binds the NF-κB DNA consensus sequence, strengthening the idea of an association between the two proteins. An inhibitor targeting ß-catenin transcriptional interactions hindered both NF-κB DNA recruitment and induced primary MCL cells apoptosis. Thus, ß-catenin likely represents another player through which BCR signaling impacts on MCL cell survival.

Caregiver Patient Reported Outcomes Measurement Information System (PROMIS) profiles in patients undergoing total joint arthroplasty and spine surgery: a prospective observational cohort study.

BACKGROUND/OBJECTIVE: The role of caregiver psychosocial characteristics and their relation to postsurgical caregiving capability remains unclear. The objective of this study was to explore caregiver psychosocial variables following surgery of patients undergoing total joint arthroplasty and spine surgery. METHODS: A prospective observational study was conducted where questionnaires were administered to caregivers preoperatively and 1 week/2 weeks/1 month postoperatively. Measures included demographics, caregiver activities and National Institutes of Health Patient Reported Outcomes Measurement Information System (NIH PROMIS) item banks. Bivariate analysis assessed differences between participants reporting baseline pain and those reporting no baseline pain. Generalized estimating equation models examined PROMIS T-scores across time. RESULTS: 190 caregivers were enrolled and completed surveys. 18% of caregivers reported experiencing a painful condition where they experienced pain during most days of the week. Across all time points, the majority of caregivers reported no worse than mild impairment across PROMIS scores. Compared with baseline, caregivers reported lower PROMIS satisfaction with social roles across all postoperative time points (p<0.001) and higher depression and fatigue at postoperative day 7 (p=0.002) and 14 (p=0.006). PROMIS sleep disturbance was only higher at day 7 (p=0.01). Caregivers reporting a baseline pain condition reported PROMIS scores indicative of higher anxiety (p=0.02), depression (p=0.003), sleep disturbances (p<0.001) and fatigue (p<0.001) and lower levels of satisfaction with social roles (p=0.002) compared with those caregivers without baseline pain. CONCLUSION: While there were transient worsening in PROMIS scores, it is unclear whether these were clinically meaningful. Postsurgical caregivers reporting baseline pain were characterized by worse functioning across all PROMIS scales.

Gain of the short arm of chromosome 2 (2p gain) has a significant role in drug-resistant chronic lymphocytic leukemia.

The different types of drug resistance encountered in chronic lymphocytic leukemia (CLL) cannot be fully accounted for by the 17p deletion (and/or TP53 mutation), a complex karyotype (CK), immunoglobulin heavy-chain variable region genes (IGHV) status and gene mutations. Hence, we sought to assess the associations between recurrent genomic abnormalities in CLL and the disease's development and outcome. To this end, we analyzed 64 samples from patients with CLL and gain of the short arm of chromosome 2 (2p+), which is frequent in late-stage and relapsed/refractory CLL. We found that fludarabine/cyclophosphamide/rituximab (a common first-line treatment in CLL) is not effective in removing the 2p+ clone - even in samples lacking a CK, the 17p deletion or unmutated IGHV. Our results suggest strongly that patients with CLL should be screened for 2p+ (using karyotyping and fluorescence in situ hybridization) before a treatment option is chosen. Longer follow-up is now required to evaluate bendamustine-rituximab, ibrutinib, and idelalisib-rituximab treatments.

Model-based Evaluation of Gene Expression Changes in Response to Leishmania Infection.

Since the development of high-density microarray technology in the late 1990s, global host gene expression changes in response to various stimuli have been extensively studied. More than a dozen peer-reviewed publications have investigated the effect of Leishmania infection in various models since 2001. This review covers the transcriptional changes in macrophage models induced by various Leishmania species and summarizes the resulting impact these studies have on our understanding of the host response to leishmaniasis in vitro. Characterization of the similarities and differences between various model systems will not only further our understanding of Leishmania-induced changes to macrophage gene expression but also identify potential therapeutic targets in the future.

Feasibility of App-Based Postsurgical Assessment of Pain, Pain Impact, and Regional Anesthesia Effects: A Pilot Randomized Controlled Trial.

OBJECTIVE: Postsurgical follow-up calls enable nurses to assess a patient's condition, provide tailored education, and improve the patient's experience. Despite the benefits, barriers to phone-based assessments may include patient nonresponse and lack of time due to demanding clinical schedules. The purpose of this trial was to examine the feasibility and utility of a smartphone app, mCare, for assessing pain, pain impact, and peripheral nerve block effects in patients. DESIGN: Pilot randomized control trial. SETTING AND PATIENTS: Eligible patients at a military treatment facility undergoing same-day surgery were randomized to the mCare group (N = 24) or the standard-of-care telephone (N = 26) group. RESULTS: Outcomes included initial response (assessment completion) rates and participant and nurse satisfaction. There were no differences in the response rates upon initial contact attempt, and patients in both groups reported similar levels of satisfaction and convenience. Nurses reported greater satisfaction with the app compared with standard-of-care telephone calls. CONCLUSIONS: Before wider implementation, further considerations of app-based assessment need to be fully explored.

Effectiveness and Safety of Potassium Replacement in Critically Ill Patients: A Retrospective Cohort Study.

BACKGROUND: Rules of thumb for potassium replacement are used in intensive care units despite minimal empirical validation. OBJECTIVE: To evaluate the effectiveness and safety of rule-of-thumb potassium replacement in critically ill patients with mild and moderate hypokalemia. METHODS: A retrospective, observational study was done of patients with mild (potassium, 3-3.9 mEq/L) and moderate (potassium, 2-2.9 mEq/L) hypokalemia admitted to a medical intensive care unit who received potassium replacement. Expected and actual frequencies of replacement that achieved target potassium concentrations (≥ 4 mEq/L) were compared by using a χ2 test. Logistic regression analysis was used to assess whether rule-of-thumb administration affected the probability of target attainment within 24 hours of replacement. RESULTS: Serum potassium concentrations were checked within 24 hours after potassium replacement on 354 of 577 days (61.4%) when replacement was provided. Concentrations were within target range in 82 instances (23.2%). Of 62 episodes of replacement expected to achieve the target according to the rule-of-thumb estimation, 22 did (35%). Rule-of-thumb administration was associated with greater likelihood of target attainment (odds ratio, 2.12; 95% CI, 1.18-3.85; P = .01). This difference in likelihood remained significant after adjustment for covariates (odds ratio, 2.18; 95% CI, 1.04-4.56; P = .04). CONCLUSION: In critically ill patients given potassium replacement without regard to a formal protocol, the target serum potassium concentration was achieved more often than expected according to the rule-of-thumb estimation but less than one-third of the time.

Effectiveness and Safety of Magnesium Replacement in Critically Ill Patients Admitted to the Medical Intensive Care Unit in an Academic Medical Center: A Retrospective, Cohort Study.

BACKGROUND: "Rules of thumb" for the replacement of electrolytes, including magnesium, in critical care settings are used, despite minimal empirical validation of their ability to achieve a target serum concentration. This study's purpose was to evaluate the effectiveness and safety surrounding magnesium replacement in medically, critically ill patients with mild-to-moderate hypomagnesemia. METHODS: This was a single-center, retrospective, observational evaluation of episodes of intravenous magnesium replacement ordered for patients with mild-to-moderate hypomagnesemia (1.0-1.9 mEq/L) admitted to a medical intensive care unit from May 2014 to April 2016. The primary effectiveness outcome, achievement of target serum magnesium concentration (≥2 mEq/L) compared to expected achievement using a "rule of thumb" estimation that 1 g intravenous magnesium sulfate raises the magnesium concentration 0.15 mEq/L, was tested using 1-sample z test. Logistic regression analysis was conducted to assess the effect of infusion rate on target achievement. RESULTS: Of 152 days on which magnesium replacements were provided for 72 patients, a follow-up serum magnesium concentration was checked within 24 hours in 89 (58.6%) episodes. Of these 89 episodes, serum magnesium concentration reached target in only 49 (59.8%) episodes compared to an expected 89 (100%; P < .0001). There was no significant association between infusion rate and achievement of the target serum magnesium concentration (odds ratio: 0.962, 95% confidence interval: 0.411-2.256). CONCLUSIONS: Medically, critically ill patients who received nonprotocolized magnesium replacement achieved the target serum magnesium concentration less frequently than the "rule of thumb" estimation predicted.

Second generation of diazachrysenes: Protection of Ebola virus infected mice and mechanism of action.

Ebola virus (EBOV) causes a deadly hemorrhagic fever in humans and non-human primates. There is currently no FDA-approved vaccine or medication to counter this disease. Here, we report on the design, synthesis and anti-viral activities of two classes of compounds which show high potency against EBOV in both in vitro cell culture assays and in vivo mouse models Ebola viral disease. These compounds incorporate the structural features of cationic amphiphilic drugs (CAD), i.e they possess both a hydrophobic domain and a hydrophilic domain consisting of an ionizable amine functional group. These structural features enable easily diffusion into cells but once inside an acidic compartment their amine groups became protonated, ionized and remain trapped inside the acidic compartments such as late endosomes and lysosomes. These compounds, by virtue of their lysomotrophic functions, blocked EBOV entry. However, unlike other drugs containing a CAD moiety including chloroquine and amodiaquine, compounds reported in this study display faster kinetics of accumulation in the lysosomes, robust expansion of late endosome/lysosomes, relatively more potent suppression of lysosome fusion with other vesicular compartments and inhibition of cathepsins activities, all of which play a vital role in anti-EBOV activity. Furthermore, the diazachrysene 2 (ZSML08) that showed most potent activity against EBOV in in vitro cell culture assays also showed significant survival benefit with 100% protection in mouse models of Ebola virus disease, at a low dose of 10 mg/kg/day. Lastly, toxicity studies in vivo using zebrafish models suggest no developmental defects or toxicity associated with these compounds. Overall, these studies describe two new pharmacophores that by virtue of being potent lysosomotrophs, display potent anti-EBOV activities both in vitro and in vivo animal models of EBOV disease.

A connectome of a learning and memory center in the adult Drosophila brain.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.