Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics.

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide 'SVAP9I' that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4-8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

Characterization of structure of peptidyl-tRNA hydrolase from Enterococcus faecium and its inhibition by a pyrrolinone compound.

In bacteria, peptidyl-tRNA hydrolase (Pth, E.C. 3.1.1.29) is a ubiquitous and essential enzyme for preventing the accumulation of peptidyl-tRNA and sequestration of tRNA. Pth is an esterase that cleaves the ester bond between peptide and tRNA. Here, we present the crystal structure of Pth from Enterococcus faecium (EfPth) at a resolution of 1.92 Å. The two molecules in the asymmetric unit differ in the orientation of sidechain of N66, a conserved residue of the catalytic site. Enzymatic hydrolysis of substrate α-N-BODIPY-lysyl-tRNALys (BLT) by EfPth was characterized by Michaelis-Menten parameters KM 163.5 nM and Vmax 1.9 nM/s. Compounds having pyrrolinone scaffold were tested for inhibition of Pth and one compound, 1040-C, was found to have IC50 of 180 nM. Antimicrobial activity profiling was done for 1040-C. It exhibited equipotent activity against drug-susceptible and resistant S. aureus (MRSA and VRSA) and Enterococcus (VSE and VRE) with MICs 2-8 µg/mL. 1040-C synergized with gentamicin and the combination was effective against the gentamicin resistant S. aureus strain NRS-119. 1040-C was found to reduce biofilm mass of S. aureus to an extent similar to Vancomycin. In a murine model of infection, 1040-C was able to reduce bacterial load to an extent comparable to Vancomycin.

Giant Faraday rotation in atomically thin semiconductors.

Faraday rotation is a fundamental effect in the magneto-optical response of solids, liquids and gases. Materials with a large Verdet constant find applications in optical modulators, sensors and non-reciprocal devices, such as optical isolators. Here, we demonstrate that the plane of polarization of light exhibits a giant Faraday rotation of several degrees around the A exciton transition in hBN-encapsulated monolayers of WSe2 and MoSe2 under moderate magnetic fields. This results in the highest known Verdet constant of -1.9 × 107 deg T-1 cm-1 for any material in the visible regime. Additionally, interlayer excitons in hBN-encapsulated bilayer MoS2 exhibit a large Verdet constant (VIL ≈ +2 × 105 deg T-1 cm-2) of opposite sign compared to A excitons in monolayers. The giant Faraday rotation is due to the giant oscillator strength and high g-factor of the excitons in atomically thin semiconducting transition metal dichalcogenides. We deduce the complete in-plane complex dielectric tensor of hBN-encapsulated WSe2 and MoSe2 monolayers, which is vital for the prediction of Kerr, Faraday and magneto-circular dichroism spectra of 2D heterostructures. Our results pose a crucial advance in the potential usage of two-dimensional materials in ultrathin optical polarization devices.

Engineering 2D Material Exciton Line Shape with Graphene/h-BN Encapsulation.

Control over the optical properties of atomically thin two-dimensional (2D) layers, including those of transition metal dichalcogenides (TMDs), is needed for future optoelectronic applications. Here, the near-field coupling between TMDs and graphene/graphite is used to engineer the exciton line shape and charge state. Fano-like asymmetric spectral features are produced in WS2, MoSe2, and WSe2 van der Waals heterostructures combined with graphene, graphite, or jointly with hexagonal boron nitride (h-BN) as supporting or encapsulating layers. Furthermore, trion emission is suppressed in h-BN encapsulated WSe2/graphene with a neutral exciton red shift (44 meV) and binding energy reduction (30 meV). The response of these systems to electron beam and light probes is well-described in terms of 2D optical conductivities of the involved materials. Beyond fundamental insights into the interaction of TMD excitons with structured environments, this study opens an unexplored avenue toward shaping the spectral profile of narrow optical modes for application in nanophotonic devices.

Structural and biophysical characterization of PadR family protein Rv1176c of Mycobacterium tuberculosis H37Rv.

Rv1176c of Mycobacterium tuberculosis H37Rv belongs to the PadR-s1 subfamily of the PadR family of protein. Rv1176c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 39.4 °C. The crystal structure of Rv1176c was determined at a resolution of 2.94 Å, with two monomers in the asymmetric unit. Each monomer has a characteristic N-terminal winged-helix-turn-helix DNA-binding domain. Rv1176c C-terminal is a coiled-coil dimerization domain formed of α-helices α5 to α7. In the Rv1176c dimer, there is domain-swapping of the C-terminal domain in comparison to other PadR homologs. In the dimer, there is a long inter-subunit tunnel in which different ligands can bind. Rv1176c was found to bind to the promoter region of its own gene with high specificity. M. smegmatis MC2 155 genome lacks homolog of Rv1176c. Therefore, it was used as a surrogate to characterize the functional role of Rv1176c. Expression of Rv1176c in M. smegmatis MC2 155 cells imparted enhanced tolerance towards oxidative stress. Rv1176c expressing M. smegmatis MC2 155 cells exhibited enhanced intracellular survival in J774A.1 murine macrophage cells. Overall, our studies demonstrate Rv1176c to be a PadR-s1 subfamily transcription factor that can moderate the effect of oxidative stress.

Mediation of Successful Reperfusion Effect through Infarct Growth and Cerebral Edema: A Pooled, Patient-Level Analysis of EXTEND-IA Trials and SELECT Prospective Cohort.

OBJECTIVE: Reperfusion therapy is highly beneficial for ischemic stroke. Reduction in both infarct growth and edema are plausible mediators of clinical benefit with reperfusion. We aimed to quantify these mediators and their interrelationship. METHODS: In a pooled, patient-level analysis of the EXTEND-IA trials and SELECT study, we used a mediation analysis framework to quantify infarct growth and cerebral edema (midline shift) mediation effect on successful reperfusion (modified Treatment in Cerebral Ischemia ≥ 2b) association with functional outcome (modified Rankin Scale distribution). Furthermore, we evaluated an additional pathway to the original hypothesis, where infarct growth mediated successful reperfusion effect on midline shift. RESULTS: A total 542 of 665 (81.5%) eligible patients achieved successful reperfusion. Baseline clinical and imaging characteristics were largely similar between those achieving successful versus unsuccessful reperfusion. Median infarct growth was 12.3ml (interquartile range [IQR] = 1.8-48.4), and median midline shift was 0mm (IQR = 0-2.2). Of 249 (37%) demonstrating a midline shift of ≥1mm, median shift was 2.75mm (IQR = 1.89-4.21). Successful reperfusion was associated with reductions in both predefined mediators, infarct growth (ß = -1.19, 95% confidence interval [CI] = -1.51 to -0.88, p < 0.001) and midline shift (adjusted odds ratio = 0.36, 95% CI = 0.23-0.57, p < 0.001). Successful reperfusion association with improved functional outcome (adjusted common odds ratio [acOR] = 2.68, 95% CI = 1.86-3.88, p < 0.001) became insignificant (acOR = 1.39, 95% CI = 0.95-2.04, p = 0.094) when infarct growth and midline shift were added to the regression model. Infarct growth and midline shift explained 45% and 34% of successful reperfusion effect, respectively. Analysis considering an alternative hypothesis demonstrated consistent results. INTERPRETATION: In this mediation analysis from a pooled, patient-level cohort, a significant proportion (~80%) of successful reperfusion effect on functional outcome was mediated through reduction in infarct growth and cerebral edema. Further studies are required to confirm our findings, detect additional mediators to explain successful reperfusion residual effect, and identify novel therapeutic targets to further enhance reperfusion benefits. ANN NEUROL 2023;93:793-804.

Thrombectomy Outcomes With General vs Nongeneral Anesthesia: A Pooled Patient-Level Analysis From the EXTEND-IA Trials and SELECT Study.

BACKGROUND AND OBJECTIVES: The effect of anesthesia choice on endovascular thrombectomy (EVT) outcomes is unclear. Collateral status on perfusion imaging may help identify the optimal anesthesia choice. METHODS: In a pooled patient-level analysis of EXTEND-IA, EXTEND-IA TNK, EXTEND-IA TNK part II, and SELECT, EVT functional outcomes (modified Rankin Scale score distribution) were compared between general anesthesia (GA) vs non-GA in a propensity-matched sample. Furthermore, we evaluated the association of collateral flow on perfusion imaging, assessed by hypoperfusion intensity ratio (HIR) - Tmax > 10 seconds/Tmax > 6 seconds (good collaterals - HIR < 0.4, poor collaterals - HIR ≥ 0.4) on the association between anesthesia type and EVT outcomes. RESULTS: Of 725 treated with EVT, 299 (41%) received GA and 426 (59%) non-GA. The baseline characteristics differed in presentation National Institutes of Health Stroke Scale score (median [interquartile range] GA: 18 [13-22], non-GA: 16 [11-20], p < 0.001) and ischemic core volume (GA: 15.0 mL [3.2-38.0] vs non-GA: 9.0 mL [0.0-31.0], p < 0.001). In addition, GA was associated with longer last known well to arterial access (203 minutes [157-267] vs 186 minutes [138-252], p = 0.002), but similar procedural time (35.5 minutes [23-59] vs 34 minutes [22-54], p = 0.51). Of 182 matched pairs using propensity scores, baseline characteristics were similar. In the propensity score-matched pairs, GA was independently associated with worse functional outcomes (adjusted common odds ratio [adj. cOR]: 0.64, 95% CI: 0.44-0.93, p = 0.021) and higher neurologic worsening (GA: 14.9% vs non-GA: 8.9%, aOR: 2.10, 95% CI: 1.02-4.33, p = 0.045). Patients with poor collaterals had worse functional outcomes with GA (adj. cOR: 0.47, 95% CI: 0.29-0.76, p = 0.002), whereas no difference was observed in those with good collaterals (adj. cOR: 0.93, 95% CI: 0.50-1.74, p = 0.82), p interaction: 0.07. No difference was observed in infarct growth overall and in patients with good collaterals, whereas patients with poor collaterals demonstrated larger infarct growth with GA with a significant interaction between collaterals and anesthesia type on infarct growth rate (p interaction: 0.020). DISCUSSION: GA was associated with worse functional outcomes after EVT, particularly in patients with poor collaterals in a propensity score-matched analysis from a pooled patient-level cohort from 3 randomized trials and 1 prospective cohort study. The confounding by indication may persist despite the doubly robust nature of the analysis. These findings have implications for randomized trials of GA vs non-GA and may be of utility for clinicians when making anesthesia type choice. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that use of GA is associated with worse functional outcome in patients undergoing EVT. TRIAL REGISTRATION INFORMATION: EXTEND-IA: ClinicalTrials.gov (NCT01492725); EXTEND-IA TNK: ClinicalTrials.gov (NCT02388061); EXTEND-IA TNK part II: ClinicalTrials.gov (NCT03340493); and SELECT: ClinicalTrials.gov (NCT02446587).

Mycobacterium tuberculosis exploits MPT64 to generate myeloid-derived suppressor cells to evade the immune system.

Mycobacterium tuberculosis (Mtb) is a smart and successful pathogen since it can persist in the intimidating environment of the host by taming and tuning the immune system. Mtb releases MPT64 (Rv1980c) protein in high amounts in patients with active tuberculosis (TB). Consequently, we were curious to decipher the role of MPT64 on the differentiating dendritic cells (DCs) and its relation to evading the immune system. We observed that pre-exposure of differentiating DCs to MPT64 (DCMPT64) transformed them into a phenotype of myeloid-derived suppressor cells (MDSCs). DCMPT64 expressed a high level of immunosuppressive molecules PD-L1, TIM-3, nitric oxide (NO), arginase 1, IDO-1, IL-10 and TGF-ß, but inhibited the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-12. DCMPT64 chemotaxis function was diminished due to the reduced expression of CCR7. DCMPT64 promoted the generation of regulatory T cells (Tregs) but inhibited the differentiation of Th1 cells and Th17 cells. Further, high lipid and methylglyoxal content, and reduced glucose consumption by DCMPT64, rendered them metabolically quiescent and consequently, reduced DCMPT64 ability to phagocytose Mtb and provided a safer shelter for the intracellular survival of the mycobacterium. The mechanism identified in impairing the function of DCMPT64 was through the increased production and accumulation of methylglyoxal. Hence, for the first time, we demonstrate the novel role of MPT64 in promoting the generation of MDSCs to favor Mtb survival and escape its destruction by the immune system.

High-Performance Broadband Faraday Rotation Spectroscopy of 2D Materials and Thin Magnetic Films.

A Faraday rotation spectroscopy (FRS) technique is presented for measurements on the micrometer scale. Spectral acquisition speeds of about two orders of magnitude faster than state-of-the-art modulation spectroscopy setups are demonstrated. The experimental method is based on charge-coupled-device detection, avoiding speed-limiting components, such as polarization modulators with lock-in amplifiers. At the same time, FRS spectra are obtained with a sensitivity of 20 µrad ( 0.001 ° \[0.001{\bm{^\circ }}\] ) over a broad spectral range (525-800 nm), which is on par with state-of-the-art polarization-modulation techniques. The new measurement and analysis technique also automatically cancels unwanted Faraday rotation backgrounds. Using the setup, Faraday rotation spectroscopy of excitons is performed in a hexagonal boron nitride-encapsulated atomically thin semiconductor WS2 under magnetic fields of up to 1.4 T at room temperature and liquid helium temperature. An exciton g-factor of -4.4 ± 0.3 is determined at room temperature, and -4.2 ± 0.2 at liquid helium temperature. In addition, FRS and hysteresis loop measurements are performed on a 20 nm thick film of an amorphous magnetic Tb20 Fe80 alloy.

Thrombolysis for stroke in elderly in the late window period.

OBJECTIVES: Safety of intravenous thrombolysis (IVT) within 3-4.5 hours of stroke onset in patients ≥80 years is still disputable. We evaluated the association of symptom onset-to-treatment time (SOTT) with the symptomatic intracranial hemorrhage (sICH), poor outcome, and mortality in patients≥80 years. MATERIALS AND METHODS: In a retrospective study, patients treated with IVT following stroke were registered. Outcomes were poor outcome (mRS>2), sICH/ECASS-2, and in-hospital mortality. We compared the patients≥80 years who received IVT within 3 hours with those receiving IVT within 3-4.5 hours. We further compared the patients who were <80 years with those ≥80 years and SOTT of 3-4.5 hours. RESULTS: Of 834 patients, 265 aged over 80. In those above 80 and in multivariable analysis, the associations of SOTT with poor outcome (aOR: 1.401, CI: 0.503-3.903, p=0.519), sICH (aOR=2.50, CI=0.76-8.26, p= 0.132) and mortality (aOR=1.12, CI=0.39-3.25, p= 0.833) were not significant. 106 patients received IVT within 3-4.5 hours. In multivariable analysis, the associations of age (≥80 versus <80) with poor outcome (aOR=1.87, CI=0.65-5.37, p=0.246), sICH (aOR=0.65, CI=0.14-3.11, p=0.590), and mortality (aOR=0.87, 95% CI=0.16-4.57, p=0.867) were not significant in patients with SOTT of 3-4.5 hours. CONCLUSION: IVT within 3-4.5 hours in patients ≥80 years is not associated with increased sICH, poor outcome, and mortality compared to the early time window, and also compared to the younger patients in 3-4.5 hours window period. The decision of IVT administration in this age group should not be made solely on the basis of stroke onset timing.

Clinical and Neuroimaging Outcomes of Direct Thrombectomy vs Bridging Therapy in Large Vessel Occlusion: Analysis of the SELECT Cohort Study.

OBJECTIVE: To evaluate the comparative safety and efficacy of direct endovascular thrombectomy (dEVT) compared to bridging therapy (BT; IV tissue plasminogen activator + EVT) and to assess whether BT potential benefit relates to stroke severity, size, and initial presentation to EVT vs non-EVT center. METHODS: In a prospective multicenter cohort study of imaging selection for endovascular thrombectomy (Optimizing Patient Selection for Endovascular Treatment in Acute Ischemic Stroke [SELECT]), patients with anterior circulation large vessel occlusion (LVO) presenting to EVT-capable centers within 4.5 hours from last known well were stratified into BT vs dEVT. The primary outcome was 90-day functional independence (modified Rankin Scale [mRS] score 0-2). Secondary outcomes included a shift across 90-day mRS grades, mortality, and symptomatic intracranial hemorrhage. We also performed subgroup analyses according to initial presentation to EVT-capable center (direct vs transfer), stroke severity, and baseline infarct core volume. RESULTS: We identified 226 LVOs (54% men, mean age 65.6 ± 14.6 years, median NIH Stroke Scale [NIHSS] score 17, 28% received dEVT). Median time from arrival to groin puncture did not differ in patients with BT when presenting directly (dEVT 1.43 [interquartile range (IQR) 1.13-1.90] hours vs BT 1.58 [IQR 1.27-2.02] hours, p = 0.40) or transferred to EVT-capable centers (dEVT 1.17 [IQR 0.90-1.48] hours vs BT 1.27 [IQR 0.97-1.87] hours, p = 0.24). BT was associated with higher odds of 90-day functional independence (57% vs 44%, adjusted odds ratio [aOR] 2.02, 95% confidence interval [CI] 1.01-4.03, p = 0.046) and functional improvement (adjusted common OR 2.06, 95% CI 1.18-3.60, p = 0.011) and lower likelihood of 90-day mortality (11% vs 23%, aOR 0.20, 95% CI 0.07-0.58, p = 0.003). No differences in any other outcomes were detected. In subgroup analyses, patients with BT with baseline NIHSS scores <15 had higher functional independence likelihood compared to those with dEVT (aOR 4.87, 95% CI 1.56-15.18, p = 0.006); this association was not evident for patients with NIHSS scores ≥15 (aOR 1.05, 95% CI 0.40-2.74, p = 0.92). Similarly, functional outcomes improvements with BT were detected in patients with core volume strata (ischemic core <50 cm3: aOR 2.10, 95% CI 1.02-4.33, p = 0.044 vs ischemic core ≥50 cm3: aOR 0.41, 95% CI 0.01-16.02, p = 0.64) and transfer status (transferred: aOR 2.21, 95% CI 0.93-9.65, p = 0.29 vs direct to EVT center: aOR 1.84, 95% CI 0.80-4.23, p = 0.15). CONCLUSIONS: BT appears to be associated with better clinical outcomes, especially with milder NIHSS scores, smaller presentation core volumes, and those who were "dripped and shipped." We did not observe any potential benefit of BT in patients with more severe strokes. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT02446587. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that for patients with ischemic stroke from anterior circulation LVO within 4.5 hours from last known well, BT compared to dEVT leads to better 90-day functional outcomes.

Immunological characterization of chimeras of high specificity antigens from Mycobacterium tuberculosis H37Rv.

Tuberculosis remains a serious global health problem. BCG is the only prophylactic TB vaccine and it shows variable protective efficacy. Chimeric protein subunit vaccines hold great potential as stand-alone vaccines or heterologous BCG prime boosters. We have designed a protein chimera, PP31, by combining Mtb ESAT-6 family antigen Rv1198 and MoCo biosynthesis family antigen Rv3111. Further, PP31 was extended by addition of latency antigen Rv1813c to yield PP43. Immunization of BALB/c mice with PP31 or PP43 with FIA adjuvant elicited strong humoral immune response. Restimulation of splenocytes of the immunized mice lead to significant proliferation of lymphocytes, secretion of cytokines IFN-γ, TNF, IL-2 of the Th1 class, IL-17A of the Th17 class, and IL-6. PP31 and PP43 also induced intracellular cytokine expression (IFN-γ, TNF, and IL-2) from both CD4+-CD44high and CD8+-CD44high T-cells. Antigen-specific IFN-γ+/IL-2+ double positive CD4+ T-cells were significantly higher in case of PP43 than PP31-immunized mice and control group. PP43 showed protection equivalent to heat-inactivated BCG in response to challenge of the immunized mice with Mtb H37Ra. Based on its immunogenicity and protective efficacy, PP43 appears to be a potential candidate for further development as a subunit vaccine against TB.

The Impact of Diabetes Mellitus in Patients with Chronic Obstructive Pulmonary Disease (COPD) Hospitalization.

(1) Background: Chronic obstructive pulmonary disease (COPD) is the leading cause of morbidity and mortality worldwide. Diabetes mellitus (DM) has been shown to have adverse inflammatory effects on lung anatomy and physiology. We investigated the impact of DM on COPD patient outcomes during inpatient hospitalization. (2) Methods: We conducted a retrospective analysis using the Nationwide Inpatient Sample (NIS) over the years 2002-2014. Three groups, COPD without diabetes, COPD with diabetes but no complication, and COPD with DM and complication, were analyzed. (3) Results: A total of 7,498,577 were COPD hospitalization; of those, 1,799,637 had DM without complications, and 483,467 had DM with complications. After adjusting for clinical, demographic, and comorbidities, the odds of increased LOS in the COPD/DM with complication were 1.37 (confidence interval (CI): 1.326-1.368), and those of DM without complication were 1.061 (1.052-1.070) when compared with COPD alone. The odds of pneumonia, respiratory failure, stroke, and acute kidney injury were also higher in COPD hospitalizations with DM. Both DM with complication (odds ratio (OR): 0.751 (CI 0.727-0.777)) and DM without complication (OR: 0.635 (CI: 0.596-0.675)) have lesser odds of mortality during hospitalization than with COPD alone. (4) Conclusions: There is a considerable inpatient burden among COPD patients with DM in the United States.

Early Infarct Growth Rate Correlation With Endovascular Thrombectomy Clinical Outcomes: Analysis From the SELECT Study.

BACKGROUND AND PURPOSE: Time elapsed from last-known well (LKW) and baseline imaging results are influential on endovascular thrombectomy (EVT) outcomes. METHODS: In a prospective multicenter cohort study of imaging selection for endovascular thrombectomy (SELECT [Optimizing Patient's Selection for Endovascular Treatment in Acute Ischemic Stroke], the early infarct growth rate (EIGR) was defined as ischemic core volume on perfusion imaging (relative cerebral blood flow<30%) divided by the time from LKW to imaging. The optimal EIGR cutoff was identified by maximizing the sum of the sensitivity and specificity to correlate best with favorable outcome and to improve its the predictability. Patients were stratified into slow progressors if EIGR2. The primary outcome was 90-day functional independence (modified Rankin Scale score =0-2). RESULTS: Of 445 consented, 361 (285 EVT, 76 medical management only) patients met the study inclusion criteria. The optimal EIGR was <10 mL/h; 200 EVT patients were slow and 85 fast progressors. Fast progressors had a higher median National Institutes of Health Stroke Scale (19 versus 15, P<0.001), shorter time from LKW to groin puncture (180 versus 266 minutes, P<0.001). Slow progressors had better collaterals on computed tomography perfusion: hypoperfusion intensity ratio (adjusted odds ratio [aOR]: 5.11 [2.43-10.76], P<0.001) and computed tomography angiography: collaterals-score (aOR: 4.43 [1.83-10.73], P=0.001). EIGR independently correlated with functional independence after EVT, adjusting for age, National Institutes of Health Stroke Scale, time LKW to groin puncture, reperfusion (modified Thrombolysis in Cerebral Infarction score of ≥2b), IV-tPA (intravenous tissue-type plasminogen activator), and transfer status (aOR: 0.78 [0.65-0.94], P=0.01). Slow progressors had higher functional independence rates (121 [61%] versus 30 [35%], P<0.001) and had 3.5 times the likelihood of achieving modified Rankin Scale score =0-2 with EVT (aOR=2.94 [95% CI, 1.53-5.61], P=0.001) as compared to fast progressors, who had substantially worse clinical outcomes both in early and late time window. The odds of good outcome decreased by 14% for each 5 mL/h increase in EIGR (aOR, 0.87 [0.80-0.94], P<0.001) and declined more rapidly in fast progressors. CONCLUSIONS: The EIGR strongly correlates with both collateral status and clinical outcomes after EVT. Fast progressors demonstrated worse outcomes when receiving EVT beyond 6 hours of stroke onset as compared to those who received EVT within 6 hours. Registration: URL: https://clinicaltrials.gov. Unique identifier: NCT02446587.

Structural and functional characterization of peptidyl-tRNA hydrolase from Klebsiella pneumoniae.

Klebsiella pneumoniae is a member of the ESKAPE panel of pathogens that are top priority to tackle AMR. Bacterial peptidyl tRNA hydrolase (Pth), an essential, ubiquitous enzyme, hydrolyzes the peptidyl-tRNAs that accumulate in the cytoplasm because of premature termination of translation. Pth cleaves the ester bond between 2' or 3' hydroxyl of the ribose in the tRNA and C-terminal carboxylate of the peptide, thereby making free tRNA available for repeated cycles of protein synthesis and preventing cell death by alleviating tRNA starvation. Pth structures have been determined in peptide-bound or peptide-free states. In peptide-bound state, highly conserved residues F67, N69 and N115 adopt a conformation that is conducive to their interaction with peptide moiety of the substrate. While, in peptide-free state, these residues move away from the catalytic center, perhaps, in order to facilitate release of hydrolysed peptide. Here, we present a novel X-ray crystal structure of Pth from Klebsiella pneumoniae (KpPth), at 1.89 Å resolution, in which out of the two molecules in the asymmetric unit, one reflects the peptide-bound while the other reflects peptide-free conformation of the conserved catalytic site residues. Each molecule of the protein has canonical structure with seven stranded ß-sheet structure surrounded by six α-helices. MD simulations indicate that both the forms converge over 500 ns simulation to structures with wider opening of the crevice at peptide-binding end. In solution, KpPth is monomeric and its 2D-HSQC spectrum displays a single set of well dispersed peaks. Further, KpPth was demonstrated to be enzymatically active on BODIPY-Lys-tRNALys3.

Investigation of HDAC8-ligands' intermolecular forces through molecular dynamics simulations: profiling of non-bonding energies to design potential compounds as new anti-cancer agents.

Histone deacetylases are zinc-dependent isoform enzymes and play important role in cellular homeostasis. Among these, HDAC8 is a potential anticancer drug target. To design new inhibitors using protein-ligand energy profiles, an all atom molecular dynamics (MD) simulations were carried out on nine HDAC8-ligand co-crystals (PDBs: 1T64, 1T69, 1T67, 3F07, 1W22, 1VKG, 5FCW, 3SFF and 3SFH). TSN, SHH, B3N, AGE, NHB, CRI, 5YA, 0DI and 1DI are ligands of PDBs, respectively. For these HDAC8-ligands, relative Gibbs binding free energy (ΔGbind) from MM/PBSA method and non-bonding energies (NBE) are in agreement with each other (r2=0.678). Therefore, the NBEs are used to analyze ligands' sub-structures, namely zinc-binding, linker and CAP groups. For linker/CAP regions, this identified carbonyl, amide, and sulfonamide moieties as desirable and alkyl/aryl moieties as electrostatically unfavourable. Using this information, systematically new compounds were designed and subjected to MD simulations. This resulted in seven compounds (NC-I to NC-VII) with encouraging energy profiles (NBE: -76.25 to -127.09 kcal/mol; ΔGbind: -17.21 to -57.42 kcal/mol) in comparison to that of the HDAC8 ligands (NBE: -46.25 to -106.29 kcal/mol; ΔGbind: -14.74 to -49.52 kcal/mol). From these, NC-VI showed best energy profile (NBE = -126.15 kcal/mol; ΔGbind = -57.42 kcal/mol) suggesting its binding affinity and thermodynamic stability. In addition to this, NC-II and NC-III have shown promising NBE and ΔGbind profiles. These may serve as lead molecules for exploration against HDAC8 in cancer therapy. This has provided a basis for designing new compounds with improved NBE and ΔGbind profiles by modifying the unfavourable or not so favourable regions of ligands. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Šresolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed ß-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors.

Impact of a Positive Viral Polymerase Chain Reaction on Outcomes of Chronic Obstructive Pulmonary Disease (COPD) Exacerbations.

INTRODUCTION: More than 15 million adults in the USA have chronic obstructive pulmonary disease. Chronic obstructive pulmonary disease (COPD) places a high burden on the healthcare system. Many hospital admissions are due to an exacerbation, which is suspected to be from a viral cause. The purpose of this analysis was to compare the outcomes of patients with a positive and negative respiratory virus panel who were admitted to the hospital with COPD exacerbations. METHODS: This retrospective cohort study was conducted in the Geisinger Healthcare System. The dataset included 2729 patient encounters between 1 January 2006 and 30 November 2017. Hospital length of stay was calculated as the discrete number of calendar days a patient was in the hospital. Patient encounters with a positive and negative respiratory virus panel were compared using Pearson's chi-square or Fisher's exact test for categorical variables and Student's t-test or Wilcoxon rank-sum tests for continuous variables. RESULTS: There were 1626 patients with a total of 2729 chronic obstructive pulmonary disease exacerbation encounters. Nineteen percent of those encounters (n = 524) had a respiratory virus panel performed during their admission. Among these encounters, 161 (30.7%) had positive results, and 363 (69.3%) had negative results. For encounters with the respiratory virus panel, the mean age was 64.5, 59.5% were female, 98.9% were white, and the mean body mass index was 26.6. Those with a negative respiratory virus panel had a higher median white blood cell count (11.1 vs. 9.9, p = 0.0076). There were no other statistically significant differences in characteristics between the two groups. Respiratory virus panel positive patients had a statistically significant longer hospital length of stay. There were no significant differences with respect to being on mechanical ventilation or ventilation-free days. CONCLUSION: This study shows that a positive respiratory virus panel is associated with increased length of hospital stay. Early diagnosis of chronic obstructive pulmonary disease exacerbation patients with positive viral panel would help identify patients with a longer length of stay.

Strain tuning of the Stokes shift in atomically thin semiconductors.

Atomically thin layers of transition metal dichalcogenides (TMDC) have exceptional optical properties, exhibiting a characteristic absorption and emission at excitonic resonances. Due to their extreme flexibility, strain can be used to alter the fundamental exciton energies and line widths of TMDCs. Here, we report on the Stokes shift, i.e. the energetic difference of light absorption and emission, of the A exciton in TMDC mono- and bilayers. We demonstrate that mechanical strain can be used to tune the Stokes shift. We perform optical transmission and photoluminescence (PL) experiments on mono- and bilayers and apply uniaxial tensile strain of up to 1.2% in MoSe2 and WS2 bilayers. An A exciton red shift of -38 meV/% and -70 meV/% is found in transmission in MoSe2 and WS2, while smaller values of -27 meV/% and -62 meV/% are measured in PL, respectively. Therefore, a reduction of the Stokes shift is observed under increasing tensile strain. At the same time, the A exciton PL line widths narrow significantly with -14 meV/% (MoSe2) and -21 meV/% (WS2), demonstrating a drastic change in the exciton-phonon interaction. By comparison with ab initio calculations, we can trace back the observed shifts of the excitons to changes in the electronic band structure of the materials. Variations of the relative energetic positions of the different excitons lead to a decrease of the exciton-phonon coupling. Furthermore, we identify the indirect exciton emission in bilayer WS2 as the ΓK transition by comparing the experimental and theoretical gauge factors.