The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Epigenetic CpG duplex marks probed by an evolved DNA reader via a well-tempered conformational plasticity.

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel 'CpG duplex marks' could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

Electrolyte under Molybdenum Disulfide Surfaces: Molecular Insights on Structure and Dynamics of Water.

Molybdenum disulfide (MoS2) is a two-dimensional (2D) material that offers molecular transport and sieving properties and might be a potential candidate for membrane technologies for energy and environmental applications. To facilitate the separation application, understanding the structural and dynamic properties of water near the substrate-aqueous solution is essential. Employing the molecular dynamics simulation, we investigate the density, local water network at the solid-liquid interface, and water dynamics in aqueous electrolyte solutions with various chloride salts confined in MoS2 nanochannels with different pore sizes and electrolyte concentrations. Our simulation results confirm that the layering of interfacial water at the hydrophilic MoS2 surface and the water density variation depends on the nature of the ions. The simulation results imply a strong attraction of cations to the surface-liquid interfaces, whereas anions are expelled from the surface due to electrostatic interaction. An examination of the dynamical property of water reveals that the confinement effect is more pronounced on water mobility when the pore width is less than 3 nm, and the salt concentration is below 1 M, whereas the electrolyte concentration greater than 1 M, ions predominantly drive the water mobility as compared to confinement one. These simulation results enhance experimental observations and provide molecular insights into the local ordering mechanism that can be crucial in controlling water dynamics in nanofiltration applications.

Genomic characterization of metastatic castration-resistant prostate cancer patients undergoing PSMA radioligand therapy: A single-center experience.

BACKGROUND: Genomic defects in DNA-damage repair (DDR) mechanisms have been proposed to affect the radiosensitivity of prostate cancers. In this study, we intended to evaluate the prevalence of genetic alterations in a cohort of metastatic castration-resistant prostate cancer (mCRPC) patients undergoing radioligand therapy (RLT) with prostate-specific membrane antigen (PSMA)-inhibitors as well as the impact of such mutations on treatment outcomes. METHODS: Data of consecutive mCRPC patients from 2017 to 2021 who were treated with PSMA-RLT and underwent next-generation sequencing (NGS) were collected and analyzed for response and survival outcomes. RESULTS: In 95 patients of mCRPC treated with PSMA-RLT, 15 patients (median age: 66 years, range: 50-73 years; [177 Lu]Lu-PSMA-617, n = 12; [225 Ac]Ac-PSMA-617, n = 3) underwent NGS. The median progression-free survival (PFS) of this cohort was 3 months (95% confidence interval: 1.6-4.4 months). On NGS, 21 genetic alterations were reported in 10/15 (67%) patients, of which 13 were DDR-associated alterations involving the genes: ATM (n = 3), BRCA2 (n = 3), TP53 (n = 2), PTEN (n = 2), FANCD2 (n = 1), FANCM (n = 1), and NBN (n = 1). Overall, 5/15 (33%) patients harbored six pathogenic variants (BRCA2, n = 2; ATM, n = 1; TP53, n = 1; PTEN, n = 2). No significant difference was noted for the biochemical response, radiological response, PFS, and overall survival between the patients with and without genetic alterations. CONCLUSIONS: Patients of mCRPC undergoing PSMA-RLT were frequently seen to harbor DDR-associated aberrations, albeit with no significant impact on treatment outcomes. Large prospective trials comparing PSMA-RLT-related outcomes in DDR-deficient and -proficient patients are required to bring out the differences, if any, in a more observable manner.

Association of CT Findings With Perineural Invasion in Gallbladder Cancer: Preliminary Assessment.

Perineural invasion (PNI) indicates a worse prognosis for patients with gallbladder cancer (GBC). This preliminary retrospective study included 19 patients with GBC who under-went contrast-enhanced CT in the 4 weeks before undergoing surgical resection. GBC showed PNI on pathologic assessment in eight of 19 patients. On CT, wall thickening morphology had sensitivity of 75.0% and specificity of 81.8% for PNI; soft-tissue stranding around the celiac plexus had sensitivity of 62.5% and specificity of 100.0% for PNI.

Synthetic Monosaccharide Channels: Size-Selective Transmembrane Transport of Glucose and Fructose Mediated by Porphyrin Boxes.

Here we report synthetic monosaccharide channels built with shape-persistent organic cages, porphyrin boxes (PBs), that allow facile transmembrane transport of glucose and fructose through their windows. PBs show a much higher transport rate for glucose and fructose over disaccharides such as sucrose, as evidenced by intravesicular enzyme assays and molecular dynamics simulations. The transport rate can be modulated by changing the length of the alkyl chains decorating the cage windows. Insertion of a linear pillar ligand into the cavity of PBs blocks the monosaccharide transport. In vitro cell experiment shows that PBs transport glucose across the living-cell membrane and enhance cell viability when the natural glucose transporter GLUT1 is blocked. Time-dependent live-cell imaging and MTT assays confirm the cyto-compatibility of PBs. The monosaccharide-selective transport ability of PBs is reminiscent of natural glucose transporters (GLUTs), which are crucial for numerous biological functions.

'Rollover' abdominal paracentesis versus standard technique: protocol of a crossover randomized comparative trial.

The sensitivity of single abdominal paracentesis for diagnosis of peritoneal carcinomatosis in patients with malignant ascites is 40-70%. Tumor cells shed from the peritoneum settle preferentially in certain recesses of the peritoneum. We aim to compare the standard technique of abdominal paracentesis versus a rollover technique in a randomized crossover study to assess the cytological yield in patients suspected to have peritoneal carcinomatosis. Each patient will serve as their own control and the outcome assessor (cytopathologist) will be blinded to the method of paracentesis performed. The primary objective will be to compare the tumor cell positivity between the standard paracentesis group and the rollover group among enrolled patients. Clinical Trial registration: CTRI/2020/06/025887 and NCT04232384.

Lay abstract Existing methods of diagnosing cancer-related ascites are dependent on microscopic evaluation of fluid obtained from the ascites. However, this may not diagnose all such cases because the fluid may not contain many tumor cells. This may be due to the settling of tumor cells in certain inaccessible locations of the peritoneum (the lining of the abdominal cavity). This trial will look at whether rolling the patient from side to side could be helpful in increasing the chances of finding tumor cells in the ascites.

The Active Site of a Prototypical "Rigid" Drug Target is Marked by Extensive Conformational Dynamics.

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off-target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly "rigid protein" that substantial active-site dynamics have generally remained unrecognized, despite thousands of medicinal-chemistry studies on this model over decades. Comparing cryogenic X-ray structures, solid-state NMR on micro-crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational-exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines.

Fast Microsecond Dynamics of the Protein-Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy.

Protein-water interactions have widespread effects on protein structure and dynamics. As such, the function of many biomacromolecules can be directly related to the presence and exchange of water molecules. While the presence of structural water sites can be easily detected by X-ray crystallography, the dynamics within functional water-protein network architectures is largely elusive. Here we use solid-state NMR relaxation dispersion measurements with a focus on those active-site residues in the enzyme human carbonic anhydrase II (hCAII) that constitute the evolutionarily conserved water pocket, key for CAs' enzymatic catalysis. Together with chemical shifts, peak broadening, and results of molecular dynamics (MD) and DFT shift calculations, the relaxation dispersion data suggest the presence of a widespread fast µs-time-scale dynamics in the pocket throughout the protein-water network. This process is abrogated in the presence of an inhibitor which partially disrupts the network. The time scale of the protein-water pocket motion coincides both with the estimated residence time of Zn-bound water/OH- in the pocket showing the longest lifetimes in earlier magnetic relaxation dispersion experiments as well as with the rate-limiting step of catalytic turnover. As such, the reorganization of the water pocket:enzyme architecture might constitute an element of importance for enzymatic activity of this and possibly other proteins.

Melting transition of Lennard-Jones fluid in cylindrical pores.

Three-stage pseudo-supercritical transformation path and multiple-histogram reweighting technique are employed for the determination of solid-liquid coexistence of the Lennard-Jones (12-6) fluid, in a structureless cylindrical pore of radius, R, ranging from 4 to 20 molecular diameters. The Gibbs free energy difference is evaluated using thermodynamic integration method by connecting solid and liquid phases under confinement via one or more intermediate states without any first order phase transition among them. The thermodynamic melting temperature, Tm, is found to oscillate for pore size, R < 8, which is in agreement with the behavior observed for the melting temperature in slit pores. However, Tm for almost all pore sizes is less than the bulk case, which is contrary to the behavior seen for the slit pore. The oscillation in Tm decays at around pore radius R = 8, and beyond that shift in the melting temperature with respect to the bulk case is in line with the prediction of the Gibbs-Thomson equation.

Use of Induced sputum to determine the prevalence of Pneumocystis jirovecii in immunocompromised children with pneumonia.

BACKGROUND: Information on prevalence of Pneumocystis jirovecii pneumonia (PCP) in immunocompromised children with pneumonia in Southeast Asia is limited. METHODS: Immunocompromised children hospitalized with radiographic pneumonia were investigated for PCP by testing induced sputum by using polymerase chain reaction (PCR). RESULTS: Ninety-four immunocompromised children (mean age 74.5 ± 43.7 months, boys 69) with pneumonia were investigated for PCP. Underlying disease included solid tumors and hematological malignancy in 57, HIV infection in 14, primary immune deficiency in 11 and other immune deficiency disorders in 12 children. PCR could detect P. jirovecii in 14 children. Prevalence of PCP in HIV-infected children was 43% (6 of 14), renal disease on immunosuppressants 45% (4 of 9), primary immune deficiency 19% (2 of 11) and malignancies on chemotherapy 4% (2 of 57). Three of 14 children died from PCP. CONCLUSIONS: PCP is responsible for pneumonia in 14% of children with underlying immunocompromised state; PCR on induced sputum improves diagnosis.

Theoretical investigation on the solid-liquid phase transition of gallium through free energy analysis.

CONTEXT: Gallium, renowned for its notably low melting point and unique property of becoming liquid at room temperature, is a valuable constituent in phase change materials. In this study, we investigate the solid-liquid phase transition of gallium using the modified embedded atom method (MEAM) potential. It addresses the technique to compute the free energy difference between the solid and liquid without using a reference state. We examine various thermodynamic and dynamic properties, including density, specific heat capacity, diffusivity, and radial distribution functions. We compute the coexistence temperature of the solid-liquid phase transitions of gallium from free energy analysis. This information is crucial for understanding the behavior of the material under different pressure conditions and can be valuable for various applications, such as materials processing and high-pressure studies. The analysis, findings, and insights of the present work will be of great significance to the broad scientific and engineering communities in the field of phase transformation of materials. METHODS: A series of molecular dynamics(MD) simulations were conducted using the LAMMPS software packages. The gallium atoms are modeled using the modified embedded atom method (MEAM) potential. To accurately predict the solid-liquid phase transitions of gallium, we calculated free energy by employing the "constrained λ integration" method, coupled with multiple histogram reweighting (MHR). The solid-liquid coexistence line is determined through the Gibbs-Duhem integration technique.

A Dynamic Water Channel Affects O2 Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.

Contrast Enhanced CT Versus MRI for Accurate Diagnosis of Wall-thickening Type Gallbladder Cancer.

Introduction: Diagnosis of wall-thickening type gallbladder cancer (GBC) is challenging. Computed tomography (CT) and magnetic resonance imaging (MRI) are commonly utilized to evaluate gallbladder wall thickening. However, there is a lack of data comparing the performance of CT and MRI for the detection of wall-thickening type GBC. Aim: We aim to compare the diagnostic accuracy of CT and MRI in diagnosis of wall-thickening type GBC. Materials and methods: This prospective study comprised consecutive patients suspected of wall-thickening type GBC who underwent preoperative contrast-enhanced CT and MRI. The final diagnosis was based on the histopathology of the resected gallbladder lesion. Two radiologists independently reviewed the characteristics of gallbladder wall thickening at CT and MRI. The association of CT and MRI findings with histological diagnosis and the interobserver agreement of CT and MRI findings were assessed. Results: Thirty-three patients (malignancy, 13 and benign, 20) were included. None of the CT findings were significantly associated with GBC. However, at MRI, heterogeneous enhancement, indistinct interface with the liver, and diffusion restriction were significantly associated with malignancy (P = 0.006, <0.001, and 0.005, respectively), and intramural cysts were significantly associated with benign lesions (P = 0.012). For all MRI findings, the interobserver agreement was substantial to perfect (kappa = 0.697-1.000). At CT, the interobserver agreement was substantial to perfect (k = 0.631-1.000). Conclusion: These findings suggest that MRI may be preferred over CT in patients with suspected wall thickening type GBC. However, larger multicenter studies must confirm our findings.

Effect of confinement on the solid-liquid coexistence of Lennard-Jones fluid.

The solid-liquid coexistence of a Lennard-Jones fluid confined in slit pores of variable pore size, H, is studied using molecular dynamics simulations. Three-stage pseudo-supercritical transformation path of Grochola [J. Chem. Phys. 120(5), 2122 (2004)] and multiple histogram reweighting are employed for the confined system, for various pore sizes ranging from 20 to 5 molecular diameters, to compute the solid-liquid coexistence. The Gibbs free energy difference is evaluated using thermodynamic integration method by connecting solid-liquid phases under confinement via one or more intermediate states without any first order phase transition among them. Thermodynamic melting temperature is found to oscillate with wall separation, which is in agreement with the behavior seen for kinetic melting temperature evaluated in an earlier study. However, thermodynamic melting temperature for almost all wall separations is higher than the bulk case, which is contrary to the behavior seen for the kinetic melting temperature. The oscillation founds to decay at around H = 12, and beyond that pore size dependency of the shift in melting point is well represented by the Gibbs-Thompson equation.

Increasing the O2 Resistance of the [FeFe]-Hydrogenase CbA5H through Enhanced Protein Flexibility.

The high turnover rates of [FeFe]-hydrogenases under mild conditions and at low overpotentials provide a natural blueprint for the design of hydrogen catalysts. However, the unique active site (H-cluster) degrades upon contact with oxygen. The [FeFe]-hydrogenase fromClostridium beijerinckii (CbA5H) is characterized by the flexibility of its protein structure, which allows a conserved cysteine to coordinate to the active site under oxidative conditions. Thereby, intrinsic cofactor degradation induced by dioxygen is minimized. However, the protection from O2 is only partial, and the activity of the enzyme decreases upon each exposure to O2. By using site-directed mutagenesis in combination with electrochemistry, ATR-FTIR spectroscopy, and molecular dynamics simulations, we show that the kinetics of the conversion between the oxygen-protected inactive state (cysteine-bound) and the oxygen-sensitive active state can be accelerated by replacing a surface residue that is very distant from the active site. This sole exchange of methionine for a glutamate residue leads to an increased resistance of the hydrogenase to dioxygen. With our study, we aim to understand how local modifications of the protein structure can have a crucial impact on protein dynamics and how they can control the reactivity of inorganic active sites through outer sphere effects.

Randomized crossover trial of 'Roll-over' technique of abdominal paracentesis versus standard technique in suspected malignant ascites.

BACKGROUND: The sensitivity of single abdominal paracentesis for diagnosis of peritoneal carcinomatosis (PC) varies from 40-70%. We hypothesized that rolling-over the patient before paracentesis might improve the cytological yield. RESEARCH DESIGN AND METHODS: This was a single center pilot study with a randomized cross-over design. We compared the cytological yield of fluid obtained by roll-over technique (ROG) with standard paracentesis (SPG) in suspected PC. In the ROG group, patients were rolled side-to-side thrice, and the paracentesis was done within 1 minute. Each patient served as their own control, and the outcome assessor (cytopathologist) was blinded. The primary objective was to compare the tumor cell positivity between SPG and ROG groups. RESULTS: Of 71 patients, 62 were analyzed. Of 53 patients with malignancy-related ascites, 39 had PC. Most of the tumor cells were adenocarcinoma (30, 94%) with one patient each having suspicious cytology and one having lymphoma. The sensitivity for diagnosis of PC was (31/39) 79.49% in SPG group and (32/39) 82.05% in ROG group (p = 1.00). The cellularity was similar between both the groups (good cellularity in 58% of SPG and 60% of ROG, p = 1.00). CONCLUSIONS: Rollover paracentesis did not improve the cytological yield of abdominal paracentesis. TRIAL REGISTRATION: CTRI/2020/06/025887 and NCT04232384.

Gastrointestinal involvement in gallbladder cancer: Computed tomography findings and proposal of a classification system.

BACKGROUND: There is relatively scarce data on the computed tomography (CT) detection of gastrointestinal (GI) involvement in gallbladder cancer (GBC). We aim to assess the GI involvement in GBC on CT and propose a CT-based classification. METHODS: This retrospective study comprized consecutive patients with GBC who underwent contrast-enhanced computed tomography (CECT) for staging between January 2019 and April 2022. Two radiologists evaluated the CT images independently for the morphological type of GBC and the presence of GI involvement. GI involvement was classified into probable involvement, definite involvement and GI fistulization. The incidence of GI involvement and the association of GI involvement with the morphological type of GBC was evaluated. In addition, the inter-observer agreement for GI involvement was assessed. RESULTS: Over the study period, 260 patients with GBC were evaluated. Forty-three (16.5%) patients had GI involvement. Probable GI involvement, definite GI involvement and GI fistulization were seen in 18 (41.9%), 19 (44.2%) and six (13.9%) patients, respectively. Duodenum was the most common site of involvement (55.8%), followed by hepatic flexure (23.3%), antropyloric region (9.3%) and transverse colon (2.3%). There was no association between GI involvement and morphological type of GBC. There was substantial to near-perfect agreement between the two radiologists for the overall GI involvement (k = 0.790), definite GI involvement (k = 0.815) and GI fistulization (k = 0.943). There was moderate agreement (k = 0.567) for probable GI involvement. CONCLUSION: GBC frequently involves the GI tract and CT can be used to categorize the GI involvement. However, the proposed CT classification needs validation.

A novel computational predictive biological approach distinguishes Integrin ß1 as a salient biomarker for breast cancer chemoresistance.

Chemoresistance is a primary cause of breast cancer treatment failure, and protein-protein interactions significantly contribute to chemoresistance during different stages of breast cancer progression. In pursuit of novel biomarkers and relevant protein-protein interactions occurring during the emergence of breast cancer chemoresistance, we used a computational predictive biological (CPB) approach. CPB identified associations of adhesion molecules with proteins connected with different breast cancer proteins associated with chemoresistance. This approach identified an association of Integrin ß1 (ITGB1) with chemoresistance and breast cancer stem cell markers. ITGB1 activated the Focal Adhesion Kinase (FAK) pathway promoting invasion, migration, and chemoresistance in breast cancer by upregulating Erk phosphorylation. FAK also activated Wnt/Sox2 signaling, which enhanced self-renewal in breast cancer. Activation of the FAK pathway by ITGB1 represents a novel mechanism linked to breast cancer chemoresistance, which may lead to novel therapies capable of blocking breast cancer progression by intervening in ITGB1-regulated signaling pathways.

Sonographic "Cervix Sign": A New Ancillary Sign of Gallbladder Neck Malignancy.

Background: The differentiation of benign and malignant gallbladder wall thickening is challenging. The purpose of this study is to evaluate a new sonographic sign, "cervix sign" for differentiation of benign and malignant gallbladder neck thickening. Methods: This retrospective study comprised consecutive patients with gallbladder neck thickening who underwent sonography between August 2019 and December 2021. The presence of "cervix sign" was assessed by two radiologists independently. Results: Sixty-five patients had gallbladder neck thickening (28 malignant and 37 benign). The sonographic "cervix sign" was present in 18 (64%) patients with malignant thickening and in only one (2.7%) patient with benign thickening (P = 0.0001). The mean wall thickness was greater, and symmetric wall thickening and liver metastases were more common in malignant thickening with "cervix sign" (without reaching statistical significance). There was substantial agreement (kappa = 0.78) between the two observers for the cervix sign. Conclusion: Sonographic "cervix sign" is a useful ancillary feature of gallbladder neck cancer.