A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator of Acinetobacter resistance and virulence.

The multidrug-resistant, nosocomial pathogen Acinetobacter baumannii is a major threat to human health. A sensor kinase-response regulator system, BfmS-BfmR, is a potential antimicrobial target in the bacterium due to its essential role in promoting drug resistance and virulence. Important questions remain, however, about how the system controls resistance and pathogenesis. Although knockout of BfmR is known to alter expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls BfmR is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and in vitro reactions using a small phosphodonor to study how phosphorylation affects BfmR function. We show that phosphorylation requires the BfmR active site Asp58 and is essential to gene regulation, antibiotic resistance, and virulence in a sepsis model. Consistent with activation of the protein, phosphorylation induces dimerization and increases its affinity for target DNA. Integrated analysis of the genome-wide binding and transcriptional profiles of BfmR led to several key findings: (1) Phosphorylation dramatically expands the number of genomic sites bound by BfmR, from 4 to >250; (2) BfmR recognizes a direct repeat motif widespread across promoters; (3) BfmR directly regulates >300 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (eg, type IV pilus machinery); (4) The regulator also directly controls a set of non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving A. baumannii resistance and pathogenicity and unravel the extensive gene regulatory network under its control.

The presence of broadly neutralizing anti-SARS-CoV-2 RBD antibodies elicited by primary series and booster dose of COVID-19 vaccine.

Antibody-mediated immunity plays a key role in protection against SARS-CoV-2. We characterized B-cell-derived anti-SARS-CoV-2 RBD antibody repertoires from vaccinated and infected individuals and elucidate the mechanism of action of broadly neutralizing antibodies and dissect antibodies at the epitope level. The breadth and clonality of anti-RBD B cell response varies among individuals. The majority of neutralizing antibody clones lose or exhibit reduced activities against Beta, Delta, and Omicron variants. Nevertheless, a portion of anti-RBD antibody clones that develops after a primary series or booster dose of COVID-19 vaccination exhibit broad neutralization against emerging Omicron BA.2, BA.4, BA.5, BQ.1.1, XBB.1.5 and XBB.1.16 variants. These broadly neutralizing antibodies share genetic features including a conserved usage of the IGHV3-53 and 3-9 genes and recognize three clustered epitopes of the RBD, including epitopes that partially overlap the classically defined set identified early in the pandemic. The Fab-RBD crystal and Fab-Spike complex structures corroborate the epitope grouping of antibodies and reveal the detailed binding mode of broadly neutralizing antibodies. Structure-guided mutagenesis improves binding and neutralization potency of antibody with Omicron variants via a single amino-substitution. Together, these results provide an immunological basis for partial protection against severe COVID-19 by the ancestral strain-based vaccine and indicate guidance for next generation monoclonal antibody development and vaccine design.

Exploring Knowledge, Awareness, and Practices Regarding Periodontal Health Assessment and Mechanical Plaque Control Among the Shillong Population of Meghalaya, India: A Descriptive Cross-Sectional Investigation.

Background Periodontal diseases are widespread oral health conditions. However, there remains a lack of comprehensive understanding regarding the knowledge, awareness, and practices related to periodontal health assessment and mechanical plaque control among specific populations, such as those residing in Shillong, Meghalaya. Shillong, being the capital city of Meghalaya in northeastern India, represents a diverse demographic and cultural landscape. Aim This study aims to evaluate the knowledge, awareness, and practices related to mechanical plaque control among the population of Shillong City. Methodology A descriptive cross-sectional online survey was conducted among the residents of Shillong City, Meghalaya. Data collection involved the administration of an 18-item, closed-ended, self-structured questionnaire. Before the main data collection, a pilot study was conducted involving 63 individuals. Data analysis was performed using IBM SPSS Statistics for Windows, Version 26.0 (Released 2019; IBM Corp., Armonk, NY, USA), employing the chi-square test and ANOVA with a significance level of 0.05. Results Study participants were categorized into five age groups spanning from 21 to 64 years old, with the age group of 41 to 50 years demonstrating the highest mean knowledge score. Age exhibited a statistically significant influence on knowledge scores. Conclusion The study reveals a commendable level of knowledge, awareness, and adherence to practices regarding the primary tool for oral hygiene maintenance, the toothbrush, among the residents of Shillong City.

Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

Clickable Granular Hydrogel Scaffolds for Delivery of Neural Progenitor Cells to Sites of Spinal Cord Injury.

Spinal cord injury (SCI) is a serious condition with limited treatment options. Neural progenitor cell (NPC) transplantation is a promising treatment option, and the identification of novel biomaterial scaffolds that support NPC engraftment and therapeutic activity is a top research priority. The objective of this study is to evaluate in situ assembled poly (ethylene glycol) (PEG)-based granular hydrogels for NPC delivery in a murine model of SCI. Microgel precursors are synthesized by using thiol-norbornene click chemistry to react four-armed PEG-amide-norbornene with enzymatically degradable and cell adhesive peptides. Unreacted norbornene groups are utilized for in situ assembly into scaffolds using a PEG-di-tetrazine linker. The granular hydrogel scaffolds exhibit good biocompatibility and do not adversely affect the inflammatory response after SCI. Moreover, when used to deliver NPCs, the granular hydrogel scaffolds supported NPC engraftment, do not adversely affect the immune response to the NPC grafts, and successfully support graft differentiation toward neuronal or astrocytic lineages as well as axonal extension into the host tissue. Collectively, these data establish PEG-based granular hydrogel scaffolds as a suitable biomaterial platform for NPC delivery and justify further testing, particularly in the context of more severe SCI.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred ram in semi-arid tropics.

A prolific three-breed (Malpura, Patanwadi, and Garole) cross Avishaan sheep has been developed in the semi-arid zone to improve farmer's income. Nutritional scarcity is a major limitation in animal husbandry during the dearth period of semi-arid tropics. Therefore, before the inaugural launch of the breed into the field, a study was designed to evaluate the effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred rams in semi-arid tropics. Thus, 16 native adapted (Malpura) and 16 native-crossbred rams were equally distributed into four groups, namely, native control (G1), native nutritional stress (G2), native-crossbred control (G3), and native-crossbred nutritional stress (G4). Both the control groups (G1 and G3) were kept on their maintenance requirement as per their body weight, whereas the nutritional stress groups (G2 and G4) were provided 30% less than their maintenance requirement. The body weight of G4 decline (P<0.05) as compared to their initial weight. The plasma glucose level of G2 and G4 reduced (P<0.05) in comparison with G1 and G3, respectively. The total motile sperm percentage, rapid motile sperm percentage, and sperm viability decrease significantly (P<0.05) within the acceptable limit in native-crossbred rams (G4) under nutritional scarcity. However, the similar blood biochemical along with acceptable seminal attributes of all the rams reflected that native-crossbred rams can cope with the nutritional scarcity in semi-arid tropics and have the potential to contribute to the sustainable small ruminant production system for livelihood security in this region.

A Review of the Functional Annotations of Important Genes in the AHPND-Causing pVA1 Plasmid.

Acute hepatopancreatic necrosis disease (AHPND) is a lethal shrimp disease. The pathogenic agent of this disease is a special Vibrio parahaemolyticus strain that contains a pVA1 plasmid. The protein products of two toxin genes in pVA1, pirAvp and pirBvp, targeted the shrimp's hepatopancreatic cells and were identified as the major virulence factors. However, in addition to pirAvp and pirBvp, pVA1 also contains about ~90 other open-reading frames (ORFs), which may encode functional proteins. NCBI BLASTp annotations of the functional roles of 40 pVA1 genes reveal transposases, conjugation factors, and antirestriction proteins that are involved in horizontal gene transfer, plasmid transmission, and maintenance, as well as components of type II and III secretion systems that may facilitate the toxic effects of pVA1-containing Vibrio spp. There is also evidence of a post-segregational killing (PSK) system that would ensure that only pVA1 plasmid-containing bacteria could survive after segregation. Here, in this review, we assess the functional importance of these pVA1 genes and consider those which might be worthy of further study.

Effect of drinking earthen pot water on physiological response and behavior of sheep under heat stress.

Heat stress is a major limiting factor for animal welfare and sheep production. Traditionally in India, the villagers used to keep their drinking water in the earthen pot to make it cold during summer. The cold drinking water (24-28 °C) during summer gives a feeling of relief from the heat. Therefore, the present study was carried out to assess the effect of drinking earthen pot water on physiological response and behavior of sheep under heat stress for one month. For this purpose, eighteen Avishaan rams were selected from the experimental animal flock and they were equally divided into three groups; viz., control (CON), heat stress (HS) and heat stress with earthen pot water (HSC). The animals of HS and HSC were exposed to higher ambient temperatures to induce heat stress inside the psychometric chamber. The animals of CON and HS were provided with ad-libitum water of their ambient temperature whereas; HSC groups were provided with ad-libitum cold water (24-28 °C) earthen pot water. All the animals were offered with 400 gm concentrate mixture and ad-libitum Cenchrus hay. The bodyweight of HS rams was significantly reduced (P < 0.05) at the end of the experimental period as compared to their initial body weight. The total roughage and dry matter intake was significantly higher (P < 0.01) in HSC rams as compared to HS rams. The plasma thyroxine concentration was significantly lower (P < 0.05) in HSC as compared with HS group. The rumination time significantly reduced (P < 0.05) in HSC group. However, The blood biochemical did not differ among the groups. Therefore, it may be concluded that Avishaan rams have the ability to adapt to heat stress. Nevertheless, the availability of earthen pot cold drinking water under heat stress reduced their body weight loss, improves their metabolic activity and ultimately improves their welfare.