Structure of MltG from Mycobacterium abscessus reveals structural plasticity between composed domains.

MltG, a membrane-bound lytic transglycosylase, has roles in terminating glycan polymerization in peptidoglycan and incorporating glycan chains into the cell wall, making it significant in bacterial cell-wall biosynthesis and remodeling. This study provides the first reported MltG structure from Mycobacterium abscessus (maMltG), a superbug that has high antibiotic resistance. Our structural and biochemical analyses revealed that MltG has a flexible peptidoglycan-binding domain and exists as a monomer in solution. Further, the putative active site of maMltG was disclosed using structural analysis and sequence comparison. Overall, this study contributes to our understanding of the transglycosylation reaction of the MltG family, aiding the design of next-generation antibiotics targeting M. abscessus.

Structural analysis of a bacterial ankyrin-like protein secreted by Acinetobacter baumannii.

In bacteria, the ankyrin-like protein AnkB helps overcome stress by regulating catalase activity when expressed under stressful conditions. As the structural properties of AnkB are largely unexplored, our understanding of various AnkB-mediated functions in bacteria remains limited. In the present study, we describe the structure of AnkB from Acinetobacter baumannii, hereafter referred to as "AbAnkB," which has a unique tertiary configuration compared with that of other ankyrin domain-containing proteins. Structural analysis revealed that AbAnkB has a relatively long loop between AKR3 and AKR4 and an oppositely positioned α8 helix. Based on amino acid conservation and protein surface analyses, we identified a hydrophobic patch that might be critical for the function of AbAnkB. To the best of our knowledge, our study is the first to report the structure of a bacterial AnkB protein; our findings will markedly enhance our understanding of its functions in bacteria.

Short Caco-2 model for evaluation of drug permeability: A sodium valerate-assisted approach.

The human colorectal adenocarcinoma cell line Caco-2, widely used for studying intestinal drug permeability, is typically grown on permeable filter supports and matures in 21 days with frequent media changes. The process is labor-intensive, prone to contamination, and has low throughput, contributing to the overall high utilization cost. Efforts to establish a low-cost, high-throughput, and short-duration model have encountered obstacles, such as weaker tight junctions causing monolayer leaks, incomplete differentiation resulting in low transporter expression, intricate and challenging protocols, and cytotoxicity, limiting the usability. Hence, this study aimed to develop a low-cost, efficient, and short-duration model by addressing the aforementioned concerns by customizing the media and finding a safe differentiation inducer. We generated a new rapid model using sodium valerate, which demonstrated sufficient transporter activity, improved monolayer integrity, and higher levels of differentiation markers than the 21-day model. Furthermore, this model exhibited consistent and reliable results when used to evaluate drug permeability over multiple days of repeated use. This study demonstrates the potential of a sodium valerate-assisted abbreviated model for drug permeability assessment with economic and practical advantages.

Emerging Immune Checkpoint Molecules on Cancer Cells: CD24 and CD200.

Cancer immunotherapy strategies are based on the utilization of immune checkpoint inhibitors to instigate an antitumor immune response. The efficacy of immune checkpoint blockade, directed at adaptive immune checkpoints, has been demonstrated in select cancer types. However, only a limited subset of patients has exhibited definitive outcomes characterized by a sustained response after discontinuation of therapy. Recent investigations have highlighted the significance of immune checkpoint molecules that are overexpressed in cancer cells and inhibit myeloid lineage immune cells within a tumor microenvironment. These checkpoints are identified as potential targets for anticancer immune responses. Notably, the immune checkpoint molecules CD24 and CD200 have garnered attention owing to their involvement in tumor immune evasion. CD24 and CD200 are overexpressed across diverse cancer types and serve as signaling checkpoints by engaging their respective receptors, Siglec-10 and CD200 receptor, which are expressed on tumor-associated myeloid cells. In this review, we summarized and discussed the latest advancements and insights into CD24 and CD200 as emergent immune checkpoint moieties, further delving into their therapeutic potentials for cancer treatment.

Ocular syphilis on the rise: a 10-year analysis from 2010 to 2020.

PURPOSE: The purpose of the study was to evaluate the incidence of ocular syphilis as well as diagnostic parameters, comorbidities, and visual outcomes over a 10-year time period in West Virginia. METHODS: A retrospective chart review included 25 eyes of 17 patients with ocular syphilis between 2010 and 2020. RESULTS: The incidence of systemic syphilis at a large tertiary referral center has increased from 27 cases in 2010 to 105 cases in 2020. Seventeen patients were identified with ocular syphilis. Bilaterality was present in 47.1% of cases. In this study, 70.6% of patients were male and 29.4% were female. The median age of presentation was 40.2 years (range 21-63). Panuveitis was the most common (60.0%) followed by isolated anterior uveitis (16.0%), chorioretinitis (12.0%), inner retinitis (4.0%), and papillitis (8.0%). Forty percent of patients had visual acuity worse than 20/400 on presentation. Post-treatment visual acuity improved in all patients. Rapid plasma reagin (RPR) and Treponema pallidum particle agglutination (TP-PA) tests were positive in 84.6% and 100% of cases, respectively. CSF venereal disease research laboratory (VDRL) was positive in 36.4%, CSF pleocytosis was present in 72.7%, and elevated CSF protein was observed in 81.8%. Human immunodeficiency virus (HIV) co-infection was present in 31.3%. A majority of patients experienced maculopapular rash and/or history of genital chancre. The anatomic classification of presenting uveitis (anterior, intermediate, posterior, and panuveitis) did not correlate with clinical variables including age, gender, HIV status, serologic test, presence of rash, or year of diagnosis (p > 0.05). CONCLUSION: Ocular syphilis is becoming increasingly prevalent and can present with a variety of ocular findings; therefore, it should be considered in the differential diagnosis for patients with ocular inflammation. Visual prognosis is excellent with timely diagnosis and treatment.

Retinitis Pigmentosa Masquerades: Case Series and Review of the Literature.

Retinitis pigmentosa (RP) displays a broad range of phenotypic variations, often overlapping with acquired retinal diseases. Timely recognition and differentiation of RP masquerades is paramount due to the treatable nature of many such conditions. This review seeks to present examples of pseudo-RP cases and provide a comprehensive overview of RP masquerades. We first present two pseudo-RP cases, including comprehensive clinical histories and multimodal retinal imaging, to highlight the important role of accurate diagnoses that subsequently steered effective intervention. Subsequently, we conduct an in-depth review of RP masquerades to provide valuable insights into their key distinguishing features and management considerations. The recent approval of ocular gene therapy and the development of investigational gene-based treatments have brought genetic testing to the forefront for RP patients. However, it is important to note that genetic testing currently lacks utility as a screening tool for inherited retinal diseases (IRDs), including RP. The integrity of a precise clinical assessment remains indispensable for the diagnosis of both RP and RP masquerade conditions, thereby facilitating prompt intervention and appropriate management strategies.

Comparison of the structure and activity of thioredoxin 2 and thioredoxin 1 from Acinetobacter baumannii.

Thioredoxin (Trx) is essential in a redox-control system, with many bacteria containing two Trxs: Trx1 and Trx2. Due to a Trx system's critical function, Trxs are targets for novel antibiotics. Here, a 1.20 Šhigh-resolution structure of Trx2 from Acinetobacter baumannii (abTrx2), an antibiotic resistant pathogenic superbug, is elucidated. By comparing Trx1 and Trx2, it is revealed that the two Trxs possess similar activity, although Trx2 contains an additional N-terminal zinc-finger domain and exhibits more flexible properties in solution. Finally, it is shown that the Trx2 zinc-finger domain might be rotatable and that proper zinc coordination at the zinc-finger domain is critical to abTrx2 activity. This study enhances understanding of the Trx system and will facilitate the design of novel antibiotics.

An Improved In Vitro Blood-Brain Barrier Model for the Evaluation of Drug Permeability Using Transwell with Shear Stress.

The development of drugs targeting the central nervous system (CNS) is challenging because of the presence of the Blood-Brain barrier (BBB). Developing physiologically relevant in vitro BBB models for evaluating drug permeability and predicting the activity of drug candidates is crucial. The transwell model is one of the most widely used in vitro BBB models. However, this model has limitations in mimicking in vivo conditions, particularly in the absence of shear stress. This study aimed to overcome the limitations of the transwell model using immortalized human endothelial cells (hCMEC/D3) by developing a novel dish design for an orbital shaker, providing shear stress. During optimization, we assessed cell layer integrity using trans-endothelial electrical resistance measurements and the % diffusion of lucifer yellow. The efflux transporter activity and mRNA expression of junctional proteins (claudin-5, occludin, and VE-cadherin) in the newly optimized model were verified. Additionally, the permeability of 14 compounds was evaluated and compared with published in vivo data. The cell-layer integrity was substantially increased using the newly designed annular shaking-dish model. The results demonstrate that our model provided robust conditions for evaluating the permeability of CNS drug candidates, potentially improving the reliability of in vitro BBB models in drug development.

Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4',6-dimethoxyisoflavone-7-O-ß-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1ß and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs.

Structural basis for the substrate specificity of an S-formylglutathione hydrolase derived from Variovorax sp. PAMC 28711.

S-Formylglutathione hydrolase was originally known to catalyze the hydrolysis of S-formylglutathione to formate and glutathione. However, this enzyme has a broader esterase activity toward substrates containing thioester and ester bonds. In a previous study, we identified a new S-formylglutathione hydrolase (VaSFGH) gene in the Antarctic bacterium Variovorax sp. PAMC 28711, and recombinant VaSFGH protein was purified and characterized. Previous enzyme activity assays showed that VaSFGH has high activity, especially toward short-chain p-nitrophenyl esters (C2-C4). In this study, we determined the crystal structure of substrate-free VaSFGH at a resolution of 2.38 Å. In addition, p-nitrophenyl ester-bound VaSFGH structure models were generated by molecular docking simulations to obtain structural evidence of its substrate specificity. Comparative structural analysis of the apo-form and p-nitrophenyl ester-bound VaSFGH model structures revealed that large substrates could not bind inside the hydrophobic substrate-binding pocket because of the intrinsically static and relatively small substrate-binding pocket size of VaSFGH. This study provides useful information for further protein engineering of SFGHs for industrial use.

The microbiological spectrum, antimicrobial resistance pattern, and visual outcomes of endogenous endophthalmitis in West Virginia 2009-2019.

PURPOSE: To report the microbiological spectrum, antimicrobial resistance patterns, and visual outcomes in patients with endogenous endophthalmitis (EE). METHODS: This was a retrospective study of 50 patients with culture-positive EE managed in a tertiary referral center between October 2009 and 2019. Clinical, microbiology analysis, and antimicrobial resistance were reviewed. A multivariable linear regression analysis was used for identifying risk factors associated with worse visual outcomes. RESULTS: Fifty organisms were identified, 62% bacterial and 38% fungal. The most common bacterial organism was Staphylococcus aureus (75% methicillin resistant), and Candida was the most common fungal species. Multidrug resistance was observed in methicillin-resistant Staphylococcus aureus (MRSA) isolates against clindamycin, daptomycin, and fluoroquinolones. The distributions of the final visual acuity (VA) between the bacterial and fungal groups were significantly different, and the visual outcomes in the bacterial group tended to be worse (p = 0.01). The distributions of enucleation status were significantly higher in bacterial EE (35%) than fungal EE (5.3%) (p = 0.02). Results from the multivariable linear regression analysis revealed that older age was significantly associated with worse visual outcome (coef = 0.03; p = 0.02), while fungal infections were associated with better outcomes (coef = - 0.87; p = 0.01). Intravenous drug use (coef = 0.87; p = 0.054) was a marginally significant factor associated with worse visual outcomes. CONCLUSION: There was a higher prevalence of bacterial organisms than fungal species among EE. Bacterial EE was associated with worse visual outcomes and higher enucleation rates than fungal EE. Multidrug resistance was prevalent among MRSA isolates. Older age and intravenous drug use may be factors associated with poor prognosis.

Phagocyte Chemoattraction Is Induced through the Mcp-1-Ccr2 Axis during Efferocytosis.

Apoptotic cells generated during development and for tissue homeostasis are swiftly and continuously removed by phagocytes via a process called efferocytosis. Efficient efferocytosis can be achieved via transcriptional modulation in phagocytes that have engulfed apoptotic cells. However, such modulation and its effect on efferocytosis are not completely understood. Here, we report that phagocytes are recruited to apoptotic cells being cleared through the Mcp-1-Ccr2 axis, which facilitates clearance of apoptotic cells. We identified Mcp-1 as a modulated transcript using a microarray and found that Mcp-1 secretion was augmented in phagocytes engulfing apoptotic cells. This augmented Mcp-1 secretion was impaired by blocking phagolysosomal degradation of apoptotic cells. Conditioned medium from wild type (WT) phagocytes promoted cell migration, but that from Mcp-1-/- phagocytes did not. In addition, blockade of Ccr2, the receptor for Mcp-1, abrogated cell migration to conditioned medium from phagocytes incubated with apoptotic cells. The intrinsic efferocytosis activity of Mcp-1-/- and Ccr2-/- phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Mcp-1-/- and Ccr2-/- mice than in that of WT mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis.

Physiological Roles of Apoptotic Cell Clearance: Beyond Immune Functions.

The clearance of apoptotic cells is known to be a critical step in maintaining tissue and organism homeostasis. This process is rapidly/promptly mediated by recruited or resident phagocytes. Phagocytes that engulf apoptotic cells have been closely linked to the release of anti-inflammatory cytokines to eliminate inflammatory responses. Defective clearance of apoptotic cells can cause severe inflammation and autoimmune responses due to secondary necrosis of apoptotic cells. Recently accumulated evidence indicates that apoptotic cells and their clearance have important physiological roles in addition to immune-related functions. Herein, we review the current understanding of the mechanisms and fundamental roles of apoptotic cell clearance and the beneficial roles of apoptotic cells in physiological processes such as differentiation and development.

BAI1 nuclear expression reflects the survival of nonsmoking non-small cell lung cancer patients.

BACKGROUND: Smoking- and nonsmoking-associated lung cancers have different mechanisms of carcinogenesis. We divided non-small cell lung cancer (NSCLC) patients into nonsmoking and smoking groups with the aim of trying to understand the utility of brain-specific angiogenesis inhibitor 1 (BAI1) expression in the separate groups. METHODS: Clinicopathological data were obtained from 148 patients who had undergone surgery for NSCLC of the lung. Tissue microarray blocks were made of samples from NSCLC patients. Two pathologists graded the intensity of BAI1 expression as high or low expression in the cancer cells of patients in the smoking and nonsmoking groups. RESULTS: NSCLC nonsmokers with higher BAI1 nuclear expression had poor disease-specific survival (DSS) (hazard ratio: 2.679; 95% confidence interval [CI]:1.022-7.022, p = 0.045). The Kaplan-Meier survival curve confirmed that higher BAI1 expression was significantly associated with poor DSS (p = 0.034) in the nonsmoking group. CONCLUSIONS: We divided NSCLC patients into nonsmoking and smoking groups and found that nuclear BAI1 expression was related to patient survival in nonsmoking NSCLC patients. We suggest BAI1 expression as a predictive marker of nonsmoking-associated NSCLC and recommend that it be evaluated as an AJCC staging criterion in the future.

Mortality risk associated with endophthalmitis in West Virginia.

PURPOSE: To explore how endophthalmitis presented from 2009 to 2019 in a West Virginia population particularly affected by the national opioid crisis. The analysis explores the relationship between the type of endophthalmitis and mortality, accounting for factors including age, gender, type of organism, and intravenous drug use (IVDU). METHODS: The electronic health record of West Virginia University (WVU) Medicine was queried for all patients managed for endophthalmitis from October 2009 to October 2019. For each of the included subjects, age, gender, history of IVDU, culture results, concomitant endocarditis, type of endophthalmitis, and the date of diagnosis were extracted. Mortality data were obtained from WVU's electronic medical record, the Social Security Death Index, and public obituaries. Mortality results were represented by a Kaplan-Meier Survival curve following each patient for one year from the date of diagnosis. Results were analyzed using unadjusted and adjusted Cox Proportional Hazard models. RESULTS: One-year mortality was 14 out of 113 endogenous cases (12.4%) compared to 6 out of 173 exogenous cases (3.5%). Endogenous endophthalmitis cases had significantly higher mortality than exogenous ones within one year of diagnosis (p = 0.0034). The unadjusted Cox proportional hazards model revealed that the type of endophthalmitis (endogenous vs. exogenous) was the only variable with a significant impact on 1-year mortality with a hazard ratio of 3.78 (p = 0.01). However, the hazard ratio for endogenous infections rose to 10.91 (CI 3.544-33.595) when the other variables of age, gender, organism, and IVDU were controlled (p < 0.01). The Cox proportional hazard ratios for age group, gender, organism type, and history of IVDU were not significantly different when adjusted for all other variables. CONCLUSION: Endogenous cases, which were significantly overrepresented in West Virginia, were associated with a significantly higher 1-year mortality rate than the exogenous ones. Age, gender, organism type, and history of IVDU have less, if any, modifying effect on mortality.

Comparison of the visual outcome between macula-on and macula-off rhegmatogenous retinal detachment based on the duration of macular detachment.

OBJECTIVE: To compare the visual outcomes between macula-on and macula-off primary rhegmatogenous retinal detachment (RRD) based on the duration of macular detachment (DMD). METHODS AND ANALYSIS: Retrospective study including 96 eyes with RRD (34 macula-on and 62 macula-off) repaired between June 2012 and March 2020. The final visual acuity (VA) was compared after the patients were divided by the status of the macula and their DMD. RESULTS: The mean final VA of patients with macula-on RRD (group A) was logarithm of the minimum angle of resolution (logMAR) 0.04±0.07, which was not statistically different from that of individuals with macula-off RRD with DMD ≤3 days (group B; logMAR 0.05±0.06) (p=0.79). There were statistically significant differences in the final VA between group A and patients with macula-off RRD with DMD of 4-7 days (group C; logMAR 0.15±0.15) (p=0.017) as well as between group A and those with macula-off RRD with DMD ≥8 days (group D; logMAR 0.36±0.29) (p<0.001). There was no significant difference in the final VA between group B and C (p=0.33). CONCLUSION: The mean final VA of patients with macula-on RRD was comparable to that of the macula-off patients with DMD ≤3 days. Our findings suggest that if macula-on RRD could not be immediately repaired, a repair within 72 hours may result in similar outcomes, even if the macula detaches within that time frame. However, once the macula detaches, we do not observe statistically significant differences in outcome for repairs done within 7 days.

Pars Plana Vitrectomy for Endophthalmitis: Microbiologic Spectrum and Clinical Outcomes.

Purpose: To report visual outcomes, microbiologic spectrum, and antibiotic resistance in endophthalmitis patients undergoing pars plana vitrectomy (PPV).Methods: Retrospective study of 32 patients who underwent PPV with microbial analysis. Linear mixed models were utilized to compare visual acuity (VA).Results: Streptococcal species and coagulase-negative staphylococcus (CoNS) were the most common organisms. No resistance to vancomycin or fluoroquinolones was observed. Culture-negative individuals had better VA and lower incidences of retinal detachment (RD) and hypotony and better VA than culture-positive group at post-surgical month 3 (p = .025) and marginally at month 12 (p = .098). CoNS endophthalmitis (final logMAR VA 0.80) was associated with better VA than Streptococcal endophthalmitis (final logMAR VA 2.36) (p = .001). Secondary RD was observed in 33.3% of non-cataract endophthalmitis.Conclusion: No organisms were resistant to vancomycin or fluoroquinolones. Culture-negative endophthalmitis had better VA and lower rates of RD and hypotony than culture-positive group. A high rate of RD was observed in non-cataract endophthalmitis.

Wide-open conformation of UDP-MurNc-tripeptide ligase revealed by the substrate-free structure of MurE from Acinetobacter baumannii.

MurE ligase catalyzes the attachment of meso-diaminopimelic acid to the UDP-MurNAc-l -Ala-d -Glu using ATP and producing UDP-MurNAc-l -Ala-d -Glu-meso-A2 pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate-free structure of MurE from Acinetobacter baumannii, which is an antibiotic-resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide-open conformation and undergoes wide-open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next-generation antibiotics targeting MurE.

Phytochemicals as Anti-Inflammatory Agents in Animal Models of Prevalent Inflammatory Diseases.

Phytochemicals are known to have anti-inflammatory effects in vitro and in vivo, such as in inflammatory disease model systems. Inflammation is an essential immune response to exogenous stimuli such as infection and injury. Although inflammation is a necessary host-defense mechanism, chronic inflammation is associated with the continuous local or systemic release of inflammatory mediators, non-cytokine mediators, such as ROS and NO, and inflammatory cytokines are strongly implicated in the pathogenesis of various inflammatory disorders. Phytochemicals that exhibit anti-inflammatory mechanisms that reduce sustained inflammation could be therapeutic candidates for various inflammatory diseases. These phytochemicals act by modulating several main inflammatory signaling pathways, including NF-κB, MAPKs, STAT, and Nrf-2 signaling. Here, we discuss the characteristics of phytochemicals that possess anti-inflammatory activities in various chronic inflammatory diseases and review the molecular signaling pathways altered by these anti-inflammatory phytochemicals, with a focus on transcription factor pathways. Furthermore, to evaluate the phytochemicals as drug candidates, we translate the effective doses of phytochemicals in mice or rat disease models into the human-relevant equivalent and compare the human-relevant equivalent doses of several phytochemicals with current anti-inflammatory drugs doses used in different types of chronic inflammatory diseases.

The crystal structure of mouse IRG1 suggests that cis-aconitate decarboxylase has an open and closed conformation.

PROTEIN DATA BANK ACCESSION CODES: Coordinate and structural factors were deposited with the Protein Data Bank under PDB ID: 7BR9.