The objective of this study was to obtain Mycobacterium tuberculosis methionyl-tRNA synthetase (MetRS) mutant forms E24A and L293A and investigate the efficiency of M. tuberculosis MetRS inhibitors toward these mutant forms in order to predict binding mechanisms. The recombinant mutant M. tuberculosis MetRS E24A and L293A were obtained using overlap PCR. The recombinant proteins MetRS E24A and L293A were expressed in BL21(DE3) cells. The activity of obtained aminoacyl-tRNA synthetases was studied with aminoacylation assay using BIOMOL® Green Reagent. It was found that the mutations of Glu24 and Leu293 with alanine in the synthetic site of M. tuberculosis MetRS lead to significant decrease of inhibitory activity of investigated compounds. These findings suggest that the amino acid residues Glu24 and Leu293 may contribute to the inhibitory activity of the studied compounds and should therefore be considered during further structural optimization.

Cryptococcus neoformans infection presenting as a rapidly reaccumulating unilateral pleural effusion in a non-immunocompromised host is a rare presentation of pulmonary cryptococcal disease. We present a rare case of recurrent unilateral pleural effusion as the initial presentation of cryptococcal disease in an immunocompetent patient. An 85-year-old male with a past medical history of hypertension, hyperlipidemia, diabetes mellitus, chronic kidney disease stage IIIb, and coronary artery disease s/p stent placement in the second obtuse marginal branch of the circumflex artery presented to the emergency department with dyspnea on exertion and an unintentional 30-pound weight loss over two to three months. His prior occupation included work in a tire factory; he denied exposure to asbestos. He chewed tobacco and had a dog. A chest radiograph showed a large right-sided pleural effusion. He had undergone repeated thoracentesis and pigtail chest tube placements over four hospital admissions. Review of the initial cytology, extensive serology, pleural fluid analysis, Gram stain, culture, HIV testing, and adenosine deaminase testing was negative. Given the recurrent hospitalizations and reaccumulation of the right pleural effusion, the decision was made to proceed with endobronchial ultrasound, which demonstrated lymphocytosis without evidence of malignancy. Bronchoalveolar lavage grew 100 colonies of Cryptococcus neoformans. The patient underwent lumbar puncture with an opening pressure of 37 and a negative cryptococcal antigen. He was started on fluconazole 400 mg daily upon discharge, with a suspected treatment course of 12 months and close outpatient pulmonology follow-up. This report highlights a rare initial presentation of Cryptococcus neoformans manifesting as recurrent unilateral pleural effusion.

Leaky gut, defined as increased intestinal permeability, has been associated with many diseases. Recent studies suggest the brain is involved in maintaining gut barrier function. Since tofogliflozin, a sodium/glucose cotransporter 2 (SGLT2) inhibitor acts centrally to increase food intake, and feeding and gut functions are deeply associated, this study aimed to clarify the hypothesis that gut barrier function is influenced by centrally administered SGLT2 inhibitors in rats using Evans blue method. Intracisternal SGLT2 inhibitor blocked colonic hyperpermeability in response to lipopolysaccharide dose-dependently (15 μg-). The blockade was cancelled by atropine, vagotomy, intracisternal orexin receptor antagonist, intracisternal compound C, an adenosine monophosphate-activated protein kinase (AMPK) inhibitor, or subcutaneously injected sulpiride, a dopamine D2 receptor antagonist. Subcutaneous SGLT2 inhibitor at much higher doses of 5mg or more could improve colonic hyperpermeability, and the improvement was also blocked by atropine or intracisternal orexin receptor antagonist or compound C. These results indicate SGLT2 inhibitor administered centrally or peripherally reduces colonic hyperpermeability through the vagal pathway although we could not exclude the possibility that peripherally administered SGLT2 inhibitor might act first in periphery. Brain orexin, AMPK and dopamine D2 signaling are involved in this process. SGLT2 inhibitors may be useful to treat leaky gut-related diseases.

In addition to cell cycle arrest, cellular senescence is characterized by disrupted mitochondrial homeostasis, development of the senescence-associated secretory phenotype (SASP), and alterations in the immune microenvironment. Mitochondrial membrane damage, disrupted mitochondrial dynamics, and impaired mitochondrial quality control (MQC) can occur during cellular senescence. These changes may cause mitochondrial DNA (mtDNA) to leak abnormally into the cytosol. Cytosolic mtDNA can then activate cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. Unlike the acute innate immune response elicited by pathogen-derived DNA, cGAS-STING signaling in senescent cells is mainly activated by the aberrant cytosolic accumulation of endogenous DNA. This signaling activates the TANK-binding kinase 1 (TBK1)-interferon regulatory factor 3 (IRF3) axis and induces a type I interferon response. It also promotes nuclear factor kappaB (NF-κB)-mediated inflammatory transcription, sustains the SASP, and modulates paracrine signaling and immune cell function. This review focuses on mitochondrial structural damage, mtDNA release, and impaired MQC. It summarizes the mechanisms that activate the mtDNA-cGAS-STING axis during cellular senescence and examines its functional changes. It also discusses the potential development of senescence-associated biomarkers. Current evidence suggests that this signaling axis mainly contributes to the maintenance and amplification of the inflammatory senescence phenotype. It is unlikely to independently determine the onset of cellular senescence. Its effects are highly dependent on cell type and pathological context, while assay standards, causal relationships, and therapeutic windows require further clarification.

Sitosterolemia is a rare lipid disorder characterized by xanthomas, premature atherosclerotic cardiovascular disease (ASCVD), and abnormally elevated circulating low-density lipoprotein cholesterol (LDL-c) measurements. Pathogenic variants in adenosine triphosphate (ATP)-binding cassette transporters lead to the accumulation of phytosterols. A child presented to the pediatric cardiology clinic with an incidentally noted presumed pathogenic variant and a variant of unknown significance in a sitosterolemia gene but had normal fasting lipids and no accumulation of circulating phytosterols. This case underscores the importance of phenotype-driven management and universal lipid screening, while highlighting the challenges of a gene-first testing/screening approach.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by aberrant fibroblast activation and excessive extracellular matrix accumulation, predominantly collagen, leading to irreversible lung remodeling. Sinigrin (SNG), a naturally occurring glucosinolate with reported anti-inflammatory and antioxidant activities, has not been investigated in the context of pulmonary fibrosis. In the present study, we evaluated the therapeutic activity of SNG using complementary in vitro and in vivo models of IPF. In the current investigation, inflammatory responses were induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages and precision-cut lung slices (PCLS), whereas fibrotic activation was elicited by TGF-β in LL29, DHLF cells, and PCLS. Treatment with sinigrin and dexamethasone significantly suppressed LPS-induced inflammatory mediators (IL-6, TNF-α, IL-1β, CCL2, MMP-9, and COX-2). In TGF-β-stimulated fibroblasts/PCLS, SNG and pirfenidone markedly attenuated collagen accumulation and downregulated the expression of key fibrotic markers (FN-1, α-SMA, TIMP-1, TIMP-3, COL1α1, and COL3α1). SNG administration in a bleomycin-induced pulmonary fibrosis mouse model significantly improved body weight, survival rate, and pulmonary index. Histopathological analyses revealed reduced collagen deposition and lowered Ashcroft scores, accompanied by decreased oxidative stress and improved lung function in SNG treatment compared to BLM and PFD groups. Consistently, gene and protein expression analyses demonstrated that SNG dose-dependently attenuated the bleomycin-induced inflammatory and fibrotic marker expression. Further mechanistic investigations, including an AMPK siRNA-based approach, demonstrated that SNG exerts its anti-inflammatory and antifibrotic effects partly through activation of AMPK signaling. Collectively, these findings suggest that SNG may represent a therapeutic candidate for the treatment of IPF.

Intratumoral tertiary lymphoid structures (TLSs) are increasingly recognized as spatially organized immune niches associated with favorable prognosis and improved responses to immune checkpoint blockade. Yet TLS positivity is not synonymous with durable antitumor immunity. Many tumors contain lymphoid aggregates that are anatomically visible but developmentally arrested, poorly vascularized, depleted of germinal center-like activity, or uncoupled from effective plasma-cell and T-cell output. This discrepancy has shifted the field from a binary TLS-positive/TLS-negative view toward a state-based interpretation of TLS competence. Here, we propose that metabolite-driven remodeling of the tumor microenvironment is a major cause of intratumoral TLS failure. However, the current literature lacks a cell-type- and spatially resolved framework linking individual metabolites to disruption of defined TLS modules and distinguishing early developmental deviation from later functional collapse. Single-cell and spatial omics now show that productive TLSs depend on coordinated interactions among stromal organizer cells, HEV-like endothelium, mature dendritic cells, T follicular helper-like cells, B-cell lineages, plasma cells, and stem-like or progenitor exhausted T cells. These cellular modules are vulnerable to oxygen limitation, nutrient competition, lactate accumulation, extracellular adenosine, tryptophan catabolism, vascular stress, and suppressive B-cell states. A central goal of this review is to establish a hierarchy of mechanistic certainty across the metabolic drivers of TLS dysfunction. Tryptophan metabolism currently has the strongest direct TLS-specific spatial and perturbational evidence, whereas lactate, adenosine, hypoxia-associated vascular stress, lipid stress, and nucleotide metabolism are supported mainly by convergent immunometabolic and spatial evidence. On this basis, we outline biomarker-guided strategies to restore TLS competence, including inhibition of tumor-derived tryptophan metabolism to promote TLS maturation and enhance anti-PD-1 efficacy.

Antibody drugs as one of the important categories of drugs in the modern biomedicine filed have been successfully applied in various fields, such as cancer diseases and immune disorders. In the early stage of monoclonal antibody drug development, scientists need to accurately select the highly functional antibodies from the numerous candidates obtained from immunized animals for subsequent development. However, due to the diverse binding sites between antigens and antibodies, the high-scoring antibodies obtained through traditional methods, such as enzyme linked immunosorbent assay (ELISA), western blot, surface plasmon resonance (SPR) affinity detection, cannot fully represent their high sealing function towards the target protein. This uncertainty prolongs the research and development cycle and increases inspection costs. This article takes ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) as an example to introduce a patented (application number 202511470362.1, China) method for direct antibody screening at the functional level. This screening method mixes the ENTPD2 enzyme, substrate ATP and antibody in a strict dosage ratio to establish precise reaction conditions; upon completion of the reaction, the residual ATP content is detected to evaluate the inhibitory effect of the antibody on the enzyme. To ensure the accuracy of the screening process, we established cell systems and mouse models to verify the function of the antibodies. Using this method, we initially screened 5 antibodies from the supernatants of 214 rabbit monoclonal B cells. These 5 antibodies all demonstrated inhibitory function in both the cell system and the mouse model. These results show that this function screening method possesses the characteristics of high accuracy, high efficiency and high throughput, which can significantly shorten the research and development time and reduce cost.

G-protein-coupled receptors (GPCRs) are essential mediators of cellular processes, and their dysfunction is implicated in various diseases. Although pharmacological approaches are powerful for elucidating GPCR functions, careful consideration of potential off-target effects remains crucial. Here, we present an off-target-free chemogenetic platform to dissect the physiological roles of target GPCRs. As a proof-of-concept, we focused on the adenosine A2A receptor (A2AR), a prototypical GPCR involved in neurodegenerative diseases. Leveraging the ligand recognition mechanisms of A2AR, we designed a prominent chemogenetic pair comprising a clinically validated A2AR antagonist and an engineered A2AR mutant that is insensitive to the antagonist but retains responsiveness to adenosine. Using this pair, we uncovered the critical role of A2AR in neurite outgrowth under hypoxic conditions in neuron-like cells, while avoiding potential off-target effects. Furthermore, we demonstrate the generalizability of this chemogenetic approach to other class A GPCRs, offering a versatile platform for receptor-specific dissection of GPCR signaling.

Allele-specific expression (ASE), the preferential expression of one gene copy, is a key mechanism of genomic regulation. However, its role in human kidney disease remains poorly understood. In this study, we generated a high-quality, genome-wide ASE map using paired whole-genome sequencing and RNA sequencing from microdissected glomerular and tubulointerstitial compartments of patients with proteinuric kidney disease. We showed that the majority of common ASE events were deterministic and sequence-mediated. We also found that diseased kidneys exhibited significantly more ASE in glomeruli (GLOM) than tubulointerstitium (TUBE) compared to controls. Unexpectedly, higher ASE in GLOM than TUBE was significantly associated with improved kidney disease outcomes in the disease cohort. Differential gene expression analysis suggested that this was the result of an active, protective transcriptional response, including ribosome and adenosine 5'-triphosphate synthesis up-regulation, rather than pathogenic dysregulation. Our work reveals glomerular ASE as a marker of adaptive transcriptional activity in proteinuric kidney disease.