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Background: The emergence of colistin-resistant Acinetobacter baumannii, particularly extensively drug-resistant carbapenem-resistant A. baumannii (XDR-CRAB), poses a critical global health threat; however, the molecular mechanisms underlying this resistance remain poorly characterized.

Methods: This study utilized integrated tandem mass tag (TMT)-based proteomics and untargeted metabolomics to compare a representative drug-sensitive (DS) strain, a colistin-sensitive multidrug-resistant (MDR-CRAB) strain, and a colistin-resistant extensively drug-resistant (XDR-CRAB) isolate from a Guangzhou hospital, China, with a primary focus on the molecular features associated with resistance in the XDR isolate.

Results: Proteomic analysis identified 260 differentially expressed proteins (DEPs) across these strains, with 98 strongly linked to the colistin-resistant phenotype. These DEPs were enriched in functions related to polysaccharide and sulfate transport, aromatic compound degradation, and cationic antimicrobial peptide (CAMP) resistance. Upregulation of the lipid A modification system, driven by increased expression of PmrA (4.2-fold), PmrB (2.9-fold), and PmrC (5.5-fold) in the XDR strain compared to the MDR strain, is consistent with this CAMP resistance. Metabolomic analysis identified 747 metabolites, with lipids and lipid-like molecules being the most altered class. The XDR strain exhibited reduced relative abundance of several glycerophospholipids and fatty acids/conjugates, when compared to the MDR strain, indicating cell membrane remodeling. Pathway enrichment analysis further implicated purine, glycerophospholipid, starch/sucrose, and glutathione metabolism in colistin resistance.

Conclusion: Colistin resistance in A. baumannii appears to involve a multifaceted strategy integrating lipid A modification, cell membrane remodeling, and metabolic reprogramming. This study provides a high-resolution molecular blueprint of colistin resistant A. baumannii and generates testable hypotheses to inform future diagnostic and therapeutic strategies against resistant infections.

Since the outbreak of the COVID-19 pandemic, SARS-CoV-2 has not stopped evolving, leading to the emergence of variants of concern (VoCs) involved in significant immune escape. Here, we compared the immunogenicity of different prime-boost vaccination regimens against SARS-CoV-2 wildtype (WT) and its Beta, Delta, and Omicron BA.1 VoCs. We used 5 databases to retrieve publications and random-effect models to estimate pooled neutralization titers. We included 11 randomized controlled trials (RCTs) and 16 non-RCTs, 10 prime-boost vaccination regimens, and 4598 subjects. We found neutralization activity against SARS-CoV-2 decreased with virus evolution. The heterologous immunization was more effective. The increase in neutralization titers against SARS-CoV-2 WT and Beta, Delta, and Omicron BA.1 VoCs after heterologous immunization was 1.41(95%CI:0.82–2.01), 0.90(95%CI:0.39–1.41), 1.23 (95%CI: 0.81–1.65), and 1.32 (95%CI: 0.99–1.65), respectively. Furthermore, the booster dose of viral vector vaccine did not show a higher increase in neutralization titers against SARS-CoV-2 WT(MD=0.48; 95%CI:−1.12-1.09), Beta (MD=0.20; 95%CI:−0.26-0.67), Delta (MD=0.35; 95%CI:−0.09-0.79), and Omicron BA.1 (MD=0.38; 95%CI:−0.14-0.89) VoCs. The combination of inactivated-recombinant protein vaccines showed a higher increase in neutralization titers (Beta: MD=1.88 and Delta: MD=1.70) than other combinations of vaccines. However, only a combination of mRNA-viral vector vaccines showed a higher increase in neutralization titers (MD:1.52; 95%CI:0.34-2.70) against Omicron BA.1 VoC. Interestingly, the viral vector-mRNA immunization regimen appears better compared to mRNA-viral vector regimen, especially against Beta and Delta VoCs. Overall, the type of combination followed by the order of administration of COVID-19 vaccines could be a potential vaccine strategy against the occurrence of SARS-CoV-2 variants.

Although COVID-19 is no longer classified as the first public health emergency, nevertheless, it still presents a serious menace to the health of the global population. Consequently, the development of COVID-19 vaccines possessing an optimal composition that can elicit broad-spectrum neutralizing responses against various SARS-CoV-2 variants is crucial. This meta-analysis aimed to compare the immunogenicity of prototype, monovalent-adapted, and bivalent COVID-19 vaccines against prototype SARS-CoV-2, Omicron BA.1 variant, and Omicron BA.4/5 subvariant in healthy adults. We utilized 4 medical databases to retrieve original studies and employed the fixed effect model to estimate pooled neutralization titers. A total of 12 studies concerning 4581 subjects were included in the meta-analysis. We found that participants who received prototype, monovalent-adapted, and bivalent vaccines as a second booster significantly developed neutralizing antibody (nAb) titers against prototype SARS-CoV-2, Omicron BA.1 variant, and Omicron BA.4/5 subvariant, with monovalent-adapted and bivalent vaccines exhibiting a higher increment. Furthermore, the bivalent(Prototype/Omicron BA.1) recombinant protein vaccine exhibited the highest increment in neutralization titers(MD = 1.95; 95 %CI:0.78–3.12; p < 0.01) against the prototype SARS-CoV-2 and Omicron BA.4/5 subvariant compared to the other vaccine regimens. Interestingly, only individuals who received the monovalent (Omicron BA.1)-adapted mRNA vaccine as a second booster showed the highest increase in neutralization titers (MD:1.37; 95 %CI:0.50–2.24; p < 0.01) against the Omicron BA.1 variant compared to the other vaccine regimens. These findings showed that bivalent recombinant protein vaccines seem more immunogenic than bivalent mRNA vaccines, and bivalent vaccines might not be superior immunogens for induced strong protective immune responses compared to monovalent-adapted vaccines.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a global health crisis complicated by immune dysregulation and T cell exhaustion. CD39, an ectonucleotidase generating immunosuppressive adenosine, is implicated in cancer and chronic infections, yet its spatiotemporal role in TB pathogenesis remains unclear.

Methods: Multiple publicly available datasets were utilized to evaluate CD39 across TB disease stages, diverse infectious diseases and anti-TB treatment. Diagnostic accuracy was evaluated via ROC curves and combined signature analysis. Immune cell infiltration were analyzed using CIBERSORTx. Cytokine profiles and age-stratified associations were examined. Pathway enrichment analysis was performed by GSEA. Single-cell analysis of non-human primate granulomas assessed CD39’s temporal dynamics, utilizing Monocle 3 for CD39+ T-cell trajectory analysis.

Results: CD39 was upregulated in active TB patients versus TB infection (TBI) and healthy controls (HC), correlating with older age, disease severity, and distinct expression patterns compared to other respiratory and systemic infections. CD39 demonstrated superior diagnostic accuracy over IFN-γ in distinguishing TB from TBI/HC and other respiratory diseases. Combining CD39 with TBX21 or GZMB further improved diagnostic specificity. High CD39 expression correlated with suppressed Th1 and elevated Th2/Th17/regulatory cytokines, alongside pronounced neutrophil infiltration. Age-stratified analysis revealed complex age-dependent associations of CD39 expression with various immune cell types. Single-cell analysis revealed declining CD39 transcriptional activity during prolonged infection despite expanded cellular distribution, linked to early T cell maturation followed by broader immunomodulatory shifts. Decreased CD39 expression with anti-TB treatment correlated with improved immune cell balance and resolved T cell exhaustion.

Conclusion: CD39 is a critical regulator of immune exhaustion and neutrophil-driven inflammation in TB, with diagnostic and therapeutic potential. Targeting CD39 may provide a novel therapeutic strategy for TB.

Cutaneous leishmaniasis, a neglected tropical disease (NTD) caused by Leishmania tropica, Leishmania major and other members of the same species, poses significant challenges in the public health sector, especially in developing countries. The limitations of current treatments, including toxicity, resistance, availability, and cost effectiveness, necessitate the development of novel therapeutics. This study investigated the leishmanicidal potential of five Thiadiazine thione derivatives (THTT) against L. tropica, at a dose range of 25–426 µM using an MTT assay. Compounds T9, T24, T25 (tris-THTT) and PG (mono-THTT) exhibited notable IC₅₀ values of 20.01, 26.94, 27.71, and 82.79 µM, respectively. Moreover, the same compounds demonstrated promising docking scores against critical leishmanial enzymes, such as Trypanothione reductase, Trypanothione synthetase, Leishmanolysin, and C24-sterol methyl transferase. Hemolytic assay revealed that all the compounds are non-toxic at lower concentrations. Mono-THTTs (PG and BG) showed higher CC₅₀ values i.e., 401.65, 375.68 as compared to tris-THTTs (T9, T24, T25) which is 104.61, 104.51 and 89.73 µM respectively. All derivatives showed high selectivity indices (SI) compared to the standard drug Amphotericin-B. Furthermore, in-silico investigations targeting COX1 and COX2 enzymes unveiled a high affinity of the tested compounds towards these enzymes, indicating their potential involvement as anti-inflammatory agents. Subsequent in-vitro HRBC membrane stabilization and egg albumin denaturation assays confirmed the anti-inflammatory potential of all the compounds. These findings validate THTT derivatives as an effective and novel class of anti-leishmanial agents against L. tropica with a favorable safety profile and potential anti-inflammatory properties. Further research is recommended to elucidate their mechanisms of action and in-vivo efficacy.

The human body employs nutritional immunity to restrict essential micronutrients, such as zinc, from invading pathogens, impeding their growth and replication. Here, we applied an advanced nanochemical imaging technique, synchrotron radiation-based X-ray fluorescence (SR-XRF), on vitrified polymorphonuclear neutrophils (PMNs) during the occurrence of phagocytosis of Saccharomyces cerevisiae. Nanoscopic SR-XRF provided trace elemental distributions at 50 nm spatial resolution, revealing the metal interplay between PMNs and S. cerevisiae. Our results were complemented with X-ray holographic nanotomography (XNH), confirming phagocytosis and providing complementary intracellular morphological information. A systematic decrease in zinc was observed between free and phagocytosed S. cerevisiae within the same XRF maps, suggesting active zinc depletion by PMNs. Other elements, such as sulfur, show an increase in the phagosome, likely indicative of the increase in proteins in the vicinity of phagocytic events. Through 2D/3D nanoimaging and time-lapse microscopy, we confirmed the reduction of zinc within phagocytosed yeast. Hence, our findings challenge the currently accepted hypothetical model that PMNs intoxicate engulfed microbes with an overwhelming influx of zinc ions into the phagosome. Furthermore, antimicrobial assays demonstrated that S. cerevisiae can cope well with sudden zinc spikes. Even high zinc concentrations imposed on S. cerevisiae grown under zinc-limiting conditions did not have adverse effects on viability. Contrarily, S. cerevisiae was more resistant to phagocytic killing by PMNs when grown under high zinc concentrations before infection. Our findings further consolidate zinc deprivation as an effective antimicrobial strategy. A better understanding of the metal deprivation mechanisms could inspire new exploitable targets for antimicrobial therapies.

Recognizing the therapeutic value of the Genus Fraxinus worldwide, this study evaluates the antioxidant potential of Fraxinus hookeri Wenz. (F. hookeri) against CCl4-induced nephrotoxicity in rats. Forty-eight rats are randomly allocated into eight groups (six rats each). Antioxidant enzymes, genotoxicity, urine and serum markers, and tissue histopathology are assessed to determine their nephroprotective effects. The Control group remains untreated, while the DMSO group receives vehicle olive oil intraperitoneally and DMSO orally (3 ml/kg). All other groups, except Control and DMSO, are given CCl4 (3 ml/kg, i.p., in 30% olive oil) twice weekly for 4 weeks. The CCl4 group receives only CCl4. The Rutin group receives reference drug Rutin orally (50 mg/kg). MEFH100 and MEFH200 groups are given MEFH at 100 and 200 mg/kg, respectively, and NHFH100 and NHFH200 receive NHFH at the same doses. Rutin and F. hookeri treatment effectively (P < 0.05) restore urine and serum markers disrupted by CCl4. CCl4 reduced (p < 0.05) antioxidant enzymes (CAT, SOD, POD) and increased TBAR levels and DNA damage, which are reversed by cotreatment with F. hookeri and Rutin. Histopathological improvements (P < 0.05) are also observed with F. hookeri. The results indicate that F. hookeri enhances antioxidant defenses, supporting its potential against CCl4-induced nephrotoxicity.

Syphilis, caused by Treponema pallidum, is resurging globally. Molecular typing allows for the investigation of its epidemiology. In Pakistan and other nations, T. pallidum subsp. pallidum has developed widespread macrolide resistance in the past decade. A study at the Peshawar Regional Blood Centre from June 2020–June 2021 analyzed serum samples from 32,812 blood donors in Khyber Pakhtunkhwa, Pakistan, to assess circulating T. pallidum strains and antibiotic resistance. Blood samples were initially screened for T. pallidum antibodies using a chemiluminescent microparticle immunoassay (CMIA). CMIA-reactive samples underwent polymerase chain reaction (PCR) targeted the polA, tpp47, bmp, and tp0319 genes. PCR-positive samples were further analyzed for molecular subtyping using a CDC-developed procedure and tp0548 gene examination. All PCR-positive samples were analyzed for the presence of point mutations A2058G and A2059G in 23S rRNA, as well as the G1058C mutation in 16S rRNA. These mutations are known to impart antimicrobial resistance to macrolides and doxycycline, respectively. Out of 32,812 serum samples, 272 (0.83%) were CMIA-reactive, with 46 being PCR-positive. Nine T. pallidum subtypes were identified, predominantly 14d/f. The A2058G mutation in 23S rRNA was found in 78% of cases, while G1058C in 16S rRNA and A2059G in 23S rRNA were absent. The research found donor blood useful for assessing T. pallidum molecular subtypes and antibiotic resistance, especially when chancres are not present. The prevalent subtype was 14d/f (51.85%), and the high macrolide resistance of 36 (78%) indicates caution in using macrolides for syphilis treatment in Khyber Pakhtunkhwa, Pakistan.

Background: The IL-12/23/ISG15-IFN-γ pathway is the main immunological pathway for controlling intra-macrophagic microorganisms such as Mycobacteria, Salmonella, and Leishmania spp. Consequently, upon mutations in genes of the IL-12/23/ISG15-IFN-γ pathway cause increased susceptibility to intra-macrophagic pathogens, particularly to Mycobacteria. Therefore, the purpose of this study was to characterize the mutations in genes of the IL-12/23/ISG15-IFN-γ pathway in severe tuberculosis (TB) patients.

Methods: Clinically suspected TB was initially confirmed in four patients (P) (P1, P2, P3, and P4) using the GeneXpert MTB/RIF and culturing techniques. The patients' Peripheral blood mononuclear cells (PBMCs) were then subjected to ELISA to measure Interleukin 12 (IL-12) and interferon gamma (IFN-γ). Flow cytometry was used to detect the surface expressions of IFN-γR1 and IFN-γR2 as well as IL-12Rβ1and IL-12Rβ2 on monocytes and T lymphocytes, respectively.The phosphorylation of signal transducer and activator of transcription 1(STAT1) on monocytes and STAT4 on T lymphocytes were also detected by flow cytometry. Sanger sequencing was used to identify mutations in the IL-12Rβ1, STAT1, NEMO, and CYBB genes.

Results: P1's PBMCs exhibited reduced IFN-γ production, while P2's and P3's PBMCs exhibited impaired IL-12 induction. Low IL-12Rβ1 surface expression and reduced STAT4 phosphorylation were demonstrated by P1's T lymphocytes, while impaired STAT1 phosphorylation was detected in P2's monocytes. The impaired IκB-α degradation and abolished H2O2 production in monocytes and neutrophils of P3 and P4 were observed, respectively. Sanger sequencing revealed novel nonsense homozygous mutation: c.191 G>A/p.W64 * in exon 3 of the IL-12Rβ1 gene in P1, novel missense homozygous mutation: c.107 A>T/p.Q36L in exon 3 of the STAT1 gene in P2, missense hemizygous mutation:: c.950 A>C/p.Q317P in exon 8 of the NEMO gene in P3, and nonsense hemizygous mutation: c.868 C>T/p.R290X in exon 8 of CYBB gene in P4.

Conclusion: Our findings broaden the clinical and genetic spectra associated with IL-12/23/ISG15-IFN-γ axis anomalies. Additionally, our data suggest that TB patients in Pakistan should be investigated for potential genetic defects due to high prevalence of parental consanguinity and increased incidence of TB in the country.

Tuberculosis (TB) is a global health problem caused by the Mycobacterium tuberculosis complex (MTBC). These bacteria secrete various proteins involved in the pathogenesis and persistence of MTBC. Among the secretory proteins, MPT64 (Rv1980C) is highly conserved and is also known as a major culture filtrate that is used in rapid diagnosis of MTBC. In the current study, we aimed to find the mutation in this highly conserved protein in isolates from the Pashtun-dominant province of Pakistan. We analyzed 470 M. tuberculosis whole-genome sequences of Khyber Pakhtunkhwa Province. Mutations in the MPT64 gene were screened through TB-Profiler and BioEdit software tools. The DynaMut web server was used to analyze the impact of the mutation on protein dynamics and stability. Among 470 MTB genomes, three non-synonymous mutations were detected in nine isolates, and one synonymous mutation (G208A) was found in four isolates. Mutation G211T (F159L), which was detected at the C-terminal domain of the protein in six isolates, was the most prominent. The second novel mutation, T480C (I70V), was detected in two isolates at the C-terminal side of the protein structure. The third novel mutation, A491C (L66R), was detected in a single isolate at the N-terminal side of the MPT64 protein. The effect of these three mutations was destabilizing on the protein structure. The molecular flexibility of the first two mutations increased, and the last one decreased. MPT64 is a highly conserved secretory protein, harboring only a few mutations. This study provides useful information for better managing the diagnosis of MTB isolates in high TB-burden countries.