Jon M. Jacobs

Undernutrition increases tuberculosis (TB) risk, but whether severe undernutrition during latent TB infection (LTBI) produces blood transcriptional changes that resemble active disease or identify future TB remains unclear. We reanalyzed whole-blood RNA sequencing from adults with LTBI in GSE152218, comparing severe undernutrition (BMI =18.5 kg/m2; n=11). Genes and Reactome pathways were evaluated. A seven-module summary was compared with active TB among severely undernourished adults and tested prospectively in GSE79362 and GC6-74/GSE94438 after pathway-representation assessment. No genes reached FDR <0.05 in the primary model; two did only in sensitivity analysis. In contrast, 241 Reactome pathways were significant and directionally concordant in both models (NES rho=0.954), including complement/Fc-receptor, B-cell, phospholipid/phagocytosis, neutrophil, vascular, autophagy, and cytokine-signaling pathways. Most changed opposite to the active-TB comparison (178/241; pathway rho=-0.390). The seven-module score was unrelated to baseline progression in GSE79362 (adjusted OR per SD=1.090; P=0.6922). In GC6, four modules met the prespecified representation criterion; their baseline score was inversely associated with progression (adjusted OR per SD=0.711; P=0.0218), but secondary and longitudinal analyses were non-confirmatory. Severe undernutrition during LTBI is associated with reproducible immune- and metabolism-related transcriptional remodeling that differs from active-TB biology and does not consistently identify future TB progression.

Recent advancements in data-independent acquisition (DIA) workflows have greatly increased the depth and throughput of mass spectrometry-based proteomics. However, many of these advancements remain undercharacterized for phosphoproteomics, particularly in bacteria that have fewer protein phosphorylation events. We evaluated the impact of instrument/gradient length (Orbitrap Astral, 15 min; Orbitrap Exploris, 90 min), analysis tools (DIA-NN 1.9.2, DIA-NN 2.3, FragPipe, Spectronaut), and library search strategies (spectral-library versus library-free) on phosphoproteomic coverage and quantification in a human cell line and bacterial lysate. The 15 min Astral analysis identified similar numbers of phosphopeptides compared to a 90 min Exploris acquisition method across all analysis tools, demonstrating a substantial advantage in throughput. Within the Astral workflow, Spectronaut provided the highest phosphoproteome coverage, whereas DIA-NN 1.9.2, DIA-NN 2.3, and FragPipe exhibited less quantitative variation. We compared library-free and spectral-library search strategies using the Mycobacterium tuberculosis (Mtb) H37Rv strain. We observed greater phosphopeptide identifications yet different phosphopeptide profiles via library-free searches in comparison to spectral-library searches. When comparing Mtb harvested at different growth phases, phosphosite fold-changes were consistent, whereas statistical significance varied between tools. This work can be informative for workflow selection in DIA phosphoproteomics studies, especially for biological samples with low phosphorylation frequencies.

Background: Although clinical biomarkers of tuberculosis (TB) relapse are well characterized, the biological mechanisms underlying different treatment outcomes remain poorly understood. Elucidating these mechanisms may reveal improved biomarkers of long-term treatment outcomes. Methods: We conducted a longitudinal, global proteomic study on 60 participants with active pulmonary TB, half who were durably cured and half who relapsed. Plasma was collected at seven time-points: at treatment initiation (baseline), during therapy, and 52 weeks postbaseline. Samples were analyzed by high-resolution LC-MS/MS. Results: 2,418 proteins were identified across all samples, with 1,756 being differentially expressed relative to baseline (p < 0.05). 956 proteins were differentially abundant between cured and relapsed participants. Relapsed participants showed heightened humoral immunity throughout treatment, as well as upregulated complement activation and HDL particles. Cured participants exhibited elevated recovery-related pathways by week 4, including downregulated epithelial invasion and upregulated oxygen transport. Conclusions: Heightened humoral and innate immune responses were associated with relapse, whereas recovery signatures were associated with durable cure. Several proteins showed potential as relapse biomarkers and warrant future validation in independent cohorts. These findings advance our understanding of host responses to treatment and provide a basis for developing blood-based biomarkers to identify patients at increased risk of relapse.

Single amino acid variants (SAAVs) in protein sequences are often a direct result of single-nucleotide polymorphisms (SNPs). Certain germline SAAVs have shown biological relevance in different disease conditions but lack precise quantification in circulation, which could hinder functional investigations and progress in biomarker development. Here, we have developed a multiplexed liquid chromatography-selected reaction monitoring (LC-SRM) assay that monitors 5 wild-type and variant peptide pairs (Complement Factor B: CFB-R32Q/R32W, Clusterin: CLU-N317H, Fetuin B: FETUB-K360R, and Kininogen: KNG1-L212P) in nondepleted human plasma. The assay was optimized for imprecision, linearity, stability, and calibration assessments with CVs of under 20%. The wild-type and variant peptide pairs were characterized in a set of healthy individual plasma samples. These target identifications were also validated by SNP genotyping with more than 99% accuracy. For all protein targets, we observed significantly lower concentrations of WT species in the presence variant peptides. In CFB, the concentration of R32Q was significantly lower than its counterpart R32W variant and WT species. Furthermore, our results distinguished phenotypes of homozygosity and heterozygosity of the SAAV presence through direct concentration level characterization. These findings provide some insights into how SAAVs affect quantitative assessments of target peptides. The assay demonstrates a platform for proteogenomic analyses with potential applications in both research and clinical settings.

Background/Objectives: Patients with epilepsy commonly experience patterns of seizures that change with sleep/wake behavior or diurnal rhythms. The cellular and molecular mechanisms that underlie these patterns in seizure activity are not well understood but may involve non-neuronal cells, such as astrocytes. Our previous studies show the critical importance of one specific astrocyte factor, the brain-type fatty acid binding protein Fabp7, in the regulation of time-of-day-dependent electroshock seizure threshold and neural activity-dependent gene expression in mice. Here, we examined whether Fabp7 influences differential seizure activity-dependent protein expression, by comparing Fabp7 knockout (KO) to wild-type (WT) mice under control conditions and after reaching the maximal electroshock seizure threshold (MEST). Methods: We analyzed the proteome in cortical–hippocampal extracts from MEST and SHAM groups of WT and KO mice using mass spectrometry (MS), followed by Gene Ontology (GO) and pathway analyses. GO and pathway analyses of all groups revealed a diverse set of up- and downregulated differentially expressed proteins (DEPs). Results: We identified 65 significant DEPs in the comparison of KO SHAM versus WT SHAM; 33 proteins were upregulated and 32 were downregulated. We found downregulation in mitochondrial-associated proteins in WT MEST compared to WT SHAM controls, including Slc1a4, Slc25a27, Cox7a2, Cox8a, Micos10, and Atp5mk. Several upregulated DEPs in the KO SHAM versus WT SHAM comparison were associated with the 20S proteasomal subunit, suggesting proteasomal activity is elevated in the absence of Fabp7 expression. We also observed 92 DEPs significantly altered in the KO MEST versus WT MEST, with 49 proteins upregulated and 43 downregulated. Conclusions: Together, these data suggest that the astrocyte Fabp7 regulation of time-of-day-mediated neural excitability is modulated by multiple cellular mechanisms, which include proteasomal pathways, independent of its role in activity-dependent gene expression.

Background: The earliest manifestation of alcohol‐associated liver disease (ALD) is steatosis characterized by deposition of fat in specialized organelles called lipid droplets (LDs). While alcohol administration causes a rise in LD numbers in the hepatocytes, little is known regarding their characteristics that allow their accumulation and size to increase. The aim of the present study is to gain insights into underlying pathophysiological mechanisms by investigating the ethanol‐induced changes in hepatic LD proteome as a function of LD size.Methods: Adult male Wistar rats (180–200 g BW) were fed with ethanol liquid diet for 6 weeks. At sacrifice, large‐, medium‐, and small‐sized hepatic LD subpopulations (LD1, LD2, and LD3, respectively) were isolated and subjected to morphological and proteomic analyses.Results: Morphological analysis of LD1‐LD3 fractions of ethanol‐fed rats clearly demonstrated that LD1 contained larger LDs compared with LD2 and LD3 fractions. Our preliminary results from principal component analysis showed that the proteome of different‐sized hepatic LD fractions was distinctly different. Proteomic data analysis identified over 2000 proteins in each LD fraction with significant alterations in protein abundance among the three LD fractions. Among the altered proteins, several were related to fat metabolism, including synthesis, incorporation of fatty acid, and lipolysis. Ingenuity pathway analysis revealed increased fatty acid synthesis, fatty acid incorporation, LD fusion, and reduced lipolysis in LD1 compared to LD3. Overall, the proteomic findings indicate that the increased level of protein that facilitates fusion of LDs combined with an increased association of negative regulators of lipolysis dictates the generation of large‐sized LDs during the development of alcohol‐associated hepatic steatosis.Conclusion: Several significantly altered proteins were identified in different‐sized LDs isolated from livers of ethanol‐fed rats. Ethanol‐induced increases in specific proteins that hinder LD lipid metabolism led to the accumulation and persistence of large‐sized LDs in the liver.