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Three NADH diaphorase phenotypes were observed in the red cells of sheep. Breeding data indicated that this polymorphism was under the control of two autosomal codominant alleles, designated DiaF and DiaS. Phenotype Dia F had significantly lower NADH diaphorase activity than phenotype Dia S. The frequency of DiaF and DiaS was determined in 9 different breeds.
The cumulus and membrana granulosa of non-atretic ovarian follicles from primordial up to a stage shortly before ovulation were studied by electron microscopy. The follicular cells of primordial follicles were undifferentiated and rested on a thick basal lamina. In secondary follicles the endoplasmic reticulum had proliferated forming an anastomosing network. In early antral and antral follicles (0.5--2.0 mm dia.) the ER was composed of short cisternae, the mitochondria had elongated and gap junctions were first observed. In late antral follicles (3.0--5.9 mm dia.) gap junctions were frequent. In the cumulus the glycogen was associated with electron lucent areas whereas in the granulosa it was invariably associated with membranes. In large antral follicles large membrane bound bodies were present in the basal cells of the cumulus. At early oestrus a distinctive mitochondrial morphology was noted in the granulosa but not elsewhere in the follicles. At mid oestrus numerous annular nexuses were present in the granulosa but not in the cumulus. At late oestrus numerous lipid droplets were formed in both cumulus and granulosa, the boundary with theca interna became indistinct and the basal lamina became incomplete.
PURPOSE: Elevated amide proton transfer-weighted (APTw) MRI signals in glioblastoma (GBM) are often linked to increased intracellular mobile proteins, but the associated molecular patterns in human tissue remain unclear. We examined the relationship between regional APTw features and cellular protein composition and profiled proteomic differences between tumor and peritumoral tissue. METHODS: In this single-center prospective study, preoperative MRI data were integrated with intraoperative neuronavigation for 12 image-guided tissue samples (8 tumor and 4 peritumoral). Total, cytoplasmic, and nuclear proteins were quantified using bicinchoninic acid (BCA) assay. Data-independent acquisition (DIA) proteomics identified exploratory differentially expressed proteins (DEPs), followed by functional enrichment and protein-protein interaction (PPI) network analyses. Transcript-level expression patterns and survival associations were queried in The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets to provide indirect external clinical context. RESULTS: Tumor regions showed higher APTw signals than peritumoral regions (p < 0.001) and increased cytoplasmic protein concentration (p < 0.05), without a corresponding increase in total or nuclear protein levels. DIA identified 654 DEPs. Further analysis highlighted 36 higher-significance DEPs, and prioritized 12 hub proteins in the PPI network. In public transcriptomic datasets, ERBB2, RUNX1, and SHC1 showed higher expression in GBM and were associated with poorer overall survival. CONCLUSION: These findings suggest that elevated APTw signal in GBM may be associated with increased cytoplasmic protein content and distinct proteomic alterations. This imaging-proteomic framework provides exploratory regional context for future mechanistic and follow-up studies, but larger, spatially matched and independently validated cohorts are required to confirm the molecular contributors to APTw contrast.
Shell matrix proteins (SMPs) are key organic components of molluscan biominerals, yet previous nacre proteomics have remained largely qualitative, limiting evaluation of the abundance and fraction association of individual SMPs. Here, we established a quantitative proteomic approach for the nacreous layer of the pearl oyster Pinctada fucata by integrating optimized shell preservation, stepwise fractionation, and data-independent acquisition (DIA) proteomics. SMPs were separated into an ethylenediaminetetraacetic acid (EDTA)-soluble matrix (ESM), an EDTA-insoluble but sodium dodecyl sulfate/dithiothreitol (SDS/DTT)-soluble matrix (SSM), and an SDS/DTT-insoluble matrix (ISM). DIA outperformed data-dependent acquisition in proteome coverage and enabled quantification of 327 SMPs across a broad dynamic range. Fraction-resolved abundance profiling showed that each fraction was characterized by distinct SMP compositions. To summarize these distributions, we introduced a solubility-based type classification that grouped SMPs into four types according to their quantitative partitioning among fractions. Well-known SMPs, including nacrein, Pif 80, and MSI60, were assigned to intuitively consistent types, whereas proteases, protease inhibitors, and tyrosinases also showed biased type distributions. These results support a three-compartment model of the nacreous layer consisting of (i) an insoluble interlamellar membrane core, (ii) a relatively extractable interfacial layer, and (iii) a soluble matrix fraction enriched in proteins potentially involved in ionic regulation and protein maturation. This study provides a quantitative framework for understanding coordinated SMP functions during nacre formation and for comparative analyses of molluscan shell proteomes.
BACKGROUND: Histone lactylation links lactate metabolism to chromatin regulation, but whether lactylation-program-associated transcriptional patterns delineate recurrent pan-cancer tumor states remains unclear. METHODS: We integrated mRNA, lncRNA, and miRNA profiles from 9712 TCGA tumors across 33 cancer types with GTEx references, six GEO cohorts, IMvigor210, and an institutional clear-cell renal cell carcinoma (ccRCC) cohort used for exploratory DIA-NN proteomic corroboration. Random-effects co-expression meta-analysis, multi-omics consensus clustering, regulon inference, immune deconvolution, TIDE, oncoPredict, and SHAP-based machine learning were applied. hsa-miR-431-5p was functionally evaluated as a proof-of-concept CS2-associated miRNA in bladder cancer models. RESULTS: LacCoEx-Atlas comprised 398,491 lactylation-related co-expression pairs across 24,667 RNA features under a random-effects framework (median I² = 88.6%). Consensus clustering identified two subtypes: CS2 showed glycolytic-mesenchymal-immune-excluded features, M2 macrophage enrichment, CD8⁺ T-cell depletion, elevated HDAC4/NSD3/KDM6B activity, and worse survival, whereas CS1 showed oxidative, sirtuin-active programs. CS2 had fewer predicted ICI responders (18.3% vs. 52.0%) and a lower observed ORR in IMvigor210 (15.3% vs. 24.0%). oncoPredict identified NU7441 as a hypothesis-generating CS2-associated sensitivity signal (Hedges' g = 1.17). DIA-NN proteomics in 50 ccRCC specimens provided exploratory support for CS2-associated hypoxia, ECM degradation, and metastasis programs. The 10-feature mRNA LARItools model achieved an apparent AUC of 0.9413, while a separate multi-omics model achieved 0.971; neither was independently validated. LARItools reproduced prognostic separation across six GEO cohorts. miR-431-5p promoted malignant phenotypes and EMT in bladder cancer cells, with concordant CMU4h expression findings. CONCLUSIONS: Lactylation-program-associated transcriptional patterns delineate a recurrent immune-excluded pan-cancer tumor state associated with adverse prognosis, reduced predicted immunotherapy responsiveness, exploratory single-cancer protein-level support, and testable DNA damage response-targeting hypotheses. LacCoEx-Atlas and LARItools provide open resources for lactylation-program-associated tumor-state stratification and future translational research.
Here we present a novel data-independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. PAMAF mode increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering, ion losses are avoided while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. In addition, by accumulating ions while the previous packet of ions is being analyzed, the PAMAF mode achieves ∼100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed ∼6× more protein group identifications compared to a standard data-dependent acquisition analysis without HRIM on the same QTOF instrument, and >100 x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by data-dependent acquisition. In addition, since precursor isolation in PAMAF mode is size-based rather than m/z-based, coeluting isobars and isomers can be resolved prior to fragmentation, eliminating chimeric spectra that compromise identification accuracy. We also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity termed DIA-PAMAF mode, which enabled the detection of over 8000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics with the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics.
Recent instrumental and computational innovations in mass-spectrometry-based proteomics offer new promise in biomarker discovery, thanks to unprecedented proteome coverage and depth. Data-independent acquisition (DIA) methods are very promising in this context as they allow improved proteome coverage, reduced missing value rates, and enhanced quantification precision. However, DIA methods also suffer from their own challenges, such as increased data complexity, cycle times, and background noise. In this work, we propose a sample-aware diaPASEF method optimization strategy for a timsTOF platform. Thorough method optimizations have first been conducted on standard HeLa lysates. Then, a ground-truth calibrated sample series, consisting of a range of UPS amounts spiked into a complex Arabidopsis background, was used to mimic differential analyses under controlled conditions. These benchmark experiments demonstrate clear benefits of using narrowPASEF for differential protein discovery. Finally, our strategy was applied to real use case biological samples to conduct a differential analysis of purified mouse astrocyte cells across two different conditions. narrowPASEF improved the proteome depth by 13%, considering proteins quantified with a coefficient of variation (CV) of <20%, and led to a 68% (435 vs 729) increase in differentially expressed proteins. These results provide an opportunity for a more precise and comprehensive analysis of the biological functions of biomarkers, offering a more profound understanding of the disease mechanisms. The benefits of our sample-aware narrowPASEF strategy demonstrated the most substantial impact on low-abundance proteins. Overall, these results show promise for more valuable and robust biomarker discoveries in the future.
Data-independent acquisition (DIA) mass spectrometry facilitates high-throughput, reproducible bottom-up proteomic analyses. Typically, DIA methods coselect multiple precursor ions within a wide selection window. These precursors are simultaneously fragmented, superimposing the product ion signals into a complex chimeric spectrum. A method for varying the quadrupole selection width over the ion accumulation period is described. This method couples the intensity of a product ion to the mass of its precursor ion. By overlapping consecutive selection windows, scan-to-scan product ion intensity profiles can be used to infer precursor mass. We assess the method's sensitivity to quadrupole width, accumulation time, and mass-to-charge range using internal fluoranthene calibrant and FlexMix calibration solution with Q-Orbitrap configured mass analyzers. Additionally, we explore usability of the described technique on a tryptic-digest monoclonal antibody sample, including both direct infusion and liquid chromatography of the sample. With direct infusion, product ions from two precursors separated by 1 thomson (Th) are resolved with this method using 10 Th windows with 5 Th overlap. The product ions are associated within 0.3 Th of their respective precursor ion's m/z. Therefore, product ion spectra have a precursor ion m/z resolving power of ∼33.
OBJECTIVE: To develop a safe and effective green therapy for triple-negative breast cancer, this study combines hydrogen-rich water with acupuncture point injection, and finds that it can prevent tumor growth and minimize cancer metastasis. METHODS: After 21 d of hydrogen rich water injection treatment on 4T1 (mouse breast cancer cells) xenograft mice, in order to systematically identify differentially expressed proteins in tumor samples between the model group and the Zusanli (ST36) group injected with hydrogen rich water at acupoints, with a focus on functional proteins or signaling pathways related to tumor occurrence and development, researchers conducted four-dimensional data independent acquisition (4D-DIA) proteomic analysis on tumor tissues. In order to further investigate the dynamic changes of metabolites after therapeutic intervention, researchers conducted liquid chromatography-tandem mass spectrometry untargeted metabolomics identification and analysis on mouse serum. The results of the joint proteomics-metabolomics analysis were validated using experimental methods such as immunofluorescence, Western blotting, and quantitative reverse transcription polymerase chain reaction detection. RESULTS: Injecting hydrogen-rich water into acupoints significantly inhibited tumor growth (P < 0.05). 4D-DIA proteomics and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses uncovered pathways such as T helper 1 cell (Th1) and T helper 2 cell (Th2) cell differentiation. The KEGG metabolic pathways identified in the metabolomics analysis included galactose metabolism along with fructose and mannose metabolism. Based on the combined proteomics and metabolomics analysis, the key pathways included the C-type lectin receptor signaling pathway. The major cancer-related differential proteins detected in Th1 and Th2 cell differentiation [interleukin 6 signal transducer, nuclear factor of activated T cells 4, recombinant mitogen activated protein kinase 10 (MAPK10), and MAPK11] were upregulated after the injection of hydrogen-rich water into the Zusanli (ST36) acupoint, whereas Linker for activation of T cells (Lat), signal transducer and activator of transcription 1, and protein kinase C, theta were downregulated. CONCLUSION: The injection of hydrogen-rich water into the Zusanli (ST36) acupoint effectively inhibited the hyperplasia of 4T1 BC cells and enhanced their apoptosis, potentially exerting a therapeutic effect through multiple pathways and targeting various sites.
The author carried out studies on male white rats and examined the effect of imidazol, papaverine and theophyline (drugs affecting the activity of phosphodiesterase), singly or in correlation with GAMA and diazepam on picrotoxic seizure threshold (PSAT). He found that imidazol had various effects on PSAT in accordance with the interval of application and antagonized the effects of GAMA. Papaverine in low doses was ineffective, but in higher doses lowered PSAT; it antagonized the effect of GAMA, elevating the threshold. Papaverine in combination with CAMF antagonized the effect of GAMA, elevating the threshold. Theophyline lowered PSAT; it did not influence the effects of the combined application of GAMA and DIA. He thinks that the obtained changes in PSAT could partly be connected with the mediating influence of imidazol, papaverine and phe theophyline on cerebral phosphodiesterase both after their single application and after examination of their correlations with GAMA or DIA, used singly or in comibination, and partly with their own pharmacologic action.
ClpB, an ATP-dependent molecular chaperone belonging to the Hsp100/Clp subfamily of AAA+ ATPases, plays a crucial role in protein disaggregation, thereby enhancing bacterial survival under stress conditions. Despite its well-conserved function in prokaryotes, the specific contributions of ClpB to the pathogenesis of the ruminant pathogen Mycoplasma bovis remain largely unexplored. In this study, we identified and functionally characterized a ClpB homolog in M. bovis. Biochemical assays confirmed that the recombinant ClpB protein exhibits intrinsic ATPase activity and, in cooperation with the DnaK chaperone system, efficiently mediates protein disaggregation in vitro. Through genome-wide transposon mutagenesis of the M. bovis HB0801 strain, we generated ClpB-deficient mutants that maintained normal growth kinetics and morphology at 37 °C but exhibited significant growth defects under thermal and oxidative stress conditions. Phenotypic analysis demonstrated that ClpB disruption attenuated key virulence traits, including impaired adhesion to host cells, marked reduction in biofilm formation, diminished pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) expression in BoMac cells. Furthermore, the reduced virulence of the ClpB mutant was investigated by DIA proteomic analyses, which revealed that the ClpB mutant strain altered distinct protein expression patterns related to proteostasis, including phosphotransferase system, serine-type peptidase activity, serine hydrolase activity, and chaperone-mediated protein folding that contribute to the stress response and virulence. These findings collectively demonstrate that ClpB serves as a multifunctional virulence determinant in M. bovis, orchestrating stress adaptation, host-pathogen interactions, and pathogenic potential through modulation of both protein quality control systems and virulence-associated pathways.
The increasing scale and complexity of proteomics data demand robust, scalable, and interpretable quality control (QC) frameworks to ensure data reliability and reproducibility. Here, we present pmultiqc, an open-source Python package that standardizes and generates web-based QC reports across multiple proteomics data analysis platforms. Built on top of the widely adopted MultiQC framework, pmultiqc offers specialized modules tailored to mass spectrometry workflows, with full initial support for quantms, DIA-NN, MaxQuant/MaxDIA, FragPipe, and mzIdentML/mzML-based pipelines. The package computes a wide range of QC metrics, including raw intensity distributions, identification rates, retention time consistency, and missing value patterns, and presents them in interactive, publication-ready reports. By leveraging sample metadata in the Sample and Data Relationship Format format, pmultiqc enables metadata-aware QC and introduces, for the first time in proteomics, QC reports and metrics guided by standardized sample metadata. Its modular architecture allows easy extension to new workflows and formats. Alongside comprehensive documentation and examples for running pmultiqc locally or integrated into existing workflows, we offer a cloud-based service that enables users to generate QC reports from their own data or public PRIDE datasets.
The Gm and Km immunoglobulin allotypes are presented, for the first time, for six South American Indian tribes (Baniwa, Kanamari, Kraho, Makiritare, Panoa, and Ticuna) and one Central American tribe (Guaymi). Additional allotype information is presented for five previously reported South American tribes (Cayapo, Piaroa, Trio, Xavante and Yanomama). The distributions of the Gm and Km allotypes among all the tribal populations tested to date are reviewed and evidence is presented for the presence of a north (high)-south(low) cline in Km frequency. The wave theory of the populating of the South American continent was tested by an examination of the distribution of six alleles (Gmax;g, Gma;b0,3,t,Dia,Rx,TFDChi, and 6PGDC), absent in some populations but with polymorphic proportions in others. The present, limited, data failed to confirm the theory.
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that is often diagnosed after curative treatment is no longer feasible. Existing biomarkers, particularly CA19-9, have limited sensitivity and specificity. Plasma proteins that capture tumor-associated biological alterations may therefore provide useful signals for earlier detection. METHODS: Plasma samples from 99 patients with PDAC and 30 healthy controls were analyzed using data-independent acquisition (DIA) proteomics. Differentially expressed proteins were identified using predefined statistical thresholds and further examined by functional enrichment analysis. Selected candidate biomarkers were validated by ELISA in an independent subset. RESULTS: Among 565 quantified plasma proteins, 52 were differentially expressed between PDAC and controls. These proteins were enriched in extracellular processes, cholesterol metabolism, complement and coagulation cascades, and pancreatic secretion pathways. ELISA validation confirmed higher plasma levels of Cathepsin S, CTRB2, MARCO, PIGR, PRDX6, REG1A, Trypsin-2, and PEP-FAP in patients with PDAC compared with healthy controls. ROC analyses showed moderate-to-good discriminatory performance for several candidates, and the MARCO + PEP-FAP model improved classification compared with either marker alone. CONCLUSION: These findings reveal circulating proteins linked to key PDAC-related biological processes and identify eight candidates for further evaluation in multi-protein diagnostic panels. Larger validation studies incorporating clinically relevant disease control groups are warranted to determine their diagnostic specificity and clinical utility.
Two cases of Noca dia asteroides infection were encountered out of 55 kidney transplant patients at Chiba University Hospital. One patient developed an extrapleural abscess and the other had a pulmonary infiltration with chest wall abscess. The patients were successfully treated by surgical drainage of the chest wall abscesses and by oral administration of minocycline. No adverse effects caused by minocycline were observed during the therapy. From 1900, when the first case of Nocardia infection was reported in Japan, there have been 60 cases reported in Japanese literature through 1973, including those we observed. This is the first report on nocardiosis in kidney transplant patients and on successful treatment of nocardiosis with minocycline in Japan.
Changes in the concentration of striatal dopamine (DA) and acetylcholine (ACh) in rats deprived of REM sleep for 10 days were compared with those obtained after a 4 day deprivation procedure. Animals placed on small (7 cm dia.) islands surrounded by water were completely deprived of REM sleep but able to obtain some slow-wave sleep. Concentration of striatal DA was significantly increased after 4 days and 10 days of REM sleep deprivation by 73 and 133%, respectively when compared to controls. Levels of ACh in the striatum were significantly enhanced (by 28%) after 10 day, but failed to show significant change after 4 day REM sleep deprivation procedure. The short term locomotor activity was significantly higher in REM sleep-deprived animals. Our data indicate that REM sleep deprivation results in marked alterations of both cholinergic and dopaminergic mechanisms in the rat striatum.
A morphological analysis was undertaken of both the dispersion characteristics and tissue content of dopamine (DA) microinjected acutely into the brain-stem of the anesthetized rat. 14C-DA, with a specific activity of 56-62 mCi/mMol, was infused unilaterally into the pars compacta of the substantia nigra in one of four test volumes: 0.5, 1.0, 4.0 or 8.0 microliters. The concentration of the 14C-DA solution was 1.0 microCi/microliter, equivalent to 3.01 micrograms/microliters, which was delivered at an injection rate of 1.0 microliter per 45 sec. At an interval of either one min or 15 min following the microinjection, the rat's brain was removed rapidly from its calvarium, flash frozen and then cut in the coronal plane on a freezing microtome in 500 micron slabs. After each of the respective serial slabs was mounted on glass, the Eik Nes-Brizzee trochar technique for the discrete removal of tissue samples was used to obtain 0.5 mm dia. cylindrical plugs of meso-diencephalic tissue at distances from the site of injection ranging from 0.5 to 2.5 mm, center to center. Each sample plug was subsequently solubilized and 14C-DA activity quantitated by liquid scintillation spectrometry. The results show that regardless of volume, the spatial patterning of the microinjected solution assumes a tear-drop or pear shape, not a sphere. Further, as the volume of the injection is increased from 0.5 to 8.0 microliters, the magnitude of the dispersion of 14C-DA is enhanced throughout the surrounding parenchyma, but not in a linear fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
A technique is described in which bundles of 25 mu dia. insulated wires can be stereotaxically implanted for single unit recording in the brain. With this method extremely stable recordings can be obtained lasting many hours and sometimes days. The unit activity recorded with these electrodes has been found comparable with that recorded from rigid etched microelectrodes in conscious animals. The fine wire electrodes have also been found to be durable for at least a month when chronically implanted in cats. The electrode assembly is easy to construct and the electrodes are easily implanted in any region of the brain.