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Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Abstract Thoracic dorsal root ganglia (tDRG) contribute to fluid secretion in the upper airways. Inflammation potentiates DRG responses, but the mechanisms remain under investigation. The receptor for advanced glycation end-products (RAGE) underlies potentiation of DRG responses in pain pathologies; however, its role in other sensory modalities is less understood. We hypothesize that RAGE contributes to electrophysiological and biochemical changes in tDRGs during inflammation. We used tDRGs and tracheas from wild types (WT), RAGE knock-out (RAGE-KO), and with the RAGE antagonist FPS-ZM1, and exposed them to lipopolysaccharides (LPS). We studied: capsaicin (CAP)-evoked currents and action potentials (AP), tracheal submucosal gland secretion, RAGE expression and downstream pathways. In WT neurons, LPS increased CAP-evoked currents and AP generation, and it caused submucosal gland hypersecretion in tracheas from WT mice exposed to LPS. In contrast, LPS had no effect on tDRG excitability or gland secretion in RAGE-KO mice or mice treated with FPS-ZM1. LPS upregulated full-length RAGE (encoded by Tv1-RAGE) and downregulated a soluble (sRAGE) splice variant (encoded by MmusRAGEv4) in tDRG neurons. These data suggest that sensitization of tDRG neurons contributes to hypersecretion in the upper airways during inflammation. And at least two RAGE variants may be involved in these effects of LPS.

Triacylglycerol synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase-2 (DGAT2). DGAT2 is an integral membrane protein that is localized to the endoplasmic reticulum and interacts with lipid droplets. Using BioId, a method to detect proximal and interacting proteins, we identified calnexin as a DGAT2-interacting protein. Co-immunoprecipitation and proximity ligation assays confirmed this finding. We found that calnexin-deficient mouse embryonic fibroblasts had reduced intracellular triacylglycerol levels and fewer large lipid droplets (>1.0 μm2 area). Despite the alterations in triacylglycerol metabolism, in vitro DGAT2 activity, localization and protein stability were not affected by the absence of calnexin.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Injuries to peripheral nerves are frequent, yet no drug therapies are available for effective nerve repair. The slow growth rate of axons and inadequate access to growth factors challenge natural repair of nerves. A better understanding of the molecules that can promote the rate of axon growth may reveal therapeutic opportunities. Molecular profiling of injured neurons at early intervals of injury, when regeneration is at the maximum, has been the gold standard for exploring growth promoters. A complementary in vitro regenerative priming model was recently shown to induce enhanced outgrowth in adult sensory neurons. In this work, we exploited the in vitro priming model to reveal novel candidates for adult nerve regeneration. We performed a whole-tissue proteomics analysis of the in vitro primed dorsal root ganglia (DRGs) from adult SD rats and compared their molecular profile with that of the in vivo primed, and control DRGs. The proteomics data generated are available via ProteomeXchange with identifier PXD031927. From the follow-up analysis, Bioinformatics interventions, and literature curation, we identified several molecules that were differentially expressed in the primed DRGs with a potential to modulate adult nerve regrowth. We then validated the growth promoting roles of mesencephalic astrocyte-derived neurotrophic factor (MANF), one of the hits we identified, in adult rat sensory neurons. Overall, in this study, we explored two growth priming paradigm and shortlisted several candidates, and validated MANF, as potential targets for adult nerve regeneration. We also demonstrate that the in vitro priming model is a valid tool for adult nerve regeneration studies., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Bautista et al.)

In both humans and cats, pancreatic carcinoma is an aggressive cancer with a grave prognosis. Proteomics techniques have successfully identified several blood-based biomarkers of human pancreatic neoplasia. Thus, this study aims to investigate whether similar biomarkers can be identified in the plasma of cats with FePAC by using liquid chromatography tandem mass spectrometry (LC-MS/MS). To facilitate evaluation of the low abundance plasma proteome, a human-based immunodepletion device (MARS-2) was first validated for use with feline plasma. Marked reduction and/or complete removal of albumin and immunoglobulins was confirmed by analysis of electrophoretograms and mass spectral data. Subsequently, plasma collected from 9 cats with pancreatic carcinoma (FePAC), 10 cats with symptomatic pancreatitis, and 10 healthy control cats was immunodepleted and subjected to LC-MS/MS. Thirty-seven plasma proteins were found to be differentially expressed (p < .05 in one-way ANOVA, FC >2 in fold change analysis). Among these proteins, ETS variant transcription factor 4 (p < .05) was overexpressed, while gelsolin (p < .01), tryptophan 2,3-dioxygenase (p < .05), serpin family F member 1 (p < .01), apolipoprotein A-IV (p < .01) and phosphatidylinositol-glycan-specific phospholipase D (p < .05) were down-regulated in cats with FePAC. Further studies on these potential biomarkers are needed to investigate their diagnostic value., (© 2022 John Wiley & Sons Ltd.)

Septic arthritis (SA) is a life-threatening condition in horses, and identifying eradication of infection in equine SA is challenging. This study explored the discovery of putative biomarkers for the eradication of joint infection in horses. We performed proteomics analysis of synovial fluid (SF) and plasma from horses with experimental SA, non-septic lipopolysaccharide-induced arthritis, and controls. The point of eradication of infection in horses with SA was determined previously. We compared spectral intensities between groups as well as before and after the eradication of infection. Twenty-six differentially abundant proteins were identified, which were upregulated in SF of horses with SA compared to the other groups, as well as compared to the same horses post-eradication of infection. In plasma, we did not identify differentially abundant proteins. Differentially abundant proteins in SF were of cellular origin and their biological functions included ubiquitination, signal transduction, apoptosis etc. The difference in their relative abundance between experimental groups was ≥10-fold compared to the abundance expected based on the difference in cell count alone (2-fold). Since most of cells in joints with bacterial infection are neutrophils, we suggest that the variable abundance of neutrophil- and cell-associated proteins represent potential biomarkers of eradication of infection in equine SA. SIGNIFICANCE: Septic arthritis is an important condition in horses, which can be life-threatening. At present, identifying eradication of infection in cases of equine septic arthritis is challenging. In this study, we performed a global proteomics analysis of synovial fluid and plasma in horses with experimental septic arthritis and identified 26 differentially abundant proteins compared to non-septic arthritis and post eradication of infection. The results of this study provide the basis for further characterization of the differentially abundant proteins and identification of clinically relevant biomarkers of septic arthritis in horses., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In eukaryotic organisms, two unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, catalyze the final step of the triacylglycerol biosynthetic pathway. Both enzymes are highly expressed in lipogenic tissues, such as adipose tissue, small intestine and the liver. DGAT2 has a prominent role in hepatocyte lipid metabolism synthesizing triacylglycerols that are utilized for very low-density lipoprotein assembly. However, due to the lack of useful antibodies to detect endogenous DGAT2 protein, it has been difficult to determine how this enzyme functions at the cellular level. We have unsuccessfully tested many commercial antibodies as well as our own "in-house" antibodies. There is currently no evidence that DGAT2 undergoes processing such that antigenic epitopes to these antibodies are removed. As an alternative, many studies have utilized epitope tagged versions of DGAT2 overexpressed in cells. These approaches can provide valuable information about a protein, but can be subject to artifacts, such as mislocalization, misregulation, protein aggregation and abnormal protein-protein interactions. In this study, we used gene editing with CRISPR/Cas9 to add three consecutive FLAG epitopes to the C-terminus of endogenous DGAT2 in HepG2 cells. HepG2 cells, derived from a human hepatocellular carcinoma, have been routinely used as a cell model to study human hepatocyte lipid and lipoprotein metabolism. Using this system allowed us to successfully detect DGAT2 expressed from its endogenous locus in HepG2 cells by immunoblotting with anti-FLAG antibodies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Seven structurally related β-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903., Objectives: To compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 β-lactamases., Methods: N. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. β-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays., Results: N. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow β-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 β-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours., Conclusions: This unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 β-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Triacylglycerol synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase-2 (DGAT2). DGAT2 is an integral membrane protein that is localized to the endoplasmic reticulum and interacts with lipid droplets. Using BioId, a method to detect proximal and interacting proteins, we identified calnexin as a DGAT2-interacting protein. Co-immunoprecipitation and proximity ligation assays confirmed this finding. We found that calnexin-deficient mouse embryonic fibroblasts had reduced intracellular triacylglycerol levels and fewer large lipid droplets (>1.0 μm2 area). Despite the alterations in triacylglycerol metabolism, in vitro DGAT2 activity, localization and protein stability were not affected by the absence of calnexin., Competing Interests: The authors have declared that no competing interests exist.

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-methoxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.