Existence and importance of Na+K+-ATPase in the plasma membrane of boar spermatozoa.

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218 ± 23% and 245 ± 38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and ß subunits (isoforms α1, α3, ß1, ß2, and ß3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0 h control (33.0 ± 2.6% vs. 19.2 ± 2.6% capacitated sperm, respectively; p = 0.014) and altered the ß2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation.

Identification of SARS-CoV-2 biomarkers in saliva by transcriptomic and proteomics analysis.

The detection of SARS-CoV-2 biomarkers by real time PCR (rRT-PCR) has shown that the sensitivity of the test is negatively affected by low viral loads and the severity of the disease. This limitation can be overcome by the use of more sensitive approaches such as mass spectrometry (MS), which has not been explored for the detection of SARS-CoV-2 proteins in saliva. Thus, this study aimed at assessing the translational applicability of mass spectrometry-based proteomics approaches to identify viral proteins in saliva from people diagnosed with COVID-19 within fourteen days after the initial diagnosis, and to compare its performance with rRT-PCR. After ethics approval, saliva samples were self-collected by 42 COVID-19 positive and 16 healthy individuals. Samples from people positive for COVID-19 were collected on average on the sixth day (± 4 days) after initial diagnosis. Viable viral particles in saliva were heat-inactivated followed by the extraction of total proteins and viral RNA. Proteins were digested and then subjected to tandem MS analysis (LC-QTOF-MS/MS) using a data-dependent MS/MS acquisition qualitative shotgun proteomics approach. The acquired spectra were queried against a combined SARS-CoV-2 and human database. The qualitative detection of SARS-CoV-2 specific RNA was done by rRT-PCR. SARS-CoV-2 proteins were identified in all COVID-19 samples (100%), while viral RNA was detected in only 24 out of 42 COVID-19 samples (57.1%). Seven out of 18 SARS-CoV-2 proteins were identified in saliva from COVID-19 positive individuals, from which the most frequent were replicase polyproteins 1ab (100%) and 1a (91.3%), and nucleocapsid (45.2%). Neither viral proteins nor RNA were detected in healthy individuals. Our mass spectrometry approach appears to be more sensitive than rRT-PCR for the detection of SARS-CoV-2 biomarkers in saliva collected from COVID-19 positive individuals up to 14 days after the initial diagnostic test. Based on the novel data presented here, our MS technology can be used as an effective diagnostic test of COVID-19 for initial diagnosis or follow-up of symptomatic cases, especially in patients with reduced viral load.

Transcriptional activation of budding yeast DDI2/3 through chemical modifications of Fzf1.

DDI2 and DDI3 (DDI2/3) are two identical genes in Saccharomyces cerevisiae encoding cyanamide (CY) hydratase. They are not only highly induced by CY, but also by a DNA-damaging agent methyl methanesulfonate (MMS), and the regulatory mechanism is unknown. In this study, we performed a modified genome-wide genetic synthetic array screen and identified Fzf1 as a zinc-finger transcriptional activator required for CY/MMS-induced DDI2/3 expression. Fzf1 binds to a DDI2/3 promoter consensus sequence CS2 in vivo and in vitro, and this interaction was enhanced in response to the CY treatment. Indeed, experimental over production of Fzf1 alone was sufficient to induce DDI2/3 expression; however, CY and MMS treatments did not cause the accumulation or apparent alteration in migration of cellular Fzf1. To test a hypothesis that Fzf1 is activated by covalent modification of CY and MMS, we performed mass spectrometry of CY/MMS-treated Fzf1 and detected a few modified lysine residues. Amino acid substitutions of these residues revealed that Fzf1-K70A completely abolished MMS-induced and reduced CY-induced DDI2/3 expression, indicating that the Fzf1-K70 methylation activates Fzf1. This study collectively reveals a novel regulatory mechanism by which Fzf1 is activated by chemical modifications and in turn induces the expression of its target genes for detoxification.

A ß-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly.

BACKGROUND: Seven structurally related ß-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 ß-lactamases. METHODS: N. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. ß-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 ß-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 ß-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 ß-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding.

Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS-PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein-protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3-hNABP1/2-C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown.

The association between endotoxin in house dust with atopy and exercise-induced bronchospasm in children with asthma.

BACKGROUND: Studies have reported protective and adverse associations between microbial exposure and childhood asthma. However, among children with asthma the relationships between endotoxin and exercise-induced bronchospasm (EIB) is less clear. OBJECTIVE: We investigated the association between exposure to endotoxin in house dust with atopy and EIB in children with asthma. METHODS: A cross-sectional survey was conducted among schoolchildren (aged 7-17 years) in the province of Saskatchewan, Canada. A subpopulation with asthma (n = 116) were identified from 335 participants using a validated asthma algorithm. We determined atopy among the asthma subpopulation by skin prick testing (SPT) while EIB was evaluated using exercise challenge testing (ECT). Dust samples were collected from mattress and play area floors, and endotoxin was measured in dust extracts. Logistic regression analyses were used to explore associations between endotoxin with atopy and EIB. RESULTS: Among the 116 children with asthma, 99 completed SPT and all had completed ECT. Of these, 71/99 (71.7%) were atopic and 26/116 (22.4%) had EIB. Exposure to high play area endotoxin concentration [adjusted odds ratio (aOR) = 0.15, 95% CI: 0.03-0.85] and load (aOR = 0.11, 95% CI: 0.02-0.73) were negatively associated with atopy. In contrast, EIB was positively associated with high mattress endotoxin concentration (aOR = 6.01, 95% CI: 1.20-30.13). CONCLUSION: Indoor microbial endotoxin exposure has varied associations with atopy and exercise-induced bronchospasm among children with asthma.

C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2.

BACKGROUND: Human Nucleic Acid Binding Protein 1 and 2 (hNABP1 and 2; also known as hSSB2 and 1, respectively) are two newly identified single-stranded (ss) DNA binding proteins (SSB). Both NABP1 and NABP2 have a conserved oligonucleotide/oligosaccharide-binding (OB)-fold domain and a divergent carboxy-terminal domain, the functional importance of which is unknown. METHODS: Recombinant hNABP1/2 proteins were purified using affinity and size exclusion chromatography and their identities confirmed by mass spectrometry. Oligomerization state was checked by sucrose gradient centrifugation. Secondary structure was determined by circular dichroism spectroscopy. Nucleic acid binding ability was examined by EMSA and ITC. RESULTS: Both hNABP1 and hNABP2 exist as monomers in solution; however, hNABP2 exhibits anomalous behavior. CD spectroscopy revealed that the C-terminus of hNABP2 is highly disordered. Deletion of the C-terminal tail diminishes the DNA binding ability and protein stability of hNABP2. Although both hNABP1 and hNABP2 prefer to bind ssDNA than double-stranded (ds) DNA, hNABP1 has a higher affinity for ssDNA than hNABP2. Unlike hNABP2, hNABP1 protein binds and multimerizes on ssDNA with the C-terminal tail responsible for its multimerization. Both hNABP1 and hNABP2 are able to bind single-stranded RNA, with hNABP2 having a higher affinity than hNABP1. CONCLUSIONS: Biochemical evidence suggests that the C-terminal region of NABP1 and NABP2 is essential for their functionality and may lead to different roles in DNA and RNA metabolism. GENERAL SIGNIFICANCE: This is the first report demonstrating the regulation and functional properties of the C-terminal domain of hNABP1/2, which might be a general characteristic of OB-fold proteins.

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

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.

Intracellular Ca2+ oscillation frequency and amplitude modulation mediate epithelial apical and basolateral membranes crosstalk.

Since the early seminal studies on epithelial solute transport, it has been understood that there must be crosstalk among different members of the transport machinery to coordinate their activity and, thus, generate localized electrochemical gradients that force solute flow in the required direction that would otherwise be thermodynamically unfavorable. However, mechanisms underlying intracellular crosstalk remain unclear. We present evidence that crosstalk between apical and basolateral membrane transporters is mediated by intracellular Ca2+ signaling in insect renal epithelia. Ion flux across the basolateral membrane is encoded in the intracellular Ca2+ oscillation frequency and amplitude modulation and that information is used by the apical membrane to adjust ion flux accordingly. Moreover, imposing experimentally generated intracellular Ca2+ oscillation modulation causes cells to predictably adjust their ion transport properties. Our results suggest that intracellular Ca2+ oscillation frequency and amplitude modulation encode information on transmembrane ion flux that is required for crosstalk.

Salivary protein homology between humans and dogs: Mass spectrometry-based proteomics analysis.

OBJECTIVE: This benchmark study aimed to investigate sex-related differences based on the identification and characterization of the salivary proteome of healthy male and female dogs using mass spectrometry (MS) technique and a homology-driven approach to analyze salivary proteins in both human and dog species utilizing protein sequence alignment technique. METHODS: Unstimulated whole saliva was collected from 10 healthy Beagles. After processing the samples and determining the total protein content, in-solution protein digestion was performed involving denaturation, reduction of disulfide bonds, alkylation, and removal of interfering compounds. Samples were analyzed using LC-ESI-MS/MS. RESULTS: LC-ESI-MS/MS analysis identified 327 and 341 unique proteins in male and female dog saliva, respectively, of which 318 (97.25 %) in male dogs and 326 (95.60 %) in female dogs were characterized. Abundant shared proteins included albumin, BPI fold-containing family A member 2, and VWFD domain-containing protein. A notable uncharacterized protein, VWFD domain-containing protein, was among the most abundant in both sexes. Comparative analysis of 69 abundant shared proteins indicated an upregulation of CES5A, EFHD, GC, IGHM, LOC100653049, KRT10, LCP1, PGD, TPI1 in male dogs, while LOC100855593 was upregulated in female dogs. In total, 84 % (n = 229/274) and 86 % (n = 235/275) salivary proteins identified in male and female dogs, respectively, were homologous to human proteins, with an overall homology of 86 % (n = 364/423), including 15 with 100 % homology. CONCLUSION: The study revealed clear differences in the salivary proteomics profile of healthy male and female dogs. However, most of the salivary proteins in both male and female dogs showed homology with human salivary proteins. CLINICAL RELEVANCE: The identification of unique salivary proteome profiles in male and female dogs, coupled with substantial homology to human proteins, provides promising biomarkers for health assessment, highlighting its clinical significance for diagnostics and therapeutic exploration not only in veterinary and human dentistry, but across mammalian species.

Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis.

OBJECTIVE: To identify protein biomarkers associated with proinflammatory high-density lipoprotein (HDL) in patients with active rheumatoid arthritis (RA) by proteomic analysis. METHODS: Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze proteins associated with immunoaffinity-purified HDL from plasma obtained from 2 sets of RA patients, 1 with antiinflammatory HDL and 1 with proinflammatory HDL. Proteins were fractionated by Offgel electrophoresis and analyzed using an LC-MS/MS system equipped with a high-capacity high-performance liquid chromatography chip incorporating C18 reverse-phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate the association between select proteins and proinflammatory HDL in a second cohort of RA patients. RESULTS: Seventy-eight proteins were identified in the HDL complexes. The levels of 12 proteins were significantly increased in RA patients with proinflammatory HDL compared to RA patients with antiinflammatory HDL. These proteins included the acute-phase proteins apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, and C9). The associations between proinflammatory HDL and 4 of the proteins were validated in a second RA cohort. CONCLUSION: Our findings indicate that proinflammatory HDL in patients with RA contains a significantly altered proteome, including increased amounts of acute-phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation in active RA, with resultant loss of its antiinflammatory function. The characterization of the biomarkers described herein may identify novel molecular connections that contribute to the higher risk of cardiovascular disease in RA patients.

Comparative Proteomics Analysis of Growth-Primed Adult Dorsal Root Ganglia Reveals Key Molecular Mediators for Peripheral Nerve Regeneration.

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.

From Extermination to Conservation: Historical Records of Shark Presence during the Early and Development Phase of the Greek Fishery.

The lack of historical data on shark presence, distribution, and status in the Eastern Mediterranean undermines efforts to manage and protect their populations. An exhaustive review of anecdotal references related to shark presence during the early and development phase of Greek fisheries (1883-1983) was conducted. In the early-20th century (1912), the first sighting of the presence of a dead shark was reported in the Ionian Sea. Later on, the presence of sharks gradually increased up to 1969, with most records being more frequent for the Aegean Sea, whereas the number of sharks being sighted declined leading up to the middle of 1980s. The increase in shark attacks during the mid-20th century led to a calling for culling of sharks in co-operation with the competent authorities promoting the permission to hunt sharks with firearms and offering rewards for killed individuals. A high number of these observations potentially resulted from shark attacks on people, whereas this is not currently evident. This is an indicator of the lower abundance of sharks in modern times and subsequently an alteration in the way that our current modern society is approaching the protection of such vulnerable species.

Identification of potential plasma protein biomarkers for feline pancreatic carcinoma by liquid chromatography tandem mass spectrometry.

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.

Discovery proteomics for the detection of putative markers for eradication of infection in an experimental model of equine septic arthritis using LC-MS/MS.

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.

An individual-based dataset of carbon and nitrogen isotopic data of Callinectessapidus in invaded Mediterranean waters.

BACKGROUND: The characterisation of functional traits of non-indigenous and invasive species is crucial to assess their impact within invaded habitats. Successful biological invasions are often facilitated by the generalist diet of the invaders which can modify their trophic position and adapt to new ecosystems determining changes in their structure and functioning. Invasive crustaceans are an illustrative example of such mechanisms since their trophic habits can determine important ecological impacts on aquatic food webs. The Atlantic blue crab Callinectessapidus is currently established and considered invasive in the Mediterranean Sea where it has been recorded for the first time between 1947 and 1949. In the last decade, the blue crab colonised most of the eastern and central Mediterranean Sea and the Black Sea and it is currently widening its distribution towards the western region of the basin. NEW INFORMATION: Stable isotope analysis is increasingly used to investigate the trophic habits of invasive marine species. Here, we collated individual measures of the blue crab δ13C and δ15N values and of its potential invertebrate prey into a geo-referenced dataset. The dataset includes 360 records with 236 isotopic values of the blue crab and 224 isotopic data of potential prey collected from five countries and 12 locations between 2014 and 2019. This dataset allows the estimation of the trophic position of the blue crab within a variety of invaded ecosystems, as well as advanced quantitative comparisons of the main features of its isotopic niche.

Proteomic analysis of surface and endosomal membrane proteins from the avian LMH epithelial cell line.

Proteins at the cell surface and within the endocytic pathway are increasingly being recognized for their roles in a wide variety of intercellular interactions. Here we used the inherent hydrophobicity and N-glycosylation of membrane proteins to enrich these proteins from the surface and endosome of avian LMH epithelial cells for mass spectrometric analysis. The cycling of many different types of proteins from the cell surface into the endosome and sometimes back to the surface again makes it appropriate to analyze these two membranous cellular components together. Stringent searches of the International Protein Index (IPI) entries for Gallus gallus identified 318 unique integral membrane proteins (IMPs) (201 bearing N-glycosylation sites), 265 unique membrane-associated proteins (MAPs), and an additional group of 784 non-membrane proteins (NMPs) among TX-114 detergent and aqueous phase-enriched proteins. Capture of N-glycosylated tryptic peptides revealed 36 additional glycoproteins most of which were CD antigens, receptors, and molecules for cell adhesion and immune response. IMPs and MAPs present at the surface and within the endosome included proteins involved in transport (255), metabolism (285), communication (108), adhesion (47), and immune responses (42). Among these were 355 putative uncharacterized and hypothetical IMPs, MAPs, and NMPs for which highly similar annotated sequences were found in standard protein-protein BLAST searches.

DGAT2 stability is increased in response to DGAT1 inhibition in gene edited HepG2 cells.

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.

Lipopolysaccharides induce a RAGE-mediated sensitization of sensory neurons and fluid hypersecretion in the upper airways.

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.

Identification of calnexin as a diacylglycerol acyltransferase-2 interacting protein.

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.