Search

Bone frequently serves as a metastatic site for breast and prostate cancers. Given the potential of low-intensity vibration (LIV) to increase bone health and reduce cancer risk, this study investigated the impact of LIV on cancer cells, as well as noncancer cells such as lymphocytes and peripheral blood mononuclear cells (PBMCs). The results revealed that LIV exposure not only suppressed cancer cell migration but also triggered the generation of induced tumor-suppressing (iTS) cells. Conditioned medium (CM) derived from LIV-treated PBMCs shrank freshly isolated breast and prostate cancer tissues, and when CM was combined with a chemotherapeutic agent, additional antitumor effects were observed. Notably, iTS cell-derived CM hindered the maturation of the receptor activator of nuclear factor-kappa B ligand (RANKL)-stimulated bone-resorbing osteoclasts while promoting the differentiation of bone-forming osteoblasts. Intriguingly, the anticancer effects induced by LIV were replicated by simply shaking a cell-containing tube with a regular tube shaker. Using mass spectrometry-based proteomics, this study revealed enrichment of tumor-suppressing proteins, including enolase 1, moesin (MSN), and aldolase A (ALDOA), which are commonly found in oncogene-activated iTS cells, in LIV-induced CM. Sad1 and UNC-84 domain containing 1 (SUN1), a core component of the linker of the nucleoskeleton and cytoskeleton (LINC) complex, exhibited heightened expression, notably enhancing the response of lymphocytes to LIV. An ex vivo bone cancer model further demonstrated the potent anticancer effects of lymphocyte-derived CM. In conclusion, this study underscores the pivotal role of LIV in preventing bone loss in the tumor microenvironment.

Abstract Circuit-based biomarkers distinguishing the gradual progression of Lewy pathology across synucleinopathies remain unknown. Here, we show that seeding of α-synuclein preformed fibrils in mouse dorsal striatum and motor cortex leads to distinct prodromal-phase cortical dysfunction across months. Our findings reveal that while both seeding sites had increased cortical pathology and hyperexcitability, distinct differences in electrophysiological and cellular ensemble patterns were crucial in distinguishing pathology spread between the two seeding sites. Notably, while beta-band spike-field-coherence reflected a significant increase beginning in Layer-5 and then spreading to Layer-2/3, the rate of entrainment and the propensity of stochastic beta-burst dynamics was markedly seeding location-specific. This beta dysfunction was accompanied by gradual superficial excitatory ensemble instability following cortical, but not striatal, preformed fibrils injection. We reveal a link between Layer-5 dendritic vulnerabilities and translaminar beta event dysfunction, which could be used to differentiate symptomatically similar synucleinopathies.

Atrazine (ATZ) is an herbicide commonly used on crops in the Midwestern US and other select global regions. The US Environmental Protection Agency ATZ regulatory limit is 3 parts per billion (ppb; µg/L), but this limit is often exceeded. ATZ has a long half-life, is a common contaminant of drinking water sources, and is indicated as an endocrine disrupting chemical in multiple species. The zebrafish was used to test the hypothesis that an embryonic parental ATZ exposure alters protein levels leading to modifications in morphology and behavior in developing progeny. Zebrafish embryos (F1) were collected from adults (F0) exposed to 0, 0.3, 3, or 30 ppb ATZ during embryogenesis. Differential proteomics, morphology, and behavior assays were completed with offspring aged 120 or 144 h with no additional chemical treatment. Proteomic analysis identified differential expression of proteins associated with neurological development and disease; and organ and organismal morphology, development, and injury, specifically the skeletomuscular system. Head length and ratio of head length to total length was significantly increased in the F1 of 0.3 and 30 ppb ATZ groups (p

Bacitracin Methylene Disalicylate (BMD), as a feed additive to poultry diets, enhances digestion, prevents Salmonella enteritidis (SE) colonization, and treats current infections. The objective of this study was to utilize a quantitative proteomic approach to determine the effect of BMD feed additive on broiler chickens challenged with SE in the spleen proteome. At 1 d of age, chicks were randomly allocated into four groups: control with and without SE challenge (CON, n = 60; CON-SE, n = 60), BMD with and without SE challenge (BMD, n = 60; BMD-SE, n = 60). Birds in the CON-SE and BMD-SE treatment were administered SE inoculum by oral gavage. On day three and day seven post-gavage, the spleen was collected aseptically from birds in each treatment group (CON, n = 4/day; CON-SE, n = 4/day; BMD, n = 4/day; BMD-SE, n = 4/day). Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed an increased abundance of 115 proteins and decreased of 77 due to the BMD. Proteins that decreased in abundance were enriched for fibrinogen complex and extracellular space, whereas proteins that increased in abundance were enriched for proteasome-mediated ubiquitin-dependent protein catabolic process and mitochondrion. Analysis of the interaction between BMD and the Salmonella challenge found 230 differentially abundant proteins including proteins associated with RNA binding, spliceosome, protein transport, and cell adhesion among the upregulated proteins, and those associated with protein folding, carbon metabolism, biosynthesis of nucleotide sugars, response to oxidative stress, positive regulation of NIK/NF-kappaB signaling, and inflammatory response among the downregulated proteins. The impact of BMD treatment on spleen proteome indicates an anti-apoptotic effect. BMD also modified the response of the spleen to the SE challenge with a marked decrease in proteins that prompt cytokine synthesis and an increase in proteins involved in the selective removal of unfolded proteins.

Abstract Background Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. Results Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of − 5.4% and − 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. Conclusions Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Despite the advance in medications in the past decade, aggressive breast cancer such as triple-negative breast cancer is difficult to treat. Here, we examined a counter-intuitive approach to converting human bone marrow-derived mesenchymal stem cells (MSCs) into induced tumor-suppressing cells by administering YS49, a PI3K/Akt activator. Notably, PI3K-activated MSCs generated tumor-suppressive proteomes, while PI3K-inactivated MSCs tumor-promotive proteomes. In a mouse model, the daily administration of YS49-treated MSC-derived CM decreased the progression of primary mammary tumors as well as the colonization of tumor cells in the lung. In the ex vivo assay, the size of freshly isolated human breast cancer tissues, including estrogen receptor positive and negative as well as human epidermal growth factor receptor 2 (HER2) positive and negative, was decreased by YS49-treated MSC-derived CM. Hsp90ab1 was enriched in CM as an atypical tumor-suppressing protein and immunoprecipitated a non-muscle myosin, Myh9. Extracellular Hsp90ab1 and Myh9 exerted the anti-tumor action and inhibited the maturation of bone-resorbing osteoclasts. Collectively, this study demonstrated that the activation of PI3K generated tumor-suppressive proteomes in MSCs and supported the possibility of using patient-derived MSCs for the treatment of breast cancer and bone metastasis.

Abstract Plant growth under spectrally-enriched low light conditions leads to adjustment in the relative abundance of the two photosystems in an acclimatory response known as photosystem stoichiometry adjustment. Adjustment of photosystem stoichiometry improves the quantum efficiency of photosynthesis but how this process perceives light quality changes and how photosystem amount is regulated remain largely unknown. By using a label-free quantitative mass spectrometry approach in Arabidopsis here we show that photosystem stoichiometry adjustment is primarily driven by the regulation of photosystem I content and that this forms the major thylakoid proteomic response under light quality. Using light and redox signaling mutants, we further show that the light quality-responsive accumulation of photosystem I gene transcripts and proteins requires phytochrome B photoreceptor but not plastoquinone redox signaling as previously suggested. In far-red light, the increased acceptor side limitation might deplete active photosystem I pool, further contributing to the adjustment of photosystem stoichiometry.

Abstract The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic β-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced IP3 receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO and IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

The objective of this study was to characterize the major proteomes and metabolites in beef exudate and determine their relationship to color and oxidative quality of beef muscles. Beef loin (LD) and tenderloin (PM) muscles were cut into sections, individually vacuum-packaged, and aged for 9, 16 and 23 days at 2 °C. Following aging, beef exudates were collected and analyzed for both proteomics and metabolomics profiles. Proteome analysis indicated clustering by muscle types, while metabolomics profiling further clustered the samples based on the aging periods. The PM exudate had a greater concentration of oxidative enzymes, while the LD exudate contained more glycolytic enzymes. Greater lipid, nucleotide, carnitine and glucoside metabolites were observed in LD and 23d exudates. HSP70 and laminin proteins, together with glucosides metabolites, were correlated to muscle oxidative stability. The results indicated that meat exudate could be a viable analytical matrix to determine changes in quality attributes of meat with aging.

Summary: Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Background Constant exposure of human gingival fibroblasts (HGFs) to oral pathogens trigger selective immune responses. Recently, the activation of immune response to cyclic dinucleotides (CDNs) via STING has come to the forefront. Reports show that other proteins outside the STING-TBK1-IRF3 axis respond to CDNs but a global view of impacted proteome in diverse cells is lacking. HGFs are constantly exposed to bacterial-derived cyclic-di-adenosine monophosphate (c-di-AMP) and cyclic-di-guanosine monophosphate (c-di-GMP). AIM To understand the response of HGFs to bacterial-derived CDNs, we carried out a global proteomics analysis of HGFs treated with c-di-AMP or c-di-GMP. Methods The expression levels of several proteins modulated by CDNs were examined. Results Interferon signaling proteins such as Ubiquitin-like protein ISG15 (ISG15), Interferon-induced GTP-binding protein Mx1 (MX1), Interferon-induced protein with tetratricopeptide repeats (IFIT) 1 (IFIT1), and (IFIT3) were significantly upregulated. Interestingly, other pathways not fully characterized to be regulated by CDNs, such as necroptosis signaling, iron homeostasis signaling, protein ubiquitination, EIF2 signaling, sumoylation and nucleotide excision repair pathways were also modulated by the bacterial-derived CDNs. Conclusion This study has added to the increasing appreciation that beyond the regulation of cytokine production via STING, cyclic dinucleotides also broadly affect many critical processes in human cells.

Background: A wide range of disorders can be detected in the urine. Tumor-modifying proteins in the urine may serve as a diagnostic tool for cancer patients and the alterations in their profiles may indicate efficacies of chemotherapy, radiotherapy, and surgery. Methods: We focused on urinary proteomes of patients with prostate cancer and identified tumor-modifying proteins in the samples before and after prostatectomy. Protein array analysis was conducted to evaluate a differential profile of tumor-promoting cytokines, while mass spectrometry-based global proteomics was conducted to identify tumor-suppressing proteins. Results: The result revealed striking differences by prostatectomy. Notably, the urine from the post-prostatectomy significantly decreased the tumorigenic behaviors of prostate tumor cells as well as breast cancer cells. We observed that angiogenin, a stimulator of blood vessel formation, was reduced in the post-prostatectomy urine. By contrast, the levels of three cell-membrane proteins such as prostasin (PRSS8), nectin 2 (PVRL2), and nidogen 1 (NID1) were elevated and they acted as extracellular tumor-suppressing proteins. These three proteins, given extracellularly, downregulated tumorigenic genes such as Runx2, Snail, and transforming growth factor beta and induced apoptosis of tumor cells. However, the role of NID1 differed depending on the location, and intracellular NID1 was tumorigenic and reduced the percent survival. Conclusions: This study demonstrated that prostatectomy remarkably altered the profile of urinary proteomes, and the post-prostatectomy urine provided tumor-suppressive proteomes. The result sheds novel light on the dynamic nature of the urinary proteomes and a unique strategy for predicting tumor suppressors.

Despite many studies on host or viral gene expression, how the cellular proteome responds to internal or external cues during the infection process remains unclear. In this study, we used a Hepatitis B Virus (HBV) replication model and performed proteomic analyses to understand how HBV evades innate immunity as a function of cell cycle progression. Specifically, we performed proteomic analyses of HBV-replicating cells in G1/S and G2/M phases, as a function of IFN-α treatment. We identified that the conserved LSm (Like-Sm1-8) proteins were differentially regulated in HBV replicating cells treated with IFN-α. Specifically, in G2/M phase, IFN-α increased protein level of LSm1, the unique subunit of cytoplasmic LSm1-7 complex involved in mRNA decay. By contrast, IFN-α decreased LSm8, the unique subunit of nuclear LSm2-8 complex, a chaperone of U6 spliceosomal RNA, suggesting the cytoplasmic LSm1-7 complex is antiviral, whereas the nuclear LSm2-8 complex is pro-viral. In HBV replication and infection models, siRNA-mediated knockdown of LSm1 increased all viral RNAs. Conversely, LSm8 knockdown reduced viral RNA levels, dependent on N6-adenosine methylation (m6A) of the epsilon stem-loop at the 5′ end of pre-Core/pregenomic (preC/pg) RNA. Methylated RNA immunoprecipitation (MeRIP) assays demonstrated reduced viral RNA methylation by LSm8 knockdown, dependent on the 5’ m6A modification, suggesting the LSm2-8 complex has a role in mediating this modification. Interestingly, splicing inhibitor Cp028 acting upstream of the LSm2-8 complex suppressed viral RNA levels without reducing the 5’ m6A modification. This observation suggests Cp028 has novel antiviral effects, likely potentiating IFN-α-mediated suppression of HBV biosynthesis.

Mechanisms governing adaptation of breast cancer to the brain metastatic microenvironment are unclear. Here, the authors use RNA-sequencing and Drosophila screening to identify Rab11b-mediated endosomal recycling as a unique mechanism for adaptation to a challenging metastatic microenvironment, which can be exploited by repurposing statins.

Photoautotrophic microorganisms are increasingly explored for the conversion of atmospheric carbon dioxide into biomass and valuable products. The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway for net CO2 fixation within oxygenic photosynthetic organisms. The cyanobacteria, Synechocystis sp. PCC 6803, is a model organism for the study of photosynthesis and a platform for many metabolic engineering efforts. The CBB cycle is regulated by complex mechanisms including enzymatic abundance, intracellular metabolite concentrations, energetic cofactors and post-translational enzymatic modifications that depend on the external conditions such as the intensity and quality of light. However, the extent to which each of these mechanisms play a role under different light intensities remains unclear. In this work, we conducted non-targeted proteomics in tandem with isotopically non-stationary metabolic flux analysis (INST-MFA) at four different light intensities to determine the extent to which fluxes within the CBB cycle are controlled by enzymatic abundance. The correlation between specific enzyme abundances and their corresponding reaction fluxes is examined, revealing several enzymes with uncorrelated enzyme abundance and their corresponding flux, suggesting flux regulation by mechanisms other than enzyme abundance. Additionally, the kinetics of 13C labeling of CBB cycle intermediates and estimated inactive pool sizes varied significantly as a function of light intensity suggesting the presence of metabolite channeling, an additional method of flux regulation. These results highlight the importance of the diverse methods of regulation of CBB enzyme activity as a function of light intensity, and highlights the importance of considering these effects in future kinetic models.

Objective: To analyze the effect of electrical pulses (EP) with tomato lipophilic extract (TLE) on human, triple negative breast cancer (TNBC) cell (MDA-MB-231) and human epithelial, non-cancerous breast cell (MCF-10A) and to gain insights into the mechanisms of action. Methods: We prepared TLE using the solvent extraction method. We identified the constituents of TLE using LC-MS analysis. TLE with the dose of 250 μg/ml was combined with 800 V/cm, 100 μs, eight electrical pulses to treat cancerous MDA-MB-231 and non-cancerous MCF-10A breast cells, and their survivability was studied. We studied the proteins responsible for TLE's anticancer activity in TNBC cells using label-free, quantitative proteomics study. To understand the relationship between changes protein and transcription level, we did a real-time quantitative PCR (RT-qPCR) study. We detected Hydrogen Peroxide (H2O2) levels of both untreated and TLE/EP treated cells to determine how oxidative stress regulates various intracellular pathways. Results: TLE comprises fatty acids that heighten MDA-MB-231 cell proliferation. However, electrical pulses application nullifies these cell proliferative effects of fatty acids, as evident by the significant MDA-MB-231 cell viability reduction for TLE+EP treatment. Tomato extract, electrical pulses, and combinational treatment did not affect normal breast MCF-10A cells. A total of 1877 proteins were identified from the proteomics study. Conclusion: Applying EP in combination with TLE suppresses and reverses back the proliferative effects caused by TLE. EP and TLE+EP treatments increase hydrogen peroxide reactive oxygen species production in TNBC to inflict cell death due to excessive oxidative stress. Proteins, such as PCNA, LDHB, and ENO1, were downregulated in EP treatment, and it was confirmed with the mRNA levels by qPCR results. Our findings render innovative perceptions into the mechanisms of electrical pulses with TLE. Significance: Our research serves as a technological platform capable of identifying the set of proteins responsible for minimizing TNBC cell proliferation when treated with TLE+EP.

Nanoparticles (NPs) interact with biomolecules by forming a biocorona (BC) on their surface after introduction into the body and alter cell interactions and toxicity. Metabolic syndrome (MetS) is a prevalent condition and enhances susceptibility to inhaled exposures. We hypothesize that distinct NP-biomolecule interactions occur in the lungs due to MetS resulting in the formation of unique NP-BCs contributing to enhanced toxicity. Bronchoalveolar lavage fluid (BALF) was collected from healthy and MetS mouse models and used to evaluate variations in the BC formation on 20 nm iron oxide (Fe3O4) NPs. Fe3O4 NPs without or with BCs were characterized for hydrodynamic size and zeta potential. Unique and differentially associated proteins and lipids with the Fe3O4 NPs were identified through proteomic and lipidomic analyses to evaluate BC alterations based on disease state. A mouse macrophage cell line was utilized to examine alterations in cell interactions and toxicity due to BCs. Exposures to 6.25, 12.5, 25, and 50 μg/mL of Fe3O4 NPs with BCs for 1 h or 24 h did not demonstrate overt cytotoxicity. Macrophages increasingly associated Fe3O4 NPs following addition of the MetS BC compared to the healthy BC. Macrophages exposed to Fe3O4 NPs with a MetS-BC for 1 h or 24 h at a concentration of 25 μg/mL demonstrated enhanced gene expression of inflammatory markers: CCL2, IL-6, and TNF-α compared to Fe3O4 NPs with a healthy BC. Western blot analysis revealed activation of STAT3, NF-κB, and ERK pathways due to the MetS-BC. Specifically, the Jak/Stat pathway was the most upregulated inflammatory pathway following exposure to NPs with a MetS BC. Overall, our study suggests the formation of distinct BCs due to NP exposure in MetS, which may contribute to exacerbated inflammatory effects and susceptibility.

Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used a multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites, of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed a significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids, such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.

Increased secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), is often associated with adipose tissue dysregulation, which often accompanies obesity. High levels of TNFα have been linked to the development of insulin resistance in several tissues and organs, including skeletal muscle and the liver. In this study, we examined the complex regulatory roles of TNFα in murine hepatocytes utilizing a combination of global proteomic and phosphoproteomic analyses. Our results show that TNFα promotes extensive changes not only of protein levels, but also the dynamics of their downstream phosphorylation signaling. We provide evidence that TNFα induces DNA replication and promotes G1/S transition through activation of the MAPK pathway. Our data also highlight several other novel proteins, many of which are regulated by phosphorylation and play a role in the progression and development of insulin resistance in hepatocytes.

Muscle tissue serves as a key nutrient reservoir that dairy cows utilize to meet energy and amino acid requirements for fetal growth and milk production. Circadian clocks act as homeostatic regulators so that organisms can anticipate regular environmental changes. The objective of this study was to use liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine how chronic circadian disruption in late gestation affected the muscle tissue proteome. At five weeks before expected calving (BEC), multiparous Holstein cows were assigned to either a control (CON, n = 8) or a 6 h forward phase shift (PS, n = 8) of the light–dark cycle every 3 days. At calving, all animals were exposed to CON light–dark cycles. Muscle biopsies were collected from longissimus dorsi muscles at 21 days BEC and at 21 days postpartum (PP). At p < 0.1, 116 and 121 proteins were differentially abundant between PS and CON at 21 days BEC and 21 days PP, respectively. These proteins regulate beta oxidation and glycolysis. Between pregnancy and lactation, 134 and 145 proteins were differentially abundant in CON and PS cows, respectively (p < 0.1). At both timepoints, PS cows exhibited an oxidative stress signature. Thus, dairy cattle management strategies that minimize circadian disruptions may ensure optimal health and production performance.

Maternal diet supplementation with pro- and prebiotics is associated with decreased incidence of diarrhea and greater piglet performance. This study investigated the impact adding whole ground oat as a prebiotic, alone or in combination with a probiotic, yeast culture (YC) (Saccharomyces cerevisiae), to sow gestation and lactation rations had on milk protein composition, piglet growth, and incidence of post-weaning diarrhea (PWD). Diets: control (CON), CON + yeast culture (YC) [5 g/kg], CON + oat (15% inclusion rate) (Oat) or CON+ YC [5 g/kg] + Oat (15%) were fed the last 30 days of gestation and throughout lactation (18–21 days). Shotgun proteome analysis of day 4 and 7 postpartum milk found 36 differentially abundant proteins (P-adj < 0.1) in both Oat and YC supplemented sows relative to CON. Notable was the increased expression of antimicrobial proteins, lactoferrin and chitinase in milk of Oat and YC sows compared to CON. The levels of IgA, IgM (within colostrum and milk) and IgG (within milk) were similar across treatments. However, colostral IgG levels in Oat-supplemented sows were significantly lower (p < 0.05) than that of the control sows, IgG from Oat-supplemented sows displayed greater reactivity to E. coli-antigens compared with CON and YC. Piglets from sows that consumed Oat alone or in combination weighed significantly more (p < 0.05) at birth compared to CON and YC. However, piglets in the Oat + YC group weighed less at weaning and had the lowest weight gain (p < 0.05) postweaning, compared with CON. Taken together with the observation that piglets of either YC- or Oat-fed sows had less PWD compared to CON and YC+ Oat suggests that Oat or YC supplementation positively impacts piglets through expression of certain milk-associated immune and antimicrobial proteins.

Proximity biotinylation was developed to detect physiologically relevant protein–protein interactions in living cells. In this method, the protein of interest is tagged with a promiscuous biotin ligase, such as BioID or BioID2, which produces activated biotin that reacts with nearby proteins; these proteins can subsequently be purified and identified by mass spectrometry. Here we report a novel modification of this technique by combining it with a self-associating split-GFP system in which we exploit the high-affinity interaction between GFP1–10 and GFP11 to recruit BioID2 to the protein of interest. As a test case, we fused GFP11 to clathrin light chain (CLTB) and BioID2 to GFP1–10. Co-expression of GFP11-CLTB and BioID2-GFP1–10 yielded a green fluorescent complex that co-localized with clathrin heavy chain. To facilitate removal of non-specifically biotinylated proteins, we generated an inducible cell line expressing BioID2-GFP1–10. Proximity biotinylation in this cell line with GFP11-CLTB yielded a higher percentage of biologically relevant interactions than direct fusion of BioID2 to CLTB. Thus, this system can be used to monitor expression and localization of BioID bait proteins and to identify protein–protein interactions.

Insulin resistance is an indication of early stage Type 2 diabetes (T2D). Insulin resistant adipose tissues contain higher levels of insulin than the physiological level, as well as higher amounts of intracellular tumor necrosis factor-α (TNF-α) and other cytokines. However, the mechanism of insulin resistance remains poorly understood. To better understand the roles played by insulin and TNF-α in insulin resistance, we performed proteomic analysis of differentiated 3T3-L1 adipocytes treated with insulin (Ins), TNF-α (TNF), and both (Ins + TNF). Out of the 693 proteins identified, the abundances of 78 proteins were significantly different (p < 0.05). Carnitine parmitoyltransferase-2 (CPT2), acetyl CoA carboxylase 1 (ACCAC-1), ethylmalonyl CoA decarboxylase (ECHD1), and methylmalonyl CoA isomerase (MCEE), enzymes required for fatty acid β-oxidation and respiratory electron transport, and β-glucuronidase, an enzyme responsible for the breakdown of complex carbohydrates, were down-regulated in all the treatment groups, compared to the control group. In contrast, superoxide dismutase 2 (SOD2), protein disulfide isomerase (PDI), and glutathione reductase, which are the proteins responsible for cytoskeletal structure, protein folding, degradation, and oxidative stress responses, were up-regulated. This suggests higher oxidative stress in cells treated with Ins, TNF, or both. We proposed a conceptual metabolic pathway impacted by the treatments and their possible link to insulin resistance or T2D.

Background Bacterial cyclic dinucleotides (CDNs), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) upregulate interferon signaling proteins of human gingival fibroblasts (HGFs). However, the simultaneous effect of bacterial CDNs and lipopolysaccharides (LPS) on the HGF proteome is unknown.Aim The aim was to apply an unbiased proteomics approach to evaluate how simultaneous exposure to CDNs and Porphyromonas gingivalis (Pg) LPS affect the global proteome of HGFs.Methods The proteomic responses of HGFs were examined under three different treatment conditions (c-di-AMP+Pg LPS, c-di-GMP+Pg LPS, and Pg LPS alone) by label-free quantitative mass spectrometry analysis.Results Simultaneous exposure to CDNs and Pg LPS significantly upregulated innate immunity-related and interferon signaling-related proteins, such as ubiquitin-like protein ISG15 (ISG15), deoxynucleoside triphosphate triphosphohydrolase (SAMHD1), interferon regulatory factor 9 (IRF-9), interferon-induced GTP-binding protein Mx (MX)1, and MX2. Interferon signaling pathway was the most significantly regulated canonical pathway in both CDN treatment groups.Conclusion Simultaneous exposure to CDNs and Pg LPS stimulates the periodontal immune response by activating the anti-microbial cellular responses of HGFs with some notable differences from individual exposures.