Changes In Protein Content Research Articles

Assessment of Serum Heat Shock Protein (HSP70) and Cortisol Concentration Change in Horses before and after an Acute Exercise

Assessment of Serum Heat Shock Protein (HSP70) and Cortisol Concentration Change in Horses before and after an Acute Exercise

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

EFFECTS OF HIGH-INTENSITY AQUATIC EXERCISE ON INFLAMMATION AND COGNITION IN OLDER ADULTS WITH MCI

Abstract Exercise shows immune benefits in older adults and can reduce the risk of late-life cognitive impairment and dementia. This study sought to explore the effects of high-intensity aquatic-based exercise on older adults with mild cognitive impairment (MCI), specifically by examining changes in inflammatory plasma protein concentrations and cognitive performance. A secondary data analysis was conducted on 20 healthy volunteers (mean age 68.2 ±7.75 years) who participated in 6 months of supervised aquatic exercise training. Participants completed a battery of neuropsychological assessments and whole blood draws at pre- and post-intervention. Plasma protein concentration levels were attained using the Olink Target96 Inflammation panel [Uppsala, Sweden]. Mixed effects models assessed the effects of protein concentration change scores on selected cognitive outcomes, while controlling for age. Results indicated that following the exercise intervention, significant changes in two inflammatory-related proteins (Extracellular newly identified receptor for advanced glycation end-products binding protein [EN-RAGE]; Interleukin-20 receptor subunit alpha [IL-20RA]) were associated with improved verbal memory and processing speed endpoints. Specifically, decreased EN-RAGE concentrations resulted in significantly improved delayed verbal memory scores (parameter estimate= -0.893, p= .023), while increased IL-20RA concentrations led to better task completion (parameter estimate= 2.145, p= .030). These findings demonstrate the cognitive benefits of aquatic-based exercise for older adults and those with MCI, and suggest these effects are mediated by alterations in pro- and anti-inflammatory protein markers such as EN-RAGE and IL-20RA. Larger studies are warranted to replicate these findings, and to further explore the role of activity-induced inflammatory changes on late-life brain health.

Factors affecting total protein and lactoferrin in human milk

The aim of this study was to investigate factors affecting total, true protein and lactoferrin (Lf) concentrations in human milk (HM) and to evaluate the changes in protein concentrations over the course of lactation (first to sixth month postpartum). HM samples were collected from exclusively breastfeeding mothers during six time periods (1–6 months postpartum); 198 breast milk samples were collected in total. The concentrations of total and true protein in HM were determined using the MIRIS human milk analyzer (HMA). The assessment of HM protein content was also performed in skim HM samples and quantified by bicinchoninic methods with the Bicinchoninic Acid Protein Assay Kit. In turn, Lf content in skim HM samples was determined by the enzyme-linked immunosorbent assay (ELISA) in accordance with a slightly modified procedure. In the first month of lactation total protein concentration was negatively correlated with maternal pre-pregnancy BMI (r = − 0.397; p = 0.022), whereas in the third month postpartum, positive correlation with maternal age was found (r = 0.399; p = 0.021). Considering Lf concentration, in the first month of lactation, it was positively correlated with baby’s birth weight (r = 0.514; p = 0.002). In the next months (from second to sixth) no relationships between Lf concentration and maternal and infants’ factors were observed. The concentration of protein and Lf in HM changes dynamically throughout lactation. Maternal and infant characteristics may impact the HM protein and Lf content, especially in the first month postpartum.

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

Evaluating the Effect of Evolving Changes in Serum Biomarkers on the Risk of Progression in Smoldering Multiple Myeloma

Introduction Smoldering multiple myeloma (SMM) is a heterogeneous condition which includes patients with varying risk of progression to active multiple myeloma (MM). The current risk stratification model is based on biomarkers reflecting disease burden at the time of diagnosis, without regard to evolution of biomarkers over time. In this study, we evaluated the dynamic changes in the monoclonal protein (MP) concentration and the free light chain ratio (FLCr), and their relation to risk of progression from SMM to MM. Methods We included all SMM patients managed at Memorial Sloan Kettering Cancer Center between 2002 and 2019, with follow up until end of 2022. Date of diagnosis and progression, as well as baseline laboratory, pathology and imaging data were manually reviewed. Serial FLC and MP were obtained through computerized data extraction. Diagnoses of SMM and MM were defined according to IMWG 2014 criteria and/or starting MM directed therapy. Time to progression (TTP) was assessed using the Kaplan-Meier method, with log-rank tests for comparison between groups. We used multivariate Cox regression to estimate the risk of progression with hazard ratios (HRs) and 95% confidence intervals (CIs). Results A total of 398 patients were included in the study; the median age was 64 years and 55% were men. At baseline, the median M-protein was 1.3 g/dL, and the median FLCr was 9.8. The overall median TTP for the cohort was 94 months during a median follow up time of 65 months. First, we stratified patients into quintiles (Q1-Q5) based on baseline M-protein and FLCr, with the highest risk of progression seen in Q5 for M-protein (defined as MP ≥2.2 g/dL, median TTP 29 months [95% CI: 24-64]) and Q5 FLCr (defined as FLCr ≥26, median TTP 36 months [29-62]), respectively (p<0.001). We compared patient trajectories during the first year of follow up based on the baseline quintiles. Interestingly, among patients who progressed from SMM to MM during the first year (11%, n=42/369), only 43% had a baseline M-protein ≥2.2 g/dL (Q5) and 43% had a FLCr ≥26 (Q5). In fact, among those who progressed, 29% had a baseline M-protein <1.6 g/dL (Q1-Q3), and 26% had baseline FLCr <11.3 (Q1-Q3). The patient trajectories during the first year of follow up based on the M-protein level are characterized in Figure 1. We then analyzed changes in M-protein and FLCr during the first year, and stratified patients into quintiles based on the rate of change. We defined evolving M-protein (eMP) and evolving FLCr (eFLCr) based on the levels of the top quintile for each group, which was an increase in ≥0.3 g/dL for eMP and ≥50% increase for eFCLr. Patients with eMP had an increased risk of progression with a median TTP of 22 months (17-not reached [NR]) after the first year (p<0.001). Patients with eFLCr had a median TTP of 48 months (21-116) after the first year (p<0.001). Recognizing that patients with high baseline M-protein level and FLCr also may have a greater increase, we developed a multivariate model adjusting for baseline levels of M-protein and FLCr. In the multivariate model, we included age, gender, and baseline M-protein ≥2g/dL and FLCr ≥20 from the Mayo 2018 risk score, as well as eMP and eFLCr. Adjusting for baseline risk, patients with eMP (Q5) had a HR of 1.8 (0.98-3.16) of progression and patients with eFLCr (Q5) had a HR of 2.9 (1.4-6) of progression using patients with little to no change in M-protein or FLCr as reference (Table 1). Finally, we did a subgroup analysis evaluating eMP and eFLCr in each baseline risk group separately. Patients with no serum risk markers per Mayo-2018 at baseline (i.e. MP <2g/dL, FLCr <20, n=127) who had both eMP (≥0.3 g/dL increase) and eFLCr (≥50% increase), had a median TTP of 25 months (0.42-NR), patients who had either eMP or eFLCr had a median TTP of 100 months (42-NR), whilst patients with neither eMP nor eFLCr had a median TTP of 201 months (130-NR), (p<0.001). Conclusion In summary, we found that when adjusting for baseline M-protein level and FLCr, evolving changes in M-protein and FLCr were associated with a higher risk of progression from SMM to MM. Interestingly, for patients with low risk by baseline stratification (M-protein <2g/dL and FLCr <20), the presence of eMP (≥0.3 g/dL increase) and eFLCr (≥50% increase) was associated with a median TTP of 25 months, similar to those with a baseline high risk. Thus, in addition to baseline disease burden, dynamic disease evolution should be considered in the risk stratification for patients with SMM.

2 Water-Based Exercise Improves Cognition and Reduces Neurological-Related Plasma Proteins in Older Adults.

Objective:In the United States, Alzheimer’s disease (AD) is the most common cause of dementia and the seventh leading cause of death. Exercise has demonstrated health benefits in older adults and reduces the risk of developing AD. Exploring underlying biological mechanisms of exercise could aid in identifying therapeutic targets to prevent AD progression, especially for high-risk individuals such as those with Mild Cognitive Impairment (MCI). Many studies of dementia focus on memory; however, executive function and processing speed are also vulnerable to the neuropathology that causes AD. This exploratory study aimed to identify potential mechanisms by which physical activity can facilitate change in cognitive functioning in older adults. This was accomplished by investigating correlations between changes in neurology-related plasma proteins and changes in measures of executive function and processing speed after participation in a water-based exercise intervention.Participants and Methods:The sample included 20 older adults with amnestic MCI, ages 55-82 years (mean 68.15 ±7.75). Participants were predominately male (90%), White (70%), and non-Hispanic (85%), with more than high school education (95%). Participants engaged in supervised high-intensity water-based exercise three times per week for six months. Neuropsychological assessments and blood samples were assessed at baseline and after completion of the exercise intervention. Cognitive measures included: the Digit Span subtests from the Wechsler Adult Intelligence Scale, 4th Edition, Trail-Making Test (TMT), Stroop Color Word Test (SCWT), and the Symbol Digit Modalities Test (SDMT). Plasma protein levels were analyzed using the Olink Target 96 Neurology assay (Uppsala, Sweden), selected a priori for the established markers linked to neurobiological processes and diseases. Changes in cognitive measures and protein levels were assessed using paired-sample t-tests, and Pearson’s correlations were calculated for significant findings.Results:Participants’ cognitive performance significantly improved on the SCWT color trial (t = -2.19, p = 0.042) and SDMT (t = -2.17, p = .043). Significant decreases in plasma proteins levels were found for GDNF family receptor alpha-1 ([GFRA1]: t =2.05, p = 0.055), neuroblastoma suppressor tumorigenicity-1 ([NBL1]: t = 2.13, p= .046), and neuropilin-2 ([NRP2]: t = 2.61 p= 0.017). Correlational analyses showed reductions in NBL1 were significantly associated with changes in both SDMT (r = -.61, p = 0.006) and the color trial of SCWT (r = .48, p = .038), and NRP2 was significantly associated with improvement on the SDMT (r = -.46, p = 0.045). GFRA1 was not significantly associated with change on any cognitive measure.Conclusions:In a sample of older adults with MCI, participation in high-intensity water-based exercise led to significant improvements in cognitive function as well as changes in neurological plasma proteome. Improved outcomes in processing speed, attention, visuospatial scanning, and working memory were associated with changes in specific plasma protein concentrations. This highlights potential activity-dependent neurobiological mechanisms that may underlie the cognitive benefits derived from physical activity. Future studies should explore these findings in Randomized Control Trials with a comparative condition and larger sample size.

54 Impact of Ileal Indigestible Protein on Nitrogen Excretion and Fecal Microbiota may be Greater Compared with Total Protein Concentration of Diets in Growing Pigs

Abstract The effect of changes in dietary ileal indigestible protein concentration may be more influential in the amount of nitrogen flowing to the large intestine compared with changes in total dietary protein concentration. For this reason, we hypothesized that an increase in dietary ileal indigestible protein concentration induces an increase in hindgut nitrogen utilization and nitrogen excretion and a shift in fecal microbiota in growing pigs, when compared with pigs given a high total protein diet. Three diets were prepared: 1) standard protein diet based on corn and soybean meal, 2) high-indigestible protein diet in which autoclaved soybean meal replaced normal soybean meal in the first diet, and 3) high protein diet where the inclusion rate of soybean meal was greater than that of the other diets. T-cannulated barrows (n = 18; initial BW = 63.4 ± 8.0 kg) were allotted to the 3 diets in a randomized complete block design with BW as a blocking factor. Pigs were individually housed in pens and the experiment lasted for 23 days. On d 7 and 21, fecal samples were collected by rectal massage for microbiota analysis. Grab samples of feces were collected on d 20 and 21, and ileal digesta were collected on d 22 and 23 for the determination of energy and nitrogen utilization. Energy and nitrogen utilization data were analyzed using the MIXED procedure of SAS using a pig as the experimental unit and a model that included diet as a fixed variable and block as a random variable. For microbiome analysis, R was used. Alpha and beta diversity metrics were analyzed and the difference in abundance of the genus was determined using the DESeq2 function. The high-indigestible protein diet achieved decreased apparent ileal digestibility of nitrogen and resulted in greater ileal indigestible nitrogen concentration (P < 0.05). Apparent total tract digestibility of nitrogen was less (P < 0.05), and correspondingly fecal nitrogen concentration and daily fecal nitrogen output were greater (P < 0.05) in the high-indigestible protein diet. Apparent post-ileal digestibility and hindgut disappearance of nitrogen and gross energy were the greatest (P < 0.05) in the high protein diet. Beta diversity metrics of feces in the high-indigestible protein diet on d 21 were different (q < 0.05) from those in the other two diets, which indicates a shift in fecal microbiota. According to the DESeq2 results, the direction of the microbiota shift induced by the high-indigestible protein diet may have reduced hindgut fiber utilization. In conclusion, an increase in dietary ileal indigestible protein increased nitrogen excretion and shifted fecal microbial communities but did not increase hindgut nitrogen utilization.

Pengaruh Lama Fermentasi Menggunakan Mikroba Ghalkoff terhadap Perubahan Konsentrasi Kandungan Kafein Kopi Robusta Organik Kabupaten Lampung Barat

Coffee is a beverage ingredient not only famous in Indonesia but also famous throughout the world. Caffeine is the most important compound found in coffee, caffeine functions as an element of taste and aroma in coffee beans. Excessive acid and caffeine content in coffee can have an impact on health. This study aims to determine the effect of fermentation time using microbial Ghalkoff on changes in concentration of caffeine, protein and acidity content in organic robusta coffee without reducing the taste of coffee. This research was conducted from July to August 2018 at PT. Ghaly Roelies Indonesia, Kecamatan Kemiling, Bandar Lampung. This study uses a laboratory and Kruskal-Wallis test Non-parametric statistical test based on questionnaire test on the level of preference. The results showed the effect of changes in the concentration of caffeine, protein and acidity levels in organic robusta coffee with control of the initial data resulting in a decrease in the concentration of caffeine by 27%. While the results of the N-Rank organoleptic test with Kruskal-WallisTest showed that administering long-acting organic coffee fermentation using microbial ghalkoff can increase the preference for coffee flavor, aroma and color. Keywords: Caffeine, Fermentation, Kruskal-Wallis Test, Organic Robusta Coffee.

Bacterial cell-size changes resulting from altering the relative expression of Min proteins

The timing of cell division, and thus cell size in bacteria, is determined in part by the accumulation dynamics of the protein FtsZ, which forms the septal ring. FtsZ localization depends on membrane-associated Min proteins, which inhibit FtsZ binding to the cell pole membrane. Changes in the relative concentrations of Min proteins can disrupt FtsZ binding to the membrane, which in turn can delay cell division until a certain cell size is reached, in which the dynamics of Min proteins frees the cell membrane long enough to allow FtsZ ring formation. Here, we study the effect of Min proteins relative expression on the dynamics of FtsZ ring formation and cell size in individual Escherichia coli bacteria. Upon inducing overexpression of minE, cell size increases gradually to a new steady-state value. Concurrently, the time required to initiate FtsZ ring formation grows as the size approaches the new steady-state, at which point the ring formation initiates as early as before induction. These results highlight the contribution of Min proteins to cell size control, which may be partially responsible for the size fluctuations observed in bacterial populations, and may clarify how the size difference acquired during asymmetric cell division is offset.

English

Acute phase proteins (APPs) have been paid attention to as sensitive and reliable biomarkers used for monitoring general health status, diagnosis, prognosis, and treatment strategies of diseases. In this study, changes in some APPs, globulin (Glb), and total protein (TP) concentrations and the correlation of these proteins with lamb weight were investigated in the neonatal period of Awassi lambs (Ovis aries). Repeated blood samples were taken from 59 lambs at 0, 1, 3, 7, 14, 21, and 28 days of age. There were statistical differences in average haptoglobin (Hp), serum amyloid-A (SAA), ceruloplasmin (Cp), and albumin (Alb) concentrations during the neonatal period. The lowest concentrations of both Hp and SAA were measured in precolostrum samples and increased to the highest level on the 3rd day of the neonatal period. Ceruloplasmin concentrations followed a constant course in the samples of the first 3 days and reached the highest level in the 14th day samples. The highest levels of Alb were measured in precolostrum samples, and the lowest levels were detected in the 14th day samples. Acute phase protein concentration changes did not differ significantly by gender except Cp. There was a negative correlation between the weights of lambs and Cp and Alb. It was concluded that the time-dependent change of APPs in the neonatal period could help in understanding the neonatal period of Awassi lambs and that its primary purpose is survival as a physiological part of the adaptation process to the extrauterine environment

A randomized controlled trial to examine the impact of a multi-strain probiotic on self-reported indicators of depression, anxiety, mood, and associated biomarkers.

To examine the efficacy of supplementing with a multi-strain probiotic (MSP) on changes associated with mood, anxiety, and neurotransmitter levels. In a randomized, double-blind, placebo-controlled fashion, 70 healthy men and women (31.0 ± 9.5 years, 173.0 ± 10.4 cm, 73.9 ± 13.8 kg, 24.6 ± 3.5 kg/m2) supplemented with a single capsule of MSP (a total daily dose of 4 × 109 colony forming units [CFU] comprised of a 1 × 109 CFU dose from each of the following strains: Limosilactobacillus fermentum LF16, Lacticaseibacillus rhamnosus LR06, Lactiplantibacillus plantarum LP01, and Bifidobacterium longum 04, Probiotical S.p.A., Novara, Italy) or a maltodextrin placebo (PLA). After 0, 2, 4, and 6 weeks of supplementation and 3 weeks after ceasing supplementation, study participants completed the Beck Depression Inventory (BDI-II), State-Trait Anxiety Inventory (STAI), and Leiden Index of Depression Sensitivity (LEIDS-R) questionnaires and had plasma concentrations of cortisol, dopamine, serotonin, and C-reactive protein determined. BDI, STAI, and total LEIDS-R scores were reduced from baseline (p < 0.05) with MSP supplementation after 4 and 6 weeks of supplementation and 3 weeks after supplementation while no changes (p > 0.05) were reported in PLA. When compared to PLA, MSP scores for state anxiety, trait anxiety, and LEIDS-R (hopeless, aggression, rumination, and total score) were significantly lower (p < 0.05) after supplementation. Plasma serotonin concentrations in MSP were increased from baseline after 6 weeks of supplementation and 3 weeks after ceasing supplementation. No changes (p > 0.05) in plasma dopamine, C-reactive protein, or cortisol concentrations were observed between groups. MSP supplementation resulted in widespread improvements in several questionnaires evaluating mood, anxiety, and depression in young, healthy men and women. MSP supplementation increased serotonin increased after 6 weeks of MSP supplementation with no change in dopamine, C-reactive protein, or cortisol. https://classic.clinicaltrials.gov/ct2/show/NCT05343533, NCT05343533.

Zinc Ions Modulate YY1 Activity: Relevance in Carcinogenesis.

YY1 is widely recognized as an intrinsically disordered transcription factor that plays a role in development of many cancers. In most cases, its overexpression is correlated with tumor progression and unfavorable patient outcomes. Our latest research focusing on the role of zinc ions in modulating YY1's interaction with DNA demonstrated that zinc enhances the protein's multimeric state and affinity to its operator. In light of these findings, changes in protein concentration appear to be just one element relevant to modulating YY1-dependent processes. Thus, alterations in zinc ion concentration can directly and specifically impact the regulation of gene expression by YY1, in line with reports indicating a correlation between zinc ion levels and advancement of certain tumors. This review concentrates on other potential consequences of YY1 interaction with zinc ions that may act by altering charge distribution, conformational state distribution, or oligomerization to influence its interactions with molecular partners that can disrupt gene expression patterns.

Single Cell-ICP-ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response.

An automated and straightforward detection and data treatment strategy for the determination of the protein relative concentration in individual human cells by single cell-inductively coupled plasma-time-of-flight mass spectrometry (sc-ICP-ToF-MS) is proposed. Metal nanocluster (NC)-labeled specific antibodies for the target proteins were employed, and ruthenium red (RR) staining, which binds to the cells surface, was used to determine the number of cell events as well as to evaluate the relative volume of the cells. As a proof of concept, the expression of hepcidin, metallothionein-2, and ferroportin employing specific antibodies labeled with IrNCs, PtNCs, and AuNCs, respectively, was investigated by sc-ICP-ToF-MS in human ARPE-19 cells. Taking into account that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was related to the cell volume (i.e., the cell volume is directly proportional to (Ru intensity)3/2), making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration. The proposed approach is of particular interest in comparing cell cultures subjected to different supplementations. ARPE-19 cell cultures under two stress conditions were compared: a hyperglycemic model and an oxidative stress model. The comparison of the control with treated cells shows not only the mass of analyzed species but also the relative changes in the cell volume and concentration of target proteins, clearly allowing the identification of subpopulations under the respective treatment.

The Effect of Dietary Protein Concentration on the Fecal Microbiome and Serum Concentrations of Gut-Derived Uremic Toxins in Healthy Adult Cats.

The purpose of this study was to evaluate the effect of feeding healthy adult cats with foods containing variable protein concentrations on the fecal microbiome and serum concentrations of the gut-derived uremic toxins indoxyl sulfate, p-cresol sulfate (pCS), and trimethylamine-n-oxide. Twenty healthy young adult cats were randomized into two groups and fed either a low-protein diet (LPD; 7.4 g/100 kcal ME) or a high-protein diet (HPD; 11.0 g/100 kcal ME) for a 12-week period. Serum uremic toxin concentrations were measured via liquid chromatography tandem mass spectrometry, and the fecal microbiome was characterized using shallow sequence shotgun metagenomics. Cats that consumed the HPD had higher pCS concentrations at 8 weeks (p = 0.028) when compared to baseline. After 12 weeks, cats fed the HPD had higher fecal alpha diversity indices at both the taxonomic and functional levels and lower fecal Bifidobacterium relative abundance compared to those cats fed the LPD. In conclusion, a change in diet and dietary protein concentration shifted the fecal microbial community and microbial function. Feeding cats a high amount of protein increased serum concentrations of the uremic toxin pCS; however, the effect was short-lived.

Serum proteome profiles in patients treated with targeted temperature management after out-of-hospital cardiac arrest

BackgroundDefinition of temporal serum proteome profiles after out-of-hospital cardiac arrest may identify biological processes associated with severe hypoxia–ischaemia and reperfusion. It may further explore intervention effects for new mechanistic insights, identify candidate prognostic protein biomarkers and potential therapeutic targets. This pilot study aimed to investigate serum proteome profiles from unconscious patients admitted to hospital after out-of-hospital cardiac arrest according to temperature treatment and neurological outcome.MethodsSerum samples at 24, 48, and 72 h after cardiac arrest at three centres included in the Target Temperature Management after out-of-hospital cardiac arrest trial underwent data-independent acquisition mass spectrometry analysis (DIA-MS) to find changes in serum protein concentrations associated with neurological outcome at 6-month follow-up and targeted temperature management (TTM) at 33 °C as compared to 36 °C. Neurological outcome was defined according to Cerebral Performance Category (CPC) scale as “good” (CPC 1–2, good cerebral performance or moderate disability) or “poor” (CPC 3–5, severe disability, unresponsive wakefulness syndrome, or death).ResultsOf 78 included patients [mean age 66 ± 12 years, 62 (80.0%) male], 37 (47.4%) were randomised to TTM at 36 °C. Six-month outcome was poor in 47 (60.3%) patients. The DIA-MS analysis identified and quantified 403 unique human proteins. Differential protein abundance testing comparing poor to good outcome showed 19 elevated proteins in patients with poor outcome (log2-fold change (FC) range 0.28–1.17) and 16 reduced proteins (log2(FC) between − 0.22 and − 0.68), involved in inflammatory/immune responses and apoptotic signalling pathways for poor outcome and proteolysis for good outcome. Analysis according to level of TTM showed a significant protein abundance difference for six proteins [five elevated proteins in TTM 36 °C (log2(FC) between 0.33 and 0.88), one reduced protein (log2(FC) − 0.6)] mainly involved in inflammatory/immune responses only at 48 h after cardiac arrest.ConclusionsSerum proteome profiling revealed an increase in inflammatory/immune responses and apoptosis in patients with poor outcome. In patients with good outcome, an increase in proteolysis was observed, whereas TTM-level only had a modest effect on the proteome profiles. Further validation of the differentially abundant proteins in response to neurological outcome is necessary to validate novel biomarker candidates that may predict prognosis after cardiac arrest.

Pregnancy related hormones increase CYP3A mediated buprenorphine metabolism in human hepatocytes: a comparison to CYP3A substrates nifedipine and midazolam.

Introduction: Pregnancy increases the clearance of CYP3A4 substrate drugs and pregnancy-related hormones (PRHs) induce hepatic CYP3A4 expression and metabolism. However, it remains unclear to what extent the magnitude of PRH-evoked changes in hepatic CYP3A metabolism varies across multiple substrates. This study quantified the impact of PRHs on CYP3A protein concentrations and buprenorphine metabolism in human hepatocytes, and compared the magnitude of these effects to nifedipine and midazolam metabolism. Methods: Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRHs, administered in combination across a range of physiologically relevant concentrations, for 72h. Absolute protein concentrations of CYP3A4, CYP3A5, and CYP3A7 in SCHH membrane fractions were quantified by nanoLC-MS/MS, and norbuprenorphine (nor-BUP), dehydro-nifedipine (dehydro-NIF), and 1-hydroxy-midazolam (1-OH-MDZ) formation was evaluated. Results: Compared to control, PRH exposure increased CYP3A4, CYP3A7, and total CYP3A protein concentrations, but not CYP3A5 concentrations, and increased nor-BUP, dehydro-NIF, and 1-OH-MDZ formation in a concentration-dependent manner. The formation of nor-BUP, dehydro-NIF, and 1-OH-MDZ each positively correlated with PRH-mediated changes in total CYP3A protein concentrations. The PRH-evoked increase in nor-BUP formation was evident in all donors; however, the PRH induction of dehydro-NIF and 1-OH-MDZ formation was diminished in a hepatocyte donor with high basal CYP3A5 expression. Discussion: These findings demonstrate that PRHs increase buprenorphine, nifedipine, and midazolam metabolism in SCHH via induction of CYP3A4 and total CYP3A protein concentrations, and the magnitude of these effects vary across hepatocyte donors in a substrate-specific manner. These data provide insight into the contribution of PRH induction of CYP3A4 metabolism to increased buprenorphine clearance during pregnancy.

Features of Changes in Exogenous Heat Shock Proteins (HSP 27 and HSP 70) in Patients with Occupational Nervous System Pathology

The aim of the study was to identify and compare changes in serum concentrations of heat shock proteins (HSP 27 and HSP 27) in patients with occupational nervous system pathology induced by exposure to physical and chemical factors. Patients with sensorineural hearing loss (SHL), chronic mercury intoxication (CMI), vibration disease (VD) formed both under the influence of local vibration and combined exposure to general and local vibration were examined. As a result of comparative evaluation and analysis of serum concentrations of heat shock proteins, peculiarities in their change were revealed. In individuals with SHL and VD due to local vibration exposure, a decrease in serum HSP 70 concentration was recorded, and in patients with CMI and VD due to combined local and general vibration exposure, high HSP 27 concentrations were recorded. The examination of patients in the linked sample at 3 years showed the absence of statistically significant differences and the preservation of changes in the content of exogenous HSPs, which confirms the progressive course of these diseases. The conjugation of the number of T-lymphocyte and B-cells subpopulations with the change in serum HSP concentrations was established, which confirms the role of HSP 27 and 70 in the regulation of the immune response in VD and allows them to be regarded as markers of cellular and tissue damage in the chronic course of diseases.

Evidence for Solution-Mediated Phase Transitions in Kidney Stones: Phase Transition Exacerbates Kidney Stone Disease

In this study, we investigated calcium oxalate (CaOx) kidney stones and showed direct evidence of the solution-mediated phase transition of calcium oxalate dihydrate (COD; the metastable phase) to calcium oxalate monohydrate (COM; the stable phase). We examined the crystal phases, crystal textures, and protein distributions within thin sections of calcium oxalate kidney stones. Observation with a polarized-light microscope showed that the outline of the mosaic texture, in which COM crystals are assembled in a mosaic pattern, roughly coincides with COD’s crystallographically stable face angles. Microfocus X-ray CT measurement captured the intermediate process of the phase transition, starting inside the COD single crystal and gradually transforming to COM crystals. In addition, the distribution of osteopontin and prothrombin fragment-1, common proteins contained in urine and visualized by multicolor fluorescence immunostaining, showed no apparent striations inside the COM single crystals with the mosaic texture, although the striation is apparent inside the COD single crystals. This is probably because the phase transition of mosaic-like COM occurred in a semiclosed system inside the COD single crystal, so the effect of periodic (day–night, seasonal, etc.) urinary protein concentration changes was small. On the other hand, striations were visible in concentrically laminated COM. This indicated that concentrically laminated COM formed in response to the changes in urinary protein concentrations. From the above, we conclude that the COD single crystals and the concentrically laminated COM seen in CaOx stones are primary structures, and the mosaic COM is a secondary structure that is a pseudomorph formed by the solution-mediated phase transition from COD single crystals.

Investigation of the Changes in Concentrations of Vitamin D-Binding Protein and Lactoferin in Plasma and Peritoneal Fluid of Patients with Endometriosis.

An evaluation of the association between the concentrations of vitamin D-binding protein and lactoferrin in the plasma and peritoneal fluid may facilitate the elucidation of molecular mechanisms in endometriosis. Vitamin D-binding protein and lactoferrin concentrations were measured by ELISA in plasma and peritoneal fluid samples from 95 women with suspected endometriosis as classified by laparoscopy into groups with (n = 59) and without endometriosis (n = 36). There were no differences (p > 0.05) in the plasma and peritoneal fluid concentrations of vitamin D-binding protein and lactoferrin between women with and without endometriosis. In women with endometriosis, there was a significant correlation between plasma and peritoneal fluid vitamin D-binding protein concentrations (r = 0.821; p = 0.000), but there was no correlation between lactoferrin concentrations in those compartments (r = 0.049; p > 0.05). Furthermore, in endometriosis, lactoferrin was found to correlate poorly with vitamin D-binding protein (r= -0.236; p > 0.05) in plasma, while in the peritoneal fluid, the correlation between those proteins was significant (r = 0.399; p = 0.002). The characteristic properties of vitamin D-binding protein and lactoferrin and the associations between their plasma and peritoneal fluid concentrations found in women with endometriosis may provide a novel panel of markers to identify high-risk patients in need of further diagnostic measures.