Serum Activity Research Articles

Correlation of the total superoxide dismutase activity between joint fluid and synovium in end-stage knee osteoarthritis.

Recently, we found significantly reduced total superoxide dismutase (SOD) activity in the cartilage of patients with end-stage knee osteoarthritis (OA). In this study, we aimed to evaluate the SOD activity in serum, joint fluid, cartilage, and synovial membrane samples collected from 52 patients with end-stage knee OA who underwent total knee arthroplasty. The relationship between the total SOD activity in each tissue was evaluated using Spearman's rank correlation coefficient. The joint fluid total SOD activity was used as the objective variable, and its association with the serum, cartilage, and synovial total SOD activities was evaluated using multiple linear regression analysis. Univariate analysis revealed that joint fluid total SOD activity was positively correlated with synovial total SOD activity. Multiple linear regression analysis using joint fluid total SOD activity as the objective variable showed a positive association with synovial total SOD activity (β = 0.493, adjusted R2 = 0.172, P < 0.01). In patients with end-stage knee OA, the state of the synovial total SOD activity is better reflected by the total SOD activity in the joint fluid than that in the cartilage. Joint fluid total SOD activity may serve as a biomarker for the treatment and prevention of synovitis.

Effects of a manganese complex with lysine and glutamic acid on growth performance, manganese deposition and emission, antioxidant capacity and metacarpal strength in weaned piglets.

The present study sought to assess the effects of manganese complexes with lysine and glutamic acid (Mn-LG) as manganese (Mn) sources on growth performance, trace element deposition, antioxidant capacity, and metacarpal strength in weaned piglets. The study involved 288 healthy Duroc × Landrace × Yorkshire piglets that were weaned at 25-28 days of age and weighed 8.66±0.96kg. These piglets were randomly divided into six groups: a control group (Mn-LG-0, receiving a basal diet without Mn supplementation), a Mn sulfate group (basal diet supplemented with 40 mg·kg-1 diet of Mn, Mn-S-40 group), and four Mn-LG groups (Mn-LG-20, Mn-LG-40, Mn-LG-60, Mn-LG-80, supplemented with 20, 40, 60, and 80 mg·kg-1 Mn from Mn-LG in the basal diet). Grouping began at weaning as the 0th day of the experiment. The corn-soybean-based basal diet during the early (d 0-14) and late (d 15-42) phases of the experiment contained 20.88 and 30.12 mg·kg-1 Mn, respectively. Blood samples were collected on days 14 and 42, and pigs were sacrificed for sample collection on day 42. The results indicated no significant differences in average daily gain, average daily feed intake, or feed-to-gain ratio among the groups (P > 0.05). The diarrhea rates of all Mn-LG groups and the Mn-S-40 group were significantly lower in the 0-14 d and during the entire experimental period than in the Mn-LG-0 group (P < 0.001). The Mn-LG-40 group exhibited a significant increase in liver Mn concentration and serum Mn superoxide dismutase (Mn-SOD) activity on d 42 (P < 0.01), as well as a significant decrease in fecal Mn concentration (P < 0.05), compared to those of the Mn-S-40 group. Significant differences (P < 0.05) were detected in the serum, liver, and fecal Mn concentrations, as well as in the serum and liver Mn-SOD activity, across the different Mn-LG groups. The serum and fecal Mn concentrations and serum Mn-SOD activity increased linearly or quadratically (P < 0.01) with increasing Mn-LG supplementation. No significant differences (P > 0.05) were found in kidney, heart, or metacarpal bone Mn concentrations or in bone strength indices. In summary, compared with the Mn-LG-0 diet, dietary supplementation with Mn-LG enhanced serum Mn deposition and Mn-SOD activity and decreased the incidence of diarrhea. Additionally, the fecal Mn concentration was lower in the Mn-LG group than in the inorganic group at equivalent dosages.

Phenylbutyrate Treatment in a Boy with MCT8 Deficiency: Improvement of Thyroid Function Tests and Possible Livertoxicity.

Monocarboxylate transporter 8 (MCT8) deficiency is a rare X-chromosomal inherited disease leading to severe cognitive impairment, muscular hypotonia and symptoms of peripheral thyrotoxicosis. Experimental approaches aiming to functionally rescue mutant MCT8 activity by the chemical chaperone phenylbutyrate (PB) demonstrated promising effects in vitro for several MCT8 missense mutations. The objective was to evaluate biochemical and clinical effects of PB in doses equivalent to those approved for the treatment of urea cycle disorders in a boy with MCT8 deficiency due to a novel MCT8 missense mutation c.703G > T (p.V235L). During a treatment period of 13 months, PB led to a significant decrease of elevated TSH and T3 serum concentrations, while fT4 increased. Weight z-score of the toddler remained remarkably stable during the treatment period. Neurodevelopmental assessments (BSID-III) revealed a slight increase of gross motor skills from developmental age 4 to 6 months. However, increasing liver enzyme serum activities and accumulation of phenylacetate (PAA) in urine led to treatment interruptions and dose alterations. In vitro analyses in MDCK1 cells confirmed the pathogenicity of MCT8 p.V235L. However, while PB increased expression of the mutant protein, it did not rescue T3 transport, suggesting a PB effect on thyroid function tests independent of restoring MCT8 activity. In a clinical attempt of PB treatment in MCT8 deficiency we observed a significant improvement of thyroid hormone function tests, tendencies towards body weight stabilization and slight neurodevelopmental improvement. Hepatotoxicity of PB may be a limiting factor in MCT8 deficiency and requires further investigation.

State of the Art in the Development of Human Serum Carnosinase Inhibitors

Human serum carnosinase is an enzyme that operates the preferential hydrolysis of dipeptides with a C-terminus histidine. Only higher primates excrete such an enzyme in serum and cerebrospinal fluid. In humans, the serum hydrolytic rate has high interindividual variability owing to gene polymorphism, although age, gender, diet, and also diseases and surgical interventions can modify serum activity. Human genetic diseases with altered carnosinase activity have been identified and associated with neurological disorders and age-related cognitive decline. On the contrary, low peripheral carnosinase activity has been associated with kidney protection, especially in diabetic nephropathy. Therefore, serum carnosinase is a druggable target for the development of selective inhibitors. However, only one molecule (i.e., carnostatine) has been discovered with the purpose of developing serum carnosinase inhibitors. Bestatin is the only inhibitor reported other than carnostatine, although its activity is not selective towards serum carnosinase. Herein, we present a review of the most critical findings on human serum carnosinase, including enzyme expression, localization and substrate selectivity, along with factors affecting the hydrolytic activity, its implication in human diseases and the properties of known inhibitors of the enzyme.

#2314 Circulating immune complexes concentration and complement activation in sensitized kidney transplant recipients

Abstract Background and Aims Chronic antibody mediated rejection of the kidney transplant is the most common cause of graft loss in the late posttransplant period. Crucial in this process seems to be the role of classical pathway complement activation due to the formation of immune complexes. The formation of immune complexes is a benign process of a normally functioning immune system. In kidney transplant recipients (KTRs), the production of donor specific IgG/IgM antibodies (DSAs) against donor antigens (MHC molecules, ABO blood group antigens) leads to the attachment of immune complexes to cell membranes and subsequent tissue damage through complement activation. The attachment of C1q complement fragment to the immune complexes holds an important part in this process. This study aims to determine the levels of immune complexes as well as classical pathway complement activation in sensitized kidney transplant recipients. Method In this cross-sectional study 3 groups of KTRs were compared as to the levels of immunocomplexes concentration and classical pathway complement activation. Fourteen KTRs with preformed or de novo donor specific antibodies were compared to 28 kidney transplant recipients with no DSAs and to 22 subjects with no history of chronic kidney disease (controls). We used MicroVue Complement CIC-C1q EIA that is designed to detect immunocomplexes that bind to C1q in human serum or plasma. In order to estimate total classical pathway complement activation in serum, we used MicroVue Complement CH50 Eq EIA. Results Kidney function (eGFR) of KTRs with and without DSAs was similar (53.1 ± vs. 60.8 ± ml/min/1.73 m2, p = 0.54) but lower in comparison to controls (106.4 ml/min/1.73 m2, p &amp;lt; 0.001). We found a significantly higher concentration of C1q-immunocomplexes in the serum of KTRs with donor specific antibodies, in comparison to controls, as well as to the group of kidney transplant recipients with no DSAs (6.78 ± 2.7 vs. 4.83 ± 1.9 vs. 5.04 ± 1.2 μgEq/ml respectively, p = 0.0078). Furthermore, there was no significant difference of C1q-immunocomplexes between kidney transplant recipients with no DSAs and controls. Finally, a significant reduction of CH50 levels was observed in KTRs with DSAs as compared to controls and KTRs with no DSAs (39.2 ± 14.9 vs. 70.6 ± 34.3 vs. 67.9 ± 39.9 U/ml respectively, p = 0.003), which suggests higher levels of classical pathway complement activation. CH50 levels in KTRs with DSAs, who were further subcategorized in those with preformed and those with de novo DSAs were similar (39.68 ± 15.6 vs 39.08 ± 16.04 U/ml). Conclusion Kidney transplant recipients with donor specific antibodies have increased serum levels of immune complexes and increased classical pathway complement activation.

#933 Serum and kidney complement and severity of ANCA associated vasculitis in the kidney

Abstract Background and Aims Kidney involvement in ANCA associated vasculitis (AAV) is commonly described as pauci-immune based on histological characteristics. However, we now know that the complement system is activated and contributes to the pathogenesis of AAV – with Avacopan (C5a inhibitor) demonstrating superior efficacy in maintenance of remission over Prednisolone in the ADVOCATE trial. Our aim was to evaluate if there is an association between kidney disease severity and the presence of serum and histological complement activation markers. Method Retrospective cohort study of 59 patients with AAV diagnosed between November 2015 and May 2019. Complement activation markers were serum complements (C3 and C4) levels and presence of complement (C3, C1q and C4) in glomerular capillary walls in immunofluorescence with intensity of 2+ and 3+. Kidney disease severity markers were peak serum creatinine value at presentation, anti-MPO or anti-PR3 levels, and number and proportion of glomeruli with crescents on kidney biopsy. Associations were evaluated using Spearman correlation or Mann Whitney U test as appropriate Results Among 59 patients with biopsy-proven AAV in the kidney, the median (25th percentile, 75th percentile) age was 68.8 (60.7, 73.4) years, serum creatinine was 263 (156, 560) µmol/L, and anti-MPO and anti-PR3 levels were 100.0 (81.5, 199.2) and 128.8 (22.9, 200) in 41 and 7 patients, respectively. respectively. The number and proportion of glomeruli with crescents were 6 (3, 12) and 33.3% (12.0%, 42.6%), respectively. Serum C3 and C4 were performed in 41 patients and the median levels were 1.00 (0.83, 1.14) and 0.24 (0.18, 0.31), respectively. Serum C3 level did not correlate with serum creatinine, anti-MPO or anti-PR3 titers, and number and proportion of glomeruli with crescents. Serum C4 level was negatively correlated with anti-MPO titer (34 patients; r = −0.393, p = 0.02) but did not correlate with serum creatinine or number or proportion of crescents. Complement in the glomerular capillary wall was present in 8 patients (13.6%): C3, C1q and C4 were present in 8, 3 and 1 patients, respectively. Serum creatinine [median 299 (IQR 133, 998) µmol/L versus 261 (156, 359) µmol/L, p = 0.39], anti-MPO titer [100 (88.3, 200) IU/ml versus 100 (79.4, 198) IU/ml, p = 0.74], number of crescents [14 (4, 21) versus 5 (3, 12), p = 0.90] and proportion of crescents [38.9% (12.5%, 42.3%) versus 31.3% (11.1%, 44.3%), p = 0.59] were not different in those with and without complement in the kidney biopsy. Conclusion The presence of complement activation markers was not associated with kidney disease severity in AAV. However, the detection of clinically significant associations may have been limited by our cohort size.

#1707 Antiphospholipid antibodies in lupus nephritis

Abstract Background and Aims Antiphospholipid antibodies (aPL) constitute a heterogeneous family of antibodies against anionic phospholipids or phospholipid-binding proteins. They may occur in association with autoimmune diseases, infections, and sometimes in the general population. Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE). The aim of this study was to investigate the association of aPL with clinical activity, renal damage, renal activity and outcomes in patients with LN. Method A total of 157 patients (11 male, 146 female, age between 17 and 58 years) with LN were included in this study. The International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of LN was used to classify the patients into LN subsets. Histopathological renal activity and damage were estimated using the Activity Index (AI) and Chronicity Index (CI), respectively. Levels of anticardiolipin (aCL) antibodies, anti-β2-glycoprotein I (anti-β2-GPI) antibodies and lupus anticoagulant (LAC) were measured, and other clinical and pathological data were also obtained. Long-term renal outcome was determined by the estimated glomerular filtration rate (eGFR) and the Chronic Kidney Disease stage, after a median follow-up of 20,5 ± 8, 7 years, counting from the occasion of the first renal biopsy. Renal and extra-renal symptoms were analyzed. Patients which have more than one class LN features, were excluded from the study. The treatment includes standard treatment for LN, inhibitors of the angiotensin system in patients with hypertension and proteinuria, steroids, anticoagulants, cyclophosphamide, mycophenolate mofetil, azathioprine. Results LN patients were classified according to the baseline renal biopsies as patients with: minimal mesangial LN (4), mesangial-proliferative LN (30), focal proliferative LN (31), diffuse proliferative LN (48), membranous LN (44). aPL antibodies were detected in 89 (56,69%) patients, aCL antibodies – in 72 (45,86%), anti-β2-GPI antibodies – in 58 (36,94%) and LAC were detected in 46 (29,30%) patients. None of these patients had a concomitant diagnosis of antiphospholipid syndrome nephropathy. Follow-up was similar between aPL-positive and aPL-negative patients. LN patients with positive aPL antibodies had significantly higher SLEDAI (p &amp;lt; 0,05), anti-dsDNA levels (p &amp;lt; 0,05), anti-ribosomal-P antibodies levels (p &amp;lt; 0,05), anti-C1q antibodies (p &amp;lt; 0,05), lower serum C3 (p &amp;lt; 0,005) and C4 (p &amp;lt; 0,05) levels, and higher intensity of complement C3 (p &amp;lt; 0,05) deposits in kidney tissue than the aCL antibody–negative LN patients. aPL-seropositivity was associated with more rapid decline in eGFR (–1,06 mL/min/year compared to –0,45 mL/min/year in aPL-seronegative patients) and showed inferior patient survival (89,8% and 67,9% at 10 and 15 years, respectively, compared to 97,3% and 76% in aPL-seronegative patients). No significant correlation was found between serum aPL antibodies levels and activity or chronicity index scores in renal biopsies at baseline, ANA levels, or age. Conclusion We concluded that aPL-seropositivity are correlated with clinical activity in patients with LN and are associated with inferior long-term patient survival.

Intracellular complement and immunometabolism: The advantages of compartmentalization.

The complement system is a proteolytic cascade triggered by pathogen and danger-associated molecular patterns, with resultant outcomes of inflammation, cellular activation, and opsonization of material for removal by phagocytosis. While first discovered as an activity in serum, it is now recognized that complement components play important roles at local and individual cell-intrinsic levels. In particular, apart from the extracellular serum activities of complement, it is now believed that complement also acts intracellularly, as part of a cellular signal transduction cascade that can stimulate cellular survival and activation, and individual immune cell phenotypes, via effects on cellular metabolism. This review will describe what is currently known about how complement functions in intracellular signal transduction, and outline the functional advantages of a compartmentalized and intracellular complement system.

Anti-aging mechanism and effect of treatment with raw and wine-steamed Polygonatum sibiricum on D-galactose-induced aging in mice by inhibiting oxidative stress and modulating gut microbiota.

Polygonatum sibiricum (PS) is a traditional Chinese medicine (TCM) first recorded in Mingyi Bielu. The book documents that PS can nourish five internal organs, be taken for a long time, relax the body and prolong lifespan. Presently, PS is widely used in TCM to prevent premature graying of hair. Based on TCM theory and clinical trials, the wine steaming processed product from PS provides a better effect. However, no published study has elucidated the anti-aging mechanism. The study aim was to investigate the anti-aging mechanism of PS and its wine steaming processed product in mice, specifically focusing on the effect of D-galactose (D-gal) surrounding the intestinal flora and the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2-antioxidant response elements (Keap1/Nrf2/ARE) pathway. The chemical components in Raw PS (RPS) and Wine-steamed PS (WPS) were identified by ultra-performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). An aging model using Kunming mice was established through intraperitoneally injected D-gal. Concentrations of RPS and WPS at 5, 10, or 15g/kg/day levels were administered intragastrically, respectively. The body weight, liver and spleen indexes, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) activities in serum and brain tissue were recorded. Hematoxylin and eosin (HE) stained brain tissue was histopathologically examined. The expressions of Keap1, Nrf2 and heme oxygenase 1 (HO-1) in the brain tissue at the mRNA and protein levels were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB). Moreover, an Illumina Hiseq platform was used for 16S ribosomal RNA (16S rRNA) high-throughput sequencing to evaluate the proportions of intestinal flora in aging mice. The proportions of saccharides, flavonoids, and triterpene acids were different between RPS and WPS. In the aging model mice, WPS outperformed RPS in improving body weight and mental state by increasing the spleen index, SOD and GSH-PX activities, decreasing the liver index and MDA activities, and restoring the histopathological morphology in D-gal-induced aging mice. At the mRNA levels, RPS and WPS significantly reduced the expression of Keap1 and increased the expressions of Nrf2 and HO-1. The trend in protein expressions was similar to that of the mRNA results, and WPS had a stronger effect than RPS. Fecal microbiota analysis showed that RPS and WPS restored intestinal microbiota proportions to normal levels. The results demonstrated that PS and its WPS had a positive effect in relieving oxidative stress in aging mice. WPS outperformed RPS, which might be related to the activation of the Keap1/Nrf2/ARE pathway and regulation of intestinal flora.

Influence of the probiotic preparation immunofloor on the immune status of broiler chickens

In conditions of intensification of the livestock industry, to combat infectious pathology, antimicrobial drugs are most often, and in some cases haphazardly, used, which accumulate in raw materials and food products and contribute to environmental pollution. Antibiotics have a detrimental and pathogenic effect on the normal microflora of the gastrointestinal tract of animals and poultry. Against this background, dysbiosis is observed, leading to metabolic disorders, decreased digestibility of essential feed nutrients, and the development of immunological deficiency. One of the leading areas of agricultural science and practice is developing and introducing drugs and feed additives that are an alternative to antibiotics into livestock farming. Probiotics have a unique role in solving this problem. Probiotic preparations ’ therapeutic and prophylactic properties are based on maintaining normal flora at a physiologically necessary level, reducing diarrhea in animals, and improving digestion processes. Probiotics have thefunctions of improving metabolic processes, immunostimulating activity, adaptogenic effects, increasing all types of productivity offarm animals and poultry, and improving the quality of livestock products. Introducing probiotic preparations into poultry and livestockfarming requiresfurther study of their biological properties and immunostimulating activity. The influence of the probiotic drug Immunoflor on the state of natural resistance factors in poultry was studied. Four groups of day-old broiler chickens of the Arbor Acres cross were formed: a control group and three experimental groups. In addition to the main diet, the birds of the experimental groups received Immunoflor in amounts of 0.5, 0.7, and 1.0 kg/t of feed. It was established that the inclusion of aprobiotic drug in the diet of broilers contributed to the strengthening of humoral factors of natural resistance due to an increase in the lysozyme and bactericidal activity of blood serum by 10.5 - 17.2 and 8.5 - 12.1% by the end of cultivation. The phagocytic properties of chicken blood leukocytes have also improved. Under the influence of Immunoflor, an increase in the mass of immunocompetent organs was observed in 42-day-old broiler chickens. The maximum indicators of poultry population safety were established in the experimental groups.

Association of Matrix Metalloproteinase-2 (MMP-2) and MMP-9 Promoter Variants, Their Serum Levels, and Activities with Aortic Valve Calcification (AVC) in a Population from Western Iran.

Background: Matrix metalloproteinase (MMP) enzyme gene polymorphisms MMP-2-1575G/A and MMP-9-1562C/T promoter polymorphism, their serum levels, and activity are associated with aortic valve calcification (AVC). Materials and Methods: The synergistic link between the risk of AVC and the alleles T and A of MMP-9 and MMP-2 was investigated, respectively. Ninety-two cases with AVC and 92 healthy individuals from the west of Iran were included, and MMP- 2-1575G/A and MMP-9-1562C/T promoter polymorphisms were detected using PCR-RFLP. The serum levels and activity of MMP-2 and -9 were assessed using ELISA and gelatin zymography methods, respectively. In addition, serum biochemical markers, including FBS, urea and creatinine, cholesterol, triglyceride, HDL, LDL, calcium, phosphorus, and blood pressure: systolic blood pressure and diastolic blood pressure were measured. Results: Heart valve calcification disease was associated with a comparatively higher frequency of the A allele of the MMP2-1575 variation (p = 0.002). In addition, the frequency of T allele of the MMP9-1562 variant was higher than the control group (p = 0.007). Conclusion: MMP-2 and MMP-9 serum levels and activities were observed to be considerably higher in the experimental group than in the control group (p < 0.001). Patients are more susceptible to cardiovascular disease than the control group due to elevated serum levels and activity of MMP-2 and MMP-9.

The sclerotic component of metabolic syndrome: Fibroblast activities may be the central common denominator driving organ function loss and death.

Fibrosis is a common feature of more than 50 different diseases and the cause of more than 35% of deaths worldwide, of which liver, kidney, skin, heart and, recently, lungs are receiving the most attention. Tissue changes, resulting in loss of organ function, are both a cause and consequence of disease and outcome. Fibrosis is caused by an excess deposition of extracellular matrix proteins, which over time results in impaired organ function and organ failure, and the pathways leading to increased fibroblast activation are many. This narrative review investigated the common denominator of fibrosis, fibroblasts, and the activation of fibroblasts, in response to excess energy consumption in liver, kidney, heart, skin and lung fibrosis. Fibroblasts are the main drivers of organ function loss in lung, liver, skin, heart and kidney disease. Fibroblast activation in response to excess energy consumption results in the overproduction of a range of collagens, of which types I, III and VI seem to be the essential drivers of disease progression. Fibroblast activation may be quantified in serum, enabling profiling and selection of patients. Activation of fibroblasts results in the overproduction of collagens, which deteriorates organ function. Patient profiling of fibroblast activities in serum, quantified as collagen production, may identify an organ death trajectory, better enabling identification of the right treatment for use in different metabolic interventions. As metabolically activated patients have highly elevated risk of kidney, liver and heart failure, it is essential to identify which organ to treat first and monitor organ status to correct treatment regimes. In direct alignment with this, it is essential to identify the right patients with the right organ deterioration trajectory for enrolment in clinical studies.

Effects of herbal dregs supplementation of Salvia miltiorrhiza and Isatidis Radix residues improved production performance and gut microbiota abundance in late-phase laying hens.

The present study was designed to evaluate the effect of a mixture of Chinese medicinal residues (CMRs) consisting of Salvia miltiorrhiza residues (SMR) and Isatidis Radix residues (IRR) on productive performance, egg quality, serum lipid and hormone levels, liver and blood antioxidant capacity, oviduct inflammation levels, and gut microbiota in the late-laying stage. A total of 288 fifty-four-week-old BaShang long-tailed hens were divided into four groups. The feed trial period was 8 weeks. The control group was fed the basic diet as a CCMR group, supplemented with 3, 4, and 6% for the experimental groups LCMR, MCMR, and HCMR. The egg production rate of the MCMR group was 8.1% higher than that of the CCMR group (p < 0.05). Serum triglyceride (TG) levels of hens of the CMR-supplemented group were significantly decreased than those of the CCMR group (p < 0.05). The group supplemented with different levels of CMR had significantly higher serum HDL-C levels compared with the control group (p < 0.05). Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were remarkably increased for the LCMR and MCMR groups and significantly decreased for the HCMR group compared to CCMR (p < 0.05). Serum and liver glutathione peroxidase (GSH-PX) activities were significantly increased, and malondialdehyde (MDA) levels were significantly decreased in the MCMR group compared to the CCMR group (p < 0.05). The expression levels of tubal inflammatory factor markers (IL-4, IL-1β, TNF-α) in the MCMR and HCMR groups were consistent with the pathological findings of the sections. As for cecal microbiota, supplementation with CMR affected the alpha diversity of the cecum microbiome at the genus level. The Shannon index was significantly higher in the MCMR group than in the CCMR and HCMR groups (p < 0.05). Supplementation with different levels of CMR mainly regulated the ratio of intestinal Firmicutes to Bacteroidetes and the abundance of phyla such as Proteobacteria. In addition, CMR supplementation at different levels in the diet enriched lipid-metabolizing bacteria, such as Bacteroides and Ruminococcus_gnavus_group. Furthermore, according to linear discriminant analysis (LDA) effect size (LEfSe) analysis, the MCMR group showed an increase in the number of short-chain fatty acid-producing bacteria Romboutsia and fiber-degrading specialized bacteria Monoglobus. Therefore, supplementation of appropriate amounts of CMR to the diet of laying hens enhanced reproductive hormone levels, hepatic antioxidant capacity, and lipid metabolism, alleviated the levels of oviductal inflammatory factors, and modulated the abundance structure of bacterial flora to improve the late-laying performance and egg quality. The results of the current study showed that CMR is a beneficial feed supplement for chickens when added in moderation.

Maternal selenium deficiency during pregnancy in association with autism and ADHD traits in children: The Odense Child Cohort

BackgroundSelenoproteins regulate pathways controlling neurodevelopment, e.g., redox signaling and thyroid hormone metabolism. However, studies investigating maternal selenium in relation to child neurodevelopmental disorders are scarce. Methods719 mother-child pairs from the prospective population-based Odense Child Cohort study in Denmark were included. Three selenium biomarkers, i.e. concentrations of serum selenium, selenoprotein P (SELENOP), and activity of glutathione peroxidase 3 (GPX3), along with serum copper, zinc and iron were measured in early third trimester (at 28.9+/-0.8 weeks of pregnancy). ADHD and ASD traits in children were assessed systematically using the established Child Behaviour Checklist at 5 years of age, based on a Danish reference cohort with cut-off at 90th percentile. Multivariable regression models adjusted for biologically relevant confounders were applied. Results155 of 719 (21.6 %) children had ASD traits and 59 of 719 (8.2 %) children had traits of ADHD at 5 years of age. In crude and adjusted models, all three selenium biomarkers associated inversely with ADHD traits. For ADHD, fully adjusted OR for 10 μg/L increment in selenium was 0.76 (95 % CI 0.60, 0.94), for one mg/L increment in SELENOP was 0.73 (0.56, 0.95), and for 10 U/L increment in GPx3 was 0.93 (0.87,1.00). Maternal total selenium was inversely associated with child ASD traits, OR per 10 μg/L increment was 0.85 (0.74, 0,98). SELENOP and GPx3 were not associated with ASD traits. The associations were specific to selenium, as other trace elements such as copper, zinc, or iron were not associated with the outcomes. ConclusionsThe results provide coherent evidence for selenium deficiency as a risk factor for ADHD and ASD traits in an environment with borderline supply, the causality of which should be elucidated in a randomized controlled trial.

Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.

H2S prevents the disruption of the blood-brain barrier in rats with prenatal hyperhomocysteinemia

Elevation of the homocysteine concentration in the plasma called hyperhomocysteinemia (hHCY) during pregnancy causes a number of pre- and postnatal developmental disorders. The aim of our study was to analyze the effects of H2S donors –NaHS and N-acetylcysteine (NAC) on blood-brain barrier (BBB) permeability in rats with prenatal hHCY. In rats with mild hHCY BBB permeability assessed by Evans Blue extravasation in brain increased markedly throughout life. Administration of NaHS or NAC during pregnancy attenuated hHCY-associated damage and increased endogenous concentrations of sulfides in brain tissues. Acute application of DL-homocysteine thiolactone induced BBB leakage, which was prevented by the NMDA receptor antagonist MK-801 or H2S donors. Rats with hHCY demonstrated high levels of NO metabolite – nitrites and proinflammatory cytokines (IL-1β, TNF-α, IL-6) in brain. Lactate dehydrogenase (LDH) activity in the serum was higher in rats with hHCY. Mitochondrial complex-I activity was lower in brain of hHCY rats. NaHS treatment during pregnancy restored levels of proinflammatory cytokines, nitrites and activity of the respiratory chain complex in brain as well as the LDH activity in serum. Our data suggest that H2S has neuroprotective effects against prenatal hHCY-associated BBB disturbance providing a potential strategy for the prevention of developmental impairments in newborns.

Simultaneous measurement of aldosterone and plasma renin activity in human serum using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for clinical research

Simultaneous measurement of aldosterone and plasma renin activity in human serum using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for clinical research

Reducing the injury of hippocampal vascular endothelial cells after stroke via targeting SIRT1 by butylphthalide

Butylphthalide (NBP) can inhibit various pathological processes of ischemic stroke. This experiment explored the mechanism of NBP and SIRT1 on damage of hippocampal vascular endothelial cells after stroke. The neurons in the hippocampus of rats were stained with HE, and morphology and density of neurons were observed. Flow cytometry, commercial kits and Western blotting detected apoptosis of endothelial cells, levels of antioxidant enzymes and apoptotic proteins, intracellular calcium level and activity of Ca2+-ATPase. The damage to rat nerve cells was alleviated by butylphthalide to varying degrees, and the lost parts of rat nerve cells were recovered with decreased Bax and cleaved caspase-3 expression after butylphthalide treatment, and increased Bcl-2 (P &lt;0.05), as well as decreased serum malondialdehyde (MDA) content and activity of catalase (CAT) decreased and elevated Superoxide dismutase (SOD) activity (P &lt;0.05). The concentration of calcium ion also decreased but activity of Ca2+-ATPase increased (P &lt;0.05) and mitochondria in the model group appeared with severe vacuolation and swelling. The vacuolation and swelling of mitochondria in the treatment group were improved. Additionally, mitochondrial membrane fluidity, potential and rat hippocampal ATPase activity in butylphthalide group were also increased. Compared to normal control group, model group, SIRT1 inhibitor group and butylphthalide+SIRT1 inhibitor group had lower levels of SIRT1 and higher p-NF-kB p65/p-IkBα levels. Butylphthalide has a protective effect on hippocampal neurons in stroke rats and can alleviate the damage degree of rat nerve cells.

A Significant Contribution of the Classical Pathway of Complement in SARS-CoV-2 Neutralization of Convalescent and Vaccinee Sera.

Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals.

Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.