1-acid Glycoprotein Research Articles

Utilization of Sodium Nitroprusside as an Intestinal Permeation Enhancer for Lipophilic Drug Absorption Improvement in the Rat Proximal Intestine.

As advanced synthetic technology has enabled drug candidate development with complex structure, resulting in low solubility and membrane permeability, the strategies to improve poorly absorbed drug bioavailability have attracted the attention of pharmaceutical companies. It has been demonstrated that nitric oxide (NO), a vital signaling molecule that plays an important role in various physiological systems, affects intestinal drug absorption. However, NO and its oxidants are directly toxic to the gastrointestinal tract, thereby limiting their potential clinical application as absorption enhancers. In this study, we show that sodium nitroprusside (SNP), an FDA-approved vasodilator, enhances the intestinal absorption of lipophilic drugs in the proximal parts of the small intestine in rats. The SNP pretreatment of the rat gastrointestinal sacs significantly increased griseofulvin and flurbiprofen permeation in the duodenum and jejunum but not in the ileum and colon. These SNP-related enhancement effects were attenuated by the co-pretreatment with dithiothreitol or c-PTIO, an NO scavenger. The permeation-enhancing effects were not observed in the case of antipyrine, theophylline, and propranolol in the duodenum and jejunum. Furthermore, the SNP treatment significantly increased acidic glycoprotein release from the mucosal layers specifically in the duodenum and jejunum but not in the ileum and colon. These results suggest that SNP increases lipophilic drug membrane permeability specifically in the proximal region of the small intestine through disruption of the mucosal layer.

Advances in chromatography in the study of drug-plasma protein interactions

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α1 -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。

Alpha 1 Acid Glycoprotein as a Marker for Diagnosis of Early-Onset Neonatal Sepsis in Full-term Neonates

Alpha 1 Acid Glycoprotein as a Marker for Diagnosis of Early-Onset Neonatal Sepsis in Full-term Neonates

Alpha-1 Acid Glycoprotein and Podocin mRNA as Novel Biomarkers for Early Glomerular Injury in Obese Children.

Introduction: Obesity, which is a serious problem in children, has a negative impact on many organs, including kidneys, and obesity-related glomerulopathy (ORG) is an increasingly common cause of ESKD (end-stage kidney disease) in adults. Early-detected and -treated glomerular lesions are reversible, so it is important to find a useful marker of early damage. The study aimed to evaluate the albumin-to-creatinine ratio (ACR), urinary alpha-1-acid glycoprotein (α1-AGP), and mRNA of podocyte-specific proteins as indicators of glomerular injury and their relationship with the degree of obesity and metabolic disorders. Materials and Methods: A total of 125 obese children and 33 healthy peers were enrolled. Patients were divided into two groups, depending on SDS BMI values. ACR, α1-AGP, mRNA expression of nephrin, synaptopodin, podocin, and C2AP protein in urine sediment were measured. Results: ACR values did not differ between groups and were within the normal range. α1-AGP and mRNA expression were significantly higher in obese children compared with controls. mRNA expression of the remaining podocyte proteins was similar in both groups. No significant differences concerning all examined parameters were found depending on the degree of obesity. There was a positive significant correlation between α1-AGP and ACR. Conclusions: Increased α1-AGP before the onset of albuminuria suggests its usefulness as a biomarker of early glomerular damage in obese children. An increased podocin mRNA expression also indicates podocyte damage and may be linked to ORG development. The lack of increase in expression of other podocyte proteins suggests that podocin mRNA may be a more specific and sensitive biomarker. The degree of obesity has no impact on the tested parameters, but further studies are needed to confirm it.

Production of bioactive recombinant ovine cysteine-rich secretory protein 1 in Escherichia coli

ABSTRACT Ovine cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymal origin under CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The aim of the present study was the optimization of bacterial production and partial characterization of putative mature ovine CRISP-1 protein. The cDNA corresponding to T23 – C242 peptide fragment of ovine CRISP-1 protein was cloned into THE pET32b(+) expression vector using E. coli DH5α. Protein expression was carried out in E. coli BL21(DE3) by inducition with 1 mM IPTG at 37°C for 4 h. The recombinant protein was expressed as inclusion bodies and purified by Ni-NTA affinity chromatography using a pH gradient. Further purification of the protein was carried out by gel extraction following zinc sulfate negative staining. SDS-PAGE analysis of the purified recombinant CRISP-1 protein revealed a 43.8 kDa band. Bioactivity of the purified CRISP-1 protein was examined on sperm motility and capacitation. The recombinant ovine CRISP-1 protein at 5 µg/ml caused significant inhibition of sperm motility, and the activity was lost following heating the protein at 100°C for 5 min. The protein also demonstrated decapacitation activity, and at a concentration of 2 µg/ml, it caused a significant (P < 0.05) reduction in sperm capacitation. In conclusion, the thioredoxin-tagged ovine CRISP-1 protein was successfully produced in E. coli and purified in the soluble form by a combination of Ni-NTA affinity chromatography, gel purification, and dialysis. The recombinant protein exhibited both motility-inhibiting and decapacitating activities. Further study is needed to elucidate the mechanism of action and evaluate it’s possible use in semen preservation. Abbreviations: CRISP-1: Cysteine-rich secretory protein-1; PCR: polymerase chain reaction; IPTG: isopropyl-β-D-thiogalactopyranoside; LB: Luria Bertani; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA: ethylene diamine tetraacetic acid; Ni-NTA: Nickel nitrilotriacetic acid.

Pharmacokinetic modeling of R and S‐Methadone and their metabolites to study the effects of various covariates in post‐operative children

Methadone is a synthetic opioid used as an analgesic and for the treatment of opioid abuse disorder. The analgesic dose in the pediatric population is not well‐defined. The pharmacokinetics (PKs) of methadone is highly variable due to the variability in alpha‐1 acid glycoprotein (AAG) and genotypic differences in drug‐metabolizing enzymes. Additionally, the R and S enantiomers of methadone have unique PK and pharmacodynamic properties. This study aims to describe the PKs of R and S methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in pediatric surgical patients and to identify sources of inter‐ and intra‐individual variability. Children aged 8–17.9 years undergoing orthopedic surgeries received intravenous methadone 0.1 mg/kg intra‐operatively followed by oral methadone 0.1 mg/kg postoperatively every 12 h. Pharmacokinetics of R and S methadone and EDDP were determined using liquid chromatography tandem mass spectrometry assays and the data were modeled using nonlinear mixed‐effects modeling in NONMEM. R and S methadone PKs were well‐described by two‐compartment disposition models with first‐order absorption and elimination. EDDP metabolites were described by one compartment disposition models with first order elimination. Clearance of both R and S methadone were allometrically scaled by bodyweight. CYP2B6 phenotype was a determinant of the clearance of both the enantiomers in an additive gene model. The intronic CYP3A4 single‐nucleotide polymorphism (SNP) rs2246709 was associated with decreased clearance of R and S methadone. Concentrations of AAG and the SNP of AAG rs17650 independently increased the volume of distribution of both the enantiomers. The knowledge of these important covariates will aid in the optimal dosing of methadone in children.

Association of plasma low-density lipoprotein receptor-related protein-1 (LRP1) with undernutrition: a case-control study in Bangladeshi adults

Purpose Studies revealed that silencing of low-density lipoprotein receptor-related protein-1 (LRP1) expression can cause inhibition of adipogenesis in animal model and contribute to reduced body size. But there is no study that has explored the association of LRP1 with body mass index (BMI) of human adults. Therefore, the aim of this study was to investigate the relationship of LRP1 with undernutrition. Methods A total of 270 Bangladeshi slum-dwelling adults were enrolled as case control design. Their socio-economic, demographic, anthropometric and biomedical data were collected. Plasma LRP1, C-reactive protein (CRP), alpha-1 acid glycoprotein (AGP) and ferritin levels were measured by ELISA, haemoglobin by HemoCue and zinc by atomic absorption spectrometry. Results The median (IQR) values of plasma LRP1 were 1673.1 (1382.5–1886.2) ng/mL in healthy participants and 707.7 (588.6–839.9) ng/mL in undernourished participants, respectively. A strong positive correlation (r = 0.70, p < 0.05) between LRP1 and BMI was found. Multivariable logistic regression analysis revealed a positive association between low plasma LRP1 (Adj. OR = 0.98, CI = 0.98, 0.99 and p < 0.05) and undernutrition. Conclusions The study found that increased level of LRP1 is associated with increased BMI, whereas lower level is associated with low BMI.

Comparison of biochemical and immunological biomarker levels of patients with COVID-19 with healthy individuals

Abstract Objectives It was aimed to compare Alpha-1 antitrypsin (AAT), Alpha-1 acid glycoprotein (AGP), Total Immunoglobulin M (Total IgM), Total Immunoglobulin G (Total IgG), Galectin-3 (Gal3), and severe acute respiratory syndrome coronavirus 2 IgG (SARS-CoV-2 IgG) levels in patients with COVID-19 and healthy individuals. Methods The study included a total of 86 participants, 44 patients diagnosed with COVID-19 by real-time reverse transcription-polymerase chain reaction (rRT-PCR) test and 42 as the control group. AAT, AGP, Total IgM, and Total IgG levels were measured using the immunoturbidimetric method. Gal3 and SARS-CoV-2 IgG levels were measured using the chemiluminescent microparticle immunoassay method. Results AAT, AGP, Total IgG, Gal3, and SARS-CoV-2 IgG levels were found to be significantly higher in the patient group compared to the control group (p&lt;0.001 for all tests). In the patient group, there was a moderate correlation between AAT-AGP and SARS-CoV-2 IgG-AAT (r=0.692; r=0.561, respectively). Conclusions High levels of AAT, AGP, Total IgG, Gal3, and SARS-CoV-2 IgG in the patient group and correlations between variables suggest that these parameters may be involved in the pathogenesis of the disease and provide an idea about the prognosis of the disease. However, new studies on this subject are needed in order to clearly reveal the laboratory tests related to the clinical course of the disease.

Pathological features in ‘humanized’ neonatal pig

Cytidine monophosphate-Nacetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) and glycoprotein, alpha1, 3-galactosyltransferase (GGTA1) double knockout (DKO) pig models were produced to reduce immune reaction for xenotransplantation. However, the role of Neu5Gc and α-Gal in pigs has not been fully elucidated and it is necessary to consider the after-effect of inactivation of GGTA1 and CMAH in pigs. Hematological profiles of DKO pigs were analyzed through complete blood count (CBC). Histology of liver and spleen of DKO were investigated, and lectin blotting and mass spectrometry (MS) were performed to explore glycosylation changes in red blood cell (RBC) membranes of DKO pigs. DKO pigs showed common clinical signs such as weakness (100%), dyspnea (90%) and constipation (65%). DKO pigs revealed a significant decrease in RBC, hemoglobin (HGB) and hematocrit (HGB), and an increase in white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and erythrocyte mean corpuscular volume (MCV). DKO piglets showed swollen liver and spleen, and exhibited raised deposition of hemosiderin and severe bleeding. Lectin assay and MS proved variations in glycosylation on RBC membranes. GGTA1/CMAH DKO pigs developed pathological features which are similar to anemic symptoms, and the variations in glycosylation on RBC membranes of DKO pigs may be attributed to the pathologies observed.

High Plains wheat mosaic virus: An enigmatic disease of wheat and corn causing the High Plains disease.

Brief historyIn 1993, severe mosaic and necrosis symptoms were observed on corn (maize) and wheat from several Great Plains states of the USA. Based on the geographical location of infections, the disease was named High Plains disease and the causal agent was tentatively named High Plains virus. Subsequently, researchers renamed this virus as maize red stripe virus and wheat mosaic virus to represent the host and symptom phenotype of the virus. After sequencing the genome of the pathogen, the causal agent of High Plains disease was officially named as High Plains wheat mosaic virus. Hence, High Plains virus, maize red stripe virus, wheat mosaic virus, and High Plains wheat mosaic virus (HPWMoV) are synonyms for the causal agent of High Plains disease.TaxonomyHigh Plains wheat mosaic virus is one of the 21 definitive species in the genus Emaravirus in the family Fimoviridae.VirionThe genomic RNAs are encapsidated in thread‐like nucleocapsids in double‐membrane 80–200 nm spherical or ovoid virions.Genome characterizationThe HPWMoV genome consists of eight single‐stranded negative‐sense RNA segments encoding a single open reading frame (ORF) in each genomic RNA segment. RNA 1 is 6,981‐nucleotide (nt) long, coding for a 2,272 amino acid protein of RNA‐dependent RNA polymerase. RNA 2 is 2,211‐nt long and codes for a 667 amino acid glycoprotein precursor. RNA 3 has two variants of 1,439‐ and 1,441‐nt length that code for 286 and 289 amino acid nucleocapsid proteins, respectively. RNA 4 is 1,682‐nt long, coding for a 364 amino acid protein. RNA 5 and RNA 6 are 1,715‐ and 1,752‐nt long, respectively, and code for 478 and 492 amino acid proteins, respectively. RNA 7 and RNA 8 are 1,434‐ and 1,339‐nt long, code for 305 and 176 amino acid proteins, respectively.Biological propertiesHPWMoV can infect wheat, corn (maize), barley, rye brome, oat, rye, green foxtail, yellow foxtail, and foxtail barley. HPWMoV is transmitted by the wheat curl mite and through corn seed.Disease managementGenetic resistance against HPWMoV in wheat is not available, but most commercial corn hybrids are resistant while sweet corn varieties remain susceptible. Even though corn hybrids are resistant to virus, it still serves as a green bridge host that enables mites to carry the virus from corn to new crop wheat in the autumn. The main management strategy for High Plains disease in wheat relies on the management of green bridge hosts. Cultural practices such as avoiding early planting can be used to avoid mite buildup and virus infections.

Preparation, structure, and properties of tea polysaccharide.

Tea polysaccharide is a kind of acid glycoprotein complex extracted from tea. Tea polysaccharide has a variety of biological activities, especially the hypoglycemic effect is outstanding. It is good for human health. Tea polysaccharides have been extensively studied over the past few decades. The advantages and disadvantages of water extraction, enzyme-assisted extraction, ultrasonic-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction were described. At the same time, the structure and biological activity of tea polysaccharide were also summarized. The development of tea polysaccharide was prospected.

Effect of calcium ions on rheological properties and structure of Lycium barbarum L. polysaccharide and its gelation mechanism

The objectives of this study were to identify the chemical composition, monosaccharide composition, and molecular weight (Mw) of the polysaccharide (LBP) extract from Lycium barbarum L., and investigate the effects of Ca2+ on the zeta potential, rheological properties, infrared spectra (IR), and structure as well as the gelation mechanism of the LBP. The results showed that the LBP was an acidic glycoprotein mixture with an Mw of 4.05 × 105 Da. The addition of Ca2+ first decreased and then increased the absolute value of zeta potential. The elastic modulus (G'), complex viscosity (η*), and stress relaxation modulus (G(t)) of LBP increased with an increase of Ca2+ concentration, and reached the maximum value at the concentration of 12 mM. IR showed that the typical peak wavenumber of the LBP has changed in the presence of Ca2+. SEM and AFM analysis confirmed that Ca2+ could improve the formation of gel network structure in LBP. In addition, the electrostatic interaction and the calcium bridges produced by the addition of Ca2+ played a key role in the formation of the gel network structure. These findings will benefit the application of LBP in the food and pharmaceutical industries.

Variations in the levels of acute-phase proteins and lactoferrin in serum and milk during bovine subclinical mastitis.

Variations in the levels of acute phase proteins and lactoferrin in serum and milk for diagnosis of subclinical mastitis in dairy cows are described in this research paper. Milking animals from two organized dairy farms in Kerala, India, were screened by California Mastitis Test (CMT), Electrical Conductivity test (EC) and Somatic Cell Count (SCC) test to identify animals affected with sub clinical mastitis (SCM). The concentrations of acute phase proteins (APP) Haptoglobin (Hp), C- reactive protein (CRP), Albumin, Lactoferrin (Lf) and α- 1 acid glycoprotein (AGP) in milk and Hp, Albumin, Serum Amyloid A (SAA) and CRP in the serum of 40 normal cows and 40 cows affected with sub clinical mastitis were assessed. Solid phase ELISA was employed for assessment of all parameters except the albumin levels, for which spectrophotometry was used. The values of Hp in milk; and SAA, AGP and Lf in serum, were significantly elevated in the group with sub clinical mastitis. Such variations were found to be independent of the specific bacterial organism causing the disease. These results show that significant variations exist in the levels of acute phase proteins Hp, AGP and Lf in milk, and SAA in serum of animals affected with subclinical bovine mastitis that are not affected by specific bacterial etiology.

The connection of alpha-1 acid glycoprotein inflammatory marker with anthropometric, hormonal, and metabolic characteristic of women with polycystic ovary syndrome.

To investigate the connection of alpha-1 acid glycoprotein inflammatory biomarker with clinical, hormonal, and metabolic characteristics in women with polycystic ovary syndrome (PCOS) and normal cycling controls. A cross-sectional study was conducted on 235 women with PCOS and 92 normal cycling controls attended between 2008 and 2018. Alpha-1 acid glycoprotein levels were correlated with clinical, anthropometric, anthropometric-metabolic indexes, and hormones of women with PCOS and controls. Simple and multivariate stepwise linear regression, matched for age and body mass index confounding variables, was performed. Alpha-1 acid glycoprotein levels were higher in women with PCOS (p=0.0016). In controls, it was positively correlated with waist circumference, fat mass, body adiposity index, and lipid accumulation product, and negatively correlated with sex hormone-binding globulin (p < 0.005 for all comparisons). In PCOS, it was positively correlated with testosterone, most biomarkers of central adiposity, homeostatic model assessment of insulin-resistant, erythrocyte sedimentation rate, and negatively correlated with sex hormone-binding globulin, high-density lipoprotein cholesterol, glucose/insulin ratio, and lymphocytes (p < 0.055 for all comparisons). After multivariate regression in women with PCOS, alpha-1 acid glycoprotein retained a significant positive correlation with erythrocyte sedimentation rate and C-reactive protein. In PCOS, alpha-1 acid glycoprotein is correlated with biomarkers of adiposity, carbohydrate metabolism, and total testosterone. This inflammatory marker is also correlated with erythrocyte sedimentation rate, neutrophils, and lymphocytes, frequent markers of an inflammation state.

Imprinted polymer and Cu2O-graphene oxide nanocomposite for the detection of disease biomarkers

α 1-Acid glycoprotein (α 1-AGp) is a critical plasma protein that acts as a biomarker for different diseases when produced in large amounts (>1.2 mg ml−1). Therefore, selective, label-free, and fast detection of α 1-AGp in human serum is essential. This article presents the development of selective coatings based on molecularly imprinted polymer (MIP) matrix with boronate-affinity and Cu2O-decorated reduced graphene oxide (Cu2O-rGO) nanomaterials. The MIP-Cu2O-rGO hybrid coatings are fabricated on quartz crystal microbalance (QCM) to develop biomimetic α 1-AGp sensors. The MIP:Cu2O-rGO ratio is optimized to enhance sensing properties. Thus-designed MIP-Cu2O-rGO/QCM sensor performs sensitive and specific detection of α 1-AGp in complex mixtures. The initial assessment of the MIP-Cu2O-rGO/QCM sensor reveals an eight-fold increase in the response toward α 1-AGp compared to non-imprinted polymer. The MIP-Cu2O-rGO/QCM sensor exhibits high sensitivity (16.28 Hz ng−1 ml−1) and a low limit of detection (0.25 ng ml−1). When compared with multiple biorelevant analytes such as bovine serum albumin, lysozyme, glucose, fructose, and uric acid, the sensor shows high selectivity due to suitably oriented imprints or interaction sites that are tailored for α 1-AGp recognition. Furthermore, the MIP-Cu2O-rGO/QCM sensor can effectively detect 150–200 ng ml−1 of α 1-AGp in spiked human serum samples with a recovery rate of ∼92%. The results achieved in this study are compared with the relevant literature. The MIP-Cu2O-rGO/QCM sensor can be suitably used for the label-free, precise detection of α 1-AGp in complex mixtures.

P151 Validation of the MaRIA Score in pediatric patients

Abstract Background To validate the Magnetic Resonance Index of Activity (MaRIA Score) in the pediatric population and determine if it would be possible to monitor inflammatory bowel disease (IBD) without invasive tests in some cases. Methods A cross-sectional and descriptive study of paediatric patients with previously diagnosed or suspected IBD who underwent upper endoscopy (EGD) and colonoscopy, blood tests, stool analysis and MR Enterography (MRE) in a 15 days range, from October 2018 to February 2020. The clinical and endoscopic situation were assessed with the activity indices PUCAI/PCDAI/shPCDAI and the activity scores UCEIS/Mayo/SES-CD respectively, according to the underlying pathologies. We considered analytical remission as FC&amp;lt; 250 mcg/g, ERS&amp;lt; 20 mm and CRP&amp;lt; 1 mg/dl. The MRE results were assessed with the MaRIA Score, that is validated for adults. Results Amongst 21 patients, 12 (57%) were males. 12 patients had Crohn ́s disease (CD) (57%), 3 had ulcerative colitis (UC) (14%), 6 had IBD unclassified (IBDu) (28%). The mean age at diagnosis was 14.2 ± 0.7 and the progression time of the disease was 3.9 ± 0.6 years. A total of 16 patients showed clinical remission (76%), 6 of them (28%) also had endoscopic and histological remission. Eight patients were receiving biological treatment (38%). The measured acute phase reactants (APR) were: CRP 0.9 ± 0.5 mg/dl, ESR 13.7 ± 2.6 mm, α1 acid glycoprotein 95.5 ± 1.1 mg/dl and fecal calprotectin (FC) of 1154.3 ± 254.8 mcg/g. Rotavirus, adenovirus, Clostridium difficile toxin analysis and stool culture were performed in 14 patients, all of them negative. The MaRIA Score values were 54.3 ± 53.3, being 0 in 11 cases. We found no differences between the MaRIA Score and the analytical remission. We found a significant correlation between the MaRIA Score and the PUCAI value (p &amp;lt; 0.05), but not with other clinical scores. Patients in clinical remission showed a tendency to have a lower MaRIA Scores, but these differences were not statistically significant (p = 0.09). The MaRIA Score showed lower values for those with endoscopic and histological remission (p &amp;lt; 0.05), All the patients with MaRIA Score&amp;gt; 0 were CD except one, diagnosed with UC. Out of the 15 patients with endoscopic/histological involvement, 10 presented a MaRIA Score&amp;gt; 0. Five of the 11 patients (45%) in whom the MaRIA Score was 0 did not have endoscopic and histological remission, only one of them had a diagnosis of CD. Conclusion The MaRIA Score was significantly correlated with endoscopic scores in pediatric IBD patients, especially in CD. However, based on the results of this study, the MRE should not replace the EGD and colonoscopy in order to thoroughly evaluate the disease activity.

Milk and serum proteomes in subclinical and clinical mastitis in Simmental cows

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SignificanceMastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

Protein binding of clindamycin in vivo by means of intravascular microdialysis in healthy volunteers.

The efficacy of an anti-infective drug is influenced by its protein binding (PB), since only the free fraction is active. We hypothesized that PB may vary in vitro and in vivo, and used clindamycin, a drug with high and concentration-dependent PB to investigate this hypothesis. Six healthy volunteers received a single intravenous infusion of clindamycin 900 mg. Antibiotic plasma concentrations were obtained by blood sampling and unbound drug concentrations were determined by means of in vivo intravascular microdialysis (MD) or in vitro ultrafiltration (UF) for up to 8 h post dosing. Clindamycin was assayed in plasma and MD fluid using a validated HPLC-UV (ultraviolet) method. Non-linear mixed effects modelling in NONMEM® was used to quantify the PB in vivo and in vitro. C max was 14.95, 3.39 and 2.32 mg/L and AUC0-8h was 41.78, 5.80 and 6.14 mg·h/L for plasma, ultrafiltrate and microdialysate, respectively. Calculated ratio of AUCunbound/AUCtotal showed values of 13.9%±1.8% and 14.7%±3.1% for UF and microdialysate, respectively. Modelling confirmed non-linear, saturable PB for clindamycin with slightly different median (95% CI) dissociation constants (Kd) for the alpha-1 acid glycoprotein (AAG)-clindamycin complex of 1.16 mg/L (0.91-1.37) in vitro versus 0.85 mg/L (0.58-1.01) in vivo. Moreover, the estimated number of binding sites per AAG molecule was 2.07 (1.79-2.25) in vitro versus 1.66 in vivo (1.41-1.79). Concentration-dependent PB was observed for both investigated methods with slightly lower levels of unbound drug fractions in vitro as compared with in vivo.

Silica embedded carbon nanosheets derived from biomass acorn cupule for non-enzymatic, label-free, and wide range detection of α 1-acid glycoprotein in biofluids

In this work, we demonstrate the first report on a low-cost, non-enzymatic, label-free electrochemical sensing of alpha-1-acid-glycoprotein (α1GP) biomarker in biofluids using silica embedded carbon nanosheets (SEC) derived from acorn cupules biomass. The SEC/GCE sensor exhibits low detection limit (LOD) of 30 ng/mL (LOD = 3 s/m) which is far below the lowest physiological concentration of α1GP and a wide linear detection range from 100 ng/mL to 10 mg/mL which covers entire clinical concentration range reported for various diseases like cancer, sepsis, etc. The sensing mechanism of the sensor relies on the direct electrooxidation of sialic acid from the α1GP structure which hinder the interfacial electron-transfer process of [Fe (CN)6]3-/4- redox probe at the electrode-electrolyte interface. The sensor exhibits excellent selectivity and stability towards detection of α1GP which is ascribed to the presence of embedded silica nanoparticles on the surface of the carbon nanosheets. The successful determination of α1GP in the simulated blood serum sample with good recovery percentage (i.e., from ∼94.03% to ∼103.50%) proves the feasibility of the sensor towards clinical applications. The overall efficacy of the sensor proves it as a promising low-cost, sustainable, and non-enzymatic platform for a wide variety of bioanalytical applications.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.