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Silver nanoparticles (Ag-NPs) are extremely useful in a diverse range of consumer goods. However, their impact on the environment is still under research, especially regarding the mechanisms involved in their effect. Aiming to provide some insight, the present work analyzes the transcriptional activity of six genes (Hsp83, Hsp17.2, Hsp19.8, SOD Cu-Zn, Mn-SOD, and BPI) in the terrestrial snail Helix aspersa in the presence of different concentrations of Ag-NPs. The animals were exposed for seven days to Lactuca sativa soaked for one hour in different concentrations of Ag-NPs (20, 50, 100 mg/L). The results revealed that the highest concentration tested of Ag-NPs (100 mg/L) led to a statistically significant induction of the Hsp83 and BPI expression in the digestive gland compared to the control group. However, a trend to upregulation with no statistical significance was observed for all the genes in the digestive gland and the foot, while in the hemolymph, the trend was to downregulation. Ag-NPs affected the stress response and immunity under the tested conditions, although the impact was weak. It is necessary to explore longer exposure times to confirm that the effect can be maintained and impact on health. Our results highlight the usefulness of the terrestrial snail Helix aspersa as a bioindicator organism for silver nanoparticle pollution biomonitoring and, in particular, the use of molecular biomarkers of pollutant effect as candidates to be included in a multi-biomarker strategy., (© 2022. The Author(s).)

Background: Fragile X syndrome (FXS) is the leading inherited cause of intellectual disability and is caused by the loss of expression of the Fragile X mental retardation protein (FMRP). In animal model of FXS, the absence of FMRP leads to an aberrant rate of neuronal protein synthesis, which in turn is believed to be at the origin of defects regarding spine morphology and synaptic plasticity. Normalisation of protein synthesis in these models has been associated with a rescue of FXS behavioral and biochemicals phenotype, thus establishing the rate of protein synthesis as one of the most promising monitoring biomarker for FXS. However, rate of protein synthesis alteration in fragile X individuals is not well characterized., Method: We applied a robust radiolabeled assay to measure rate of protein synthesis in freshly extracted peripheral blood mononuclear cells (PBMCs) and blood platelets. We ultimately settle on PBMCs to measure and compare rate of protein synthesis in 13 males with fragile X and 14 matched controls individuals., Results: Using this method, we measured a 26.9% decrease (p = 0,0193) in the rate of protein synthesis in fragile X individuals PBMCs. Furthermore, the rate of protein synthesis measurements obtained were highly reproducible, highlighting the robustness of the method., Conclusion: Our work presents the first evidence of a diminution of the rate of protein synthesis in a human peripheral model of fragile X. Our results also support the finding of previous studies using brain PET imaging in Fragile X individuals. Since our assay only requires a simple venous puncture, it could be used in other cases of intellectual disability in order to determine if an aberrant rate of protein synthesis is a common general mechanism leading to impairment in synaptic plasticity and to intellectual disability., Competing Interests: The authors declare that they have no competing interest.

Preeclampsia (PE) is a serious pregnancy-related disease, and patients usually present with a high inflammatory response. Previous studies have suggested that aspirin (ASP) may have a role in alleviating the pathogenesis of preeclampsia. However, whether ASP can improve kidney damage and the mechanism for improving it is currently unclear. Here we optimized a lipopolysaccharide (LPS)-induced PE mouse model to identify the role of ASP in renal protection. We found that ASP treatment ameliorated LPS-induced renal failure and pathological changes, the tubular injury was significantly attenuated by ASP. Administration of ASP decreased the renal expression of pro-inflammatory factors, resulting in reduced kidney inflammation. The number of GALECTIN-3-positive cells was reduced, and the up-regulation of IL-6 and TNF-α was decreased. In addition, ASP also suppressed renal cell apoptosis and oxidative stress. An in vitro study indicated that ASP relieved LPS-induced HK-2 cell damage by inhibiting WNT5A/NF-κB signaling. Collectively, our data suggest that ASP is a useful therapeutic option for PE-related kidney injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Wei, Suo, Zhang, Yu, Liu, Guo, Zhen and Yu.)

Understanding the molecular and cellular processes in skin wound healing can pave the way for devising innovative concepts by turning the identified natural effectors into therapeutic tools. Based on the concept of broad‑scale engagement of members of the family of galactoside‑binding lectins (galectins) in pathophysiological processes, such as cancer or tissue repair/regeneration, the present study investigated the potential of galectins‑1 (Gal‑1) and ‑3 (Gal‑3) in wound healing. Human dermal fibroblasts, which are key cells involved in skin wound healing, responded to galectin exposure (Gal‑1 at 300 or Gal‑3 at 600 ng/ml) with selective changes in gene expression among a panel of 84 wound‑healing‑related genes, as well as remodeling of the extracellular matrix. In the case of Gal‑3, positive expression of Ki67 and cell number increased when using a decellularized matrix produced by Gal‑3‑treated fibroblasts as substrate for culture of interfollicular keratinocytes. In vivo wounds were topically treated with 20 ng/ml Gal‑1 or ‑3, and collagen score was found to be elevated in excisional wound repair in rats treated with Gal‑3. The tensile strength measured in incisions was significantly increased from 79.5±17.5 g/mm 2 in controls to 103.1±21.4 g/mm 2 after 21 days of healing. These data warrant further testing mixtures of galectins and other types of compounds, for example a combination of galectins and TGF‑β1.

Diabetes-related macrovascular and microvascular complications lead to poor prognosis. Insulin receptor substrate p53 (IRSp53) is known to act as a substrate for the insulin receptor tyrosine kinase, but its role in endothelial dysfunction remains unclear. Human umbilical vein endothelial cells (HUVECs) treated with D-glucose at different concentrations and a streptozocin-induced rat diabetes mellitus (DM) model were used to investigate the effects of hyperglycemia on the expression levels of IRSp53 and galectin-3 (gal-3) and the inflammatory state and mobility of HUVECs. Thereafter, IRSp53-overexpressing HUVECs and IRSp53-knockdown HUVECs were established using IRSp53-overexpressing lentivirus or IRSp53-siRNA to explore the role of IRSp53 in the HUVEC inflammatory state and HUVEC mobility. D-glucose at high concentration (HG) and hyperglycemia were found to induce downregulation of IRSp53 and upregulation of gal-3 in vitro and in vivo . Treatment with HG resulted in activation of NF- κ B in HUVECs and impaired HUVEC mobility. Insulin restored HG-induced changes in the expression levels of IRSp53 and gal-3 in HUVECs and protected the cells from NF- κ B activation and impaired mobility. Overexpression of IRSp53 inhibited the activation of NF- κ B in HUVECs and strengthened HUVEC migration. Knockdown of IRSp53 facilitated the activation of NF- κ B in HUVECs and decreased HUVEC migration. However, neither overexpression nor knockdown of IRSp53 altered the effects of insulin on HG-induced detrimental changes in HUVECs. HG and hyperglycemia resulted in downregulation of IRSp53 in vitro and in vivo . IRSp53 is concluded to inhibit the activation of NF- κ B in HUVECs and to strengthen HUVEC migration., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Fen Liu et al.)

Galectin-3 is supposed as a prognostic factor and therapeutic target for many cancers. In a previous study, we have reported that galectin-3 was related to the development of renal cell cancer and served a therapeutic target for renal cell carcinoma (RCC). However, the mechanisms underlying the regulation of galectin-3 in RCC are still not known. In this study, we detected the expression of galectin-3 and hypoxia-inducible factor 1 (HIF-1) α in RCC using immunohistochemistry, and then conducted in vitro experiments to verify the regulation of galectin-3 by hypoxia in RCC. Our results showed that the expression of galectin-3 and HIF-1α were remarkably high in RCC tissues compared with those in the paracancerous tissues. Interestingly, hypoxia significantly promoted cytoplasmic and nuclear HIF-1α and galectin-3 expression in renal carcinoma cell lines, but not in renal tubular epithelial cell (HK-2). Renal carcinoma cell line (Caki-1), but not HK-2 showed significant increase of luciferase reporter activity of galectin-3 encoding the fragment from the site of -845 to +50 upon hypoxic insult. Moreover, HIF-1α overexpression vector promoted, while HIF-1α silencing vector reduced luciferase reporter activity of galectin-3 in Caki-1 and HK-2 cells in both normal and hypoxia conditions. A direct interaction of HIF-1α with Gal-3 promoter was also verified by electrophoretic mobility shift assay and chromatin immunoprecipitation. Together, our data indicated that hypoxia was critical for galectin-3 expression in RCC in a HIF-1α-dependent manner., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Thyroid hormones have important roles in normal development and energy regulating mechanisms as well as signaling mechanisms that affect energy consumption through central and peripheral pathways. The aim of this study was to determine the effects of thyroid dysfunction on adropin, asprosin and preptin levels in rat., Methods: The study was performed on the 38 male Wistar-albino rats. Experiment groups were designed as follows. 1-Control, 2-Hypothyroidism; To induce hypothyroidism PTU was applied by intraperitoneal as 10 mg/kg/day for 2 weeks. 3-Hypothyroidism + Thyroxine; Previously animals were made with hypothyroidism by 1 week PTU application and then 1 week l-thyroxine was given by intraperitoneal as 1.5 mg/kg/day. 4-Hyperthyroidism; Rats were made with hyperthyroidism by 3 weeks l-thyroxine (0.3 mg/kg/day). 5-Hyperthyroidism + PTU; Animals were made hyperthyroisim by l-thyroxine as groups 4, then 1 week PTU was applied to treatment of hiperthyrodism. At the end of supplementation animals were sacrificed and blood samples were collected for FT3, FT4, adropin, asprosin, preptin analysis., Results: FT3 ve FT4 levels were reduced significantly in hypothyroidism while increased in hyperthyroidism (p<0.001). Hipothyrodism led to reduces adropin, asprosin and preptin levels. And also hyperthyroidism reduced adropin and preptin levels (p<0.001)., Conclusions: The results of study show that experimental hypothyroidism and hyperthyroidism lead to significantly change to adropin, asprosin and preptin levels. However, correction of thyroid function caused to normals levels in asprosin and preptin., (© 2020 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Heart failure (HF) is the most common long-term complication of acute myocardial infarction (MI). Understanding plasma proteins associated with post-MI HF and their gene expression may identify new candidates for biomarker and drug target discovery., Methods: We used aptamer-based affinity-capture plasma proteomics to measure 1305 plasma proteins at 1 month post-MI in a New Zealand cohort (CDCS [Coronary Disease Cohort Study]) including 181 patients post-MI who were subsequently hospitalized for HF in comparison with 250 patients post-MI who remained event free over a median follow-up of 4.9 years. We then correlated plasma proteins with left ventricular ejection fraction measured at 4 months post-MI and identified proteins potentially coregulated in post-MI HF using weighted gene co-expression network analysis. A Singapore cohort (IMMACULATE [Improving Outcomes in Myocardial Infarction through Reversal of Cardiac Remodelling]) of 223 patients post-MI, of which 33 patients were hospitalized for HF (median follow-up, 2.0 years), was used for further candidate enrichment of plasma proteins by using Fisher meta-analysis, resampling-based statistical testing, and machine learning. We then cross-referenced differentially expressed proteins with their differentially expressed genes from single-cell transcriptomes of nonmyocyte cardiac cells isolated from a murine MI model, and single-cell and single-nucleus transcriptomes of cardiac myocytes from murine HF models and human patients with HF., Results: In the CDCS cohort, 212 differentially expressed plasma proteins were significantly associated with subsequent HF events. Of these, 96 correlated with left ventricular ejection fraction measured at 4 months post-MI. Weighted gene co-expression network analysis prioritized 63 of the 212 proteins that demonstrated significantly higher correlations among patients who developed post-MI HF in comparison with event-free controls (data set 1). Cross-cohort meta-analysis of the IMMACULATE cohort identified 36 plasma proteins associated with post-MI HF (data set 2), whereas single-cell transcriptomes identified 15 gene-protein candidates (data set 3). The majority of prioritized proteins were of matricellular origin. The 6 most highly enriched proteins that were common to all 3 data sets included well-established biomarkers of post-MI HF: N-terminal B-type natriuretic peptide and troponin T, and newly emergent biomarkers, angiopoietin-2, thrombospondin-2, latent transforming growth factor-β binding protein-4, and follistatin-related protein-3, as well., Conclusions: Large-scale human plasma proteomics, cross-referenced to unbiased cardiac transcriptomics at single-cell resolution, prioritized protein candidates associated with post-MI HF for further mechanistic and clinical validation.

Objective: Iron overload and oxidative stress are the major causes of serious complications and mortality in thalassemic patients. Our previous work supports the synergistic effects of antioxidant cocktails (curcuminoids or vitamin E, N-acetylcysteine, and deferiprone) in treatment of β-thalassemia/Hb E patients. This further 2-DE-based proteomic study aimed to identify the plasma proteins that expressed differentially in response to antioxidant cocktails., Methods: Frozen plasma samples of ten normal subjects and ten β-thalassemia/Hb E patients at three-time points (baseline, month 6, and month 12) were reduced the dynamic range of proteome using ProteoMiner kit and separated proteins by two-dimensional gel electrophoresis. Differentially expressed proteins were identified using tandem mass spectrometry. Several plasma proteins were validated by ELISA and Western blot analysis., Results: Thirteen and 11 proteins were identified with altered expression levels in the curcuminoids- and vitamin E cocktail groups, respectively. The associations between vitronectin (VTN) expression and total bilirubin levels, as well as between serum paraoxonase/arylesterase 1 (PON1) expression and blood reactive oxygen species were observed. Validation results were consistent with proteomics results., Discussion and Conclusions: These plasma proteins may provide better understanding of the mechanisms underlying the therapeutic effects of antioxidant cocktails in thalassemic patients.

Aims/objectives: We aimed to analyse the molecular backgrounds and red blood cell (RBC) antigen expression of a male blood donor with Rh mod phenotype and his family members., Background: Rh deficiency phenotypes are rarely found worldwide and are characterised by the lack of Rh antigen expression on RBCs. During routine screening, we found a blood donor who seemingly lacked Rh antigens. Therefore, we recruited the donor and his family for further investigation., Methods: RBC serotyping and antibody screening/identification were performed for each sample. A routine blood examination was also conducted. RHD, RHCE and RHAG were sequenced at the genomic DNA or RNA level. Eleven antigens or proteins associated with Rh complex were tested using flow cytometry analysis., Results: The proband and one of his brothers showed extremely weak D antigen and Rh expression levels but did not manifest anaemia. Most of the expressed RBC antigens of the two Rh-deficient individuals were similar to the previously reported cases but with some exceptions. Molecular analyses demonstrated homozygous expression of a novel RHAG allele, namely, c.[572G>A;707A>C], both in the proband and one of his brothers., Conclusions: To our knowledge, we identified the second double-variant RHAG allele and the first one related to Rh mod phenotype. The novel allele was also confirmed to be heritable by family analyses., (© 2019 British Blood Transfusion Society.)

Objectives: The aim of this study was to characterise a novel mutation in the gene encoding RhAG in order to elucidate a molecular mechanism for Rh antigen expression and spherocytosis., Background: Rhesus-associated glycoprotein (RhAG) is critical for maintaining the structure and stability of erythrocytes. Single missense mutations in the gene encoding RhAG are sufficient to induce spherocytosis and deficiencies in Rh complex formation. We report a novel missense mutation that incompletely disrupts Rh antigen expression and selectively knocks out RhD antigen expression., Methods: Blood samples were taken from a 38-year-old male, his brother, his wife and his daughter in Xi'an, China. To detect the proband's RhAG and D antigen expression, the RBC were stained with anti-D and anti-RhAG and analysed by flow cytometry. Red blood cell morphology was detected with atomic force microscopy (AFM). Genomic DNA was isolated from whole blood samples, and the RHD, RHCE and RHAG alleles were sequenced and analysed. The mutation was mapped onto a predicted crystal structure of RhAG by the I-TASSER server and visualised using PyMOL., Results: Morphological testing by AFM found clear evidence of spherocytosis in the proband's erythrocytes. RHAG gene sequencing identified the mutation at sequence 236G > A, resulting in a serine to asparagine substitution at residue 79 (S79N). Family survey indicated that inheriting this allele is necessary and sufficient to cause the condition. Mapping the mutation onto a predicted crystal structure of RhAG revealed the proximity of the mutation to the critical structural elements of the protein., Conclusions: A novel RHAG mutation significantly lowers RhAG antigen expression and antigen-mediated agglutination intensity., (© 2018 British Blood Transfusion Society.)

We previously showed that mice with knockout of Cytl1, a functionally uncharacterized cytokine candidate, exhibit normal endochondral ossification and long-bone development. Here, we investigated the potential functions of CYTL1 in bone homeostasis. We found that Cytl1 -/- mice exhibited higher bone mass than wild-type littermates and resisted ovariectomy-induced bone resorption. This led us to investigate the functions of CYTL1 in the osteogenesis and osteoclastogenesis of bone marrow-derived stem cells. CYTL1 was down-regulated during the osteogenesis of human mesenchymal stem cells (hMSCs). The osteogenesis of hMSCs was inhibited by overexpression or exogenous treatment of CYTL1, but enhanced by CYTL1 knockdown. CYTL1 decreased osteogenesis by inhibiting RUNX2 and promoted proliferation among undifferentiated hMSCs, but stimulated apoptosis among osteogenically differentiating cells. Finally, Cytl1 -/- mice exhibited inhibition of osteoclast activity and the osteoclastogenesis of bone marrow-derived macrophages. Our results collectively suggest that CYTL1 negatively regulates the osteogenesis of MSCs and positively regulates osteoclastogenesis to modulate bone mass in mice.

Objective: The study aimed to explore some novel diagnostic biomarkers for papillary thyroid carcinoma (PTC) by identifying the different expression of TROP-2, SLP-2 and CD56 in benign and malignant thyroid lesions., Methods: We evaluated the mRNA expressions of TROP-2 and SLP-2 in fine needle aspirates (FNAs) which contained 10 PTCs and 10 benign follicular adenomas (FAs) using quantitative real-time PCR (qRT-PCR). Immunohistochemical (IHC) staining of TROP-2, SLP-2 and CD56 was also performed on postoperative samples of 30 PTCs and 29 FAs. Membranous or cytoplasmic staining in > 10% of cells was considered as positive. Diagnostic sensitivity, specificity, positive predictive value, negative predictive value (NPV) and diagnostic accuracy of these three biomarkers were carried out. We further analyzed the associations between the clinical features and the expressions of markers in PTCs., Results: The mRNA expressions of both TROP-2 and SLP-2 were increased substantially in PTCs in comparison with those in FAs (P < 0.05). Similarly, IHC for these two proteins demonstrated higher positive staining in PTCs than in FAs (96.5% vs. 12.5% for TROP-2, 83.3% vs. 20.7% for SLP-2, P < 0.05). Conversely, CD56 expression was lost with 86.7% of PTCs. In identifying malignancy, TROP-2 was the most sensitive marker and CD56 was the most specific one. When the markers were combined, the sensitivity and NPV increased to 100% and had better diagnostic accuracy. However, no association was found between biomarker expressions and clinicopathological factors in PTCs., Conclusions: We found that TROP-2, SLP-2 and CD56 were effective diagnostic markers for PTC, especially when they were combined to use.

Background: Lung adenocarcinoma is characterized by early asymptomatic progression and metastasis. Appearance of respiratory symptoms usually means the disease is aggravated. The aim of this study was to identify the protein profile in plasma of lung adenocarcinoma of stages I-IV, and look for novel diagnostic biomarkers., Methods: Isobaric tags for relative and absolute quantification (iTRAQ) coupled with two dimensional liquid chromatography - tandem mass spectrometry technology (2D LC-MS/MS) was applied to separate and identify differential expression of proteins in plasma specimens from 10 healthy individuals, 10 patients with pneumonia, 7 patients with lung adenocarcinoma of stages I-II, respectively, and 10 patients with lung adenocarcinoma of stages III-IV, then analyze the functions of the differential expression of proteins by bioinformatics., Results: ADAMTS-like protein 4, Fibrinogen-like protein 1 precursor, secretoglobin family 3A member 2 and haptoglobin were up-regulated in patients with lung adenocarcinoma by proteomics analysis, and the expression levels of SCGB3A2 and Hp by ELISA were consistent. Pathway analysis of identified differential expression of proteins showed they were mainly involved in chemokine/p53/TGF-beta signaling pathway., Conclusions: Plasma SCGB3A2 is a potential maker for diagnosis of primary pulmonary adenocarcinoma. Abnormal post-translational protein modification may be associated with the progression of lung cancer., (© 2018 John Wiley & Sons Ltd.)

Essentials Platelets express retinoic acid receptor (RAR)α protein, specifically binding target mRNAs. mRNAs under RARα control include MAP1LC3B2, SLAIN2, and ANGPT1. All-trans retinoic acid (atRA) releases RARα from its target mRNA. RARα expressed in human platelets exerts translational control via direct mRNA binding., Summary: Background Translational control mechanisms in platelets are incompletely defined. Here, we determined whether the nuclear transcription factor RARα controls protein translational events in human platelets. Methods Isolated human platelets were treated with the pan-RAR agonist all-trans-retinoic acid (atRA). Global and targeted translational events were examined. Results Stimulation of platelets with atRA significantly increased global protein expression. RARα protein bound to a subset of platelet mRNAs, as measured by next-generation RNA-sequencing. In-depth analyses of 5' and 3'-untranslated regions of the RARα-bound mRNAs revealed consensus RARα binding sites in microtubule-associated protein 1 light chain 3 beta 2 (MAP1LC3B2), SLAIN motif-containing protein 2 (SLAIN2) and angiopoietin-1 (ANGPT1) transcripts. When platelets were treated with atRA, binding interactions between RARα protein and mRNA for MAP1LC3B2, SLAIN2 and ANGPT1 were significantly decreased. Consistent with the release of bound RARα protein from MAP1LCB2mRNA, we observed an increase in the synthesis of MAP1LC3B2 protein. Conclusions These findings provide the first evidence that RARα, a nuclear transcriptional factor, regulates synthetic events in anucleate human platelets. They also reveal an additional non-genomic role for RARα in platelets that may have implications for the vitamin A-dependent signaling in humans., (© 2017 International Society on Thrombosis and Haemostasis.)

Objective: To identify biomarkers associated with the development of progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS) patients treated with natalizumab (NTZ)., Methods: Relapsing-remitting MS patients who developed PML under NTZ therapy (pre-PML) and non-PML NTZ-treated patients (NTZ-ctr) were included in the study. Cryopreserved peripheral blood mononuclear cells and serum samples collected at baseline, at 1- and 2-year treated time points, and during PML were analyzed for gene expression by RNA sequencing and for serum protein levels by Luminex and enzyme-linked immunosorbent assays, respectively., Results: Among top differentially expressed genes in the RNA sequencing between pre-PML and NTZ-ctr patients, pathway analysis revealed a high representation of genes belonging to the following categories: proangiogenic factors (MMP9, VEGFA), chemokines (CXCL1, CXCL5, IL8, CCL2), cytokines (IL1B, IFNG), and plasminogen- and coagulation-related molecules (SERPINB2, PLAU, PLAUR, TFPI, THBD). Serum protein levels for these candidates were measured in a 2-step manner in a screening cohort and a validation cohort of pre-PML and NTZ-ctr patients. Only matrix metalloproteinase 9 (MMP9) was validated; in pre-PML patients, MMP9 protein levels were significantly reduced at baseline compared with NTZ-ctr patients, and levels remained lower at later time points during NTZ treatment., Interpretation: The results from this study suggest that the proangiogenic factor MMP9 may play a role as a biomarker associated with the development of PML in MS patients treated with NTZ. Ann Neurol 2017;82:186-195., (© 2017 American Neurological Association.)

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700-800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

Renal proximal tubule epithelial cells (PTECs) are known to reabsorb salts and small plasma proteins filtered through Bowman's capsule. Following acute kidney injury, PTECs assume some characteristics of hepatocytes in producing various plasma proteins. We now demonstrate that even at a resting state, a PTEC cell line, HK2 expresses mRNAs for and synthesizes and secretes plasma proteins in a complex with complement C3, an α 2 -macroglobulin family chaperone, including albumin, transferrin, α 1 -antitrypsin, α 1 -antichymotrypsin, α 2 -HS-glycoprotein, ceruloplasmin, haptoglobin, C1-inhibitor, secreted phosphoprotein-24, and insulin-like growth factor-1. When grown on transwell inserts, HK2 cells predominantly secrete (∼90%) plasma proteins into the apical side and a smaller fraction into the basolateral side as determined by ELISA assays. When cultured in the presence of exogenous cytokines such as IL1β, IL6, TNFα, BMP2, or TGFβ1, HK2 cell mRNA expressions for plasma proteins were variably affected whereas basolateral secretions were elevated to or in excess of those of the apical level. In addition, HK2 cells produce proTGFβ1 with its intact N-terminal latency associated peptide and latent-TGF-β-binding proteins. The complex cannot be dissociated under conditions of SDS, heating, and electrophoresis. Moreover, HK2 cells maintain their ability to quickly uptake exogenously added serum proteins from the culture medium, as if they are recognized differently by the endocytic receptors. These results provide new insight into the hepatization of PTECs. In addition to their unique uptake of plasma proteins and salts from the filtrate, they are a source of urinary proteins under normal conditions as wells as in chronic and acute kidney diseases. J. Cell. Biochem. 118: 924-933, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Tunicates have been used as primitive models for understanding cell-mediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn 2+ ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, β-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn 2+ added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn 2+ import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn 2+ has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.

Introduction: Red blood cells (RBC) are the most abundant host cells in the human body. Mature erythrocytes are devoid of nuclei and organelles and have always been regarded as circulating 'bags of hemoglobin'. The advent of proteomics has challenged this assumption, revealing unanticipated complexity and novel roles for RBCs not just in gas transport, but also in systemic metabolic homeostasis in health and disease. Areas covered: In this review we will summarize the main advancements in the field of discovery mode and redox/quantitative proteomics with respect to RBC biology. We thus focus on translational/clinical applications, such as transfusion medicine, hematology (e.g. hemoglobinopathies) and personalized medicine. Synergy of omics technologies - especially proteomics and metabolomics - are highlighted as a hallmark of clinical metabolomics applications for the foreseeable future. Expert commentary: The introduction of advanced proteomics technologies, especially quantitative and redox proteomics, and the integration of proteomics data with omics information gathered through orthogonal technologies (especially metabolomics) promise to revolutionize many biomedical areas, from hematology and transfusion medicine to personalized medicine and clinical biochemistry.

Background: To identify potential biomarkers in the 1st trimester of pregnancy for the identification of women destined to develop early onset preeclampsia (EOPE)., Methods: Blood samples were obtained from pregnant women at 11-13 weeks of gestation. Women were followed up until delivery. Five samples from EOPE complicated pregnancies and 5 from unaffected ones were analysed using 2-DE and MALDI-TOF-TOF MS/MS. The altered expression of selected proteins was verified by ELISA in an extended sample cohort., Results: Twelve proteins were differentially expressed in the plasma of women who subsequently developed EOPE as compared to controls. Alpha-1-antitrypsin (A1AT), CD5 antigen-like molecule (CD5L) Keratin, type I cytoskeletal 9 (K1C9), Myeloid cell nuclear differentiation antigen (MNDA), Transferrin (TRFE) and Vitamin D-binding protein (VTDB) were up-regulated with fold changes 3.14, 2.18, 1.53, 1.53, 4.26 3.38 respectively, whereas Alpha-2-HS-glycoprotein (FETUA), Beta-2-glycoprotein 1 (APOH), Complement factor B (CFAB), Haptoglobin (HPT), Vitronectin (VTNC) and Zinc-alpha-2-glycoprotein (ZA2G) were down-regulated with fold changes -0.38, -0.76, -0.24, -0.47, -0.23, and -0.50 respectively. The down-regulation of APOH, VTNC and HPT was verified using ELISA., Conclusions: The differentially expressed proteins represent potential biomarkers for the early screening for EOPE. Follow-up experiments however are necessary for evaluation.

Current nutrition research is focusing on health promotion, disease prevention, and performance improvement for individuals and communities around the world. The humans with required nutritional ingredients depend on both how well the individual is provided with balanced foods and what state of gut microbiota the host has. Studying the mutually beneficial relationships between gut microbiome and host is an increasing attention in biomedical science. The purpose of this study is to understand the role of gut microbiota and to study interactions between gut microbiota and host. In this study, we used a shotgun proteomic approach to reveal the serum and liver proteomes in gut-microbiota-lacking mice. For serum, 15 and 8 proteins were uniquely detected in specific-pathogen-free (SPF) and germ-free (GF) mice, respectively, as well as the 3 and 20 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Among the proteins of the serum, major urinary protein 1 (MUP-1) of GF mice was significantly decreased compared to SPF mice. In addition, MUP-1 expression is primarily regulated by testosterone. Lacking in gut flora has been implicated in many adverse effects, and now we have found its pathogenic root maybe gut bacteria can regulate the sex-hormone testosterone levels. In the liver, 8 and 22 proteins were uniquely detected in GF mice and SPF mice, respectively, as well as the 14 and 30 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Furthermore, ingenuity pathway analysis (IPA) indicated that gut microbiota influence the host in cancer, organismal injury and abnormalities, respiratory disease; cell cycle, cellular movement and tissue development; cardiovascular disease, reproductive system disease; and lipid metabolism, molecular transport and small molecule biochemistry. Our findings provide more detailed information of the role of gut microbiota and will be useful to help study gut bacteria and disease prevention., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan-related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N-glycans, free oligosaccharides (fOSs), glycosaminoglycans, O-glycans, and glycosphingolipid (GSL)-glycans, were quantified. In both serum samples, N-glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N- and O-glycosylation, while GSL-glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Doxorubicin and Cyclophosphamide (AC protocol) combination is usually considered as a first line therapy in newly diagnosed breast cancer patients. Thus, a retrospective observational study was conducted to monitor the effect of AC protocol on liver synthetic functions and production of plasma proteins in breast cancer patients, reporting to specialized cancer care hospital of Lahore, Pakistan. A total of 75 patients (n=75) on AC protocol with breast cancer were observed in this study. The patient data including age, gender, body surface area, dosage, disease status and laboratory biochemical values were recorded by reviewing historical treatment records. Pre-treatment values were taken as baseline values for albumin, globulin, blood urea nitrogen (BUN), albumin/globulin (A/G) ratio and total proteins. The baseline values were compared after each cycle of by applying ANOVA using statistical tool SPSS® version 21. The plasma levels of blood urea nitrogen (BUN), total protein and globulin dropped significantly (p<0.05) in patients of all age groups. However, the albumin levels were not significantly changed (p>0.05). The A/G ratio level increased (p<0.05) as a result of reduction in globulin levels. Significant changes in plasma protein levels were observed in the elderly patients (50 to 65 years) than patients between 20 to 50 years of age. AC protocol impairs liver synthetic functions as observed by decreased blood urea nitrogen (BUN) and plasma protein levels.

Purpose: Pathogen inactivation technologies (PITs) were introduced into blood banking to further improve the safety of blood products. However, the UV light used in PITs to terminate pathogen growth might alter the functionality of the cells in the blood product as well as the protein profile of the blood components. This study employed proteomic approaches to assess changes in the platelet proteome and translatome., Experimental Design: Apheresis-derived platelet concentrates treated with riboflavin/UV light or untreated controls were analyzed throughout blood bank storage by quantitative proteomics using iTRAQ and puromycin-associated nascent chain (PUNCH) proteomics., Results: Quantitative proteomic analysis identified 408 individual proteins including 26 unique proteins that changed in the treated arm during storage. Proteomic results were confirmed using immunoblot analyses and results suggested a translational control of the protein expression profile. PUNCH proteomic analysis of day 7 samples from illuminated units identified 52 unique platelet proteins that incorporated puromycin, including proteins involved in the cytoskeleton, metabolism, and signaling., Conclusion and Clinical Relevance: This study demonstrates for the first time that platelets can synthesize proteins despite the riboflavin and UV treatment and suggests that platelets may possess a mechanism to protect their mRNA from damage by the PI treatment., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Aided by the advent of advanced mass spectrometry (MS)-based technologies and methodologies, quantitative proteomics has emerged as a viable technique to capture meaningful data for candidate biomarker evaluation. To aid clinical translation, these methods generally utilize a bottom-up strategy with isotopically labeled standards and a targeted form of MS measurement., Areas Covered: This article reviews the status, challenges, requirements, and potential of translating current, MS-based methods to the clinical laboratory. The described methods are discussed and contrasted within a fit-for-purpose approach, while different resources for quality control, quantitative analysis, and data interpretation are additionally provided. Expert commentary: Although great strides have been made over the past five years in developing reliable quantitative assays for plasma protein biomarkers, it is crucial for investigators to have an understanding of the clinical validation process, a major roadblock in translational research. Continued progress in method design and validation of protein assays is necessary to ultimately achieve widespread adoption and regulatory approval.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins., (Copyright © 2016. Published by Elsevier B.V.)

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

The multifactorial chronic inflammatory disease periodontitis, which is characterized by destruction of tooth-supporting tissues, has also been implicated as a risk factor for various systemic diseases. Although periodontitis has been studied extensively, neither disease-specific biomarkers nor therapeutic targets have been identified, nor its link with systemic diseases. Here, we analyzed the global transcriptome of periodontitis and compared its gene expression profile with those of other inflammatory conditions, including cardiovascular disease (CVD), rheumatoid arthritis (RA), and ulcerative colitis (UC). Gingival biopsies from 62 patients with periodontitis and 62 healthy subjects were subjected to RNA sequencing. The up-regulated genes in periodontitis were related to inflammation, wounding and defense response, and apoptosis, whereas down-regulated genes were related to extracellular matrix organization and structural support. The most highly up-regulated gene was mucin 4 (MUC4), and its protein product was confirmed to be over-expressed in periodontitis. When comparing the expression profile of periodontitis with other inflammatory diseases, several gene ontology categories, including inflammatory response, cell death, cell motion, and homeostatic processes, were identified as common to all diseases. Only one gene, pleckstrin (PLEK), was significantly overexpressed in periodontitis, CVD, RA, and UC, implicating this gene as an important networking link between these chronic inflammatory diseases.

Serum composition is linked to metabolic diseases not only to understand their pathogenesis but also for diagnostic purposes. Quality and quantity of nutritional intake can affect disease risk and serum composition. It is then possible that diet derived serum components directly affect pathogenetic mechanisms. To identify involved factors, we evaluated the effect on gene expression of direct addition of dyslipidemic human serum samples to cultured human hepatoma cells (HepG2). Sera were selected on the basis of cholesterol level, considering this parameter as mostly linked to dietary intake. Cells were treated with 32 sera from hypercholesterolemic and normocholesterolemic subjects to identify differentially regulated mRNAs using DNA microarray analysis. We identified several mRNAs with the highest modulations in cells treated with dyslipidemic sera versus cells treated with normal sera. Since the two serum groups had variable polyunsaturated fatty acids (PUFAs) contents, selected mRNAs were further assessed for their regulation by docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA). Four genes resulted both affected by serum composition and PUFAs: 3-hydroxy-3-methylglutaryl-CoenzymeA synthase 2 (HMGCS2), glutathione S-transferase alpha 1 (GSTA1), liver expressed antimicrobial peptide 2 (LEAP2) and apolipoprotein M (ApoM). HMGCS2 expression appears the most relevant and was also found modulated via transcription factors peroxysome proliferator activated receptor α (PPARα) and forkhead box O1 (FoxO1). Our data indicate that expression levels of the selected mRNAs, primarily of HMGCS2, could represent a reference of nutritional intake, PUFAs effects and dyslipidemic diseases pathogenesis., (© 2015 Wiley Periodicals, Inc.)

Purpose: Exploratory gene expression array analyses suggested multimerin-1 (MMRN1) to be a predictive biomarker in acute myelogenous leukemia (AML). Following up on these studies, we evaluated the role of MMRN1 expression as outcome predictor in two recent Children's Oncology Group trials., Experimental Design: We retrospectively quantified MMRN1 expression in 183 participants of AAML03P1 and 750 participants of AAML0531 by reverse-transcriptase PCR and correlated expression levels with disease characteristics and clinical outcome., Results: In AAML03P1, the highest quartile of MMRN1 expression (expression ≥0.5 relative to β-glucuronidase; n = 45) was associated with inferior event-free survival (EFS; P < 0.002) and higher relapse risk (P < 0.004). In AAML0531, in which we quantified MMRN1 mRNA for validation, patients with relative MMRN1 expression ≥0.5 (n = 160) less likely achieved remission (67% vs. 77%, P = 0.006), and more frequently had minimal residual disease (43% vs. 24%, P = 0.001) after one induction course. They had inferior overall survival (OS; 44% ± 9% vs. 69% ± 4% at 5 years; P < 0.001) and EFS (32% ± 8% vs. 54% ± 4% at 5 years; P < 0.001) and higher relapse risk (57% ± 10% vs. 35% ± 5% at 5 years; P < 0.001). These differences were partly attributable to the fact that patients with high MMRN1 expression less likely had cytogenetic/molecular low-risk disease (P < 0.001) than those with low MMRN1 expression. Nevertheless, after multivariable adjustment, high MMRN1 expression remained statistically significantly associated with shorter OS (HR, 1.57; 95% confidence interval, 1.17-2.12; P = 0.003) and EFS (HR, 1.34; 1.04-1.73; P = 0.025), and higher relapse risk (HR, 1.40; 1.01-1.94; P = 0.044)., Conclusions: Together, our studies identify MMRN1 expression as a novel biomarker that may refine AML risk stratification., (©2015 American Association for Cancer Research.)

Previously, we demonstrated that inhibition of proteasomal chymotrypsin-like (CT-like) activity in human prostate cancer (PCa) PC-3 cultures and PC-3 xenografts results in accumulation of ubiquitinated proteins, followed by induction of cell death. Studies have shown that plasma CT-like proteasomal activity may be a powerful biomarker for risk stratification in hematologic malignancies. We hypothesized that circulating proteasomes could also be used to stratify risk for patients with PCa. A total of 109 patients with suspected PCa underwent prostatic biopsies were enrolled. Subjects were divided into non-cancer, low-risk PCa, and high-risk PCa groups. Three different proteasomal activity markers (CT-like, caspase-like, and trypsin-like) were measured and compared among the three groups. The proteasomal target proteins, Ub-prs, Hsp70, Bax, and P27 in plasma and prostate tissues were also evaluated. Multivariate analysis was used to assess whether CT-like activity was a predictor of PCa progression. Only proteasomal CT-like activity in the high-risk group was statistically higher than in the non-cancer group (P < 0.05). The expression of Ub-prs, Hsp70, Bax, and P27 protein was decreased in both plasma and PCa tissue of high-risk patients. CT-like activity was found to be an independent predictor of high-risk PCa. Subjects with CT-like activity ≥55 had a 2.15-fold higher risk of having high-risk PCa as compared to those with a CT-like activity of <55 (P = 0.021). We found CT-like activity to be an independent predictor of high-risk PCa, and as such, it may be a good candidate as a biomarker for high-risk PCa detection and stratification.

Newly discovered intrinsic regulators, the miRNAs regulate gene expression by binding to the 3'-untranslated regions of the genome. Accumulating studies have indicated that miRNAs are aberrantly expressed in various human cancers. We found that miRNA-1207-5p (miR‑1207-5p) was markedly downregulated in esophageal carcinoma (EC) tissues, and was correlated with EC differentiation, pathological stage and lymph node metastasis. Rates of apoptosis were increased and cell invasion ability was decreased in EC9706 and EC-1 cells transfected with a miR‑1207-5p mimic. Stomatin-like protein 2 (STOML-2) was predicted to be a potential target of miR‑1207-5p by bioinformatics analysis and this was confirmed by luciferase assay and western blotting. Our study showed that STOML-2 was negatively regulated by miR‑1207-5p. Furthermore, overexpression of STOML-2 abolished the miR‑1207-5p anti-invasion function. Based on these results, we proposed that miR‑1207-5p might act as a potential therapeutic target in the treatment of EC.

Intrauterine growth restriction (IUGR) is associated with increased morbidity and metabolic anomalies in adults. The serum proteome of venous blood was compared in 43 IUGR and 45 adequate gestational age (AGA) infants, separated into three gestational age groups, "Very Preterm" 29-32weeks, "Moderate Preterm" 33-36w, and, "Term" ≥37weeks, in samples drawn three times from birth to 1month of life. After depleting the abundant serum proteins (ProteoMiner(TM)), expression changes were studied by 2-DE, image analysis (Proteomweaver 4.0(TM)), and identification by MALDI-TOF/TOF. Significant expression differences were found in thirty-four proteins, and thirty-three were identified. Lysophospholipid acyltransferase 7 (MBOAT7), was detected exclusively in IUGR of all gestational ages and sampling times; seven other proteins were found only in AGA. Another twenty-five proteins had intensity changes ≥2.5 folds in IUGR: twenty were upregulated and five downregulated. Western blots confirmed the identification of several proteins: MBOAT7 increased 20.5-fold in IUGR, while AGA had 11.2-fold higher levels of SUMO3 and sumoylated proteins and 13.7-fold higher levels of APOL1. Upregulation of MBOAT7 in IUGR neonates could be an adaptive response to protect the brain from an adverse environment., Biological Significance: There are significant protein expression differences between IUGR and AGA at different gestational age groups and blood extraction times. The extensive upregulation of lysophospholipid acyltransferase 7 in all IUGR gestational ages and extraction times might be an adaptative response to an adverse fetal environment, reminiscent of Barker's fetal programming theory. Two serotransferrins were also upregulated in IUGR of all gestational ages. Just at birth "Very Preterm" IUGR showed nine upregulated proteins, including five albumins, apolipoprotein E, keratin type I cytoskeletal 10, solute carrier family member 2 fragment, and anaphase-promoting complex subunit 2., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The natural abundance of ¹⁵N in animal proteins (δ¹⁵Nanimal) is greater than that in the diet consumed by the animals (δ¹⁵Ndiet), with a discrimination factor (Δ¹⁵N = δ¹⁵Nanimal - δ¹⁵Ndiet) that is known to vary according to nutritional conditions. The objectives of the present study were to test the hypothesis that Δ¹⁵N variations depend on the efficiency of nitrogen utilisation (ENU) in growing beef cattle, and to identify some of the physiological mechanisms responsible for this N isotopic fractionation in ruminants. Thus, we performed the regression of the Δ¹⁵N of plasma proteins obtained from thirty-five finishing beef cattle fed standard and non-conventional diets against different feed efficiency indices, including ENU. We also performed the regression of the Δ¹⁵N of different ruminant N pools (plasma and milk proteins, urine and faeces) against different splanchnic N fluxes obtained from multi-catheterised lactating dairy cows. The Δ¹⁵N of plasma proteins was negatively correlated with feed efficiency indices in beef cattle, especially ENU (body protein gain/N intake) and efficiency of metabolisable protein (MP) utilisation (body protein gain/MP intake). Although Δ¹⁵N obtained from different N pools in dairy cows were all negatively correlated with ENU, the highest correlation was found when Δ¹⁵N was calculated from plasma proteins. Δ¹⁵N showed no correlation with urea-N recycling or rumen NH₃ absorption, but exhibited a strong correlation with liver urea synthesis and splanchnic amino acid metabolism, which points to a dominant role of splanchnic tissues in the present N isotopic fractionation study.

Recent studies have demonstrated that the DNA methylome changes with age. This epigenetic drift may have deep implications for cellular differentiation and disease development. However, it remains unclear how much of this drift is functional or caused by underlying changes in cell subtype composition. Moreover, no study has yet comprehensively explored epigenetic drift at different genomic length scales and in relation to regulatory elements. Here we conduct an in-depth analysis of epigenetic drift in blood tissue. We demonstrate that most of the age-associated drift is independent of the increase in the granulocyte to lymphocyte ratio that accompanies aging and that enrichment of age-hypermethylated CpG islands increases upon adjustment for cellular composition. We further find that drift has only a minimal impact on in-cis gene expression, acting primarily to stabilize pre-existing baseline expression levels. By studying epigenetic drift at different genomic length scales, we demonstrate the existence of mega-base scale age-associated hypomethylated blocks, covering approximately 14% of the human genome, and which exhibit preferential hypomethylation in age-matched cancer tissue. Importantly, we demonstrate the feasibility of integrating Illumina 450k DNA methylation with ENCODE data to identify transcription factors with key roles in cellular development and aging. Specifically, we identify REST and regulatory factors of the histone methyltransferase MLL complex, whose function may be disrupted in aging. In summary, most of the epigenetic drift seen in blood is independent of changes in blood cell type composition, and exhibits patterns at different genomic length scales reminiscent of those seen in cancer. Integration of Illumina 450k with appropriate ENCODE data may represent a fruitful approach to identify transcription factors with key roles in aging and disease.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Background: d-Ribose is a popular dietary supplement for humans and the equine because of its crucial role in cellular bioenergetics. However, as a reducing sugar, it has been suggested that ingestion of d-ribose might promote the formation of glycated proteins in vivo with potential adverse consequences., Objective: The aim of this study was to examine if d-Ribose would promote the formation of glycated proteins in vivo following exercise in training thoroughbred racehorses., Methods: Two groups of horses received the supplement (30 and 50 g d-Ribose daily) for 17 weeks, during which period the horses were subjected to low-intensity exercises followed by high-intensity exercises. Blood samples were analyzed for glycated plasma proteins at baseline and following the 2 exercise regimens., Results: This study shows that long-term ingestion of d-Ribose at 30-50 g a day does not promote the formation of glycated plasma proteins in thoroughbred racehorses. Ribose supplementation also protected the horses from cramping while enhancing muscle recovery at the same time. No adverse effects were reported., Conclusion: Ribose supplementation is safe and does not cause glycation in vivo. This investigation also establishes safety of d-Ribose in thoroughbred racehorses, suggesting similar implications in humans as well.

Background and Objective: Looking for tumor markers by using protein chip technology is one of the hot topics, but many studies are still limited on short term detection of differential expressed proteins before and after surgery among patients with RCC. This study analyzed differential expressed serum protein and its clinical significance with clear-cell renal cell carcinoma to further measurement of the rule of variable expressing., Methods: Eighty-nine patients with clear-cell renal cell carcinoma who underwent surgery from November 2013 to 2014 and postoperatively confirmed by pathology were entered in RCC group, 100 healthy volunteers and patients without RCC who underwent medical examination in the same period were entered in control group. The serum protein were analyzed in both group before surgery and every regular follow-up period in 1 year after surgery with RCC group. The surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and weak cation exchange protein chip (CM10) technology systems were used for identifying differential expressed serum protein in RCC group and controls. The linear support vector machine (SVM) was applied to establish the diagnostic model of protein fingerprints and the leave-one-out cross validation was used for determining model discriminating effect. The differential expressed proteins were analyzed by ZUCI-PDAS protein spectral data analysis system., Results: Five kinds of proteins were identified as potential biomarker, ultimately. The M/Z of these proteins was 15953, 7987, 9304, 8948, 5911, respectively. There were significant difference on expression level of these proteins with two groups preoperatively (P< 0.05). Comparison of all postoperative expression levels to preoperative one and each differential level mutually between a year in postoperative period also revealed statistical significance (P< 0.05). With taking identified proteins as biomarker, the sensitivity and specificity in predicting clear-cell renal cell carcinoma was 88.8% (79/89) and 91.0% (91/100), respectively., Conclusion: The corresponding specific protein was Bcl-2 family apoptosis regulatory proteins, WAP four-disulfide core protein, Krueppel-like factor 8, monocyte chemotactic protein-1, serum amyloid β -protein-4, respectively, and will may serve as tumor markers of kidney cancer. These proteins manifests high predictive value for clear-cell renal cell carcinoma, and may contribute to therapeutic evaluation, prognosis and targeted therapy for clear-cell renal cell carcinoma.

The delicate balance between eukaryotic pathogens and their human hosts during the initiation and development of infection is a complex process involving many diverse interactions. Different infectious agents, including pathogenic fungi, parasitic protozoa and multicellular parasites, directly interact through their cell surface with epithelial or endothelial cells of the human host as well as various proteinaceous host ligands such as extracellular matrix or plasma proteins. Eukaryotic pathogens possess a number of virulence factors but a relatively recently recognized and particularly interesting group of factors capable of enhancing virulence is the set of so-called 'moonlighting proteins'. This term was coined for a relatively large collection of housekeeping enzymes lacking special targeting motifs that would determine their extracellular localization, but that are often present at the cell surface of pathogen. Several such enzymes with key metabolic functions in glycolysis, the pentose phosphate cycle or other fundamental intracellular processes perform entirely new, non-catalytic roles often associated with adhesion to host ligands. Our current study summarizes some of the current knowledge of interesting moonlighting proteins which play putative or confirmed roles as virulence factors in pathogenic fungi, parasitic protozoa and multicellular parasites., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Objectives: Low availability of oxygen at high altitudes has a great impact on the human life processes. There is a widespread interest and need to find out protein(s) that are possibly involved in mediating tolerance to hypobaric hypoxia. We undertook this study to identify and characterize protein expression in plasma of hypoxia susceptible and tolerant rats., Methods: Male albino Sprague Dawley rats were segregated into susceptible and tolerant groups on the basis of their gasping time when exposed to simulated hypobaric hypoxia of 32,000 ft (9,754 m) at 32°C. Comparative proteome profiling of blood plasma of hypoxia susceptible and tolerant individuals was performed using 2-dimentional (2-D) gel electrophoresis., Results: Three proteins with higher expression levels were selected separately from tolerant and susceptible samples. Characterization of these proteins from tolerant sample using MALDI-TOF/TOF and MASCOT search indicated their homology with two different super-families viz. NADB-Rossmann superfamily (Rab GDP dissociation inhibitor β) and Transferrin superfamily (two Serotransferrins), having potential role in imparting tolerance against hypoxia. Three high level upregulated proteins were characterized from blood plasma of hypoxia susceptible animals showing similarity with threonine tRNA ligase (mitochondrial), carbohydrate sulphotransferase 7 and aspartate tRNA ligase (cytoplasmic) that play a role in ATP binding, carbohydrate metabolism and protein biosynthesis, respectively., Interpretation & Conclusions: Our results indicated that rats segregated into hypoxia sensitive and tolerant based on their gasping time showed differential expression of proteins in blood plasma. Characterization of these differentially expressed proteins will lead to better understanding of molecular responses occurring during hypoxia and subsequently development of biomarkers for categorization of hypoxia susceptible and tolerant individuals.

Venomous and non-venomous snakes possess phospholipase A2 (PLA2) inhibitory proteins (PLIs) in their blood serum. This study shows the expression and biochemical and functional characterization of a recombinant alpha inhibitor from Bothrops alternatus snake, named rBaltMIP. Its expression was performed in Pichia pastoris heterologous system, resulting in an active recombinant protein. The expressed inhibitor was tested regarding its ability to inhibit the phospholipase activity of different PLA2s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA2:PLI). rBaltMIP was also effective in decreasing the myotoxic activity of the tested toxins at molar ratios greater than 1:0.4 (myotoxin:PLI). The inhibition of the myotoxic activity of different Asp49 (BthTX-II and PrTX-III) and Lys49 (BthTX-I and PrTX-I) myotoxins was also performed without the prior incubation of myotoxins/inhibitor in order to analyze the real possibility of using snake plasma inhibitors or recombinant inhibitors as therapeutic agents for treating envenomations. As a result, rBaltMIP was able to significantly inhibit the myotoxicity of Lys49 myotoxins. Histopathological analysis of the gastrocnemius muscles of mice showed that the myotoxins are able to induce severe damage to the muscle fibers of experimental animals by recruiting a large number of leukocyte infiltrates, besides forming an intense accumulation of intercellular fluid, leading to local edema. When those myotoxins were incubated with rBaltMIP, a reduction of the damage site could be observed. Furthermore, the cytotoxic activity of Asp49 PLA2s and Lys49 PLA2-like enzymes on C2C12 cell lines was decreased, as shown by the higher cell viabilities after preincubation with rBaltMIP. Heterologous expression would enable large-scale obtainment of rBaltMIP, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of snake PLA2s, which have not yet been fully clarified., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Aim: To evaluate the expression of atherosclerosis-associated genes in patients with hypertension and type 2 diabetes mellitus., Material and Methods: Twenty-seven patients (14 men, 13 women), mean age 43.26 ± 11.69 years, were included in the study, which was divided into three groups: group 1 - patients with newly diagnosed hypertension and normal glucose tolerance (n = 9), group 2 - normotensive individuals with newly diagnosed type 2 diabetes (n = 9), and control group - normotensive individuals with normal glucose tolerance (n = 9). Gene expression analysis was performed with Human Atherosclerosis RT2 Profiler PCR Array., Results: In patients with hypertension, we found eight genes with increased expression - FABP3, FAS, FN1, IL1R2, LPL, SERPINE1, TGFB1, and VCAM1. Decreased expression was observed for two genes - SELPLG and SERPINEB2. In patients with type 2 diabetes we found seven up-regulated genes - APOE, BAX, MMP1, NFKB1, PDGFB, SPP1, and TGFB2, whereas no specifically down-regulated genes were observed. Three genes - KLF2, PDGFRB, and PPARD were found to be expressed only in groups 1 and 2., Conclusion: Hypertension is associated with increased expression of FABP3, FAS, FN1, IL1R2, LPL, SERPINE1, TGFB1, and VCAM1 and decreased expression of SELPLG and SERPINEB2. The up-regulation of FAS, FN1, SERPINE1, TGFB1, and VCAM1 might be associated with an increased cardiovascular risk. Type 2 diabetes is associated with increased expression of APOE, BAX, MMP1, NFKB1, PDGFB, SPP1, and TGFB2. KLF2 and PPARD might be part of protective mechanisms that limit target organ damage in both disease conditions. Expression of PDGFRB might play an important role in the pathogenesis of both hypertension and type 2 diabetes.

Background: The yolk sac (YS) is an extra-embryonic tissue that surrounds the yolk and absorbs, digests and transports nutrients during incubation of the avian embryo as well as during early term mammalian embryonic development. Understanding YS functions and development may enhance the efficient transfer of nutrients and optimize embryo development. To identify temporal large-scale patterns of gene expression and gain insights into processes and mechanisms in the YS, we performed a transcriptome study of the YS of chick embryos on embryonic days (E) E13, E15, E17, E19, and E21 (hatch)., Results: 3547 genes exhibited a significantly changed expression across days. Clustering and functional annotation of these genes as well as histological sectioning of the YS revealed that we monitored two cell types: the epithelial cells and the erythropoietic cells of the YS. We observed a significant up-regulation of epithelial genes involved in lipid transport and metabolism between E13 and E19. YS epithelial cells expressed a vast array of lipoprotein receptors and fatty acid transporters. Several lysosomal genes (CTSA, PSAP, NPC2) and apolipoproteins genes (apoA1, A2, B, C3) were among the highest expressed, reflecting the intensive digestion and re-synthesis of lipoproteins in YS epithelial cells. Genes associated with cytoskeletal structure were down-regulated between E17 and E21 supporting histological evidence of a degradation of YS epithelial cells towards hatch. Expression patterns of hemoglobin synthesis genes indicated a high erythropoietic capacity of the YS between E13 and E15, which decreased towards hatch. YS histological sections confirmed these results. We also observed that YS epithelial cells expressed high levels of genes coding for plasma carrier proteins (ALB, AFP, LTF, TTR), normally produced by the liver., Conclusions: Here we expand current knowledge on developmental, nutritional and molecular processes in the YS. We demonstrate that in the final week of chick embryonic development, the YS plays different roles to support or replace the functions of several organs that have not yet reached their full functional capacity. The YS has a similar functional role as the intestine in digestion and transport of nutrients, the liver in producing plasma carrier proteins and coagulation factors, and the bone marrow in synthesis of blood cells.

Background: As diagnosis of nasopharyngeal carcinoma at an early disease stage is important, we attempted to distinguish between patients with nasopharyngeal carcinoma and noncancer controls by using serum protein profiles., Methods: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and CM10 protein chip were used to detect the serum proteomic patterns of 65 patients with nasopharyngeal carcinoma before radiotherapy and 93 noncancer controls. Proteomic spectra of serum samples from 50 nasopharyngeal carcinoma patients and 60 noncancer controls were used as a training set. The validity of the classification tree was then challenged with a blind test set which included another 15 patients with nasopharyngeal carcinoma and 33 noncancer controls. Biomarker Wizard 3.01 and Biomarker Pattern 5.01 were used in combination to analyze the data and to develop diagnostic models., Results: 21 protein peaks were significantly different between nasopharyngeal carcinoma and controls. 4 mass peaks (M4182, M5343, M5913 and M8702 mass/charge ratio) were chosen automatically to construct a classification tree. The classification tree correctly determined 93.8 % (45/48) of the test samples with 93.3 % (14/15) of the nasopharyngeal carcinoma samples and 93.9 % (31/33) of the noncancer samples. Using a combination of serum protein profiles and Epstein-Barr viral capsid antigen immunoglobulin A antibody tests, the diagnostic sensitivity and specificity were increased to 100 and 97 %, respectively., Conclusions: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry could correctly distinguish nasopharyngeal carcinoma from noncancer individuals and showed great potential for the development of a screening test for the detection of nasopharyngeal carcinoma.

This study was designed to identify genes whose expression in peripheral blood may serve as early markers for treatment response to lithium (Li) in patients with bipolar disorder. Although changes in peripheral blood gene-expression may not relate directly to mood symptoms, differences in treatment response at the biochemical level may underlie some of the heterogeneity in clinical response to Li. Subjects were randomized to treatment with (n=28) or without (n=32) Li. Peripheral blood gene-expression was measured before and 1 month after treatment initiation, and treatment response was assessed after 6 months. In subjects treated with Li, 62 genes were differentially regulated in treatment responders and non-responders. Of these, BCL2L1 showed the greatest difference between Li responders and non-responders. These changes were specific to Li responders (n=9), and were not seen in Li non-responders or patients treated without Li, suggesting that they may have specific roles in treatment response to Li.

Blood and plasma viscosity are the major factors affecting blood flow and normal circulation. Whole blood viscosity is mainly affected by plasma viscosity, red blood cell deformability/aggregation and hematocrit, and other physiological factors. Thirty patients (twenty males + ten females) with age range 50-65 years, normotensive with history of cerebrovascular disorders, were selected according to the American Heart Stroke Association. Blood viscosity and other rheological parameters were measured after two-day abstinence from any medications. Dual effects of vinpocetine and pyritinol exhibit significant effects on all hemorheological parameters (P < 0.05), especially on low shear whole blood viscosity (P < 0.01), but they produced insignificant effects on total serum protein and high shear whole blood viscosity (P > 0.05). Therefore, joint effects of vinpocetine and pyritinol improve blood and plasma viscosity in patients with cerebrovascular disorders.

Pericardial fluid (PF) is often considered to be reflection of the serum by which information regarding the physiological status of the heart can be obtained. Some local and systemic disorders may perturb the balance between synthesis and discharge of PF and may cause its aberrant accumulation in the pericardial cavity as pericardial effusion (PE). PE may then lead to an increased intrapericardial pressure from which the heart function is undesirably affected. For some cases, the causes for the perturbance of fluid balance are well understood, but in some other cases, they are not apparent. It may, thus, be helpful to understand the molecular mechanisms behind this troublesome condition to elucidate a clinical approach for therapeutic uses. In this study, protein profiles of PEs from idiopathic pericarditis patients were analyzed. Control samples from patients undergoing elective cardiac surgery (ECS) were included for comparison. In addition to high abundant serum-originated proteins that may not hold significance for understanding the molecular mechanisms behind this disease, omentin-1 was identified and its level was higher for more than two-fold in PE of IP patients. Increased levels of omentin-1 in PE may open a way for understanding the molecular mechanisms behind idiopathic pericarditis (IP).