70-kD Protein Research Articles

Association of Antibodies to Prevotella copri in Anti-Cyclic Citrullinated Peptide-Positive Individuals At Risk of Developing Rheumatoid Arthritis and in Patients With Early or Established Rheumatoid Arthritis.

Prevotella copri (P copri), a gut commensal, has been reported to be an immune-relevant organism in individuals with rheumatoid arthritis (RA). This study sought to evaluate anti-P copri (anti-Pc) antibody responses in our participant cohorts and to determine when in the natural history of RA such responses develop. We analyzed serum levels of immunoglobulin A (IgA) and IgG antibodies from a 27-kd protein of P copri (anti-Pc-p27), an immunogenic P copri protein, in study participants at risk of developing RA, participants who transitioned to RA, participants with early RA (<1 year of disease), and participants with established RA, with comparisons made to their matched controls. We also evaluated anti-Pc-p27 antibody levels in individuals stratified by RA-related autoantibody status. Overall, participants with RA had significantly higher IgA anti-Pc-p27 antibody levels and trended toward higher IgG anti-Pc-p27 antibody levels compared with matched controls. When stratified by early versus established RA, participants with early RA had median IgG anti-Pc-p27 antibody levels that were overall higher, whereas median IgA anti-Pc-p27 antibody levels were statistically significantly higher in participants with established RA compared with their matched controls. In the autoantibody-specific analyses, the at-risk population with anti-cyclic citrullinated peptide (anti-CCP) antibodies, but not rheumatoid factor (RF), trended toward increased levels of IgG anti-Pc-p27. Additionally, RA participants who were seropositive for both CCP and RF had significantly increased levels of IgA anti-Pc-p27 antibodies and trended toward higher levels of IgG anti-Pc-p27 antibodies compared with matched controls. Our findings support a potential etiologic role for P copri in both RA preclinical evolution and the subsequent pathogenesis of synovitis.

Proteomics study of mitochondrial proteins in tilapia red meat and their effect on color change during storage

The underlying mechanism of the role of mitochondria in color changing of tilapia fillet during 0–4 d storage is not completely clear. A total of 209 differentially significant expressed proteins (DSEPs) were identified by using label-free mitochondrial proteomics, with 56 proteins up-regulated in T2 and 61 proteins (up-regulated) in T3. Protein-Protein interaction reveled proteins which participate in TCA cycles (Citrate synthase (cs)), Oxidoreductase (Malate dehydrogenase (mdh1, mdh2), Succinyl-CoA (Oxct1), Hydroxyacyl-coenzyme a dehydrogenase (hadh), Dehydrogenase/reductase (SDR family) member 1 (dhrs1)) interacted strongly with each other. In turn, they can increase the level of mitochondrial respiration and mitochondrial function, leading to color changing of tilapia fillet. The heat shock 60kD protein 1 (chaperonin, hspd1) interacted with metabolic enzymes (cs and mdh2) and had important effects on color. These results could help researchers better understand the color changing mechanism on the surface of tilapia fillet during the storage.

Narrative medicine to investigate the quality of life and emotional impact of inherited retinal disorders through the perspectives of patients, caregivers and clinicians: an Italian multicentre project

ObjectivesAlthough inherited retinal disorders (IRDs) related to the gene encoding the retinal pigment epithelium 65kD protein (RPE65) significantly impact the vision-related quality of life (VRQoL), their emotional and social aspects...

Mitochondrial Protein Homeostasis and Cardiomyopathy.

Human mitochondrial disorders impact tissues with high energetic demands and can be associated with cardiac muscle disease (cardiomyopathy) and early mortality. However, the mechanistic link between mitochondrial disease and the development of cardiomyopathy is frequently unclear. In addition, there is often marked phenotypic heterogeneity between patients, even between those with the same genetic variant, which is also not well understood. Several of the mitochondrial cardiomyopathies are related to defects in the maintenance of mitochondrial protein homeostasis, or proteostasis. This essential process involves the importing, sorting, folding and degradation of preproteins into fully functional mature structures inside mitochondria. Disrupted mitochondrial proteostasis interferes with mitochondrial energetics and ATP production, which can directly impact cardiac function. An inability to maintain proteostasis can result in mitochondrial dysfunction and subsequent mitophagy or even apoptosis. We review the known mitochondrial diseases that have been associated with cardiomyopathy and which arise from mutations in genes that are important for mitochondrial proteostasis. Genes discussed include DnaJ heat shock protein family member C19 (DNAJC19), mitochondrial import inner membrane translocase subunit TIM16 (MAGMAS), translocase of the inner mitochondrial membrane 50 (TIMM50), mitochondrial intermediate peptidase (MIPEP), X-prolyl-aminopeptidase 3 (XPNPEP3), HtraA serine peptidase 2 (HTRA2), caseinolytic mitochondrial peptidase chaperone subunit B (CLPB) and heat shock 60-kD protein 1 (HSPD1). The identification and description of disorders with a shared mechanism of disease may provide further insights into the disease process and assist with the identification of potential therapeutics.

Identification of Novel, Immunogenic HLA-DR-Presented Prevotella copri Peptides in Patients With Rheumatoid Arthritis.

We previously identified HLA-DR-presented epitopes from a 27-kd protein of Prevotella copri (Pc) obtained from peripheral blood mononuclear cells (PBMCs) from 1 rheumatoid arthritis (RA) patient. Herein, we sought to identify other HLA-DR-presented Pc peptides and source proteins in PBMCs from additional patients to better understand Pc immune responses and RA disease pathogenesis. Using tandem mass spectrometry, we searched for HLA-DR-presented Pc peptides in PBMCs from RA and Lyme arthritis (LA) patients. The identified peptides and source proteins were tested for reactivity in RA patients, those with other arthritides, and the general population. These results were assessed for correlation with clinical findings. Including Pc-p27, we identified 5 HLA-DR-presented Pc peptides, each derived from a different Pc protein, in 3 of 4 RA patients, but none in 2 LA patients. When tested in our RA cohort, 14 of 19 patients (74%) had T cell responses, and 47 of 89 patients (53%) had IgG or IgA responses to ≥1 of the 5 Pc peptides or proteins, most commonly IgA reactivity with Pc-p27. Additionally, 74% of RA patients with IgA antibodies to ≥1 Pc protein had anti-citrullinated protein antibodies (ACPAs) compared with 49% of patients who lacked IgA Pc antibody responses (P = 0.05), and IgA Pc antibody levels correlated with ACPA values. The majority of the RA patients had Pc immune responses. The correlation of IgA Pc antibody responses, particularly to Pc-p27, with ACPA supports the hypothesis that specific microbial antigens in the mucosa have a role in shaping or amplifying immune responses in RA joints.

Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy.

Identification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%-20% of patients. A multipronged approach, using traditional and modern technologies, converged on a novel target antigen, and capitalized on the temporal variation in autoantibody titer for biomarker discovery. Immunoblotting of human glomerular proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection followed by mass spectrometry, elution of immune complexes from renal biopsy specimen tissue, and autoimmune profiling on a protein fragment microarray. These approaches identified serine protease HTRA1 as a novel podocyte antigen in a subset of patients with primary MN. Sera from two patients reacted by immunoblotting with a 51-kD protein within glomerular extract and with recombinant human HTRA1, under reducing and nonreducing conditions. Longitudinal serum samples from these patients seemed to correlate with clinical disease activity. As in PLA2R- and THSD7A- associated MN, anti-HTRA1 antibodies were predominantly IgG4, suggesting a primary etiology. Analysis of sera collected during active disease versus remission on protein fragment microarrays detected significantly higher titers of anti-HTRA1 antibody in active disease. HTRA1 was specifically detected within immune deposits of HTRA1-associated MN in 14 patients identified among three cohorts. Screening of 118 "quadruple-negative" (PLA2R-, THSD7A-, NELL1-, EXT2-negative) patients in a large repository of MN biopsy specimens revealed a prevalence of 4.2%. Conventional and more modern techniques converged to identify serine protease HTRA1 as a target antigen in MN.

Leptin improves the in vitro development of preimplantation rabbit embryos under oxidative stress of cryopreservation.

Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

The Protective Effect of PK-11195 on Cognitive Impairment in Rats Survived of Polymicrobial Sepsis.

Sepsis is an organ dysfunction caused by a host's unregulated response to infection, causing long-term brain dysfunction with microglial activation, the release of inflammatory components, and mitochondrial changes. Neuroinflammation can increase the expression of the 18-kD translocator protein (TSPO) in the mitochondria, leading to the activation of the microglia and the release of inflammatory components. The antagonist PK-11195 can modulate TSPO and reduce microglial activation and cognitive damage presented in an animal model of sepsis. The aim of this was to evaluate the effects of PK-11195 on long-term brain inflammation and cognitive impairment in an animal model of sepsis. Wistar rats, 60 days old, were submitted to cecal ligation and puncture (CLP) surgery, divided into groups control/saline, control/PK-11195, sepsis/saline, and sepsis/PK-11195. Immediately after surgery, the antagonist PK-11195 was administered at a dose of 3 mg/kg. Ten days after CLP surgery, the animals were submitted to behavioral tests and determination of brain inflammatory parameters. The sepsis/saline group presented cognitive damage. However, there was damage prevention in animals that received PK-11195. Besides, the sepsis increased the levels of cytokines and M1 microglia markers and caused oxidative damage. However, PK-11195 had the potential to decrease inflammation. These events show that the modulation of neuroinflammation during sepsis by PK-11195, possibly related to changes in TSPO, improves mitochondrial function in the animals' brains. In conclusion, the antagonist PK-11195 attenuated brain inflammation and prevented cognitive impairment in animals subjected to sepsis.

Heat stress alters muscle protein and amino acid metabolism and accelerates liver gluconeogenesis for energy supply in broilers

Heat stress impairs growth performance and alters body protein and amino acid metabolism. This study was investigated to explore how body protein and amino acid metabolism changed under heat stress (HS) and the stress adaptation mechanism. A total of 144 broilers (28 d old) were divided into 3 treatment groups for 1 wk: HS group (32°C), normal control group (22°C), and pair-feeding group (22°C). We found that HS elevated the feed-to-gain ratio, reduced the ADFI and ADG, decreased breast muscle mass and plasma levels of several amino acids (glycine, lysine, threonine, and tyrosine), and increased serum glutamic oxaloacetic transaminase (GOT) activity and corticosterone (CORT) level and liver GOT and glutamic pyruvic transaminase activities. Heat stress elevated muscle atrophy F-box mRNA expression and reduced mRNA expression of the 70-kD ribosomal protein S6 kinase in the breast muscle of broilers. Broilers in the HS group exhibited striking increases of mRNA expressions of solute carrier family 1 member 1, family 3 member 1, family 7 member 1, and family 7 member-like in the liver and liver gluconeogenesis genes (PCKc, PCKm, PC, and FBP1) in comparison with the other 2 groups. In conclusion, HS increased the circulating CORT level and subsequently caused muscle protein breakdown to provide amino acid substrates to liver gluconeogenesis responsible for energy supply.

Cyclophilin A (CyPA) as a Novel Biomarker for Early Detection of Diabetic Nephropathy in an Animal Model.

Background and AimType 2 diabetes mellitus (DM) is the most common single cause of the end-stage renal disease (ESRD). Cyclophilin A (CyPA) is an 18-kD protein. The connection between diabetic nephropathy (DN) and the secreted form of CyPA (sCyPA) has been elucidated in this study that aims to investigate sCyPA correlation with renal dysfunction.Materials and MethodsThirty-four male adult Wistar rats weighing 180–220 g were used. Animals were divided into a study group and a control group, 17 rats in each. Streptozotocin (STZ) and nicotine amide were used to damage some pancreatic cells for induction of type 2 DM. Comparison was made between the study and the control groups. Moreover, a comparison was made between the members of the study group before and after induction of DN.ResultsThe rat model that exhibited a higher concentration of urinary sCyPA was detected early in the eighth week. There was a significantly higher level of 24-h urinary CyPA in the study group compared to the control group (p-value=0.004) and there was a significant elevation in the 24-h urinary Cyp-A in the study group after injection of STZ compared to the values before injection (p-value <0.001). Immunohistochemical analysis of renal tissue revealed that the mean expression of CyPA was higher in the study group than in the control group. For the urinary 24-h CYP-A, using a cutoff of 1.15 ng/mL, the accuracy was 72.4%, sensitivity was (77.8%) and specificity was (67%).ConclusionAccording to this animal study, we proved that CyPA is a valuable marker for DN. It is a more sensitive, noninvasive and rapid biomarker for early detection of any renal affection in human diabetic patients.

Urinary podocalyxin and cyclophilin A: markers for early detection of type 2 diabetic nephropathy

Objective To investigate urinary podocalyxin (PCX) and cyclophilin A (CypA) as useful markers for early detection of type 2 diabetic nephropathy (DN). Background PCX is a transmembrane protein that localizes to the apical cell of glomerular podocytes. PCX maintains podocytes shape. PCX is shed from injured podocytes into urine as small vesicles. CypA is an 18-kD protein that is distributed in the cytoplasm and facilitates protein folding and trafficking. It acts as a cellular receptor for cyclosporine A. The expression of CypA is quite high in the kidney, where proximal tubular epithelial cells contain more CypA than other kidney tissues. CypA was detected in diabetic patients' plasma and secreted by monocytes in response to hyperglycemia, indicating that CypA could be a potential secretory marker in type 2 DN. Patients and methods This case–control study was conducted at the Clinical Pathology Department of Menoufia University Hospitals from September 2017 to February 2018 on 66 type 2 diabetic patients and 20 healthy participants as a control group. They all underwent full history, clinical examination, routine renal functions, urinary creatinine and albumin, urinary albumin creatinine ratio, glycated hemoglobin (%), and quantification of urinary PCX and CypA by enzyme-linked immunosorbent assay. Results Urinary PCX/creatinine ratio and urinary CypA/creatinine ratio were highly significant (P Conclusion Urinary PCX and CypA may be markers for early stage of DN, with more specificity of CypA.

Isolation and Purification of 18KD Protein of Sika Deer Antler Plate and its Antibacterial Activity

Isolation and Purification of 18KD Protein of Sika Deer Antler Plate and its Antibacterial Activity

The fungus-derived retinoprotectant theissenolactone C improves glaucoma-like injury mediated by MMP-9 inhibition

BackgroundElevated intraocular pressure (IOP) is a major risk factor for glaucoma that has been found to induce matrix metalloproteinase-9 (MMP-9) activation and result in eventual retinal dysfunction. Proinflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β (IL-1β) were also found to be involved in disease progression by mediating MMP-9 production. We previously reported that fungal derivative theissenolactone C (LC53) could exert ocular protective effects by suppressing neuroinflammation in experimental uveitis. PurposeThe aim of this study was to investigate the retinoprotective effects of natural compound LC53 on the high IOP-induced ischemia/reperfusion (I/R)-injury model of glaucoma and its cellular mechanisms. MethodsA high IOP-induced I/R-injury model was manipulated by normal saline injection into the anterior chamber of the rat eye. MCP-1-stimulated monocytes and IL-1β-activated primary astrocytes were used to investigate the cellular mechanisms of LC53. Retinal function was evaluated with the scotopic threshold response (STR) and combined rod–cone response by electroretinography (ERG). As a positive control, rats were treated with memantine. MMP-9 gelatinolysis, mRNA expression and protein expression were analyzed by gelatin zymography, RT-PCR, and Western Blot, respectively. The phosphorylation levels of MAPKs and NF-κB p65 were tested by Western Blot. Additionally, the levels of inflammatory MCP-1 and IL-1β were determined by ELISA. ResultsThe present study revealed that LC53 preserved the retina functional deficiency assessed by scotopic threshold response (STR) and combined rod–cone response of ERG after high IOP-induced I/R injury. These retinal protective effects of LC53 were positively correlated with inhibitory activities in I/R injury-elicited ocular MMP-9 activation and expression. The increased level of MCP-1 was not affected, and the enhanced IL-1β production was partially reduced by LC53 in the retina after I/R injury. According to cellular studies, LC53 significantly and concentration-dependently abrogated MMP-9 activation and expression in MCP-1-stimulated THP-1 monocytes. We found the inhibitory activities of LC53 were through the ERK- and NF-κB-dependent pathways. In addition, LC53 dramatically suppressed IL-1β-induced MMP-9 activation and expression in primary astrocytes. The phosphorylation of 65-kD protein (p65) of NF-κB was substantially blocked by LC53 in IL-1β-stimulated primary astrocytes. ConclusionLC53 exerted a retinal protective effect through NF-κB inhibition and was highly potent against MMP-9 activities after high IOP-induced I/R injury, suggesting that LC53 would be a promising drug lead for glaucoma or related medical conditions attributed to retinal ischemia.

Expression of codon-optimized TgMIC16 in three Escherichia coli strains.

In a previous study, we found that rabbit anti-Toxoplasma gondii serum was capable of recognizing truncated T. gondii microneme protein 16 (TgMIC16), indicating that TgMIC16 is an essential antigenic T. gondii protein. However, the broad application of this recombinant protein is limited by its low expression level. In this study, we performed codon optimization of TgMIC16 by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence and expressed the optimized gene in three different Escherichia coli strains, followed by comparison of soluble recombinant-protein expression and yield. Our results showed that the recombinant protein rTgMIC16 was expressed as inclusion bodies in all three strains following optimization of induction parameters, and western blot analysis revealed the presence of a ~72-kD recombinant protein as a specific band following purification. A shuffle-expression strain was selected to amplify incubation products and induce expression, resulting in an overall rTgMIC16 yield of ~20mg/L. These findings provide a basis for further investigation of TgMIC16 to elucidate its functions and interaction partners.

Evidence of the Immune Relevance of Prevotella copri, a Gut Microbe, in Patients With Rheumatoid Arthritis.

Prevotella copri, an intestinal microbe, may overexpand in stool samples from patients with new-onset rheumatoid arthritis (RA), but it is not yet clear whether the organism has immune relevance in RA pathogenesis. HLA-DR-presented peptides (T cell epitopes) from P copri were sought directly in the patients' synovial tissue or peripheral blood mononuclear cell (PBMC) samples using tandem mass spectrometry. The antigenicity of peptides or their source proteins was examined in samples from the RA patients or comparison groups. T cell reactivity was determined by enzyme-linked immunospot assay; antibody responses were measured by enzyme-linked immunosorbent assay, and cytokine/chemokine determinations were made by bead-based assays. Serum and synovial fluid samples were examined for 16S ribosomal DNA for P copri using nested polymerase chain reaction analysis. In PBMCs, we identified an HLA-DR-presented peptide from a 27-kd protein of P copri (Pc-p27), which stimulated Th1 responses in 42% of patients with new-onset RA. In both new-onset RA patients and chronic RA patients, 1 subgroup had IgA antibody responses to either Pc-p27 or the whole organism, which correlated with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPAs). The other subgroup had IgG P copri antibodies, which were associated with Prevotella DNA in synovial fluid, P copri-specific Th1 responses, and less frequent ACPAs. In contrast, P copri antibody responses were rarely found in patients with other rheumatic diseases or in healthy controls. Subgroups of RA patients have differential IgG or IgA immune reactivity with P copri, which appears to be specific for this disease. These observations provide evidence that P copri is immune-relevant in RA pathogenesis.

Assessment of safety, efficacy, and dosimetry of a novel 18-kDa translocator protein ligand, [11C]CB184, in healthy human volunteers

BackgroundN,N-di-n-propyl-2-[2-(4-[11C]methoxyphenyl)-6,8-dichloroimidazol[1,2-a]pyridine-3-yl]acetamide ([11C]CB184) is a novel selective radioligand for the 18-kD translocator protein (TSPO), which is upregulated in activated microglia in the brain, and may be useful in positron emission tomography (PET). We examined the safety, radiation dosimetry, and initial brain imaging with [11C]CB184 in healthy human volunteers.ResultsDynamic [11C]CB184 PET scans (90 min) were performed in five healthy male subjects. During the scan, arterial blood was sampled at various time intervals, and the fraction of the parent compound in plasma was determined with high-performance liquid chromatography. No serious adverse events occurred in any of the subjects throughout the study period. [11C]CB184 was metabolized in the periphery: 36.7% ± 5.7% of the radioactivity in plasma was detected as the unchanged form after 60 min. The total distribution volume (VT) was estimated with a two-tissue compartment model. The VT of [11C]CB184 was highest in the thalamus (5.1 ± 0.4), followed by the cerebellar cortex (4.4 ± 0.2), and others. Although regional differences were small, the observed [11C]CB184 binding pattern was consistent with the TSPO distribution in the normal human brain. Radiation dosimetry was determined in three healthy male subjects using a serial whole-body PET scan acquired over 2 h after [11C]CB184 injection. [11C]CB184 PET demonstrated high uptake in the gallbladder at a later time (>60 min). In urine obtained approximately 100 min post-injection, 0.3% of the total injected radioactivity was recovered, indicating hepatobiliary excretion of radioactivity. The absorbed dose (μGy/MBq) was highest in the kidneys (21.0 ± 0.5) followed by the lungs (16.8 ± 2.7), spleen (16.6 ± 6.6), and pancreas (16.5 ± 2.2). The estimated effective dose for [11C]CB184 was 5.9 ± 0.6 μSv/MBq.ConclusionsThis initial evaluation indicated that [11C]CB184 is feasible for imaging of TSPO in the brain.

Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro.

Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.

Glial Activation and Glucose Metabolism in a Transgenic Amyloid Mouse Model: A Triple-Tracer PET Study.

Amyloid imaging by small-animal PET in models of Alzheimer disease (AD) offers the possibility to track amyloidogenesis and brain energy metabolism. Because microglial activation is thought to contribute to AD pathology, we undertook a triple-tracer small-animal PET study to assess microglial activation and glucose metabolism in association with amyloid plaque load in a transgenic AD mouse model. Groups of PS2APP and C57BL/6 wild-type mice of various ages were examined by small-animal PET. We acquired 90-min dynamic emission data with (18)F-GE180 for imaging activated microglia (18-kD translocator protein ligand [TSPO]) and static 30- to 60-min recordings with (18)F-FDG for energy metabolism and (18)F-florbetaben for amyloidosis. Optimal fusion of PET data was obtained through automatic nonlinear spatial normalization, and SUVRs were calculated. For the novel TSPO tracer (18)F-GE180, we then calculated distribution volume ratios after establishing a suitable reference region. Immunohistochemical analyses with TSPO antisera, methoxy-X04 staining for fibrillary β-amyloid, and ex vivo autoradiography served as terminal gold standard assessments. SUVR at 60-90 min after injection gave robust quantitation of (18)F-GE180, which correlated well with distribution volume ratios calculated from the entire recording and using a white matter reference region. Relative to age-matched wild-type, (18)F-GE180 SUVR was slightly elevated in PS2APP mice at 5 mo (+9%; P < 0.01) and distinctly increased at 16 mo (+25%; P < 0.001). Over this age range, there was a high positive correlation between small-animal PET findings of microglial activation with amyloid load (R = 0.85; P < 0.001) and likewise with metabolism (R = 0.61; P < 0.005). Immunohistochemical and autoradiographic findings confirmed the in vivo small-animal PET data. In this first triple-tracer small-animal PET in a well-established AD mouse model, we found evidence for age-dependent microglial activation. This activation, correlating positively with the amyloid load, implies a relationship between amyloidosis and inflammation in the PS2APP AD mouse model.

Using Smudge Cells Percentage on Routine Blood Smear in Chronic Lymphoytic Leukemia As Prognostic Factor: Senegalese Experience

Introduction/ Background : Chronic lymphocytic leukemia (CLL) is a heterogeneous disease which can present as an aggressive and life threatening leukemia or as an indolent form that will not require treatment for decades. This heterogeneity has important consequences which will impact on clinical approaches, treatment strategies, and survival times from diagnosis. Prognostic markers such as expression of specific proteins in or on CLL cells (ie, CD38, 70-kD ζ-associated protein or CD49d), cytogenetic abnormalities (del 13q, del 11q, del 17p and trisomy 12) quantified by FISH and immunoglobulin heavy chain (IgVH) gene mutation have all been very useful. Futhermore, patients with early-stage disease, with biologically aggressive disease and shorter survival times can be distiguished. However, these prognostic tests are expensive and require considerable technical expertise and equipment and thus are not available to many patients with CLL living in developing countries. Therefore less expensive prognostic markers are needed. In this study, we evaluated the prognostic significance of smudge cells percentage on a blood smear in CLL patients.Patients and Methods : In this prospective study, 42 untreated patients with CLL have participated after signing a consent form. Patients were seen at our center between July 2011 and May 2015. Patients were diagnosed on the basis of an absolute lymphocyte count greater than 5.109/L and a demonstration of monoclonality using flow cytometry (panel comprising CD19, CD5, CD22, CD23, FMC7, and surface Ig). Staging was done according to the Binet staging system. CD38 surface expression was determined by flow cytometry in all patients. The cytogenetic abnormalities : del 13q, del 11q, del 17p and trisomy 12, were performed by FISH and available for 25 patients.Smudge cells were defined as broken cells with no intact cytoplasm and a disrupted nuclear membrane (Figure 1). The smudge cell percentage is estimated by counting 200 lymphocytes and/or smudge cells; the smudge cell number is then divided by total number of cells counted (smudge cells + intact lymphocytes) and multiplied by 100. Each slide was evaluated by 2 hematologists and the blood smears were prepared using a manual wedge method. Categorical variables were analyzed using the χ2 test or Fisher exact test (p<0.05). The statistical analysis was performed using STATA version 13.Results : The mean age was 63 years ranging from 48 to 85. There were 31 males and 11 females (sex ratio=2.81). At the time of diagnosis 81% of the patients were classified as having advanced Binet stages B or C. The median lymphocyte count was 189.9 109/L (ranges, 5.2 - 887 109/L). The prognostic marker CD38 was positive in 28 patients, while 11 patients had 13q del, 7 patients Trisomy 12, 3 del 11d and 3 del 17p. The median smudge cell percentage was 20.1% (range, 4 - 80%) and 30 patients had a percentage less than 30. We found no correlation of proportion of smudge cells with age (p=0.19) and sex (p=0.76). However there was a significant correlation with the Binet stage (p=0.0002), the CD38 expression (p=0.04) and a negative correlation with the lymphocyte count (p=0,01) (r= -0,38).Conclusion:A low percentage of smudge cells (less than 30%) is associated with high lymphocyte count, advanced Binet stage (B or C) and positive CD38. Simple, accessible and inexpensive, the percentage of smudge cells on a routine blood smear could be a prognostic test available to all patients with CLL especially those in countries with limited resources. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interpretation of an Extended Autoantibody Profile in a Well-Characterized Australian Systemic Sclerosis (Scleroderma) Cohort Using Principal Components Analysis.

To determine the relationships between systemic sclerosis (SSc)-related autoantibodies, as well as their clinical associations, in a well-characterized Australian patient cohort. Serum from 505 Australian SSc patients were analyzed with a commercial line immunoassay (EuroLine; Euroimmun) for autoantibodies to centromere proteins CENP-A and CENP-B, RNA polymerase III (RNAP III; epitopes 11 and 155), the 90-kd nucleolar protein NOR-90, fibrillarin, Th/To, PM/Scl-75, PM/Scl-100, Ku, topoisomerase I (topo I), tripartite motif-containing protein 21/Ro 52, and platelet-derived growth factor receptor. Patient subgroups were identified by hierarchical clustering of the first 2 dimensions of a principal components analysis of quantitative autoantibody scores. Results were compared with detailed clinical data. A total of 449 of the 505 patients were positive for at least 1 autoantibody by immunoblotting. Heatmap visualization of autoantibody scores, along with principal components analysis clustering, demonstrated strong, mutually exclusive relationships between CENP, RNAP III, and topo I. Five patient clusters were identified: CENP, RNAP III strong, RNAP III weak, topo I, and other. Clinical features associated with CENP, RNAP III, and topo I were consistent with previously published reports concerning limited cutaneous and diffuse cutaneous SSc. A novel finding was the statistical separation of RNAP III into 2 clusters. Patients in the RNAP III strong cluster had an increased risk of gastric antral vascular ectasia, but a lower risk of esophageal dysmotility. Patients in the other cluster were more likely to be male and to have a history of smoking and a history of malignancy, but were less likely to have telangiectasia, Raynaud's phenomenon, and joint contractures. Five major autoantibody clusters with specific clinical and serologic associations were identified in Australian SSc patients. Subclassification and disease stratification using autoantibodies may have clinical utility, particularly in early disease.