Vibrio alginolyticus is a Gram-negative halophilic bacterium and has been recognized as an opportunistic pathogen in both humans and marine animals. It is the causative agent of food-borne diseases, such as gastroenteritis, and it invades through wounds in predisposed individuals. In this study, we present the completed genome of V. alginolyticus ATCC 17749T through high-throughput sequencing.
To identify tumor suppressor genes (TSGs) silenced by hypermethylation and discover new epigenetic biomarkers for early cancer detection. ADAMTS18, located at 16q23.1, has been reported to be a critical TSG in multiple primary tumors; however, this has not yet been verified in clear cell renal cell carcinoma (ccRCC). We explored epigenetic alterations in this gene in ccRCC and analyzed possible clinicopathological associations. We examined ADAMTS18 gene expression and methylation by semi-quantitative reverse transcription PCR (RT-PCR) and methylation-specific polymerase chain reaction (MSP) in 5 ccRCC-derived cell lines before and after treatment with 5-aza-2'-deoxycytidine (5-AzaC). MSP was further performed for 101 ccRCC primary tumors and 20 adjacent normal tissues. Some cell lines and specimens were examined by subsequent bisulfite genomic sequencing (BGS) and real-time PCR. Further, we analyzed the relationship between the ADAMTS18 gene methylation and clinicopathological features, including short-term disease-free survival (DFS), in patients with ccRCC. ADAMTS18 down-regulation and hypermethylation were detected in the ccRCC-derived cell lines using RT-PCR and MSP. Treatment with 5-AzaC reversed the hypermethylation of the ADAMTS18 gene and restored its expression. Hypermethylation was further detected in 44 of 101 (43.6%) primary tumors and 3 of 20 (15.0%) adjacent normal tissues. However, a significant difference between both groups was observed (p = 0.02). BGS analysis and real-time PCR were subsequently performed to confirm the results of RT-PCR and MSP. Furthermore, the methylation status of ADAMTS18 was not significantly associated with gender, age, location, tumor diameter, pathological stage, nuclear grade or short-term DFS in patients with ccRCC (p > 0.05). The ADAMTS18 gene is often down-regulated by hypermethylation in ccRCC-derived cell lines and primary tumors, indicating its critical role as a TSG in ccRCC. We conclude that ADAMTS18 gene hypermethylation may be involved in the tumorigenesis of ccRCC and may serve as a novel biomarker for this disease.
hypermethylation; ADAMTS18 gene; clear cell renal cell carcinoma; tumor suppressor gene
To establish longitudinal validation of a new tool, the Asthma Symptom Tracker (AST). AST combines weekly use of the Asthma Control Test with a color-coded graph for visual trending.
Prospective cohort study of children age 2 to 18 years admitted for asthma. Parents or children (n = 210) completed baseline AST assessments during hospitalization, then over 6 months after discharge. Concurrent with the first 5 AST assessments, the Asthma Control Questionnaire (ACQ) was administered for comparison.
Test–retest reliability (intraclass correlation) was moderate, with a small longitudinal variation of AST measurements within subjects during follow-ups. Internal consistency was strong at baseline (Cronbach’s α 0.70) and during follow-ups (Cronbach’s α 0.82–0.90). Criterion validity demonstrated a significant correlation between AST and ACQ scores at baseline (r = −0.80, P < .01) and during follow-ups (r = −0.64, −0.72, −0.63, and −0.69). The AST was responsive to change over time; an increased ACQ score by 1 point was associated with a decreased AST score by 2.65 points (P < .01) at baseline and 3.11 points (P < .01) during follow-ups. Discriminant validity demonstrated a strong association between decreased AST scores and increased oral corticosteroid use (odds ratio 1.13, 95% confidence interval, 1.10–1.16, P < .01) and increased unscheduled acute asthma visits (odds ratio 1.23, 95% confidence interval, 1.18–1.28, P < .01).
The AST is reliable, valid, and responsive to change over time, and can facilitate ongoing monitoring of asthma control and proactive medical decision-making in children.
asthma control; pediatrics; self-monitoring; self-management
Both post-contrast myocardial T1 and extracellular volume (ECV) measurements have been associated with interstitial fibrosis. The cardiovascular magnetic resonance (CMR) field is migrating towards ECV, because it is largely insensitive to confounders that affect post-contrast myocardial T1. Despite the theoretical advantages of myocardial ECV over post-contrast myocardial T1, systematic experimental studies comparing the two measurements are largely lacking. We sought to measure the temporal changes in post-contrast myocardial T1 and ECV in an established canine model with chronic atrial fibrillation.
Seventeen mongrel dogs, implanted with a pacemaker to induce chronic atrial fibrillation via rapid atrial pacing, were scanned multiple times for a total of 46 CMR scans at 3T. These dogs with different disease durations (0–22 months) were part of a separate longitudinal study aimed at studying the relationship between AF and patho-physiology. In each animal, we measured native and post-contrast T1s and hematocrit. Temporal changes in post-contrast myocardial T1 and ECV, as well as other CMR parameters, were modeled with linear mixed effect models to account for repeated measurements over disease duration.
In 17 animals, post-contrast myocardial T1 decreased significantly from 872 to 698 ms (p< 0.001), which corresponds to a 24.9% relative reduction. In contrast, ECV increased from 21.0 to 22.0% (p=0.38), which corresponds to only a 4.5% relative increase. To partially investigate this discrepancy, we quantified collagen volume fraction (CVF) in post-mortem heart tissues of 6 canines sacrificed at different disease duration (0–22 months). CVF quantified by histology increased from 0.9 to 1.9% (p=0.56), which agrees more with ECV than post-contrast myocardial T1. This study shows that post-contrast myocardial T1 and ECV may disagree in a longitudinal canine study. A more comprehensive study, including histologic, cardiac, and renal functional analyses, is warranted to test rigorously which CMR parameter (ECV or post-contrast myocardial T1) agrees more with CVF.
Diffuse myocardial fibrosis; post-contrast myocardial T1; extracellular volume fraction; MRI; heart failure; atrial fibrillation; collagen volume fraction
There is a paucity of literature describing metabolic and histological data in adult-onset autoimmune diabetes. This subgroup of diabetes mellitus affects at least 5% of clinically diagnosed type 2 diabetic patients (T2DM) and it is termed Latent Autoimmune Diabetes in Adults (LADA). We evaluated indexes of insulin secretion, metabolic assessment, and pancreatic pathology in clinically diagnosed T2DM patients with and without the presence of humoral islet autoimmunity (Ab). A total of 18 patients with at least 5-year duration of clinically diagnosed T2DM were evaluated in this study. In those subjects we assessed acute insulin responses to arginine, a glucose clamp study, whole-body fat mass and fat-free mass. We have also analyzed the pancreatic pathology of 15 T2DM and 43 control cadaveric donors, using pancreatic tissue obtained from all the T2DM organ donors available from the nPOD network through December 31, 2013. The presence of islet Ab correlated with severely impaired β-cell function as demonstrated by remarkably low acute insulin response to arginine (AIR) when compared to that of the Ab negative group. Glucose clamp studies indicated that both Ab positive and Ab negative patients exhibited peripheral insulin resistance in a similar fashion. Pathology data from T2DM donors with Ab or the autoimmune diabetes associated DR3/DR4 allelic class II combination showed reduction in beta cell mass as well as presence of autoimmune-associated pattern A pathology in subjects with either islet autoantibodies or the DR3/DR4 genotype. In conclusion, we provide compelling evidence indicating that islet Ab positive long-term T2DM patients exhibit profound impairment of insulin secretion as well as reduced beta cell mass seemingly determined by an immune-mediated injury of pancreatic β-cells. Deciphering the mechanisms underlying beta cell destruction in this subset of diabetic patients may lead to the development of novel immunologic therapies aimed at halting the disease progression in its early stage.
The role of proximal tubule (PT) angiotensinogen (AGT) in modulating blood pressure has previously been examined using mice expressing PT human AGT and human renin, or rat AGT. These animals are hypertensive; however, the question remains whether alterations in mouse PT AGT alone affects arterial pressure.
Mouse AGT cDNA was knocked-in to the endogenous kidney androgen protein (KAP) gene using an internal ribosomal entry site (IRES)-based strategy.
The KAP-mAGT animals showed kidney-specific KAP-AGT mRNA expression; renal in situ hybridization detected KAP-AGT mRNA only in PT. Urinary AGT was markedly increased in KAP-mAGT mice. On a high Na diet, radiotelemetric arterial pressure showed a systolic pressure elevation; no significant difference in arterial pressure was observed on a normal diet. Plasma renin concentration (PRC) was reduced in KAP-mAGT animals given a high Na diet, but was not different between mouse lines during normal Na intake. Plasma AGT concentration was not altered by overexpression of PT mouse AGT.
In summary, PT overexpression of mouse AGT leads to salt-sensitive hypertension without recruitment of the systemic renin–angiotensin system.
angiotensinogen; blood pressure; hypertension; kidney; proximal tubule; renin; transgene
What are the outcomes of acupuncture for back pain? According to well-regarded trials, acupuncture is little better for back pain than biomedicine, and active acupuncture is no better than sham acupuncture. These trials occurred in the West. Patients are inside the clinic a miniscule amount of time in relation to the time they are outside the clinic and enmeshed in the wider sociocultural context. Nevertheless, trials have largely overlooked potential effects of sociocultural context. The main objective of this article is to draw attention to designated features of sociocultural context that, as compared with outcomes obtained in the West, may enhance outcomes of acupuncture for back pain in China. Additional objectives of the article are to reconceptualize “sociocultural context” so that it is measurable, and to measure pre-existing acquaintance with acupuncture and other forms of Traditional Chinese Medicine (TCM) within the sociocultural context of China.
Back pain patients (N=86) were recruited from the Acupuncture Clinic and Pain Clinic of West China Hospital (Chengdu, Sichuan Province). Patients completed questionnaires on their use of TCM before they came to the Acupuncture Clinic and their families' use of TCM.
Most patients had used TCM, and those who did so likely used it repeatedly, which indicated substantial acquaintance with TCM beliefs in the cultural context. Patients whose families used TCM were also likely to use it themselves, which indicated that TCM use was anchored in the social context of the family.
Although multiple studies substantiate biologic mechanisms of acupuncture, there is not necessarily a fixed relationship between those mechanisms and people's experience of them. Rather, sociocultural context may interact with biologic mechanisms and mediate this experience. The theory proposed here explains why outcomes of acupuncture for back pain will potentially be more pronounced in the sociocultural context of China than in the West.
Acute kidney injury (AKI) remains to be an independent risk factor for mortality and morbidity. Inflammation is believed to play a major role in the pathophysiology of AKI. Exogenous mesenchymal stem cells (MSCs) are now under extensive investigation as a potential therapy for AKI. Various preclinical studies indicated the beneficial effects of MSCs in alleviating renal injury and accelerating tissue repair. However the mechanisms responsible for these effects are incompletely understood. In the recent years, anti-inflammatory/immunoregulatory properties of MSCs have become one of the important issues in the treatment of AKI. This review will summarize the current literature on the regulation of inflammatory mediators via exogenous MSCs contributing to the recovery from AKI.
Aim. It was aimed to monitor early treatment response of Sunitinib in U87MG models mimicking glioblastoma multiforme by longitudinal 18F-FLT microPET/CT imaging in this study. Methods. U87MG tumor mice were intragastrically injected with Sunitinib at a dose of 80 mg/kg for consecutive 7 days. 18F-FLT microPET/CT scans were acquired on days 0, 1, 3, 7, and 13 after therapy. Tumor sizes and body weight were measured. Tumor samples were collected for immunohistochemical analysis of proliferation and microvessel density (MVD) with anti-Ki67 and anti-CD31, respectively. Results. The uptake ratios of tumor to the contralateral muscle (T/M) of 18F-FLT in the Sunitinib group decreased from baseline to day 3 (T/M0 = 2.98 ± 0.33; T/M3 = 2.23 ± 0.36; P < 0.001), reached the bottom on day 7 (T/M7 = 1.96 ± 0.35; P < 0.001), and then recovered on day 13. The T/M of 18F-FLT uptake in the control group remained around 3.0. There was no difference for the tumor size between both groups until day 11. 18F-FLT uptakes of tumor were correlated with Ki67 staining index and MVD. Conclusion. Early therapy response to Sunitinib could be predicted via 18F-FLT PET, which will contribute to monitoring antiangiogenesis treatment.
Angiotensinogen (AGT) is synthesized in the liver and proximal tubule. AGT overexpression at either site might increase blood pressure (BP). We used transgenic mice with AGT overexpression in proximal tubule (K), liver (L), or both sites (KL) to determine the relative contributions of hepatic- and proximal tubule–derived AGT in modulating BP.
Hepatic AGT overexpression was obtained using the albumin enhancer promoter; the kidney androgen protein gene was used for proximal tubule AGT overexpression. BP and renin angiotensin system parameters were examined in male KL, K, L, and wild-type mice on normal and high-sodium diets.
Compared with wild-type mice, K and KL mice had higher BP on normal and high-sodium diets. L mice had similar BP to wild-type mice on a normal-sodium diet, but high sodium intake caused hypertension. There were no differences in plasma AGT, plasma renin concentration, urine volume, or urine sodium excretion between the groups. Urine AGT and angiotensin II (Ang II) excretion were higher in KL and K mice than in L or wild-type mice on a normal-sodium diet and increased with high sodium intake. During high sodium intake, urine AGT and Ang II were higher in all transgenic mice vs wild-type mice.
Mice with liver AGT overexpression manifest salt-sensitive hypertension, whereas mice with renal AGT overexpression are hypertensive regardless of salt intake. Systemic AGT may stimulate endogenous renal AGT synthesis during high sodium intake, leading to hypertension in L mice. This suggests that systemic and renal AGT may interact to modulate BP.
angiotensinogen; blood pressure; hypertension; kidney; liver; proximal tubule; transgene.
Autophagy is a catabolic process that functions in recycling and degrading cellular proteins, and is also induced as an adaptive response to the increased metabolic demand upon nutrient starvation. However, the prosurvival role of autophagy in response to metabolic stress due to deprivation of glutamine, the most abundant nutrient for mammalian cells, is not well understood. Here, we demonstrated that when extracellular glutamine was withdrawn, autophagy provided cells with sub-mM concentrations of glutamine, which played a critical role in fostering cell metabolism. Moreover, we uncovered a previously unknown connection between metabolic responses to ATG5 deficiency and glutamine deprivation, and revealed that WT and atg5−/− MEFs utilized both common and distinct metabolic pathways over time during glutamine deprivation. Although the early response of WT MEFs to glutamine deficiency was similar in many respects to the baseline metabolism of atg5−/− MEFs, there was a concomitant decrease in the levels of essential amino acids and branched chain amino acid catabolites in WT MEFs after 6 h of glutamine withdrawal that distinguished them from the atg5−/− MEFs. Metabolomic profiling, oxygen consumption and pathway focused quantitative RT-PCR analyses revealed that autophagy and glutamine utilization were reciprocally regulated to couple metabolic and transcriptional reprogramming. These findings provide key insights into the critical prosurvival role of autophagy in maintaining mitochondrial oxidative phosphorylation and cell growth during metabolic stress caused by glutamine deprivation.
ATG5; autophagy; glutamine; ATP; transcriptional reprogramming; altered metabolism
Vibrio alginolyticus is a Gram-negative halophilic bacterium with worldwide distribution. In this work, we report the draft genome sequence of a V. alginolyticus strain (E0666) isolated from Epinephelus coioides ascites in the Shantou city of Guangdong Province, China.
To determine the prevalence and distribution of human enteroviruses (HEVs) among healthy children in Shenzhen, China.
Clinical specimens were obtained from 320 healthy children under 5 years old in Shenzhen, China from 2010 to 2011. The specimens were evaluated using real-time PCR and cell cultures. The positive specimens were further tested using reverse transcription-seminested PCR (RT-snPCR). Molecular typing and phylogenetic analysis were based on the sequence determined.
Among the 320 samples, 34 were tested positive for HEVs (10.6%) and 22 different serotypes were identified using RT-snPCR. PV1 and PV2 were also detected. The predominant serotype observed was EV71 (17.6%), followed by CV-B4 (14.7%). HEV-B was detected most frequently, with an overall prevalence of 47.1%. HEV-A and HEV-C were found in 32.3% and 20.6% of the samples, respectively. No HEV-D was identified. Molecular phylogeny indicated that all EV71 strains were of C4 genotype.
Although a variety of HEVs was detected in healthy children, HEV-B was relatively more prevalent than other HEV species. Considering HEV-A is more prevalent than HEV-B among patients with hand-foot-mouth disease, additional long-term surveillance of HEV is warranted in both asymptomatic and symptomatic populations.
Several studies suggest that mesenchymal stem cells (MSCs) possess antitumor properties; however, the exact mechanisms remain unclear. Recently, microvesicles (MVs) are considered as a novel avenue intercellular communication, which may be a mediator in MSCs-related antitumor effect. In the present study, we evaluated whether MVs derived from human umbilical cord Wharton’s jelly mesenchymal stem cells (hWJMSCs) may inhibit bladder tumor T24 cells growth using cell culture and the BALB/c nu/nu mice xenograft model. CCK-8 assay and Ki-67 immunostaining were performed to estimate cell proliferation in vitro and in vivo. Flow cytometry and TUNEL assay were used to assess cell cycle and apoptosis. To study the conceivable mechanism by which hWJMSC-MVs attenuate bladder tumor T24 cells, we estimated the expression of Akt/p-Akt, p-p53, p21 and cleaved Caspase 3 by Western blot technique after exposing T24 cells to hWJMSC-MVs for 24, 48 and 72h. Our data indicated that hWJMSC-MVs can inhibit T24 cells proliferative viability via cell cycle arrest and induce apoptosis in T24 cells in vitro and in vivo. This study showed that hWJMSC-MVs down-regulated phosphorylation of Akt protein kinase and up-regulated cleaved Caspase 3 during the process of anti-proliferation and pro-apoptosis in T24 cells. These results demonstrate that hWJMSC-MVs play a vital role in hWJMSC-induced antitumor effect and may be a novel tool for cancer therapy as a new mechanism of cell-to-cell communication.
Whether PET scan maximum standard uptake value (SUVmax) could differentiate luminal A from luminal B and help predict the survival of metastatic breast cancer (MBC) patients with luminal subtype is still unknown and need to be investigated.
305 MBC patients with luminal subtypes were screened with PET/CT. Eligible patients were prospectively followed up.
In total, 134 patients were eligible for this study. SUVmax was significantly related to the number of metastatic sites and presence of visceral metastasis on univariate analysis. SUVmax could not effectively differentiate patients with luminal A from luminal B subtype. Although luminal subtype at diagnosis could predict the relapse-free interval, it could not predict progression-free survival (PFS) or overall survival (OS) after developing relapse. In contrast, SUVmax was predictive of both PFS and OS and this effect was maintained in multivariate COX regression model.
SUVmax of MBC did not correlate with molecular subtypes of primary tumor. While molecular subtype may be a valuable prognostic factor at primary diagnosis of breast cancer, the SUVmax, rather than molecular subtype, does have a potential to predict independently in multivariate analysis for the PFS and OS in patients with metastatic disease of luminal subtype.
Metastatic breast cancer; Luminal subtype; PET/CT; SUVmax; Prognosis
One new alkaloid, 3-((6-methylpyrazin-2-yl)methyl)-1H-indole (1) was obtained from the deep-sea actinomycete Serinicoccus profundi sp. nov., along with five known compounds (2–6). Their structures were determined on the basis of detailed analysis of the 1D and 2D NMR as well as MS data. The new indole alkaloid displayed weak antimicrobial activity against Staphylococcus aureus ATCC 25923 with an MIC value of 96 μg/mL. It showed no cytotoxicity on a normal human liver cell line (BEL7402) and a human liver tumor cell line (HL-7702).
deep-sea sediment; actinomycete; Serinicoccus profundi sp. nov.; alkaloid; antibacterial activity; cytotoxicity
Aim of the study
This study aims to investigate the expression of human kallikrein 6 (hK6) in gastric cancer, gastric ulcer and normal gastric mucosa tissues and its biological significance.
Material and methods
The expression of hK6 in 15 normal gastric mucosa (NGM) tissues, 15 gastric ulcer (GU) tissues and 55 gastric carcinoma (GC) tissues was respectively detected by immunohistochemistry. The correlations between the expression of hK6 and the clinical pathological parameters of gastric cancer were also analyzed.
Human kallikrein 6 was mainly expressed in cytoplasm. The positive rate of hK6 was significantly higher in gastric cancer tissues than that in gastric ulcer or normal gastric mucosa tissues (70.9%, 40% and 20%, respectively, p < 0.01). With the increase of the invasion depth of gastric cancer cells, aggravation of TNM stage and development of lymph node metastasis, the expression of hK6 increased significantly (p < 0.05 and p < 0.01). There was no obvious correlation between the expression of hK6 and sex, age, tumor diameter, histodifferentiation degree or primary pathological location of gastric cancer (p > 0.05).
The overexpression of hK6 is related to the depth of invasion, lymph node metastasis and clinical stage of gastric carcinoma, which suggests that hK6 may act as a new marker of gastric cancer biological behavior.
expression; gastric cancer; gastric neoplasm; human kallikrein 6; immunohistochemistry
Esophageal squamous cell carcinoma (ESCC) is a prevalent and fatal cancer in China and other Asian countries. Epigenetic silencing of key tumor suppressor genes (TSGs) is critical to ESCC initiation and progression. Recently, many novel TSGs silenced by promoter methylation have been identified in ESCC, and these genes further serve as potential tumor markers for high-risk group stratification, early detection, and prognosis prediction. This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC, providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.
Tumor suppressor gene; CpG island; promoter methylation; esophageal squamous cell carcinoma; tumor marker
In 2003 the Accreditation Council for Graduate Medical Education mandated work hour restrictions. Violations can results in a residency program being cited or placed on probation. Recurrent violations could results in loss of accreditation. We wanted to determine specific intern and workload factors associated with violation of a specific mandate, the 30-hour duty period requirement.
Retrospective review of interns’ performance against the 30-hour duty period requirement during inpatient ward rotations at a pediatric residency program between June 24, 2008 and June 23, 2009. The analytical plan included both univariate and multivariable logistic regression analyses.
Twenty of the 26 (77%) interns had 80 self-reported episodes of continuous work hours greater than 30 hours. In multivariable analysis, noncompliance was inversely associated with the number of prior inpatient rotations (odds ratio: 0.49, 95% confidence interval (0.38, 0.64) per rotation) but directly associated with the total number of patients (odds ratio: 1.30 (1.10, 1.53) per additional patient). The number of admissions on-call, number of admissions after midnight and number of discharges post-call were not significantly associated with noncompliance. The level of noncompliance also varied significantly between interns after accounting for intern experience and workload factors. Subject to limitations in statistical power, we were unable to identify specific intern characteristics, such as demographic variables or examination scores, which account for the variation in noncompliance between interns.
Both intern and workload factors were associated with pediatric intern noncompliance with the 30-hour duty period requirement during inpatient ward rotations. Residency programs must develop information systems to understand the individual and experience factors associated with noncompliance and implement appropriate interventions to ensure compliance with the duty hour regulations.
The molecular and neural mechanisms regulating human social-emotional behaviors are fundamentally important but largely unknown; unraveling these requires a genetic systems neuroscience analysis of human models. Williams Syndrome (WS), a condition caused by deletion of ∼28 genes, is associated with a gregarious personality, strong drive to approach strangers, difficult peer interactions, and attraction to music. WS provides a unique opportunity to identify endogenous human gene-behavior mechanisms. Social neuropeptides including oxytocin (OT) and arginine vasopressin (AVP) regulate reproductive and social behaviors in mammals, and we reasoned that these might mediate the features of WS. Here we established blood levels of OT and AVP in WS and controls at baseline, and at multiple timepoints following a positive emotional intervention (music), and a negative physical stressor (cold). We also related these levels to standardized indices of social behavior. Results revealed significantly higher median levels of OT in WS versus controls at baseline, with a less marked increase in AVP. Further, in WS, OT and AVP increased in response to music and to cold, with greater variability and an amplified peak release compared to controls. In WS, baseline OT but not AVP, was correlated positively with approach, but negatively with adaptive social behaviors. These results indicate that WS deleted genes perturb hypothalamic-pituitary release not only of OT but also of AVP, implicating more complex neuropeptide circuitry for WS features and providing evidence for their roles in endogenous regulation of human social behavior. The data suggest a possible biological basis for amygdalar involvement, for increased anxiety, and for the paradox of increased approach but poor social relationships in WS. They also offer insight for translating genetic and neuroendocrine knowledge into treatments for disorders of social behavior.
This study aims to address hypothesis that the high mobility group A2 (HMGA2), an oncofetal protein, relates to survivability and serves as a prognostic biomarker for colorectal cancer (CRC).
This is a retro-prospective multiple center study. The HMGA2 expression level was determined by performing immunohistochemistry (IHC) on surgical tissue samples of 89 CRCs from a training set and 191 CRCs from a validation set. The Kaplan-Meier analysis and COX proportional hazard model were employed to analyze survivability.
Multivariate logistic analysis indicated that the expression of HMGA2 significantly correlates with distant metastasis in training set (odd ratio, OR=3.53, 95% CI 1.37-9.70) and validation set (OR=6.38, 95% CI 1.47-43.95). Survival analysis revealed that the overexpression of HMGA2 is significantly associated with poor survival of CRC patients (p < 0.05). The adjusted hazard ratios (HRs) for overall survival were 2.38 (95% CI 1.30-4.34) and 2.14 (95% CI 1.21-3.79) in training and validation sets, respectively. Further investigation revealed that HMGA2 delays the clearance of γ-H2AX in HCT-116 and SW480 cells post γ-irradiation, which supports our finding that CRC patients with HMAG2 positive staining in primary tumors had augmented efficacy of adjuvant radiotherapy (HR=0.18, 95% CI 0.04-0.63).
Overexpression of HMGA2 is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC. Therefore, HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator for better response of radiotherapy.
high mobility group A2 protein; colorectum; adenocarcinoma; survival; metastasis
The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus.
Hepatitis C virus (HCV) is a highly diverse virus and a significant challenge for vaccine development is to identify protective epitopes conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope E1E2 proteins, the targets for neutralizing antibody response, are two of the most variable proteins of the virus. Modified E2 antigens were constructed that are not bound by antibodies to previously recognized clusters of highly immunogenic epitopes on E2. Their employment as screening antigens has led to the isolation of a novel panel of human monoclonal antibodies to HCV E2. Functional and biochemical studies revealed that these antibodies bind and neutralize HCV of different genotypes and subtypes. Several of these antibodies neutralized cell culture infectious HCV with genotypes 1–6 envelope proteins. Furthermore, when virus was passaged in culture in the presence of each of these antibodies, virus escape was not observed. Thus, these epitopes are relevant in vaccine design for this virus.
AIM: To investigate the expression and clinical significance of Wnt member 5a (Wnt5a) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) in hepatocellular carcinoma (HCC).
METHODS: In HCC tissues obtained from 85 patients, the protein expressions of Wnt5a, Ror2, β-catenin, and Ki-67 via immunohistochemical staining using the Envision Plus System. The antibody binding was visualized with 3, 3’-diaminobenzidine tetrahydrochloride (DAB) before brief counterstaining with Mayer’s hematoxylin. The degree of immunohistochemical staining was recorded using a semiquantitative and subjective grading system. The mRNA expression of Ror2 was examined by real-time reverse transcription polymerase chain reaction, including nineteen of the 85 HCC and three normal liver tissues. The ratios of Ror2 to the housekeeping gene GAPDH represented the normalized relative levels of Ror2 expression. To determine the prognostic factor, the outcome of the 82 patients was determined by reviewing their medical charts. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models.
RESULTS: Compared to nontumorous (hepatitis or cirrhotic) tissues, Ror2 mRNA expression was clearly decreased in HCC. Ror2 and Wnt5a protein expressions in the majority of HCC patients (63% and 77%, respectively) was significantly less in tumor tissues, as compared to adjacent nontumorous tissues, and this reduction was correlated with increasing serum α-fetoprotein and tumor stage. In 68% (58/85) of the HCC cases, the expression of β-catenin in tumor tissues was either downregulated in the cellular membrane, upregulated in the cytoplasm, or both. Survival analysis indicated that Wnt5a and Ror2 protein expressions could be regarded as independent prognostic factors for HCC; HCC patients with decreased Wnt5a or Ror2 protein expression had a poorer prognosis than those with elevated Wnt5a and Ror2 expression (P = 0.016, P = 0.007, respectively).
CONCLUSION: Wnt5a and Ror2 may serve as tumor suppressor genes in the development of HCC, and may serve as clinicopathologic biomarkers for prognosis in HCC patients.
Hepatocellular carcinoma; Wnt5a; Receptor tyrosine kinase-like orphan receptor 2; β-catenin; Prognosis
Mechanical hyperalgesia is a common and potentially disabling complication of many inflammatory and neuropathic conditions. Activation of the enzyme PKCε in primary afferent nociceptors is a major mechanism that underlies mechanical hyperalgesia, but the PKCε substrates involved downstream are not known. Here, we report that in a proteomic screen we identified the NaV1.8 sodium channel, which is selectively expressed in nociceptors, as a PKCε substrate. PKCε-mediated phosphorylation increased NaV1.8 currents, lowered the threshold voltage for activation, and produced a depolarizing shift in inactivation in wild-type — but not in PKCε-null — sensory neurons. PKCε phosphorylated NaV1.8 at S1452, and alanine substitution at this site blocked PKCε modulation of channel properties. Moreover, a specific PKCε activator peptide, ψεRACK, produced mechanical hyperalgesia in wild-type mice but not in Scn10a–/– mice, which lack NaV1.8 channels. These studies demonstrate that NaV1.8 is an important, direct substrate of PKCε that mediates PKCε-dependent mechanical hyperalgesia.