Search

The number of circulating endothelial progenitor cells (EPCs) was found to increase in patients with breast cancer, but the alteration in EPC function remains to be elusive. We conducted this study to evaluate the number and function of peripheral EPCs of breast cancer patients and its possible underlying mechanism. Besides, the vascular endothelial growth factor (VEGF), VCAM-1, IL-6, and IL-34 levels were measured in blood samples and also in vitro in a medium of EPCs. We found that the number of circulating EPCs in breast cancer patients was significantly higher than that in normal control and remarkably augmented in a stage-dependent manner. Meanwhile, a similar enhancement was observed in the migratory, proliferative, and adhesive activity of circulating EPCs originating from breast cancer patients. More importantly, the VEGF level in blood samples was dramatically elevated in comparison to the control, which was correlated positively with the number and activity of circulating EPCs from breast cancer patients. Moreover, in vitro medium of EPCs from breast cancer patients highly expressed VEGF compared with that from the control, which also had a positive correlation with the number and activity of circulating EPCs from breast cancer patients. This is the first time to demonstrate that the number and function of circulating EPCs are promoted in breast cancer patients, which are positively related to an enhanced VEGF production. These may provide a novel target for improving the outcome of breast cancer.

Depression is associated with abnormal lipid metabolism, and omega (n)-3 polyunsaturated fatty acids (PUFAs) can effectively treat depression. However, mechanism of lipid metabolism involved in the depressive attenuation remains poorly understood. Olfactory bulbectomy (OB)-induced changes in animal behavior and physiological functions are similar to those observed in depressed patients. Therefore, the present study used wild type (WT) and Fat-1 mice with or without OB to explore whether endogenous n-3 PUFA treatment of depression was through rectifying lipid metabolism, and to discover the possible lipid metabolic pathways. In WT mice, OB enhanced locomotor activity associated with up-regulation of lipid metabolites in the serum, such as phosphatidylcholines, L-a-glutamyl-L-Lysine and coproporphyrinogen III (Cop), which were involved in anti-inflammatory lipid metabolic pathways. OB also increased microglia activation marker CD11b and pro-inflammatory cytokines in the hippocampus. In one of the lipid pathways, increased Cop was significantly correlated with the hyper-activity of the OB mice. These OB-induced changes were markedly attenuated by endogenous n-3 PUFAs in Fat-1 mice. Additionally, increased expressions of anti-inflammatory lipid genes, such as fatty acid desaturase (Fads) and phospholipase A2 group VI (Pla2g6), were found in the hippocampus of Fat-1 mice compared with WT mice. Furthermore, Cop administration increased the production of pro-inflammatory cytokines and nitric oxide in a microglial cell line BV2. In conclusion, endogenous n-3 PUFAs in Fat-1 mice attenuated abnormal behavior in the depression model through restoration of lipid metabolism and suppression of inflammatory response.

Coronavirus disease 2019 (COVID-19) and tuberculosis (TB) are major public health and social issues worldwide. The long-term follow-up of COVID-19 with pulmonary TB (PTB) survivors after discharge is unclear. This study aimed to comprehensively describe clinical outcomes, including sequela and recurrence at 3, 12, and 24 months after discharge, among COVID-19 with PTB survivors.From January 22, 2020 to May 6, 2022, with a follow-up by August 26, 2022, a prospective, multicenter follow-up study was conducted on COVID-19 with PTB survivors after discharge in 13 hospitals from four provinces in China. Clinical outcomes, including sequela, recurrence of COVID-19, and PTB survivors, were collectedThirty-two COVID-19 with PTB survivors were included. The median age was 52 (45, 59) years, and 23 (71.9%) were men. Among them, nearly two-thirds (62.5%) of the survivors were moderate, three (9.4%) were severe, and more than half (59.4%) had at least one comorbidity (PTB excluded). The proportion of COVID-19 survivors with at least one sequela symptom decreased from 40.6% at 3 months to 15.8% at 24 months, with anxiety having a higher proportion over a follow-up. Cough and amnesia recovered at the 12-month follow-up, while anxiety, fatigue, and trouble sleeping remained after 24 months. Additionally, one (3.1%) case presented two recurrences of PTB and no re-positive COVID-19 during the follow-up period.The proportion of long symptoms in COVID-19 with PTB survivors decreased over time, while nearly one in six still experience persistent symptoms with a higher proportion of anxiety. The recurrence of PTB and the psychological support of COVID-19 with PTB after discharge require more attention.

Objective Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. Methods A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients’ medical records. Multivariable logistic regression models were used to estimate the odds ratio (OR) and 95% confidence interval (95% CI) of the associations between comorbidities (cardiometabolic or non-cardiometabolic diseases), clinical severity, and treatment outcomes of COVID-19. Results Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30–6.32), type 2 diabetes (T2DM) (3.57, 2.32–5.49), cardiovascular disease (CVD) (3.78, 1.81–7.89), fatty liver disease (7.53, 1.96–28.96), hyperlipidemia (2.15, 1.26–3.67), other lung diseases (6.00, 3.01–11.96), and electrolyte imbalance (10.40, 3.00–26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89–12.75), CVD (8.47, 6.03–11.89), and electrolyte imbalance (19.44, 11.47–32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25–9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46–29.64) within two weeks. Conclusion Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.

ImportanceThe E-cadherin gene, CDH1, and the α-E-catenin gene, CTNNA1, were previously identified as hereditary diffuse gastric cancer (HDGC) susceptibility genes, explaining 25% to 50% of HDGC cases. The genetic basis underlying disease susceptibility in the remaining 50% to 75% of patients with HDGC is still unknown.ObjectiveTo assess the incidence rate of CDH1 germline alterations in HDGC, identify new susceptibility genes that can be used for screening of HDGC, and provide a genetic landscape for HDGC.Design, Setting, and ParticipantsThis cohort study conducted retrospective whole-exome and targeted sequencing of 284 leukocyte samples and 186 paired tumor samples from Chinese patients with HDGC over a long follow-up period (median, 21.7 [range, 0.6-185.9] months). Among 10 431 patients diagnosed with gastric cancer between January 1, 2002, and August 31, 2018, 284 patients who met the criteria for HDGC were included. Data were analyzed from August 1 to 30, 2020.Main Outcomes and MeasuresIncidence rate of CDH1 germline alterations, identification of new HDGC susceptibility genes, and genetic landscape of HDGC.ResultsAmong 284 Chinese patients, 161 (56.7%) were female, and the median age was 35 (range, 20-75) years. The frequency of CDH1 germline alterations was 2.8%, whereas the frequency of CDH1 somatic alterations was 25.3%. The genes with the highest incidence (>10%) of private germline alterations (including insertions and deletions) in the HDGC cohort were MUC4, ABCA13, ZNF469, FCGBP, IGFN1, RNF213, and SSPO, whereas previously reported germline alterations of CTNNA1, BRCA2, STK11, PRSS1, ATM, MSR1, PALB2, BRCA1, and RAD51C were observed at low frequencies (median, 4 [range, 1-12] cases). Furthermore, enrichment of the somatic variant signature of exposure to aflatoxin suggested potential interaction between genetics and environment in HDGC. Double-hit events in genes such as CACNA1D were observed, which suggested that these events might serve as important mechanisms for HDGC tumorigenesis. In addition, germline variants of FSIP2, HSPG2, and NCKAP5 and somatic alterations of FGFR3, ASPSCR1, CIC, DGCR8, and LZTR1 were associated with poor overall survival among patients with HDGC.Conclusions and RelevanceThis study provided a genetic landscape for HDGC. The study’s findings challenged the previously reported high germline alteration rate of CDH1 in HDGC and identified new potential susceptibility genes. Analyses of variant signatures and double-hit events revealed potentially important mechanisms for HDGC tumorigenesis. Findings from the present study may provide helpful information for further investigations of HDGC.

Bisulfite and hydrazine are harmful to the environment safety and human health. Therefore, it is of great value to develop a smart fluorescent probe with high selectivity for detection of bisulfite and hydrazine. In our report, a dual-response fluorescent probe EDBI with high selectivity, rapid response, and low detection limit for discriminative determination HSO

With the objective of investigating the characteristics influencing high-risk sexual behaviours in elderly men (60–74 years of age) in Chongqing, China, a total of 1433 healthy elderly men with sexual intercourse frequencies of one to six times/month who were willing to participate in the questionnaires were studied at four hospitals. We measured serum testosterone levels and performed follow-ups every six months, with a total of 1128 elderly men followed up after two years. We also investigated socio-economic and demographic characteristics (age, education, income, location, marital status and number of marriages), types of sexual partners, age differences with fixed sexual partners, frequency of sexual intercourse, combined basic age-related diseases, sexually transmitted infections (STIs) education, elderly self-care ability and high-risk sexual behaviours (frequency of sexual intercourse and number of sexual partners) using questionnaires. We analysed the influencing factors of high-risk sexual behaviours in elderly men using a univariate analysis, multivariate logistic regression analysis, BP neural network prediction and cluster analysis. Finally, we found that serum total testosterone, age, types of sexual partners, age differences with fixed partners and frequency of sexual intercourse are five factors that influence high-risk sexual behaviours in elderly men.

Background Natural killer/T-cell lymphoma (NKTCL) is a disease with limited treatment options and poor outcomes. Daratumumab monotherapy demonstrated clinical activity in a single-patient case report. We present data from the primary analysis of a phase 2 study of daratumumab monotherapy in relapsed or refractory (R/R) NKTCL. Methods This phase 2 study with Simon’s two-stage design evaluated daratumumab in patients with histologically confirmed extranodal NKTCL, nasal type, per WHO classification that was refractory to or relapsed after ≥ 1 line of chemotherapy, who were not candidates for other treatment modalities. All patients received daratumumab 16 mg/kg intravenously once weekly for Cycles 1 and 2, every other week for Cycles 3 through 6, and every 4 weeks thereafter until progression or unacceptable toxicity; all cycles were 28 days. The primary end point was objective response rate (ORR) based on blinded independent central review per Revised Criteria for Response Assessment of Hodgkin and non-Hodgkin Lymphoma (Lugano classification). Results In total, 32 Asian patients received daratumumab. The ORR was 25.0% (95% confidence interval [CI] 11.5–43.4); all 8 responders had a partial response; and the median duration of response was 55.0 days (95% CI 29–339). At 10.2 months of median follow-up, median progression-free survival (PFS) was 53.0 days (95% CI 43–106); the 4-month PFS rate was 13.0%. Median overall survival (OS) was 141.0 days (95% CI 94–438); the 6-month OS rate was 42.9%. Nineteen (59.4%) patients had grade 3/4 treatment-emergent adverse events (TEAEs); the most common was thrombocytopenia (25.0%; n = 8). TEAEs leading to death occurred in 4 patients (death, respiratory failure, septic shock, and pneumonia); all were unrelated to daratumumab. Conclusions In patients with R/R NKTCL, daratumumab monotherapy was well tolerated with no new safety concerns and achieved an ORR of 25.0%. However, no patients achieved complete response, and duration of response was short. Trial registration ClinicalTrials.gov, NCT02927925. Registered 7 October 2016.

Psoriasis is a chronic inflammatory skin disease that affects adults and children. The most common subtype is psoriasis vulgaris. This article analyzes the characteristics and clinical features of children with psoriasis vulgaris to strengthen the understanding, treatment, and management for children with psoriasis.A total of 208 children with psoriasis vulgaris, who were first admitted to the Department of Dermatology, Xiangya Hospital, Central South University from October 2012 to December 2018, were retrospectively analyzed. Their clinical characteristics, results of laboratory examination, treatment options and efficacy were summarized.The age of the 208 children with psoriasis vulgaris was (11.19±3.97) years old, the peak incidence was 12 years old, the disease duration was (27.46±31.30) months, and the male-female ratio was 1∶0.96. The most common site of the first attack was the scalp (37.98%), followed by the trunk (26.44%) and the limbs (22.12%). The causes leading to exacerbation were more common in infections and diets. There were 33 patients (15.87%) with a family history of psoriasis, showing the higher score of Psoriasis Area and Severity Index (PASI) and the higher Dermatological Quality of Life Index (DLQI) (bothChildren with psoriasis vulgaris are mainly caused by infection and diet. Patients with family history have more serious illness, lower quality of life, and are more likely to have metabolic abnormalities such as overweight and obesity. The serum 25-OH-VD levels in children with psoriasis vulgaris are negatively correlated with the score of PASI.

The efficacy of susceptible variants derived from genome-wide association studies (GWAs) optimizing discriminatory accuracy of colorectal cancer (CRC) in Chinese remains unclear. In the present validation study, we assessed 75 recently identified variants from GWAs. A risk predictive model combining 19 variants using the least absolute shrinkage and selection operator (LASSO) statistics offered certain clinical advantages. This model demonstrated an area under the receiver operating characteristic (AUC) of 0.61 during training analysis and yielded robust AUCs from 0.59 to 0.61 during validation analysis in three independent centers. The individuals carrying the highest quartile of risk score revealed over 2-fold risks of CRC (ranging from 2.12 to 2.90) compared with those who presented the lowest quartile of risk score. This genetic model offered the possibility of partitioning risk within the average risk population, which might serve as a first step toward developing individualized CRC prevention strategies in China.

Background Dysphagia is common after stroke. Patients with dysphagia have a higher risk of stroke-associated pneumonia (SAP) and poor outcomes. Early detection of dysphagia is necessary to identify and manage patients at high risk of aspiration. The aim of the study was to assess the impact of the systematic administration of the volume-viscosity swallow test (V-VST) in patients with acute ischaemic stroke. Methods This was a retrospective observational study that enrolled patients with acute ischaemic stroke in two consecutive time periods: pre-V-VST, when the 30-mL water-swallowing test (WST) was systematically administered, and V-VST, when all patients underwent the WST and the V-VST test was systematically administered if the patient failed the WST. Results Two hundred and 42 patients were enrolled. The mean age of the participants was 68.8 ± 10.88 years, 61.2% were male, and the median National Institutes of Health Stroke Scale score was 3 (IQR, 1–6). A total of 147 patients were enrolled during the pre-V-VST period and 95 were enrolled during the V-VST period. There was a significant difference in the occurrence of SAP (21.8% vs. 10.5%, p = 0.024) and the rate of nasogastric tube feeding (25.9% vs. 14.7%, p = 0.040) between the two groups, and no differences were found in the length of hospital stay (p = 0.277) or the total cost of hospitalization (p = 0.846). Conclusions The V-VST was a better clinical screening tool, and it can also provide detailed suggestions regarding dietary modifications to prevent aspiration and SAP.

In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.

Herein, we construct an ingenious spatially localized amplification reaction (SLAR) by colocalizing the entropy-driven reaction (EDR) in a nanometer space, which greatly accelerates target conversion and realizes the sensitive detection of microRNA-21 (miRNA-21). A large number of EDR complex are hybridized with the prefabricated DNA scaffold via a DNA self-assembly strategy to form the SLAR nanoprobe. Target miRNA-21 triggers interval EDR along the long DNA scaffold, resulting in fluorescence recovery with high signal gain because of the fast release of reporter. Compared with reactions with diffusible components, spatial arrangement of all components of EDR on a nanoscale scaffold can increase the local concentration of reactants, accelerating the interaction between adjacent components, and can also avoid the influence of stochastic diffusion to reduce the unintentional binding interaction between further separated components. Therefore, this SLAR assay displayed an excellent analytical performance for miRNA-21 detection with a detection limit of 6 pM and showed good specificity in distinguishing miRNA-21 from similar miRNAs. In addition, the proposed assay has been experimentally demonstrated for estimation of miRNA-21 in MCF-7 and HeLa cells lysates, which exhibited great promise in the sensitive detection of biomarkers in early diagnosis.

This research examines the level of pre-exposure prophylaxis (PrEP) self-efficacy among HIV-negative men who have sex with men (MSM) in China and identifies the influencing factors associated with the level of PrEP self-efficacy in terms of social-demographic characteristics and social psychological factors. The data were gathered from a baseline assessment of a longitudinal randomized controlled intervention trial. From April 2013 to March 2015, nonprobability sampling was used to recruit HIV-negative MSM at Chongqing, Guangxi, Xinjiang, and Sichuan in west China. A total of 1884 HIV-negative MSM were analyzed. Chi-square test and nonparametric rank sum test were used for univariate analysis. Multivariable linear regression analysis was used to discuss the factors that influence the level of PrEP self-efficacy. Overall levels of PrEP self-efficacy were low, and five factors were found to effect PrEP self-efficacy: age, residence, AIDS-related knowledge, PrEP-related motivation, and anxiety. Age and anxiety score were negatively related to PrEP self-efficacy. The higher the age and anxiety score, the lower the PrEP self-efficacy. AIDS-related knowledge and PrEP-related motivation were actively related to PrEP self-efficacy. The higher the knowledge and motivation score, the higher the PrEP self-efficacy. In addition, the PrEP self-efficacy level of MSM in rural areas is lower than that in urban areas. The lower level of self-efficacy in the MSM population needs to be improved. Pertinent interventions should be taken to promote the self-efficacy of PrEP in MSM, to enhance their willingness to take medicine, improve their medication adherence, and thus reduce HIV infection among MSM.

Objective Previous studies mainly reported the clinical characteristics of novel coronavirus 2019 (COVID-19) infections, but the research on clinical characteristics and treatment outcomes of COVID-19 patients with stroke is still rare. Methods A multi-center retrospective study was conducted at 11 hospitals in 4 provinces of China, and COVID-19 patients with stroke were enrolled from February 24 to May 4, 2020. We analyzed epidemiological, demographic, and clinical characteristics of cases as well as the laboratory test results, treatment regimens and outcomes, and the clinical characteristics and therapeutic outcomes were compared between severe and nonsevere patients, and by age group, respectively. Results A total of 27 patients [mean age: 66.41 (SD 12.1) years] were enrolled. Among them, 9 (33.3%) were severe patients and 18 (66.7%) were nonsevere patients; 17 (63.0%) were female; 19 (70.4%) were aged 60 years and above. The most common symptoms were fever [19 (70.4%)], fatigue [12 (44.4%)] and cough [11 (40.7%)], respectively. Abnormal laboratory findings of COVID-19 patients with stroke included high levels of C-reactive protein [19 (73.1%)], D-dimer [14 (58.3%)], blood glucose [14 (53.8%)], fibrinogen [13 (50.0%)], and decreased lymphocytes [12 (44.4%)]. Comparing to nonsevere cases with stroke, severe patients with stroke were likely to be older, susceptible to receiving oxygen inhalation, and had more complications (p < 0.05). In addition, there were significant differences in lymphocytes, neutrophils, lactate dehydrogenase, C-reactive protein, creatine kinase between the severe cases and nonsevere cases (p < 0.05). The older patients had a decreased platelet count and elevated fibrinogen, compared with the younger (p < 0.05). All patients (100%) received antiviral treatment, 12 (44.4%) received antibiotics treatment, 26 (96.3%) received Traditional Chinese Medicine (Lung cleansing & detoxifying decoction), and oxygen inhalation was in 18 (66.7%). The median duration of hospitalization was 16 days. By May 4, 2020, a total of 26 (96.3%) patients were cured and discharged, and 1 (3.7%) patients died. Conclusion COVID-19 patients with stroke had poor indicators of coagulation system, and severe and older patients might have a higher risk of complications and unfavorable coagulation system. However, the overall treatment outcome is favorable., Graphical abstract Image, graphical abstract

The aim of the present study was to report experiences with invasive prenatal diagnosis of α-thalassemia for the prevention of Hb Bart’s hydrops fetalis syndrome in the Guangxi Zhuang Autonomous Region, China. Pregnant women and their partners who tested positive for α0-thalassemia or were diagnosed with HbH diseases were counseled and suggested to undergo a prenatal diagnostic procedure for α-thalassemia. Fetal material was obtained by chorionic villus sampling (CVS) between 9 and 13 weeks of gestation, by amniocentesis between 16 and 24 weeks of gestation and by cordocentesis after 24 weeks of gestation. The α0-thalassemia gene types were detected by gap polymerase chain reaction (Gap-PCR). All results were finally confirmed by DNA analysis after delivery or termination of pregnancy. An invasive prenatal α-thalassemia diagnosis was performed in 3155 cases at risk for Hb Bart’s hydrops fetalis syndrome at our hospital from 2002 to 2016. CVS was performed in 1559 cases (49.4%), amniocentesis in 1240 cases (39.3%) and cordocentesis in 356 cases (11.3%). In total, 786 fetuses were diagnosed as Hb Bart’s hydrops fetalis syndrome. Among these cases, the α-thalassemia genotype was --SEA/--SEA in 784 cases and --SEA/--THAI in 2 cases. All affected pregnancies were terminated in time. This extensive experience suggests that carrier screening, molecular diagnostics, genetic counselling, and prenatal diagnosis are effective measures to prevent Hb Bart’s hydrops fetalis syndrome.

The purpose of this study was to evaluate the diagnostic value of ultrasonic elastography (UE) and conventional ultrasonography (CUS) in the differential diagnosis of non-mass-like (NML) breast lesions. Static sonograms of 39 pathologically diagnosed NML breast lesions were reviewed. Lesions were evaluated by CUS and UE using CUS subjective ratings (benign, malignant or indeterminate), a 5-point subjective elasticity scoring system and the quantitative strain ratio (SR). Receiver operating characteristic curves and diagnostic tests were used to assess the diagnostic value of CUS and UE. Areas under the receiver operating characteristic curves (Az) of the CUS, 5-point elasticity and SR methods were 0.848 (p < 0.001), 0.895 (p < 0.001) and 0.943 (p < 0.001), respectively. In diagnosing NML breast lesions, there was no significant difference between the 5-point elasticity and CUS methods. The combination of UE and CUS helps to improve the accuracy of the ultrasonic diagnosis of NML breast lesions.

The number of axillary lymph nodes involved and retrieved are important prognostic factors in breast cancer. The purpose of our study was to investigate whether the lymph node ratio (LNR) is a better prognostic factor in predicting disease-free survival (DFS) for breast cancer patients as compared with pN staging. The analysis was based on 804 breast cancer patients who had underwent axillary lymph node dissection between 1999 and 2008 in Sun Yat-Sen University Cancer Center. Optimal cutoff points of LNR were calculated using X-tile software and validated by bootstrapping. Patients were then divided into three groups (low-, intermediate-, and high-risk) according to the cutoff points. Predicting risk factors for relapse were performed according to Cox proportional hazards analysis. DFS was estimated using the Kaplan-Meier method and compared by the log-rank test. The 5-year DFS rate decreased significantly with increasing LNRs and pN. Univariate analysis found that the pT , pN, LNR, molecule type, HER2, pTNM stage and radiotherapy well classified patients with significantly different prognosis. By multivariate analysis, only LNR classification was retained as an independent prognostic factor. Furthermore, there was a significant prognostic difference among different LNR categories for pN2 category, but no apparent prognostic difference was seen between different pN categories in any LNR category. Therefore, LNR rather than pN staging is preferable in predicting DFS in node positive breast cancer patients, and routine clinical decision-making should take the LNR into consideration.

We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC 50 ], 1 to 4 μM; and 50% cytotoxic concentration [CC 50 ], >40 μM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or nonflaviviruses (Western equine encephalomyelitis virus, Chikungunya virus, and vesicular stomatitis virus). A mode-of-action study suggested that the compound inhibits viral RNA synthesis. Replicons resistant to the inhibitor were selected in cell culture. Sequencing of the resistant replicons revealed two mutations (P104L and A119T) in the viral NS4B protein. Genetic analysis, using DENV-2 replicon and recombinant viruses, demonstrated that each of the two NS4B mutations alone confers partial resistance and double mutations confer additive resistance to the inhibitor in mammalian cells. In addition, we found that a replication defect caused by a lethal NS4B mutation could be partially rescued through trans complementation. The ability to complement NS4B in trans affected drug sensitivity when a single cell was coinfected with drug-sensitive and drug-resistant viruses. Mechanistically, NS4B was previously shown to interact with the viral NS3 helicase domain; one of the two NS4B mutations recovered in our resistance analysis—P104L—abolished the NS3-NS4B interaction (I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006). Collectively, the results suggest that the identified inhibitor targets the DENV NS4B protein, leading to a defect in viral RNA synthesis.

The role of the α-helical domain (MH) of dengue virus (DENV) precursor membrane protein in replication was investigated by site-directed mutagenesis. Proline substitutions of three residues (120, 123 and 127) at the C-terminus, but not those at the N-terminus of MH domain, reduced the virus-like particles of DENV1, DENV2 and DENV4 detected in supernatants. In a DENV2 replicon trans-packaging system, these three mutations suppressed particles detected; two of them (I123P and V127P) also affected viral entry. In the context of DENV2 genome-length RNA, all three mutations reduced virion assembly and virus spreading in cell culture. Analysis of revertants showed that mutation A120P could partially support viral infection cycle; in contrast, mutations I123P and V127P were lethal, and adaptations of I123P → I123L and V127P → V127L were required to restore the viral infection cycle. These findings demonstrate that the C-terminus of the MH domain is involved in both assembly and entry of DENV.

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2′- C -acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3′- and 5′-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3′,5′- O -diisobutyryl-2′- C -acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.

Objective: To evaluate the predictive value of three-dimensional (3D)-power Doppler sonography on recurrent miscarriage. Materials and methods: The study patients were divided into a recurrent miscarriage group (30 cases) and a normal pregnancy group (21 cases). Measurement of endometrial thickness was performed using two-dimensional transvaginal ultrasound in the midluteal phase. The endometrial volume, vascularization index (VI), flow index (FI), and vascularization-flow index (VFI) in midluteal and placenta volume, as well as the VI, FI, and VFI of early pregnancy were measured using Virtual Organ Computer-aided Analysis of 3D-power Doppler ultrasound. Results: Endometrial thickness, endometrial volume, endometrial vascular data, VI, FI, and VFI of the midluteal phase were lower in the recurrent miscarriage group compared with the normal pregnancy group (p 0.05). The predictive accuracy of endometrial thickness, endometrial volume, VI, FI, and VFI in the midluteal phase, and placenta volume, VI, FI, and VFI in early pregnancy as measured by the receiver operating characteristic curve to predict miscarriage before 12 gestational weeks in participants was 0.681, 0.876, 0.770, 0.720, 0.879, 0.771, 0.907, 0.592, respectively. Conclusion: The 3D-power Doppler ultrasound is a more comprehensive and sensitive method for evaluating endometrial receptivity. Endometrial volume, VI, FI, and VFI in the midluteal phase, as well as VI in early pregnancy, can be considered as predictive factors for recurrent miscarriage.

Background Low tyrosine-protein phosphatase nonreceptor type 12 (PTPN12) expression may be associated with breast cancer growth, proliferation, and metastasis. However, the prognostic value of PTPN12 in breast cancer has not been clearly identified. Patients and methods 51 triple-negative breast cancer (TNBC) patients and 83 non-TNBC patients with a histopathology diagnosis from October 2001 to September 2006 were included in this study. Immunohistochemical staining for PTPN12 on tissue microarrays was conducted. Results High PTPN12 expression was seen in 39.2% of TNBC and 60.2 % of non-TNBC cases. Low PTPN12 expression was associated with lymph node status (p = 0.002) and distant metastatic relapse (p = 0.002) in TNBC patients. Similarly, low PTPN12 expression in non-TNBC patients was significantly correlated with lymph node status (p = 0.002), stage (p = 0.002) and distant metastatic relapse (p = 0.039). The high PTPN12 expression group was associated with longer DFS and OS compared with low PTPN12 expression group only in TNBC cases (p = 0.005, p = 0.015), according to univariate Cox regression analysis. Conclusion These findings provide evidence that low expression of PTPN12 is associated with worse prognosis and may be used as a potential prognostic biomarker in TNBC patients.

Background Studies on the risk of chromosomal abnormalities in early spontaneous abortion after assisted reproductive technology (ART) are relatively controversial and insufficient. Thus, to obtain a more precise evaluation of the risk of embryonic chromosomal abnormalities in first-trimester miscarriage after ART, we performed a meta-analysis of all available case–control studies relating to the cytogenetic analysis of chromosomal abnormalities in first-trimester miscarriage after ART. Methods Literature search in the electronic databases MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) based on the established strategy. Meta-regression, subgroup analysis, and Galbraith plots were conducted to explore the sources of heterogeneity. Results A total of 15 studies with 1,896 cases and 1,186 controls relevant to the risk of chromosomal abnormalities in first- trimester miscarriage after ART, and 8 studies with 601 cases and 602 controls evaluating frequency of chromosome anomaly for maternal age≥35 versus

This case—control study explored the relationship between early recurrent spontaneous abortion (RSA) and the expression of two genes: VEGFA, the gene encoding vascular endothelial growth factor (VEGF); and fms-related tyrosine kinase 1 (FLT1), the gene encoding the soluble VEGF receptor-1 (sFlt-1). Women experiencing RSA or undergoing induced abortions in the early stage of normal pregnancy were recruited to the study ( n = 30 per group). There were no significant between-group differences in maternal age or duration of pregnancy. The levels of VEGF and sFlt-1 mRNA in chorionic villus tissue samples were examined by quantitative reverse transcription—polymerase chain reaction. Levels of sFlt-1 and VEGF mRNA in the chorionic villus tissue of women with RSA were significantly higher than levels in the control group. This study demonstrated that there is a relationship between early RSA and VEGF and sFlt-1 levels, suggesting that overexpression of the FLT1 and VEGFA genes may be associated with the pathogenesis of RSA.

We describe a novel translation inhibitor that has anti-dengue virus (DENV) activity in vitro and in vivo . The inhibitor was identified through a high-throughput screening using a DENV infection assay. The compound contains a benzomorphan core structure. Mode-of-action analysis indicated that the compound inhibits protein translation in a viral RNA sequence-independent manner. Analysis of the stereochemistry demonstrated that only one enantiomer of the racemic compound inhibits viral RNA translation. Medicinal chemistry was performed to eliminate a metabolically labile glucuronidation site of the compound to improve its in vivo stability. Pharmacokinetic analysis showed that upon a single subcutaneous dosing of 25 mg/kg of body weight in mice, plasma levels of the compound reached a C max (maximum plasma drug concentration) above the protein-binding-adjusted 90% effective concentration (EC 90 ) value of 0.96 μM. In agreement with the in vivo pharmacokinetic results, treatment of DENV-infected mice with 25 mg/kg of compound once per day reduced peak viremia by about 40-fold. However, mice treated with 75 mg/kg of compound per day exhibited adverse effects. Collectively, our results demonstrate that the benzomorphan compounds inhibit DENV through suppression of RNA translation. The therapeutic window of the current compounds needs to be improved for further development.

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC 50 ) of 2.4 nM and a 50% cytotoxic concentration (CC 50 ) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC 90 s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo , consistent with observations in DEN patients.

We recently reported that (2 R ,3 R ,4 R ,5 R )-2-(4-amino-pyrrolo[2,3- d ]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC 50 ) and cytotoxic concentration (CC 50 ) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14 C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo . Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.

Although flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of flavivirus replication complex and could be targeted for potential antiviral development.

Type III interferons (IFN), IFN-lambda or IL-28/29, are new members of the IFN super-family. Except for using distinct receptors, type I and type III IFNs share the same major post receptor signaling components to activate the transcription of a similar set of IFN-stimulated genes (ISGs). To examine the antiviral effects of the new type IFNs against West Nile virus (WNV), we compared the antiviral effects of IFN-alpha and IFN-lambda on WNV virus-like particle (VLP) infection and replicon replication in Huh7.5 and Hela cells. The results revealed that (i) both types of IFNs could efficiently prevent the WNV infection, but IFN-alpha demonstrated a stronger antiviral efficacy; (ii) WNV genome replication in VLP-infected cells and replicon-containing cell lines could only be inhibited by IFN-alpha, but not IFN-lambda; (iii) in agreement with the observed antiviral effects, only IFN-lambda-induced activation of JAK-STAT signaling pathway and induction of ISG expression were completely inhibited in WNV replicon-containing cell lines, but IFN-alpha signal transduction was either unaffected or only partially inhibited in Huh7.5 or Hela cells by the virus. Hence, the differential inhibition of WNV on IFN-alpha and IFN-lambda signal transduction implies that the receptors of the two types of IFNs, but not the common post receptor signaling components, could be selectively targeted either directly by WNV nonstructural proteins or indirectly by the cellular responses induced by the virus infection to inhibit the signal transduction of the cytokines.

Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway.

To evaluate the value of diagnosis of alpha-thalassemia by analyzing fetal DNA in maternal plasma.Ten families were screened, the husbands being alpha-thalassemia Southeast Asia deletion (SEA alpha-thalassemia-1) heterozygotes and the pregnant women being alpha-thalassemia-2 heterozygotes. Fluorescent polymerase chain reaction (PCR) and gene scanning were used to detect the paternally inherited genotypes of SEA alpha-thalassemia-1 gene mutation and short tandem repeats (STRs) in the maternal plasma fetal DNA. The results were compared to those of conventional prenatal diagnosis of fetal DNA in amniotic fluid, chorionic villus or cord blood.Paternally derived STR genotypes were detected in all specimens of plasma fetal DNA. Paternally inherited SEA alpha-thalassemia-1 gene mutation was detected in 4 cases, while the other 6 cases did not inherit the paternal mutation. The results were completely concordant with those of the conventional prenatal diagnosis.Noninvasive prenatal diagnostic method, the technique using fluorescent PCR and gene scanning to detect the fetal DNA and paternally inherited SEA alpha-thalassemia-1 gene mutation in maternal plasma helps exclude the fetuses with hemoglobin H diseases.

To conduct prenatal diagnosis on the couples carrying Thailand deletion (--THAI) alpha-thalassemia 1 and at high risk of having fetus with alpha-thalassemia.Genotypes of couples and fetuses were analyzed by PCR and DNA sequencing.Four pregnant women were patients with Hb H diseases of --THAI compounding with alpha-thalassemia 2, while their husbands were heterozygote of the Southeast Asian type alpha-thalassemia 1 (--SEA). Another 5 families, either husbands or wives were heterozygote of --THAI or --SEA. The genotypes of their fetuses were as follows: 2 cases with Hb Bart's hydrops fetalis syndrome, 1 Hb H disease, 4 alpha-thalassemia heterozygote and 2 normal. The DNA sequencing approved the PCR results.The study on prenatal diagnosis of Thailand deletion alpha-thalassemia 1 is of importance to the genetic counseling and prenatal diagnosis of alpha-thalassemia.