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Urtica cannabina L. (UL) has been used clinically for centuries because of its anti-inflammatory properties. This study aimed to investigate the underlying mechanisms and anti-inflammatory effects of different UL concentrations in zebrafish. To elucidate UL's anti-inflammatory properties, two inflammation zebrafish models were designed 1) by severing the zebrafish's caudal fin to assess the repairing effect of UL on the tail inflammation, and 2) by inducing lipopolysaccharides (LPS)-mediated intestinal inflammation to assess the protective and reparative effects of UL on intestinal inflammation at the histological and genetic levels. Furthermore, the effect of UL on the LPS-induced intestinal flora changes was also assessed. After caudal fin resection, a scar formed on the tail of the zebrafish, and the area of the caudal fin increased by 1.30 times as much as that of the control group (P < 0.01). Moreover, this tail scar was alleviated after 10 mg/g UL supplementation but not after 30 mg/g UL dose. LPS decreased the feed intake and body weight of the zebrafish; however, these effects were reversed after 10 and 30 mg/g doses of UL. In addition, the LPS treatment also reduced the intestinal goblet cells by 49% in the zebrafish when compared with the control, which was significantly restored after 10 and 30 mg/g UL treatments. At the genetics level, the expression of the pro-inflammatory cytokine genes (TNF-α, IL6, and IL8) showed that 10 and 30 mg/g UL doses could rescue LPS-induced expression. The gut microbiota analysis revealed changes in the abundance of four major bacterial phyla in the 10 and 30 mg/g UL-treated groups, with an increased probiotic Bacteroidota and decreased pathogenic bacteria. These results indicate that UL strongly inhibits inflammation caused by caudal fin removal and LPS-induced inflammatory changes in the zebrafish intensity, suggesting that UL is a feed additive that could be developed to improve resistance to inflammation in livestock., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic postsurgical pain may have a substantial impact on patient's quality of life, and has highly heterogenous presentation amongst sufferers. We aimed to explore the risk factors relating to chronic pain and the related miRNA phenotypes in patients with lung adenocarcinoma after video-assisted thoracoscopic lobectomy to identify potential biomarkers. Our prospective study involved a total of 289 patients with early invasive adenocarcinoma undergoing thoracoscopic lobotomy and a follow-up period of 3 months after surgery. Blood was collected the day before surgery for miRNA detection and patient information including operation duration, duration of continuous drainage of the chest, leukocyte count before and after operation, and postoperative pain scores were recorded. Using clinical and biochemical information for each patient, the risk factors for chronic postsurgical pain and related miRNA phenotypes were screened. We found that chronic postsurgical pain was associated with higher body mass index; greater preoperative history of chronic pain; longer postoperative drainage tube retention duration; higher numerical rating scale scores one, two, and three days after surgery; and changes in miRNA expression, namely lower expression of miRNA 146a-3p and higher expression of miRNA 550a-3p and miRNA 3613-3p in peripheral blood (p < 0.05). Of these factors, patient body mass index, preoperative history of chronic pain, average numerical rating scale score after operation, and preoperative peripheral blood miRNA 550a-3P expression were independent risk factors for the development of chronic postsurgical pain. Identification of individual risk markers may aid the development and selection of appropriate preventive and control measures., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: The purpose of this study is to examine the efficacy of BETTER (Brain Injury, Education, Training, and Therapy to Enhance Recovery) vs. usual transitional care management among diverse adults with traumatic brain injury (TBI) discharged home from acute hospital care and families., Methods: This will be a single-site, two-arm, randomized controlled trial (N = 436 people, 218 patient/family dyads, 109 dyads per arm) of BETTER, a culturally- and linguistically-tailored, patient- and family-centered, TBI transitional care intervention for adult patients with TBI and families. Skilled clinical interventionists will follow a manualized protocol to address patient/family needs. The interventionists will co-establish goals with participants; coordinate post-hospital care, services, and resources; and provide patient/family education and training on self- and family-management and coping skills for 16 weeks following hospital discharge. English- and Spanish-speaking adult patients with mild-to-severe TBI who are discharged directly home from the hospital without inpatient rehabilitation or transfer to other settings (community discharge) and associated family caregivers are eligible and will be randomized to treatment or usual transitional care management. We will use intention-to-treat analysis to determine if patients receiving BETTER have a higher quality of life (primary outcome, SF-36) at 16-weeks post-hospital discharge than those receiving usual transitional care management. We will conduct a descriptive, qualitative study with 45 dyads randomized to BETTER, using semi-structured interviews, to capture perspectives on barriers and facilitators to participation. Data will be analyzed using conventional content analysis. Finally, we will conduct a cost/budget impact analysis, evaluating differences in intervention costs and healthcare costs by arm., Discussion: Findings will guide our team in designing a future, multi-site trial to disseminate and implement BETTER into clinical practice to enhance the standard of care for adults with TBI and families. The new knowledge generated will drive advancements in health equity among diverse adults with TBI and families., Trial Registration: NCT05929833., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Oyesanya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Parkinson disease (PD) psychosis (PDP) is a disabling non-motor symptom. Pharmacologic treatment is limited to pimavanserin, quetiapine, and clozapine, which do not worsen parkinsonism. A Food and Drug Administration black box warning exists for antipsychotics, suggesting increased mortality in elderly patients with dementia. However, the reasons for higher mortality are unknown., Aim: Expanding on prior work exploring mortality in treated PDP patients, we conducted a retrospective comparison to understand the links between treatment regimen, clinical characteristics, and negative outcomes., Methods: Electronic medical record data extraction included clinically diagnosed PD patients between 4/29/16-4/29/19 and excluded patients with primary psychiatric diagnoses or atypical parkinsonism. Mortality and clinical characteristics during the study period were compared between untreated patients and those receiving pimavanserin, quetiapine, or both agents (combination). Mortality analyses were adjusted for age, sex, levodopa equivalent daily dose (LEDD), and dementia., Results: The pimavanserin group (n = 34) had lower mortality than the untreated group (n = 66) (odds ratio = 0.171, 95% confidence interval: 0.025-0.676, p = 0.026). The untreated group had similar mortality compared to the quetiapine (n = 147) and combination (n = 68) groups. All treated groups had a higher LEDD compared to the untreated group, but no other differences in demographics, hospitalizations, medical comorbidities, medications, or laboratory values were found between the untreated and treated groups., Conclusions: PDP patients receiving pimavanserin had lower mortality than untreated patients. We found no other clear differences in clinical characteristics to explain the mortality risk. Prospective randomized trials are needed to definitively identify the optimal PDP treatment regimen and associated risks., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Longardner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Compound screening by in silico approaches has advantages in identifying high-activity leading compounds and can predict the safety of the drug. A key challenge is that the number of observations of drug activity and toxicity accumulation varies by target in different datasets, some of which are more understudied than others. Owing to an overall insufficiency and imbalance of drug data, it is hard to accurately predict drug activity and toxicity of multiple tasks by the existing models. To solve this problem, this paper proposed a two-stage transfer learning workflow to develop a novel prediction model, which can accurately predict drug activity and toxicity of the targets with insufficient observations. We built a balanced dataset based on the Tox21 dataset and developed a drug activity and toxicity prediction model based on Siamese networks and graph convolution to produce multitasking output. We also took advantage of transfer learning from data-rich targets to data-poor targets. We showed greater accuracy in predicting the activity and toxicity of compounds to targets with rich data and poor data. In Tox21, a relatively rich dataset, the prediction model accuracy for classification tasks was 0.877 AUROC. In the other five unbalanced datasets, we also found that transfer learning strategies brought the accuracy of models to a higher level in understudied targets. Our models can overcome the imbalance in target data and predict the compound activity and toxicity of understudied targets to help prioritize upcoming biological experiments., Competing Interests: The authors have declared that no competing interests exist.

Background: Schistosomiasis is a neglected tropical parasitic and chronic disease affecting hundreds of millions of people. Adult schistosomes reside in the blood stream of the definitive mammalian host. These helminth parasites possess two epithelial surfaces, the tegument and the gastrodermis, both of which interact with the host during immune evasion and in nutrient uptake., Methods: Female ARC Swiss mice (4-6 weeks old) were infected percutaneously with Schistosoma japonicum cercariae freshly shed from Oncomelania hupensis quadrasi snails (Philippines strain). Fluorescent in situ hybridisation (FISH) was performed by using fresh adult S. japonicum perfused from those infected mice. Adult S. japonicum worms were processed to isolate the tegument from the carcass containing the gastrodermis; blood and bile were collected individually from infected and uninfected mice. Total DNA extracted from all those samples were used for microbiome profiling., Results: FISH and microbiome profiling showed the presence of bacterial populations on two epithelial surfaces of adult worms, suggesting they were distinct not only from the host blood but also from each other. Whereas microbial diversity was reduced overall in the parasite epithelial tissues when compared with that of host blood, specific bacterial taxa, including Anoxybacillus and Escherichia, were elevated on the tegument. Minimal differences were evident in the microbiome of host blood during an active infection, compared with that of control uninfected blood. However, sampling of bile from infected animals identified some differences compared with controls, including elevated levels of Limnohabitans, Clostridium and Curvibacter., Conclusions: Using FISH and microbial profiling, we were able to demonstrate, for the first time, that bacteria are presented on the epithelial surfaces of adult schistosomes. These schistosome surface-associated bacteria, which are distinct from the host blood microenvironment, should be considered as a new and important component of the host-schistosome interaction. The importance of individual bacterial species in relation to schistosome parasitism needs further elucidation., Competing Interests: The authors have declared that no competing interests exist.

Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism's medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia., Competing Interests: The authors have declared that no competing interests exist.

Background: Postpartum Family Visits (PFVs) have been advocated as a way to improve health outcomes for puerperal women and their newborns. This study aimed to identify individual factors associated with the utilization of PFVs in rural Jiangsu Province, China., Methods: We employed responses of the household survey in Jiangsu province, part of the National Health Service Survey (NHSS), a nationally representative survey in China. The data analysis framework was designed based on Andersen's behavioral model. The outcome variables included nonuse and deficient use of PFVs, and the explanatory variables were organized into three hierarchical levels: predisposing, enabling and need factors. Univariate analysis and multivariate logistic regression analysis were conducted to examine the impact of the three hierarchical levels on PFVs utilization., Results: A total of 884 rural women who had a childbearing history in the prior five years answered PFVs questions. About 23.4% of them had never received any PFVs, and 40.4% received <3 visits. In the results of multivariate logistic regression, educational level (OR = 0.43, 95% CI: 0.24-0.77), income (OR = 0.62, 95% CI: 0.43-0.88), the distance from the nearest hospital (OR = 1.49, 95% CI: 1.07-2.07) and parity (OR = 2.17, 95% CI: 1.54-3.05) had significant relationship with nonuse. Factors significantly associated with deficient use of PFVs included employment (OR = 0.62, 95% CI: 0.39-0.98), the distance from the nearest hospital (OR = 1.73, 95% CI: 1.26-2.36), level of delivery institution (OR = 1.57, 95% CI: 1.14-2.17), and parity (OR = 1.45, 95% CI: 1.03-2.05)., Conclusion: The study found lower Social Economic Status (SES), long distance with primary health institutions, and the increased need for services stemming from multi-parity reduced the likelihood of nonuse or deficient use of PFVs in rural areas. Multiparous, low SES women and those living far away from primary health institutions should be paid more attention to assure the coverage of postpartum care.

Introduction: Diabetes affects both the peripheral and central nervous systems. The aim of this study was to explore the changes in brain activity in response to thermal stimuli in diabetic patients with and without diabetic peripheral neuropathy (DPN) using functional magnetic resonance imaging (fMRI)., Methods: A total of 36 right-handed volunteers were enrolled: eight patients with Type-2 diabetes mellitus and DPN, 13 patients with Type-2 diabetes mellitus lacking DPN (NDPN patients), and 15 healthy volunteers (HV). Blood oxygenation level-dependent baseline scans were performed, first without any stimuli, and then with four sessions of thermal stimuli (0, 10, 34, and 44°C, in a random order) applied to the lateral side of the right lower extremity. There was a 240-s rest interval between each thermal stimulation. Each stimulation session consisted of three cycles of 30 s of stimulation followed by 30 s of rest. After each stimuli session, the participant rated pain and itch perception on a visual analog scale. The fMRI data series were analyzed by using Statistical Parametric Mapping 8 and Data Processing Assistant for Resting-State fMRI., Results: In response to temperature stimuli, DPN patients showed stronger activation than HV and NDPN patients, not only in brain areas that participate in somatosensory pathways (right insula, left caudate nucleus, frontal gyrus, and cingulate cortex), but also in the cognition-related cerebral areas (right temporal lobe, left hippocampus, and left fusiform gyrus). Activation of vermis 1-3 was greater in NDPN patients than in HV in response to 0°C stimulation., Conclusions: fMRI may be useful for the early detection of central nervous system impairment caused by DPN. Our results indicate that central nervous system impairment related to diabetic neuropathy may not be limited to motion- and sensation-related cortical regions. Cognition-associated cerebral regions such as the hippocampus and fusiform gyrus are also affected by functional changes caused by DPN. This suggests that fMRI can detect the early stages of cognitive impairment in DPN patients before the symptoms become clinically significant.

Di-(2-ethylhexyl) phthalate (DEHP), a commonly used plasticizer, has an adjuvant effect in combination with ovalbumin (OVA). The adjuvant effect of DEHP has already been verified in our previous studies. In this study, to further investigate whether thymic stromal lymphopoietin (TSLP) was involved in the DEHP-adjuvant effect, DEHP was administered through a daily gavage exposure route. Mice were sensitized with ovalbumin (OVA) to trigger allergic responses, and an anti-TSLP monoclonal antibody was used to neutralize the effect of TSLP. Biomarkers including cytokines in bronchoalveolar lavage fluid (BALF), serum total IgE and TSLP content in the lung were detected. In addition, airway hyperreactivity and lung sections were examined. Collectively, these data indicated a salient Th2 response which was characterized by the upregulation of Th2-type cytokines, such as interleukin 4 (IL-4), IL-5 and IL-13. Moreover, the eosinophil number in BALF and the eosinophil cationic protein (ECP) in the lung were seen to have increased significantly. However, neutralization of TSLP with an anti-TSLP mAb reversed the adjuvant effect of DEHP on airway inflammation, structural alterations in the airway wall and increased airway hyperresponsiveness (AHR) to methacholine induced by the OVA allergen, suggesting that TSLP was an effective target site for suppressing the adjuvant effect of DEHP co-exposure.

Schistosoma japonicum insulin receptors (SjIRs) have been identified as encouraging vaccine candidates. Interrupting or blocking the binding between host insulin and the schistosome insulin receptors (IRs) may result in reduced glucose uptake leading to starvation and stunting of worms with a reduction in egg output. To further understand how schistosomes are able to exploit host insulin for development and growth, and whether these parasites and their mammalian hosts compete for the same insulin source, we identified insulin binding sites on the SjIRs. Based on sequence analysis and the predicted antigenic structure of the primary sequences of the SjIRs, we designed nine and eleven peptide analogues from SjIR-1 and SjIR-2, respectively. Using the Octet RED system, we identified analogues derived from SjIR-1 (10) and SjIR-2 (20, 21 and 22) with insulin-binding sequences specific for S. japonicum. Nevertheless, the human insulin receptor (HIR) may compete with the SjIRs in binding human insulin in other positions which are important for HIR binding to insulin. However, no binding occurred between insulin and parasite analogues derived from SjIR-1 (2, 7 and 8) and SjIR-2 (14, 16 and 18) at the same locations as HIR sequences which have been shown to have strong insulin binding affinities. Importantly, we found two analogues (1 and 3), derived from SjIR-1, and two analogues (13 and 15) derived from SjIR-2, were responsible for the major insulin binding affinity in S. japonicum. These peptide analogues were shown to have more than 10 times (in KD value) stronger binding capacity for human insulin compared with peptides derived from the HIR in the same sequence positions. Paradoxically, analogues 1, 3, 13 and 15 do not appear to contain major antigenic determinants which resulted in poor antibody responses to native S. japonicum protein. This argues against their future development as peptide-vaccine candidates.

Background: The New Rural Cooperative Medical Scheme (NCMS) includes a maternal care benefits package that is associated with increasing maternal health services. The local compensation policies have been frequently adjusted in recent years. This study examined the association between the NCMS maternal-services policy adjustment and expense reimbursement in Yuyao, China., Methods: Two household surveys were conducted in Yuyao in 2008 and 2011 (before and after the NCMS policy adjustment, respectively). Local women (N = 154) who had delivery history in the past three years were recruited. A questionnaire was used to collect information about delivery history, maternal health services utilization (prenatal care, postnatal care, and the grade of delivery institutions), NCMS participation, and reimbursement status. Logistic regression analyses were used to predict the association between policy adjustment and maternal health utilization and the association between policy adjustment and out-of-pocket proportion. Next, t-tests and covariance analyses adjusting for household income were used to compare the out-of-pocket proportion between 2008 and 2011., Results: Results revealed that compensation policy adjustment was associated with an increase in postnatal visits (adjusted OR = 3.32, p = 0.009) and the use of second level or above institutions for delivery (adjusted OR = 2.32, p = 0.03) among participants. In 2008, only 9.1% of pregnant women received reimbursement from the NCMS; however, this rate increased to 36.8% in 2011. After policy adjustment, there were no significant changes in the proportion of out-of-pocket expenses shared in delivery fee (F = 0.24, p = 0.63) and in household income (F = 0.46, p = 0.50)., Conclusions: Financial compensation increase improved maternal health services utilization; however, this effect was limited. Although the reimbursement rate was raised, the out-of-pocket proportion was not significant changed; therefore, the compensation design scheme must be adjusted in practice.

The latent reservoirs of HIV represent a major impediment to eradication of HIV/AIDS. To overcome this problem, agents that can activate latent HIV proviruses have been actively sought after, as they can potentially be used in combination with the highly active antiretroviral therapy (HAART) to eliminate the latent reservoirs. Although several chemical compounds have been shown to activate latency, they are of limited use due to high toxicity and poor clinical outcomes. In an attempt to identify natural products as effective latency activators from traditional Chinese medicinal herbs that have long been widely used in human population, we have isolated procyanidin C-13,3',3"-tri-O-gallate (named as REJ-C1G3) from Polygonum cuspidatum Sieb. et Zucc., that can activate HIV in latently infected Jurkat T cells. REJ-C1G3 preferentially stimulates HIV transcription in a process that depends on the viral encoded Tat protein and acts synergistically with prostratin (an activator of the NF-κB pathway) or JQ1 (an inhibitor of Brd4) to activate HIV latency. Our mechanistic analyses further show that REJ-C1G3 accomplishes these tasks by inducing the release of P-TEFb, a host cofactor essential for Tat-activation of HIV transcription, from the cellular P-TEFb reservoir 7SK snRNP.

Our recent publication showed that a small bioactive pigment epithelium derived factor (PEDF) peptide (P78-PEDF) prevents the development of diabetic nephropathy (DN). However, its effects on the progression of established DN were not clear. Therefore, the purpose of this study was to determine the effect of P78-PEDF in the progression of DN and to compare the effects of P78-PEDF and an ACE inhibitor (ACEi), a standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with P78-PEDF or captopril starting at 6 wks of age for 12 wks (early treatment) or starting at 12 wks of age for 6 wks (late treatment). We first established the optimal dose of the P78-PEDF peptide to ameliorate DN in Ins2(Akita) mouse for a 6 wk study period and found that the peptide was effective at 0.1- 0.5 µg/g/day. We next showed that early or late treatment with P78-PEDF resulted in protection from DN as indicated by reduced albuminuria, kidney macrophage recruitment, histological changes, inflammatory cytokines and fibrotic markers (kidney TNF-α, fibronectin, VEGFA and EGFR), and restored nephrin expression compared with vehicle-treated Ins2(Akita) mice. Interestingly, only early but not late treatment with captopril was as effective as P78-PEDF in reducing most DN complications, despite its lack of effect on nephrin, VEGFA and EGFR expression. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in both the development and progression of diabetic renal injury.

Background: c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance., Methods: We utilized the human MHCC97-H c-Met positive (c-Met+) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses., Results: We have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone., Conclusion: c-Met inhibition monotherapy is not sufficient to eliminate c-Met+ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met+ HCC.

Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

The study investigated the effect of oxytetracycline (OTC) on the anti-oxidative defense system, the structure (hemolysis rate and morphology) and function (ATP enzyme activity) of human red blood cells (hRBCs) to investigate the possible toxic mechanism of OTC to hRBCs. The experimental results indicate that OTC can cause a decline in the function of the antioxidant defense system of hRBCs, resulting in oxidative stress. OTC can bring about morphological changes to hRBCs, and further leads to hemolysis, when the concentration of OTC is over 8×10(-5) M (about 164 µg/ml). At a low OTC concentration, below 4×10(-5) M (82 µg/ml), OTC can enhance the activity of ATP enzyme of hRBCs, known as hormesis. However, at a high concentration, above 4×10(-5) M (about 82 µg/ml), the ATP enzymatic activity was inhibited, affecting the function of hRBCs. The estalished mechanism of toxicity of OTC to hRBCs can facilitate a deeper understanding of the toxicity of OTC in vivo.

Background: Tuberculosis is a serious global health problem. Its paradigms are shifting through time, especially in rapidly developing countries such as China. Health providers in China are at the forefront of the battle against tuberculosis; however, there are few empirical studies on health providers' perspectives on the challenges they face in tuberculosis control at the county level in China. This study was conducted among health providers to explore their experiences with tuberculosis control in order to identify bottlenecks and emerging challenges in controlling tuberculosis in rural China., Methods: A qualitative approach was used. Semi-structured, in-depth interviews were conducted with 17 health providers working in various positions within the health system of one rural county (ZJG) of China. Data were analyzed based on thematic content analysis using MAXQDA 10 qualitative data analysis software., Results: Health providers reported several problems in tuberculosis control in ZJG county. Migrant workers and the elderly were repeatedly documented as the main obstacles in effective tuberculosis control in the county. At a personal level, doctors showed their frustration with the lack of new drugs for treating tuberculosis patients, and their opinions varied regarding incentives for referring patients., Conclusion: The results suggest that several problems still remain for controlling tuberculosis in rural China. Tuberculosis control efforts need to make reaching the most vulnerable populations a priority and encourage local health providers to adopt innovative practices in the local context based on national guidelines to achieve the best results. Considerable changes in China's National Tuberculosis Control Program are needed to tackle these emerging challenges faced by health workers at the county level.

Background: The newly diagnosed rate of HCV infection is increasing in China. However, the risk factors have not been fully identified. Here, a survey was performed in Yanbian Prefecture, a high-endemic area in China., Methods: We identified newly diagnosed HCV infection in 2007-2011, using the local National Disease Supervision Information Management System from the Chinese Center for Disease Control and Prevention. We determined the risk factors using a case-control survey by questionnaire., Results: Yanbian Prefecture had a rapid increase in the yearly newly diagnosed rate of HCV infection from 32.6 to 72.1/100.000 from the year 2007 to 2011. People aged 50-64 years had a high HCV infection of 43.4%, but only 0.3% of cases were reported in those aged less than 20 years. Cosmetic treatment, family history, blood transfusion, and dental treatment were independent risk factors for HCV infection. Unexpectedly, cosmetic treatments [odd ratio (OR) = 5.15, 95% confidence interval (CI) = 2.31-11.48, P = 0.00] and family history (OR = 4.68, 95% CI = 2.67-8.75, P = 0.00) showed a higher risk than the conventional risk factors of blood transfusion (OR = 4.49, 95% CI = 1.95-10.37, P = 0.001) and dental treatment (OR = 2.98, 95% CI = 1.42-6.25, P = 0.00). To further analyze the intrafamilial transmission, we found that spouses of HCV patients had an increased risk for acquiring HCV (OR = 5.75, 95% CI: 1.94-17.07), without significant association between either HCV RNA viral load (P = 0.29) or genotype (P = 0.43)., Conclusions: HCV infection was increased in Yanbian Prefecture. Cosmetic treatment was a higher risk factor than medical procedure. HCV infection had a clear family clustering phenomenon, especially between spouses.

Nijmegen breakage syndrome (NBS) with NBS1 germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The NBS1 gene codes for a protein, Nbs1(p95/Nibrin), involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous NBS1 mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC). Eight missense NBS1 mutations were identified in six of 64 (9.4%) HCCs and two of 18 (11.1%) ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified NBS1 mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with NBS1 mutations. Moreover, seven of the eight tumors with NBS1 mutations had at least one genetic alteration in the TP53 pathway, including TP53 mutation, MDM2 amplification, p14ARF homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of NBS1, a DNA repair associated gene.

Background: To examine the characteristics of oxidative stress in patients with acute kidney injury (AKI) and investigate the association between plasma nitrotyrosine levels and 90-day mortality in patients with AKI., Methodology/principal Findings: 158 patients with hospital-acquired AKI were recruited to this prospective cohort study according to RIFLE (Risk, Injury, Failure, Lost or End Stage Kidney) criteria. Twelve critically ill patients without AKI and 15 age and gender-matched healthy subjects served as control. Plasma 3-nitrotyrosine was analyzed in relation to 90-day all cause mortality of patients with AKI. The patients with AKI were followed up for 90 days and grouped according to median plasma 3-nitrotyrosine concentrations. Highest 3-NT/Tyr was detected in patients with AKI compared with healthy subjects, and critically ill patients without AKI (ANOVA p<0.001). The 90-day survival curves of patients with high 3-NT/Tyr showed significant differences compared with the curves of individuals with low 3-NT/Tyr (p = 0.001 by log rank test). Multivariate analysis (Cox regression) revealed that 3-NT/Tyr (p = 0.025) was independently associated with mortality after adjustment for age, gender, sepsis and Acute Physiology and Chronic Health Evaluation (APACHE) II score., Conclusions/significance: There is excess plasma protein oxidation in patients with AKI, as evidenced by increased nitrotyrosine content. 3-NT/Tyr level was associated with mortality of AKI patients independent of the severity of illness.

Although cellular signaling pathways that affect lentivirus infection have been investigated, the role of cell-cell interactions in lentiviral gene delivery remains elusive. In the course of our studies we observed that lentiviral gene transfer was a strong function of the position of epithelial cells within colonies. While peripheral cells were transduced efficiently, cells in the center of colonies were resistant to gene transfer. In addition, gene delivery was enhanced significantly under culture conditions that disrupted adherens junctions (AJ) but decreased upon AJ formation. In agreement, gene knockdown and gain-of-function approaches showed that α-catenin, a key component of the AJ complex prevented lentivirus gene transfer. Using a doxycycline regulatable system we showed that expression of dominant negative E-cadherin enhanced gene transfer in a dose-dependent manner. In addition, dissolution of AJ by doxycycline increased entry of lentiviral particles into the cell cytoplasm in a dose-dependent manner. Taken together our results demonstrate that AJ formation renders cells non-permissive to lentiviral gene transfer and may facilitate development of simple means to enhance gene delivery or combat virus infection.

Introduction: Regional citrate anticoagulation (RCA) is gaining popularity in continous renal replacement therapy (CRRT) for critically ill patients. The risk of citrate toxicity is a primary concern during the prolonged process. The aim of this study was to assess the pharmacokinetics of citrate in critically ill patients with AKI, and used the kinetic parameters to predict the risk of citrate accumulation in this population group undergoing continuous veno-venous hemofiltration (CVVH) with RCA., Methods: Critically ill patients with AKI (n = 12) and healthy volunteers (n = 12) were investigated during infusing comparative dosage of citrate. Serial blood samples were taken before, during 120 min and up to 120 min after infusion. Citrate pharmacokinetics were calculated and compared between groups. Then the estimated kinetic parameters were applied to the citrate kinetic equation for validation in other ten patients' CVVH sessions with citrate anticoagulation., Results: Total body clearance of citrate was similar in critically ill patients with AKI and healthy volunteers (648.04±347.00 L/min versus 686.64±353.60 L/min; P = 0.624). Basal and peak citrate concentrations were similar in both groups (p = 0.423 and 0.247, respectively). The predicted citrate curve showed excellent fit to the measurements., Conclusions: Citrate clearance is not impaired in critically ill patients with AKI in the absence of severe liver dysfunction. Citrate pharmacokinetic data can provide a basis for the clinical use of predicting the risk of citrate accumulation., Trial Registration: ClinicalTrials.gov Identifier NCT00948558.

Background: Low circulating vitamin D levels have been suggested to potentially contribute to acute complications in critically ill patients. However, in patients with acute kidney injury (AKI), whether vitamin D deficiency occurs and is a potential contributor to worse early outcomes at the time of AKI diagnosis remains unclear., Methodology/principal Findings: Two hundred patients with AKI were enrolled in our study. Healthy subjects and critically ill patients without AKI served as controls. Serum vitamin D concentrations were measured in the three groups. The patients with AKI were followed up for 90 days and grouped according to median serum vitamin D concentrations. In addition, vitamin D receptor polymorphisms (BsmI and FokI) were measured in these patients; they were also followed up for 90 days and grouped according to vitamin D receptor gene mutations. Low serum 1,25-dihydroxyvitamin D levels (59.56±53.00 pmol/L) were detected in patients with AKI and decreased with increasing severity of AKI. There were no significant findings with respect to 25-hydroxyvitamin D. The 90-day survival curves of individuals with high vitamin D concentrations showed no significant differences compared with the curves of individuals with low concentrations. The survival curves of patients with BB/Bb or FF/Ff genotypes also showed no significant differences compared with patients with bb or ff genotypes. In Cox regression analysis, the vitamin D status in patients with AKI was not an independent prognostic factor as adjusted by age, sex, Sequential Organ Failure Assessment score, or vitamin D receptor polymorphisms., Conclusions/significance: Patients with AKI manifested a marked decrease in the 1,25-dihydroxyvitamin D level at the time of AKI diagnosis, and the degree of 1,25-dihydroxyvitamin D deficiency increased with the severity of AKI. No association between the serum vitamin D level at the time of AKI diagnosis and 90-day all-cause mortality was found in patients with AKI.

Objective: Asthma is a complex pulmonary inflammatory disease characterized by the hyper-responsiveness, remodeling and inflammation of airways. Formaldehyde is a common indoor air pollutant that can cause asthma in people experiencing long-term exposure. The irritant effect and adjuvant effect are the two possible pathways of formaldehyde promoted asthma., Methodology/principal Findings: To explore the neural mechanisms and adjuvant effect of formaldehyde, 48 Balb/c mice in six experimental groups were exposed to (a) vehicle control; (b) ovalbumin; (c) formaldehyde (3.0 mg/m(3)); (d) ovalbumin+formaldehyde (3.0 mg/m(3)); (e) ovalbumin+formaldehyde (3.0 mg/m(3))+HC-030031 (transient receptor potential ankyrin 1 antagonist); (f) ovalbumin+formaldehyde (3.0 mg/m(3))+ capsazepine (transient receptor potential vanilloid 1 antagonist). Experiments were conducted after 4 weeks of combined exposure and 1-week challenge with aerosolized ovalbumin. Airway hyper-responsiveness, pulmonary tissue damage, eosinophil infiltration, and increased levels of interleukin-4, interleukin-6, interleukin-1β, immunoglobulin E, substance P and calcitonin gene-related peptide in lung tissues were found in the ovalbumin+formaldehyde (3.0 mg/m(3)) group compared with the values seen in ovalbumin -only immunized mice. Except for interleukin-1β levels, other changes in the levels of biomarker could be inhibited by HC-030031 and capsazepine., Conclusions/significance: Formaldehyde might be a key risk factor for the rise in asthma cases. Transient receptor potential ion channels and neuropeptides have important roles in formaldehyde promoted-asthma.

Aims: We aimed to replicate the association of the rs290487 (IVS3C/T) and rs7903146 (IVS3C/T) polymorphisms of transcription factor 7-like 2 (TCF7L2) and type 2 diabetes mellitus (T2DM) in Han Chinese people in Henan province, China., Methods: In all, 1,842 patients with T2DM and 7,777 normal glucose-tolerant controls underwent genotyping for the T2DM-associated variants rs7903146 (IVS3C/T) and rs290487 (IVS3C/T). W performed a meta-analysis of the association of the risk alleles of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) in TCF7L2 and T2DM in Han Chinese by combining previous studies with the present study., Results: We found that T2DM was associated with the CC genotype (1.364, 1.137-1.636, p  = 0.001), the recessive model (1.457, 1.156-1.838, p  = 0.001) of rs290487 (IVS3C/T) and haplotype CC (1.116, 1.034-1.204, p  = 0.004) in Han Chinese. Moreover, our meta-analyses supported the association of the T allele (IVS3C/T) of rs7903146 (1.36, 1.24-1.48; p  = 6.404×10(-12)) and T2DM but not the C allele of rs290487 (IVS3C/T) (0.99, 0.85-1.15, p  = 0.890) in Han Chinese. We found no interactions between behavioral risk factors (smoking, alcohol drinking, and physical activity) and rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) polymorphisms., Conclusions: The CC genotype and the recessive model of the variant rs290487 (IVS3C/T) and CC haplotype of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) in TCF7L2 may be associated with T2DM in Han Chinese people in Henan province, China.

Background: Postpartum visits (PPVs) have been advocated as a way to improve health outcomes for mothers and their infants, but the rate of PPVs is still low in rural China. This study aims to investigate the utilization of PPVs and to explore the factors associated with PPVs in rural China. Parity is the most concerned factor in this study., Methods: A cross-sectional household survey was performed in two counties of Zhejiang province. Questions include socio-economic, health services and women's delivery data. Chi-square tests and multivariate logistic regression analyses were performed to identify factors associated with PPVs., Results: 223 women who had a delivery history in the recent five years were enrolled in analyses. 173 (78%) of them were primiparous. Among the primiparous women, 43 (25%) had not received any PPVs. The majority, 27 (55%) of the 49 multiparous women, had not received any PPVs. Multiparous women were less likely to receive PPVs than primiparous women. Among 223 puerperal women, 47 (21%) had been compensated for delivery fee expenses. Women who received compensation were found to be more likely to receive standard (at least 3) PPVs., Conclusions: It was found that women with "second babies" were less likely to use PPVs. This could be an unintended consequence of the "one-child policy", due to fear that contact with public health facilities could result in sanctions. This phenomenon should be taken seriously by government in order to improve the health of babies and their mothers. Financial compensation for delivery fee charges can improve the use of PPVs, thus free-of-charge delivery should be promoted.

Background: Asthma is a complex pulmonary inflammatory disease, which is characterized by airway hyperresponsiveness, variable airflow obstruction and inflammation in the airways. The majority of asthma is allergic asthma, which is a disease caused by type I hypersensitivity mediated by IgE. Exposures to a number of environmental chemicals are suspected to lead to asthma, one such pollutant is di-(2-ethylheyl) phthalate (DEHP). DEHP is a manufactured chemical that is commonly added in plastic products to make them flexible. Epidemiological studies have revealed a positive association between DEHP exposure and asthma prevalence., Methodology/principal Findings: The present study was aimed to determine the underlying role of DEHP exposure in airway reactivity, especially when combined with allergen exposure. The biomarkers include pulmonary histopathology, airway hyperresponsiveness (lung function), IgE, IL-4, IFN-γ and eosinophils. Healthy balb/c mice were randomly divided into eight exposure groups (n = 8 each): (1) saline control, (2) 30 µg/(kg•d) DEHP, (3) 300 µg/(kg•d) DEHP, (4) 3000 µg/(kg•d) DEHP, and (5) ovalbumin (OVA)-sensitized group, (6) OVA-combined with 30 µg/(kg•d) DEHP, (7) OVA-combined with 300 µg/(kg•d) DEHP, and (8) OVA-combined with 3000 µg/(kg•d) DEHP. Experimental tests were conducted after 52-day DEHP exposure and subsequently one week of challenge with aerosolized OVA. The principal findings include: (1) Strong postive associations exist between OVA-combined DEHP exposure and serum total IgE (T-IgE), as well as histological findings. These positive associations show a dose-dependent low dose sensitive effect of DEHP. (2) IL-4, eosinophil recruitment and lung function are also indicators for adjuvant effect of DEHP., Conclusions/significance: Our results suggest that except the significant changes of immunological and inflammatory biomarkers (T-IgE, IL-4, IFN-γ and eosinophils), the pulmonary histological (histopathological examination) and physiological (lung function) data also support that DEHP may promote and aggravate allergic asthma by adjuvant effect.

Background: Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed., Methodology/principal Findings: A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC(50)=106 ng/mL), the direct hapten coated format (IC(50)=14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples., Conclusions/significance: The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples., (© 2011 Wei et al.)

Background: Schistosomes depend for growth and development on host hormonal signals, which may include the insulin signalling pathway. We cloned and assessed the function of two insulin receptors from Schistosoma japonicum in order to shed light on their role in schistosome biology., Methodology/principal Findings: We isolated, from S. japonicum, insulin receptors 1 (SjIR-1) and 2 (SjIR-2) sharing close sequence identity to their S. mansoni homologues (SmIR-1 and SmIR-2). SjIR-1 is located on the tegument basal membrane and the internal epithelium of adult worms, whereas SjIR-2 is located in the parenchyma of males and the vitelline tissue of females. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are close to Echinococcus multilocularis insulin receptor (EmIR), suggesting that SjIR-2, SmIR-2 and EmIR share similar roles in growth and development in the three taxa. Structure homology modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells., Conclusions: Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. A complete understanding of the role of SjIRs in the biology of S. japonicum may result in their use as new targets for drug and vaccine development against schistosomiasis.

Most of the individuals infected with SARS coronavirus (SARS-CoV) spontaneously recovered without clinical intervention. However, the immunological correlates associated with patients' recovery are currently unknown. In this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in SARS-CoV-specific antibody responses as well as cytotoxic T cell (CTL) responses. We have found persistence of robust antibody and CTL responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. We have also identified two potential major CTL epitopes in N proteins based on ELISPOT analysis of pooled peptides. However, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. In summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and CTL responses in the natural history of SARS-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. Our findings have direct implications for future design and development of effective therapeutic agents and vaccines against SARS-CoV infection.