The combination of Butyricicoccus pullicaecorum and 3-hydroxyanthranilic acid prevents postmenopausal osteoporosis by modulating gut microbiota and Th17/Treg.

BACKGROUND: Postmenopausal osteoporosis (PMO) is a chronic condition characterized by decreased bone strength. This study aims to investigate the effects and mechanisms of the combination of Butyricicoccus pullicaecorum (Bp) and 3-hydroxyanthranilic acid (3-HAA) on PMO. METHODS: The effects of Bp and 3-HAA on PMO were evaluated in ovariectomized (OVX) rats by assessing stereological parameters, femur microstructure, and autophagy levels. The T helper (Th) 17/Regulatory T (Treg) cells of rats were detected using flow cytometric analysis. Furthermore, the impact of Bp and 3-HAA on the gut microbiota of rats was assessed using 16S rRNA gene sequencing. The correlation between the gut microbiota of rats and Th17/Treg immune factors, as well as femoral stereo parameters, was separately assessed using Spearman rank correlation analysis. RESULTS: Bp and 3-HAA treatments protected OVX rats by promoting osteogenesis and inhibiting autophagy. Compared to the Sham group, OVX rats showed an increase in Th17 cells and a decrease in Treg cells. Bp and 3-HAA reversed these changes. Enterorhabdus and Pseudomonas were significantly enriched in OVX rats. Bp and 3-HAA regulated the gut microbiota of OVX rats, enriching pathways related to nutrient metabolism and immune function. There was a correlation between the gut microbiota and the Th17/Treg, as well as femoral stereo parameters. The concurrent administration of Bp and 3-HAA medication facilitated the enrichment of gut microbiota associated with the improvement of PMO. CONCLUSION: The combination therapy of Bp and 3-HAA can prevent PMO by modulating the gut microbiota and restoring Th17/Treg immune homeostasis.

Education level as a predictor of survival in patients with multiple myeloma.

BACKGROUND: Disparities in multiple myeloma (MM) prognosis based on sociodemographic factors may exist. We investigated whether education level at diagnosis influenced Chinese MM patient outcomes. METHODS: We performed a multicenter retrospective analysis of data from 773 MM patients across 9 centers in China from 2006 to 2019. Sociodemographic and clinical factors at diagnosis and treatment regimens were recorded, and univariate and multivariate analyses were performed. RESULTS: Overall, 69.2% of patients had low education levels. Patients with low education levels differed from those with high education levels in that they were more likely to be older, and a higher proportion lived in rural areas, were unemployed, had lower annual incomes and lacked insurance. Additionally, compared to patients with high education levels, patients with low education levels had a higher proportion of international staging system (ISS) stage III classification and elevated lactate dehydrogenase (LDH) levels and underwent transplantation less often. Patients with high education levels had a median progression-free survival (PFS) of 67.50 (95% confidence interval (CI): 51.66-83.39) months, which was better than that of patients with low education levels (30.60 months, 95% CI: 27.38-33.82, p < 0.001). Similarly, patients with high education levels had a median overall survival (OS) of 122.27 (95% CI: 117.05-127.49) months, which was also better than that of patients with low education levels (58.83 months, 95% CI: 48.87-62.79, p < 0.001). In the multivariable analysis, patients with high education levels had lower relapse rates and higher survival rates than did those with low education level in terms of PFS and OS (hazard ratio (HR) = 0.50 [95% CI: 0.34-0.72], p < 0.001; HR = 0.32 [0.19-0.56], p < 0.001, respectively). CONCLUSIONS: Low education levels may independently predict poor survival in MM patients in China.

Proteomic and transcriptomic studies of HBV-associated liver fibrosis of an AAV-HBV-infected mouse model.

BACKGROUND: Human hepatitis B virus (HBV) infection is an important public health issue in the Asia-Pacific region and is associated with chronic hepatitis, liver fibrosis, cirrhosis and even liver cancer. However, the underlying mechanisms of HBV-associated liver fibrosis remain incompletely understood. RESULTS: In the present study, proteomic and transcriptomic approaches as well as biological network analyses were performed to investigate the differentially expressed molecular signature and key regulatory networks that were associated with HBV-mediated liver fibrosis. RNA sequencing and 2DE-MALDI-TOF/TOF were performed on liver tissue samples obtained from HBV-infected C57BL/6 mouse generated via AAV8-HBV virus. The results showed that 322 genes and 173 proteins were differentially expressed, and 28 HBV-specific proteins were identified by comprehensive proteomic and transcriptomic analysis. GO analysis indicated that the differentially expressed proteins were predominantly involved in oxidative stress, which plays a key role in HBV-related liver fibrosis. Importantly, CAT, PRDX1, GSTP1, NXN and BLVRB were shown to be associated with oxidative stress among the differentially expressed proteins. The most striking results were validated by Western blot and RT-qPCR. The RIG-I like receptor signaling pathway was found to be the major signal pathway that changed during HBV-related fibrosis. CONCLUSIONS: This study provides novel insights into HBV-associated liver fibrosis and reveals the significant role of oxidative stress in liver fibrosis. Furthermore, CAT, BLVRB, NXN, PRDX1, and IDH1 may be candidates for detection of liver fibrosis or therapeutic targets for the treatment of liver fibrosis.

Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer.

A major hindrance in gene therapy trials with adeno-associated virus (AAV) vectors is the presence of neutralizing antibodies (NAbs) that inhibit AAV transduction. In this study, we used directed evolution techniques in vitro and in mouse muscle to select novel NAb escape AAV chimeric capsid mutants in the presence of individual patient serum. AAV mutants isolated in vitro escaped broad patient-specific NAb activity but had poor transduction ability in vivo. AAV mutants isolated in vivo had enhanced NAb evasion from cognate serum and had high muscle transduction ability. More importantly, structural modeling identified a 100 amino acid motif from AAV6 in variable region (VR) III that confers this enhanced muscle tropism. In addition, a predominantly AAV8 capsid beta barrel template with a specific preference for AAV1/AAV9 in VR VII located at threefold symmetry axis facilitates NAb escape. Our data strongly support that chimeric AAV capsids composed of modular and nonoverlapping domains from various serotypes are capable of evading patient-specific NAbs and have enhanced muscle transduction.

The genomic and seroprevalence of human bocavirus in healthy Chinese plasma donors and plasma derivatives.

BACKGROUND: Human bocavirus (HBoV) is a novel parvovirus identified in 2005. It has mostly been detected in respiratory and enteric infections and has not been studied large scale in blood products in relation to transfusion. STUDY DESIGN AND METHODS: An in-house quantitative polymerase chain reaction (Q-PCR) was developed to test HBoV DNA in plasma and plasma derivatives. Plasma samples (n = 6096) collected from healthy donors, 241 plasma pools, and 326 plasma derivatives were screened for HBoV DNA by Q-PCR. Positive samples were confirmed by nested PCR and further amplified for sequence analysis and phylogenetic studies. The prevalence of immunoglobulin (Ig)G and IgM specific to HBoV structural proteins was measured by enzyme-linked immunosorbent assay in 209 samples grouped according to virus load (Group 1, HBoV DNA >10(4) copies/mL; Group 2, HBoV DNA >5 × 10(2) copies/mL but below 10(4) copies/mL; Group 3,HBoV DNA negative). RESULTS: The genomic prevalence of HBoV in the plasma donors was 9.06%, ranging from 5.01 × 10(2) to 3.02 × 10(6) copies/mL. HBoV-specific IgG and IgM were detected at 20.00 and 7.50% in Group 1, at 20.29 and 2.90% in Group 2, and at 13.00 and 4.0% in Group 3, respectively. Phylogenetic analyses proved that HBoV Genotype 1 was the prevalent genotype in Chinese plasma donors. CONCLUSION: Low levels of HBoV DNA were detectable at high prevalence in Chinese plasma donors and plasma derivatives. Further study is needed to determine whether HBoV screening is necessary.

Identification of a prognostic signature based on five ferroptosis-related genes for diffuse large B-cell lymphoma.

BACKGROUND: Therapies for diffuse large B-cell lymphoma (DLBCL) are limited due to the diverse gene expression profiles and complicated immune microenvironments, making it an aggressive lymphoma. Beyond this, researches have shown that ferroptosis contributes to tumorigenesis, progression, and metastasis. We thus are interested to dissect the connection between ferroptosis and disease status of DLBCL. We aim at generating a valuable prognosis gene signature for predicting the status of patients of DLBCL, with focus on ferroptosis-related genes (FRGs). OBJECTIVE: To examine the connection between ferroptosis-related genes (FRGs) and clinical outcomes in DLBCL patients based on public datasets. METHODS: An expression profile dataset for DLBCL was downloaded from GSE32918 (https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=gse32918), and a ferroptosis-related gene cluster was obtained from the FerrDb database (http://www. zhounan.org/ferrdb/). A prognostic signature was developed from this gene cluster by applying a least absolute shrinkage and selection operator (LASSO) Cox regression analysis to GSE32918, followed by external validation. Its effectiveness as a biomarker and the prognostic value was determined by a receiver operator characteristic curve mono factor analysis. Finally, functional enrichment was evaluated by the package Cluster Profiler of R. RESULTS: Five ferroptosis-related genes (FRGs) (GOP1, GPX2, SLC7A5, ATF4, and CXCL2) associated with DLBCL were obtained by a multivariate analysis. The prognostic power of these five FRGs was verified by TCGA (https://xenabrowser.net/datapages/?dataset=TCGA.DLBC.sampleMap%2FHiSeqV2_PANCAN&host=https%3A%2F%2Ftcga.xenahubs.net&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A44) and GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse 32918) datasets, with ROC analyses. KEGG and GO analyses revealed that upregulated genes in the high-risk group based on the gene signature were enriched in receptor interactions and other cancer-related pathways, including pathways related to abnormal metabolism and cell differentiation. CONCLUSION: The newly developed signature involving GOP1, GPX2, SLC7A5, ATF4, and CXCL2 has the potential to serve as a prognostic biomarker. Furthermore, our results provide additional support for the contribution of ferroptosis to DLBCL.

Incidence and survival of second primary non-Hodgkin lymphoma: A Surveillance, Epidemiology, and End Results-based cohort study.

BACKGROUND: The aim of this study was to investigate patient survival and factors associated with survival in second primary non-Hodgkin lymphoma (NHL) compared with the first primary NHL. METHODS: The retrospective cohort study used data from the Surveillance, Epidemiology, and End Results (SEER) database between 2000 and 2014. Demographic characteristics, histological types, Ann Arbor stage, and treatment information were collected. Cox proportional hazard models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for factors associated with overall survival (OS) and cancer-specific survival (CSS) in the first and second primary NHLs. RESULTS: Of 318,168 cases followed for 5 years, 299,248 patients developed the first primary NHL and 18,920 patients developed the second primary NHL. This study identified a rising incidence of first and second primary NHL from 2000 to 2014. For the second primary NHL, the OS risk was higher when compared to the first primary NHL (HR: 1.13, 95% CI: 1.11 to 1.15, P <0.001). Risk factors that negatively affected OS in the first primary NHL included being male, over 40 years of age, certain marital statuses, specific histological types, and advanced disease stages. In contrast, being of White race and having histological types such as Follicular Lymphoma (FL), Marginal Zone Lymphoma (MZL), and mantle B-cell NHL were associated with better OS outcomes. Treatments like surgery, radiation therapy, and chemotherapy were associated with a lower risk of OS and CSS in the first primary NHL. For the second primary NHL, the detrimental risk factors were similar but also included being over the age of 60. Certain histological types showed a lower OS risk relative to diffuse Large B-cell Lymphoma (DLBCL). While surgery and chemotherapy were beneficial for OS, radiation therapy did not improve survival in second primary NHL cases. Notably, undergoing chemotherapy for the first primary cancer increased the OS risk in the second primary NHL, whereas surgery and radiation seemed to offer a protective effect against OS risk in the second primary NHL (all P <0.05). CONCLUSION: Our findings emphasize the need for tailored strategies in managing the second primary NHL, given the distinct survival patterns and risk factor profiles compared to the first primary NHL. Future research should aim to further elucidate these differences to improve prognosis and treatment approaches for second primary NHL patients.

Study on the treatment of postmenopausal osteoporosis with quercetin in Liuwei Dihuang Pill based on network pharmacology.

BACKGROUND: Liuwei Dihuang Pill (LP) was verified to alleviate postmenopausal osteoporosis (PMOP) development. Nevertheless, the major constituent of LP and the related network pharmacology study remain unexplored. METHODS: Protein-protein interaction was established to identify the downstream target of LP in PMOP, and the related signaling pathway was investigated by bioinformatics analysis. MC3T3-E1 cells were added to ferric ammonium citrate (FAC) to mimic osteoporosis in vitro. The osteoblasts were identified by Alizarin red staining. Western blot was applied to evaluate protein levels. In addition, Cell Counting Kit-8 (CCK8) assay was applied to assess cell viability, and cell apoptosis was assessed by flow cytometry. RESULTS: Quercetin was the major constituent of LP. In addition, quercetin significantly reversed FAC-induced inhibition of osteogenic differentiation in MC3T3-E1 cells. In addition, quercetin notably abolished the FAC-induced upregulation of Bax, Caspase-3, FOS, JUN, TGFB1 and PPARD. In contrast, Bcl-2, p-mTOR/mTOR, p-AKT/AKT and p-PI3K/PI3K levels in MC3T3-E1 cells were reduced by FAC, which was restored by quercetin. Meanwhile, FAC notably inhibited the viability of MC3T3-E1 cells via inducing apoptosis, but this impact was abolished by quercetin. Furthermore, quercetin could reverse pcDNA3.1-FOS-mediated growth of FAC-treated osteoblasts by mediating PI3K/AKT/mTOR signaling. CONCLUSION: Quercetin alleviated the progression of PMOP via activation of PI3K/AKT/mTOR signaling. Hence, this study would shed novel insights into discovering new methods against PMOP.

Parvovirus B19V DNA contamination in Chinese plasma and plasma derivatives.

BACKGROUND: To ensure the safety of plasma derivatives, screening for human parvovirus B19V genomic DNA in donated plasma using a pooling strategy is performed in some countries. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Chinese plasma pools, plasma derivatives and plasma donations to evaluate the risk posed by B19V. METHODS: Using a Q-PCR assay developed in-house, we tested for B19V genomic DNA in 142 plasma pools collected between January 2009 and June 2011 from two Chinese blood products manufacturers. Plasma derivatives collected between 1993-1995 (10 batches of albumin, 155 batches of intravenous immunoglobulin, IVIG) and 2009-2011 (50 batches of albumin, 54 batches of IVIG, 35 batches of factor VIII, 7 batches of fibrinogen, and 17 batches of prothrombin complex concentrate, PCC) were also tested for B19V contamination. In addition, B19V genome prevalence in minipools(including 90 individual donations) of 49680 individual plasma samples collected between August 2011 and March 2012 by a single Chinese manufacturer was investigated. IgM/IgG was also investigated in plasma pools/derivatives and in minipools with B19V-DNA titers above 1x10(4) and 1x10(6) geq/mL using B19 ELISA IgM/IgG assay(Virion-Serion, Würzburg, Germany), respectively. RESULTS: B19V-DNA was detected in 54.2% of plasma pools from two Chinese blood product manufacturers; among recently produced blood products, B19V was detected in 21/54 IVIG samples, 19/35 factor VIII samples, 6/7 fibrinogen samples, and 12/17 PCC samples, but not in albumin samples. The levels of B19V-DNA in these samples varied from 10(2)-10(7) geq/mL. In samples with >10(4) geq/mL genome DNA, B19V-specific IgG was also found in all corresponding plasma pools and IVIG, whereas none was detected in the majority of other plasma derivatives. Screening of plasma donations indicated that most minipools were contaminated with B19V-DNA (10(2)-10(8) geq/mL) and one donation had 1.09 × 10(10) geq/mL B19V genomic DNA along with a non-classical IgG/IgM profile. CONCLUSIONS: Despite the implementation of some inactivation/removal methods designed to prevent viral contamination, B19V DNA was detectable in Chinese plasma pools and plasma derivatives. Thus, the introduction of B19V screening and discard donation with high viramic concentration for Chinese plasma donors would be desirable.

Prevalence and prevalence trends of transfusion transmissible infections among blood donors at four Chinese regional blood centers between 2000 and 2010.

BACKGROUND: In China, high prevalence of HBV and HCV parallels with the growing epidemic of syphilis and HIV in the general population poses a great threat to blood safety. This study investigated the prevalence of serologic markers for transfusion transmissible infections (TTIs) among four Chinese blood centers. METHODS: We examined whole blood donations collected from January 2000 through December 2010 at four Chinese blood centers. Post-donation testing of TTIs (HIV, HBV, HCV and syphilis) were conducted using two different enzyme-linked immunosorbent assay kits for each seromarker. The prevalence of serologic markers for TTIs (%) was calculated and additional analysis was conducted to examine donor characteristics associated with positive TTIs serology. RESULTS: Of the 4,366,283 donations, 60% were from first-time donors and 40% were from repeated donors. The overall prevalence of HIV, HBsAg, HCV and syphilis was 0.08%, 0.86%, 0.51% and 0.47%, respectively. The prevalence profile of TTIs varied among different blood centers and appeared at relatively high levels. Overall, the prevalence of HBsAg and HCV demonstrated a decline trend among four blood centers, while the prevalence of HIV and syphilis displayed three different trends: constantly steady, continually increasing and declining among different centers. CONCLUSIONS: This study reflects the risk of TTIs has been greatly reduced in China, but blood transfusion remains an ongoing risk factor for the spread of blood-borne infections, and further work and improvements are needed to strengthen both safety and availability of blood in China.

Prevalence, emotional and follow-up burden of insulin injection-related needle-stick injuries among clinical nurses in Shaanxi Province, west of China: A cross-sectional study.

AIMS AND OBJECTIVES: The aim of this study was to investigate the prevalence, emotional and follow-up burden of insulin injection-related needle-stick injuries among clinical nurses. BACKGROUND: needle-stick injures introduce statistically significant occupational hazards to healthcare workers. Although the large proportion of the needles injuries attributed to insulin injection, research evidence about the prevalence, emotional and follow-up burden of such injures is lacking. DESIGN: Cross-sectional study. METHODS: 5389 nurses were recruited from 45 hospitals in Shaanxi, China, from November 2018 to July 2019. Participants were administrated with a questionnaire specifically developed for this study. Descriptive statistics were used to present the findings. RESULTS: All 5,389 nurses responded to the survey, of which 396 (7.4%) participants experienced 620 insulin injection-related needle-stick injuries in the past year, representing an annual prevalence of 115.0 per 1000 nurses. The annual prevalence of infection caused by the injuries was 18.7 per 1000 nurses. The injuries occurred most frequently when nurses were recapping the needle (42.4%). In the majority (98.4%) of the injuries, the hurt nurses took proper immediate actions. However, only 30.3% of nurses reported the injuries to the administrative staff, and in 43.2% of the injuries, the nurses refused or discontinued the suggested follow-up. A large proportion (58.6%) of the hurt nurses experienced emotional changes. Multivariate logistic regression showed that department, removing and/or setting back needle caps with bare hands, frequency of insulin pen and syringes are associated with the incidence of insulin injection-related needle-stick injuries. This paper is reported following the STROBE recommendations. CONCLUSIONS: This survey demonstrated a considerably high prevalence of insulin injection-related needle-stick injuries among clinical nurses. Even though the majority of the hurt nurses took proper immediate actions, a large quantity of them failed to report the accidents to the administrative staff and complete the suggested follow-up. Nurses who suffered from insulin injection-related needle-stick injuries were subject to various negative emotional changes. It portends a statistically significant risk to occupational health management for nurses. RELEVANCE TO CLINICAL PRACTICE: Scientific preventive and management strategies are desirable in order to minimize the consequences of insulin injection-related needle-stick injuries.

The prevalence of human parvovirus B19 DNA and antibodies in blood donors from four Chinese blood centers.

BACKGROUND: Human parvovirus B19 is a common human pathogen that causes a variety of diseases with outcomes ranging from asymptomatic to severe, especially in immunocompromised patients. The B19 virus can be transmitted via blood and/or blood products and its resistance to common viral inactivation and/or removal methods raises the importance of B19-related blood safety. However, the existence, variation, and loading of B19 in Chinese blood donors have not been determined. STUDY DESIGN AND METHODS: Quantitative polymerase chain reaction (PCR) was developed to detect all three genotypes of the human erythrovirus DNA in plasma samples. In total, 3957 donations from four Chinese blood centers were screened for B19 by real-time minipool nucleic acid amplification technology (NAT). The positive samples were then confirmed by nested PCR and subjected to sequence analysis and alignment for phylogenetic studies. An enzyme-linked immunosorbent assay-based experiment was also performed to identify the prevalence of immunoglobulin (Ig)G and/or IgM antibodies specific to the B19 structural proteins in acquired samples. RESULTS: Of 3957 blood donors, 23 (0.58%) specimens were found positive for B19 DNA. The quantitative DNA levels ranged from 2.48 × 10(2) to 6.38 × 10(4) copies/mL. The phylogenic analyses showed that the prevalent genotypes in Chinese blood donors belong to B19 Genotype 1. A total of 448 samples from Chinese blood donors were investigated for the seroprevalence of B19 antibodies, among which 24.6 and 6.9% of specimens were seropositive for B19 IgG and IgM antibodies, respectively. A total of 2.5% of these samples were positive for both antibody isotypes. CONCLUSIONS: Whether B19 NAT screening of blood and blood products should be launched in China, larger studies are needed to facilitate an informed decision.

Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB).

DNA shuffling and directed evolution were employed to develop a novel adeno-associated virus (AAV) vector capable of crossing the seizure-compromised blood-brain barrier (BBB) and transducing cells in the brain. Capsid DNA from AAV serotypes 1-6, 8, and 9 were shuffled and recombined to create a library of chimeric AAVs. One day after kainic acid-induced limbic seizure activity in rats, the virus library was infused intravenously (i.v.), and 3 days later, neuron-rich cells were mechanically dissociated from seizure-sensitive brain sites, collected and viral DNA extracted. After three cycles of selection, green fluorescent protein (GFP)-packaged clones were administered directly into brain or i.v. 1 day after kainic acid-induced seizures. Several clones that were effective after intracranial administration did not transduce brain cells after the i.v. administration. However, two clones (32 and 83) transduced the cells after direct brain infusion and after i.v. administration transduced the cells that were localized to the piriform cortex and ventral hippocampus, areas exhibiting a seizure-compromised BBB. No transduction occurred in areas devoid of BBB compromise. Only one parental serotype (AAV8) exhibited a similar expression profile, but the biodistribution of 32 and 83 diverged dramatically from this parental serotype. Thus, novel AAV vectors have been created that can selectively cross the seizure-compromised BBB and transduce cells.

MYCN contributes to the sensitization of acute myelogenous leukemia cells to cisplatin by targeting SRY-box transcription factor 4.

Little was known about the role of MYCN in drug resistance in acute myeloid leukemia (AML). We investigated impacts of MYCN on sensitization of AML cell to cisplatin, and its interaction with SRY-box transcription factor 4 (SOX4). In vitro, human AML cell lines HL60 and KG-1 were transfected with the overexpression plasmids and specific small interfering RNA of MYCN (siMYCN), or siSOX4, and then treated with 2 µg/mL cisplatin. MTT and flow cytometry assays were performed to estimate the viability and apoptosis of AML cell. The expressions of MYCN and SOX4 in AML patients and the associations between these two were analyzed by bioinformatics analysis, luciferase assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR). In vivo, transfected HL60 cells were injected into BALB/c nude mice. The tumor size and weight were recorded, and the expressions of MYCN and SOX4 were determined with immunohistochemistry. MYCY and SOX4 were highly-expressed in AML, and their expressions were positively correlated. In AML cells, the treatment using cisplatin induced higher MYCN expression and augmented apoptosis, whereas decreasing the viability. Silencing of MYCN enhanced the tumor-suppressive effects of cisplatin on AML by decreasing cell viability and increasing apoptosis. The promoter of SOX4 was targeted by MYCN. Lower cell viability and higher apoptosis rate were seen in AML cells transfected with siSOX4, the effects of which were partially reversed by overexpressed MYCN. Silencing of MYCN retarded the tumor growth and SOX4 expression in tumor in vivo. Collectively, MYCN sensitized AML cells to cisplatin by targeting SOX4.

Generation of novel AAV variants by directed evolution for improved CFTR delivery to human ciliated airway epithelium.

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

Short hairpin RNA attenuates liver fibrosis by regulating the PPAR­Î³ and NF­κB pathways in HBV­induced liver fibrosis in mice.

Progressive liver fibrosis, caused by chronic viral infection and metabolic disorders, results in the development of cirrhosis and hepatocellular carcinoma. However, no antifibrotic therapies have been approved to date. In our previous study, adeno­associated virus (AAV) short hairpin RNAs (shRNAs) targeting hepatitis B virus (HBV) and transforming growth factor (TGF)­ß administration could persistently inhibit HBV replication and concomitantly prevent liver fibrosis. However, the differentially expressed proteins and critical regulatory networks of AAV­shRNA treatment remain unclear. Accordingly, in the present study, we aimed to analyze differentially expressed proteins in the liver of AAV­shRNA­treated mice with HBV infection and liver fibrosis using isobaric tags for relative and absolute quantitation (iTRAQ)­based quantitative proteomics and to elucidate the underlying antifibrotic mechanisms. In total 2,743 proteins were recognized by iTRAQ­based quantitative proteomics analysis. Gene Ontology analysis revealed that the differentially expressed proteins mostly participated in peptide metabolism in the biological process category, cytosolic ribosomes in the cell component category, and structural constituents of ribosomes in the molecular function category. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that oxidative stress and the peroxisome proliferator­activated receptor (PPAR) signaling pathway were activated after treatment. Verification studies revealed that AAV­shRNAs inhibited hepatic stellate cell activation and inflammation by suppressing nuclear factor­κB p65 phosphorylation and α­smooth muscle actin expression via upregulation of PPAR­Î³. Hepatocytes steatosis was also decreased by activating the PPAR signaling pathway and improving lipid metabolism. The expression level of TGF­ß was decreased due to upregulation of PPAR­Î³ expression and direct inhibition using AAV­shRNA targeting TGF­ß. TGF­ß­induced oxidative stress was suppressed by increasing glutathione S­transferase Pi 1 and reducing peroxiredoxin 1. Collectively, the present results indicated that AAV­shRNAs were effective in modulating liver fibrosis by reducing oxidative stress, inflammation and activating the PPAR signaling pathway.

The association between ambient temperature and clinical visits for inflammation-related diseases in rural areas in China.

BACKGROUND: The association between temperature and mortality has been widely reported. However, it remains largely unclear whether inflammation-related diseases, caused by excessive or inappropriate inflammatory reaction, may be affected by ambient temperature, particularly in low-income areas. OBJECTIVES: To explore the association between ambient temperature and clinical visits for inflammation-related diseases in rural villages in the Ningxia Hui Autonomous Region, China, during 2012─2015. METHODS: Daily data on inflammation-related diseases and weather conditions were collected from 258 villages in Haiyuan (161 villages) and Yanchi (97 villages) counties during 2012─2015. A Quasi-Poisson regression with distributed lag non-linear model was used to examine the association between temperature and clinical visits for inflammation-related diseases. Stratified analyses were performed by types of diseases including arthritis, gastroenteritis, and gynecological inflammations. RESULTS: During the study period, there were 724,788 and 288,965 clinical visits for inflammation-related diseases in Haiyuan and Yanchi, respectively. Both exposure to low (RR: 2.045, 95% CI: 1.690, 2.474) and high temperatures (RR: 1.244, 95% CI: 1.107, 1.399) were associated with increased risk of total inflammation-related visits in Haiyuan county. Low temperatures were associated with increased risks of all types of inflammation-related diseases in Yanchi county (RR: 4.344, 95% CI: 2.887, 6.535), while high temperatures only affected gastroenteritis (RR: 1.274, 95% CI: 1.040, 1.561). Moderate temperatures explained approximately 26% and 33% of clinical visits due to inflammation-related diseases in Haiyuan and Yanchi, respectively, with the burden attributable to cold exposure higher than hot exposure. The reference temperature values ranged from 17 to 19 in Haiyuan, and 12 to 14 in Yanchi for all types of clinical visits. CONCLUSIONS: Our findings add additional evidence for the adverse effect of suboptimal ambient temperature and provide useful information for public health programs targeting people living in rural villages.

Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity.

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.

Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles.

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.