Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library.

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

Serum hyaluronic acid and procollagen III, N-terminal propeptide levels are highly associated with disease severity and predict the progression of COVID-19.

Background: The coronavirus disease 2019 (COVID-19) pandemic is a rapidly evolving global emergency and continuously poses a serious threat to public health, highlighting the urgent need of identifying biomarkers for disease severity and progression. In order to early identify severe and critical patients, we retrospectively analyze the clinical characteristics and risk indicators of severe disease in patients with corona virus disease 2019 (COVID-19). Methods: A total of 420 confirmed COVID-19 patients were included in the study. According to the "Diagnosis and Treatment of novel coronavirus Pneumonia (10th Edition)", the cases were divided into mild group (n = 243) and severe group (n =177). Laboratory parameters were analyzed in combination with clinical data. Results: Male patients over 46 years who have smoking habits were more likely to suffer from severe COVID-19. Critically ill patients had lower lymphocyte counts and red blood cell counts, and higher white blood cell counts (P<0.05). Expectedly, serum inflammatory factors (NLR, PLR, LMR, CLR, PCT, CRP), coagulation markers (APTT, PT, TT, FIB, D-Dimer), Myocardial damage markers (hs-TNT, LDH) were significantly increased (P<0.05) in severe COVID-19 patients. Surprisedly, those patients showed obviously elevated levels of common tumor markers (ProGRP, CYFRA21-1, SCC, NSE) (P<0.05). In this case, the levels of tumor marker reflected more the condition of inflammation than the growth of tumor. More importantly, HA and PIIIN-P were highly associated with COVID-19 severity. The AUC of the ROC curve for the diagnosis of severe COVID-19 by HA and PIIIN-P was 0.826. Meanwhile, HA was positively correlated with myocardial damage markers (hs-TNT, LDH). PIIIN-P was positively correlated with myocardial damage markers (hs-TNT, LDH) and inflammatory factors (NLR, PLR, LMR, CLR, ProGRP, SCC, PCT, CRP). On the contrary, PIIIN-P was negatively correlated with pulmonary function indexes (oxygenation index and oxygen saturation of hemoglobin). Conclusion: HA and PIIIN-P are highly associated with disease severity and progression of COVID-19 and can be used as new markers for the prediction of severe COVID-19.

[Solid-solution Partitioning Coefficients and Environmental Risk of Cd and Pb in Soil in Chang-Zhu-Tan Area].

The leaching risk of heavy metals in soil has a large spatial variability on a regional scale. Taking the Chang-Zhu-Tan area as the research object, this work studied the distribution and influencing factors of available contents and solid-solution partition coefficient (Kd) of Cd and Pb in soil with land uses and clarified the environmental risk of heavy metals in soil based on Kd values measured by CaCl2 (soil-to-water ratio, 1:0.5). The results showed that the contents of available Cd and Pb in soil followed the order of forest land>suburban farmland>urban green space>industrial green space. The average Kd of Cd in soil was 449.79 L·kg-1, and that of Pb was 27604.07 L·kg-1, indicating that the mobility of Cd in the soil was significantly higher than that of Pb. The Kd values of forest soil were significantly lower than that in the other land uses. The Kd values were mainly affected by soil pH and the total content of heavy metals in soil. Adopting the available content of heavy metals measured by CaCl2 (soil-to-water ratio, 1:10) as a dependent variable, the multiple regressions effectively predicted the Kd values of Cd and Pb in soil, with R2 values of 84.2% and 67.6%, respectively. The environmental risk assessment indicated that the leaching risk in 93.8%-96.1% of the sampling sites could be ignored, whereas a few sampling sites near factories with low pH may pose a risk to the groundwater environment. The mobility of heavy metals in soil and the distribution of pollution sources determined the leaching risk of heavy metals. The results provide a method and theoretical support for preventing the environmental risk of heavy metals in soil on a regional scale.

Loss of DJ-1 function contributes to Parkinson's disease pathogenesis in mice via RACK1-mediated PKC activation and MAO-B upregulation.

Parkinson's disease (PD) is a common neurodegenerative motor disorder characterized by a dramatic reduction in pars compacta of substantia nigra dopaminergic neurons and striatal dopamine (DA) levels. Mutations or deletions in the PARK7/DJ-1 gene are associated with an early-onset familial form of PD. DJ-1 protein prevents neurodegeneration via its regulation of oxidative stress and mitochondrial function as well as its roles in transcription and signal transduction. In this study, we investigated how loss of DJ-1 function affected DA degradation, ROS generation and mitochondrial dysfunction in neuronal cells. We showed that loss of DJ-1 significantly increased the expression of monoamine oxidase (MAO)-B but not MAO-A in both neuronal cells and primary astrocytes. In DJ-1-knockout (KO) mice, MAO-B protein levels in the substantia nigra (SN) and striatal regions were significantly increased. We demonstrated that the induction of MAO-B expression by DJ-1 deficiency depended on early growth response 1 (EGR1) in N2a cells. By coimmunoprecipitation omics analysis, we found that DJ-1 interacted with receptor of activated protein C kinase 1 (RACK1), a scaffolding protein, and thus inhibited the activity of the PKC/JNK/AP-1/EGR1 cascade. The PKC inhibitor sotrastaurin or the JNK inhibitor SP600125 completely inhibited DJ-1 deficiency-induced EGR1 and MAO-B expression in N2a cells. Moreover, the MAO-B inhibitor rasagiline inhibited mitochondrial ROS generation and rescued neuronal cell death caused by DJ-1 deficiency, especially in response to MPTP stimulation in vitro and in vivo. These results suggest that DJ-1 exerts neuroprotective effects by inhibiting the expression of MAO-B distributed at the mitochondrial outer membrane, which mediates DA degradation, ROS generation and mitochondrial dysfunction. This study reveals a mechanistic link between DJ-1 and MAO-B expression and contributes to understanding the crosslinks among pathogenic factors, mitochondrial dysfunction and oxidative stress in PD pathogenesis.

Effects of salt stress on interspecific competition between an invasive alien plant Oenothera biennis and three native species.

Biological invasions and soil salinization have become increasingly severe environmental problems under global change due to sea-level rise and poor soil management. Invasive species can often outcompete native species, but few studies focus on whether invasive alien species are always superior competitors under increasing stressors. We grew an invasive grass species, Oenothera biennis L., and three native grass species (Artemisia argyi Lévl. et Vant., Chenopodium album L., and Inula japonica Thunb.) as a monoculture (two seedlings of each species) or mixture (one seedling of O. biennis and one native species seedling) under three levels of salt treatments (0, 1, and 2 g/kg NaCl) in a greenhouse. We found that invasive O. biennis exhibited greater performance over native C. album and I. japonica, but lower performance compared to A. argyi, regardless of the soil salinity. However, salinity did not significantly affect the relative dominance of O. biennis. Interspecific competition enhanced the growth of O. biennis and inhibited the growth of I. japonica. Although O. biennis seedlings always had growth dominance over C. album seedlings, C. album was not affected by O. biennis at any salt level. At high salt levels, O. biennis inhibited the growth of A. argyi, while A. argyi did not affect the growth of O. biennis. Salt alleviated the competitive effect of O. biennis on I. japonica but did not mitigate the competition between O. biennis and the other two native species. Therefore, our study provides evidence for a better understanding of the invasive mechanisms of alien species under various salinity conditions.

Functional analysis of flower development related gene SvGLOBOSA from Senecio vulgaris.

The unique capitulum of Asteraceae has important ornamental and research value. Few studies have described the complex molecular mechanism of flower development. In this study, SvGLOBOSA(SvGLO), the MADS-box gene of Senecio vulgaris, was identified by screening the transcriptome data, and its function was examined. The gene structure was analyzed and its function was predicted through bioinformatics. The relative expression levels in different tissues of wild-type S. vulgaris were analyzed by qRT-PCR. SvGLO was overexpressed in Solanum nigrum and morphological observations were made. Histological staining was used in analyzing the histological changes in the ovary of transgenic S. nigrum. The results showed that the open reading frame of SvGLO was 591 bp long, encoding 196 amino acids. It has typical MADS-box and K-box domains and contains a PI motif at the C-terminal. SvGLO belongs to the PI/GLO subfamily of class B MADS-box genes. qRT-PCR results showed that SvGLO was highly expressed in inflorescence tissues but not in vegetative organs. In SvGLO-overexpressed S. nigrum, the sepals showed some characteristics of petals, carpels transformed into stamen-like organs, and fruit development was abnormal. Histological staining revealed that the morphology of ovary wall cells of transgenic S. nigrum was similar to that of anther wall cells of the stamen of wild-type S. vulgaris. Therefore, SvGLO may be involved in the regulation of petal and stamen development in S. vulgaris.

Clinical and Pathological Implications of Increases in Tonsillar CD19+CD5+ B Cells, CD208+ Dendritic Cells, and IgA1-positive Cells of Immunoglobulin A Nephropathy.

OBJECTIVE: Several studies indicated that tonsillectomy can improve the prognosis of patients with immunoglobulin A nephropathy (IgAN). However, the relationship between tonsillar immunity and IgAN is still unclear. METHODS: A total of 14 IgAN patients were recruited in the current study from May 2015 to April 2016 in Tongji Hospital. B cells, dendritic cells (DCs), and IgA1 positive cells in human tonsils were detected using immunofluorescence and immunohistochemistry. Correlations between these cells and clinicopathologic features were evaluated. RESULTS: CD19+CD5+ B cells were predominantly located in germinal centers and mantle zones of lymphoid follicles, the CD208+ DCs were distributed in the interfollicular and subepithelial area, and IgA1-positive cells were predominantly detected in mantle zones of lymphoid follicles and subepithelial tissues. The numbers of CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues from IgAN patients were significantly higher than those in the normal controls (P<0.01, respectively). CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues were significantly associated with 24-h proteinuria levels and tubular atrophy/interstitial fibrosis of IgAN. CONCLUSION: CD19+CD5+ B cells, CD208+ DCs, and IgA1-positive cells in tonsillar tissues might be involved in the pathogenesis of IgAN.

Interleukin-7 Is Associated with Clinical and Pathological Activities in Immunoglobulin A Nephropathy and Protects the Renal Proximal Tubule Epithelium from Cellular Fibrosis.

OBJECTIVE: Diagnosis of immunoglobulin A nephropathy (IgAN) requires the evaluation of renal biopsy specimens. However, renal biopsy is an invasive procedure and is not frequently performed for various reasons. Thus, recognized noninvasive biomarkers for predicting IgAN progression are urgently needed. METHODS: In the present study, we included 86 IgAN patients with renal biopsy from June 2015 to May 2016 and had their plasma interleukin-7 (IL-7) level measured with ELISA. The association between the plasma IL-7 level and clinico-pathological characteristics was analyzed. Immunohistochemical staining was used to assay the in situ expression of IL-7 in vivo. Western blotting was performed to examine the production of extracellular matrix, p-mTOR and the markers of autophagy under the treatment of IL-7 after TGF-ß1 stimulation in renal tubular epithelial cells. RESULTS: IL-7 was significantly decreased in patients with IgAN compared to healthy subjects (2.3077 vs. 8.6294 pg/mL, P<0.0001). There was a significant difference in the plasma IL-7 level between tubular atrophy/interstitial fibrosis T0 and T2 classes (P=0.0064). A lower plasma IL-7 value in patients at the time of biopsy indicated a poor renal outcome. In addition, IL-7 was over-expressed in renal tubular epithelial cells and significantly attenuated transforming growth factor ßl-induced extracellular matrix production by suppression of cellular autophagy via activation of mTOR1 signaling. CONCLUSION: These results suggested that IL-7 might be a noninvasive biomarker for predicating IgAN. It protected renal proximal tubular epithelial cells from cellular fibrosis by inhibiting autophagy via mTORl signaling.

Discovery of synergistic anti-inflammatory compound combination from herbal formula GuGe FengTong Tablet.

Multi-components in herbal formulae exert holistic effects in synergistic or additive manners. However, appropriate strategies and supportive evidences are still lacking to uncover the synergistic or additive combinations. The present investigation aimed at seeking a screening strategy to identify the targeted combinations in GuGe FengTong Tablet (GGFTT), an herbal formula. Two compounds, belonging to different chemical classes, were combined with different concentration ratios and their anti-inflammation effects were investigated. The most significant anti-inflammatory combinations were evaluated by combination index (CI) method (additive effect, CI = 1; synergism, CI < 1; antagonism, CI > 1). The modulating effects of candidate combinations on pro-inflammatory cytokines and MAPKs signaling pathway were also detected. Two combinations, "biochanin A + 6-gingerol" (Bio-6G) and "genistein + 6-gingerol" (Gen-6G), showed synergistic effects (CI < 1), and Bio-6G was selected for further study. Compared with single compound, Bio-6G could synergistically inhibit the production of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and the activation of MAPKs signaling pathway in LPS-stimulated RAW264.7 cells. The combined results showed that Bio-6G was a synergistic anti-inflammatory combination in GGFTT. Our results could provide a useful strategy to screen the synergistic combinations in herbal formulae.

Bacterial community shaped by heavy metals and contributing to health risks in cornfields.

Scientists are increasingly aware that heavy metal contamination in soils, especially in farmland ecosystems, can negatively affect human health and alter the bacterial community that plays a critical role in plant growth and heavy metal accumulation. The goal of the present paper was to uncover how various heavy metals and non-metallic elements affect human health and bacterial diversity in cornfields and to explore the contribution of soil bacteria to heavy metal accumulation in crops. Soil samples were collected from five counties in Shandong Province, China, where abnormally high levels of heavy metals and metalloids were caused by mining and heavy industry. We calculated a hazard quotient (HQ) to evaluate the health risk these heavy metals cause and analyzed the soil bacterial community using 16S rRNA gene sequencing. The HQ results showed that As posed the greatest threat to human health followed by Pb although concentrations of all metals did not reach the health risk threshold. Meanwhile, principal component analysis (PCA) and redundancy analysis (RDA) revealed soil bacterial richness was significantly influenced by As, Ni, and Cr as well as pH and phosphorus, but not by the species diversity of aboveground weeds. The most abundant bacteria in our study region were heavy metal tolerant groups, specifically Actinobacteria and Proteobacteria. Moreover, correlation analysis suggested that Actinobacteria might reduce the phytoaccumulation of Cr, Cu, Zn, and Hg in corn, while Proteobacteria might weaken phytoaccumulation of Pb, Ni, As, and Cd. Our results verified that heavy metals play an important role in shaping the soil bacterial community. Using native bacteria in farmland provides a potential biological strategy for reducing the health risk posed by heavy metals related to food consumption.

An UHPLC-MS/MS method for simultaneous determination of quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-rutinoside, bilobalide and ligustrazine in rat plasma, and its application to pharmacokinetic study of Xingxiong injection.

The present study was designed to develop and validate a rapid, sensitive, and reliable ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of five major active constituents in the traditional Chinese medicinal preparation Xingxiong injection (XXI) in rat plasma, including quercetin 3-O-rutinoside (QCR), kaempferol 3-O-rutinoside (KFR), isorhamnetin 3-O-rutinoside (ISR), bilobalide (BB), and ligustrazine (LGT). The plasma samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was achieved on a Waters Symmetry C18 analytical column (2.1 mm × 100 mm, 3.5 µm) with a mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B). Quantitation of the five bioactive constituents was achieved. Naringin was used as the internal standard (IS). All the calibration curves showed good linearity (r > 0.996) over the concentration range, with the lowest limit of quantification (LLOQ) between 2-18 ng·mL-1. The intra- and inter-day accuracy and precision of the analytes were both within acceptable limits. Moreover, satisfactory extraction recoveries (90.92%-104.03%) were obtained by protein precipitation. The validated method was successfully applied to a pharmacokinetic study of XXI in rats after intravenous administration at three doses. The pharmacokinetic parameters of the five compounds varied in a dose-dependent manner within the tested dosage range. The present study was the first report of pharmacokinetic study for XXI.

A new compound, methylbergenin along with eight known compounds with cytotoxicity and anti-inflammatory activity from Ardisia japonica.

A new compound, methylbergenin (1), was isolated from the whole plants of Ardisia japonica (Thunb.) Bl. (Myrsinaceae), along with eight known compounds: demethoxybergenin (2), bergenin (3), afzelin (4), quercitrin (5), bauerenol (6), bauerenone (7), α-spinasterol (8) and chondrillasterone (9). Compounds 1, 7 and 9 were isolated from A. japonica for the first time. The structure of compound 1 was determined on the basis of NMR (1D and 2D) and mass spectroscopic analysis. The absolute configuration of 1 was assessed by single-crystal X-ray diffraction. Compounds 1, 4, 5, 7 and 9 showed potential inhibitory effects against nitric oxide production in LPS stimulated RAW 264.7 murine macrophages. In addition, Compounds 7 and 9 exhibited cytotoxicity against A549 and HepG-2 cells.

Quality standardization of herbal medicines using effective compounds combination as labeled constituents.

Selection of suitable labeled constituents is vital for the quality standardization of herbal medicines (HMs). However, discovery of labeled constituents that can account for the whole efficacy of original HMs is a challenging issue. Taking tanshinones extract (TE) as an example, a strategy to establish reasonable quality control method using effective compounds combination (ECC) as labeled constituents was proposed. The strategy consists of three core steps, including chemical profiling of TE, discovery and in vivo process research of ECC, and quality standardization based on ECC. Using this strategy, a combination of four tanshinones (tanshinones IIA, cryptotanshinone, tanshinone I and dihydrotanshinone I), which was as effective as TE in cell models and in a rat model of myocardial ischemia-reperfusion, was identified as ECC in TE. Furthermore, quality standardization of TE was established based on proposed ECC using single standard for determination of multiple components (SSDMC) method. In conclusion, the presently developed ECC-based approach not only offers new insight into the understanding of the holistic effects of HMs, but also provides efficacy-associated labeled constituents for quality control of botanicals.

Surgery for left ventricular aneurysm after myocardial infarction: techniques selection and results assessment.

BACKGROUND: The most appropriate surgical approach for patients with post-infarction left ventricular (LV) aneurysm remains undetermined. We compared the efficacy of the linear versus patch repair techniques, and investigated the mid-term changes of LV geometry and cardiac function, for repair of LV aneurysms. METHODS: We reviewed the records of 194 patients who had surgery for a post-infarction LV aneurysm between 1998 and 2010. Short-term and mid-term outcomes, including complications, cardiac function and mortality, were assessed. LV end-diastolic and systolic dimensions (LVEDD and LVESD), LV end-diastolic and end-systolic volume indexes (LVEDVI and LVESVI) and LV ejection fraction (LVEF) were measured on pre-operative and follow-up echocardiography. RESULTS: Overall in-hospital mortality was 4.12%, and major morbidity showed no significant differences between the two groups. Multivariate analysis identified preoperative left ventricular end diastolic pressure > 20 mmHg, low cardiac output and aortic clamping time > 2 hours as risk factors for early mortality. Follow-up revealed that LVEF improved from 37% pre-operation to 45% 12 months post-operation in the patch group (P = 0.008), and from 44% pre-operation to 40% 12 months postoperation in the linear group (P = 0.032). In contrast, the LVEDVI and LVESVI in the linear group were significantly reduced immediately after the operation, and increased again at follow-up. However, in the patch group, the LVEDVI and LVESVI were significantly reduced at follow-up. And there were significant differences in the correct value changes of LVEF and left ventricular remodeling between linear repair and patch groups. CONCLUSIONS: Persistent reduction of LV dimensions after the patch repair procedure seems to be a procedure-related problem. The choice of the technique should be tailored on an individual basis and surgeon's preference. The patch remodeling technique results in a better LVEF improvement, further significant reductions in LV dimensions and volumes than does the linear repair technique. The results suggest that LV patch remodeling is a better surgical choice for patients with post-infarction LV aneurysm.

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis.

Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale). In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 µM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA) suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

Small molecular glasses based on multiposition encapsulated phenyl benzimidazole iridium(III) complexes: toward efficient solution-processable host-free electrophosphorescent diodes.

Three electrophosphorescent small molecular Ir(3+) complexes, Ir(HexPhBI)(3) 1 (HexPhBI = 1-Hexyl-2-phenyl-1H-benzo[d]imidazole), Ir(CzPhBI)(3) 2 (CzPhBI = 9-(6-(2-phenyl-1H-benzo[d]imidazol-1-yl)hexyl)-9H-carbazole), and Ir(Cz(2)PhBI)(3) 3 (Cz(2)PhBI = 9-(6-(4-(1-(6-(9H-carbazol-9-yl)hexyl)-1H-benzo[d]imidazol-2-yl)phenoxy)hexyl)-9H-carbazole), were synthesized in which 3 was designed with the structure of multiposition encapsulation. Compared to the hexyl-substituted 1, 2 and 3 end-capped with the conjugated carbazole moieties have improved thermal stability. X-ray diffraction analysis proved the amorphous state of 2 and 3. High-photoluminescent efficiencies of 3 are achieved as 72% in solution and 61% in solid. It indicates that the peripheral carbazoles not only facilitate the separation of triplet-emission cores and reduce the intermolecular aggregation but also supply a routine for the intermolecular energy transfer. Electrochemical analysis showed the more oxidation states of 3, which might be anticipated to make it superior to 1 and 2 in hole injection and transporting. The important role of the peripheral carbazole moieties in carrier injection/transporting and the optical properties of the complexes were further investigated by Gaussian simulation. A dramatic electroluminescent (EL) performance, including external quantum efficiency of nearly 6%, low turn-on voltage of 2.5 V, and high brightness over 6000 cd m(-2), from the host-free spin-coated device of 3 was achieved. The superiority of multiencapsulation in EL was proved by comparing the EL performance of 2 and 3. By making comparison between the host-free and phosphor-doping devices, it indicated that the combined modification of the aliphatic chains and functional groups in multipositions is a feasible approach to realize the high-efficiency small molecular phosphorescent materials.