Transcriptome characteristics of epithelial cells from advanced non-small cell lung cancer were revealed by single-cell RNA sequencing.

BACKGROUND: Lung adenocarcinoma (LUAD) is the prevalent malignancy worldwide. The aim is to explore differentially expressed genes (DEGs) associated with immune infiltration and survival time of LUAD patients, and predict transcriptional factors for shedding new light on molecular mechanisms and individual therapy of LUAD. METHOD: ScRNA-seq data of LUAD patients was downloaded from GSE148071 and analyzed by R packages. The clustering and protein-protein interaction network were constructed for screening DEGs. Gene Set Enrichment Analysis (GSEA) and GO enrichment analysis were performed in epithelial cell subgroups with high differentiation potential. Potential regulatory transcription factors were predicted. RESULTS: Sixteen epithelial cell types were required and top 20 genes were identified on cell subgroup Epi4 with the highest differentiation potential associated with poor prognosis of LUAD in PPI network. GSEA and GO annotation results showed that cell subgroup Epi4 was enriched in the biological processes of cell proliferation and energy metabolism, and positively regulated the function of cell proliferation. TPI1 was significantly highly expressed in LUAD samples (p < .0001). TPI1 demonstrated a negative correlation with the infiltration levels of CD8+ T cells, CD4+ T cells, B cells, and activated mast cells, whilst manifesting a positive correlation with the infiltration levels of resident mast cells, Th2 cells, and MDSC. Epi4 was regulated by transcription factors MXD3 and GATA4. CONCLUSION: Overexpression of TPI1 was identified as a novel biomarker for LUAD, and potential regulatory transcription factors MXD3 and GATA4 regulated the proliferation of LUAD with the poor prognosis, which may serve as potential targets to suppress the proliferation of LUAD.

Cinchonine and cinchonidine alleviate cisplatin-induced ototoxicity by regulating PI3K-AKT signaling.

AIM: Cinchonine (CN) and its isomer cinchonidine (CD), two of the common cinchona alkaloids, are wildly used as antimalarial drugs. However, the effects of CN and CD on the auditory system are unknown. METHODS: Molecular docking and molecular dynamics (MD) simulation were used for predicting effective drugs. The CCK-8 assay was conducted for assessing cell viability in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. MitoSox Red staining revealed reactive oxygen species (ROS) amounts. TMRM staining was used to assess the mitochondrial membrane potential (ΔΨm). Immunofluorescence staining of myosin 7a was used to examine hair cells (HCs) in cisplatin-treated neonatal mouse cochlear explants, while TUJ-1 immunostaining was used for the detection of spiral ganglion neurons (SGNs). Cleaved caspase-3 and TUNEL immunostaining were utilized for apoptosis assessment. Immunoblot was carried out to detect PI3K-AKT signaling effectors. RESULTS: Pretreatment with CN or CD significantly increased cell viability and reduced mitochondrial dysfunction and ROS accumulation in cisplatin-treated HEI-OC1 cells. Immunofluorescent staining of cochlear explants showed that CN and CD attenuated cisplatin-induced damage to SGNs and HCs. Immunoblot revealed that CN and CD downregulated the expression of cleaved caspase-3 and activated PI3K-AKT signaling in cisplatin-injured HEI-OC1 cells. CONCLUSION: CD and CN can reduce ototoxicity caused by cisplatin and might help treat cisplatin-associated hearing loss.

Characterization of tumor microenvironment infiltration and therapeutic responses of cell cycle-related genes' signature in breast cancer.

BACKGROUND: It is unknown how the cell cycle plays a role in breast cancer (BC). This study aimed to establish a clinically applicable predictive model to predict the therapeutic responses and overall survival in BC patients. MATERIALS AND METHODS: Cell cycle-related genes (CCGs) were identified within the Cancer Genome Atlas cohort (n is equal to 1001) and the Gene Expression Omnibus cohort (n is equal to 3265). An analysis of univariate and multivariate Cox was then conducted to develop a nomogram based on CCGs. After validating the nomogram, risk cohort stratification was established and the predictive value was examined. Finally, immune cell infiltration and therapeutic responses were analysed. RESULTS: Based on 15 CCGs, 4 prognostic predictors were identified and entered into the nomogram. According to the curves of calibration, the estimated and observed value of the nomogram is in optimal agreement. Subsequently, stratification into two risk cohorts showed that the predictive value, immune cell infiltration and overall survival were better among patients with low risk. Immune checkpoint expression in patients with BC at higher risk was downregulated. Furthermore, the results of the study revealed that doxorubicin, paclitaxel, docetaxel, cisplatin and vinorelbine all had higher IC50 values in patients with high-risk BC. CONCLUSION: The nomogram based on CCG could assess tumour immune micro-environment regulation and therapeutic responses of immunotherapy in BC. Moreover, it may provide novel information on the control of immune micro-environment infiltration in BC and aid in the development of targeted immunotherapy.

Regulating the thickness of the carbon coating layer in iron/carbon heterostructures to enhance the catalytic performance for oxygen evolution reaction.

The exploration of high-performance electrocatalysts for the oxygen evolution reaction (OER) is crucial and urgent for the fast development of green and renewable hydrogen energy. Herein, an ultra-fast and energy-efficient preparation strategy (microwave-assisted rapid in-situ pyrolysis of organometallic compounds induced by carbon nanotube (CNT)) is developed to obtain iron/carbon (Fe/C) heterogeneous materials (Fe/Fe3C particles wrapped by carbon coating layer). The thickness of the carbon coating layer can be adjusted by changing the content and form of carbon in the metal sources during the fast preparation process. Fe/Fe3C-A@CNT using iron acetylacetonate as metal sources possesses unique Fe/C heterogeneous, small Fe/Fe3C particles encapsulated by the thin carbon coating layer (1.77 nm), and obtains the optimal electron penetration effect. The electron penetration effect derives from the redistribution of charge between the surface carbon coating layer and inner Fe/Fe3C nanoparticles efficiently improving both catalytic activity and stability. Therefore, Fe/Fe3C-A@CNT shows efficient OER catalytic activity, just needing a low overpotential of 292 mV to reach a current density of 10 mA cm-2, and long-lasting stability. More importantly, the unique control strategy for carbon thickness in this work provides more opportunity and perspective to prepare robust metal/carbon-based catalytic materials at the nanoscale.

5,7-Dihydroxy-4-methylcoumarin modulates the JNK/FoxO1 signaling pathway to attenuate cisplatin-induced ototoxicity by suppressing oxidative stress and apoptosis in vitro.

5,7-Dihydroxy-4-methylcoumarin (D4M) is attributed to free radical scavenging effects, with wide application for anti-oxidation. This work aimed to assess D4M's impact on cisplatin-induced ototoxicity. The cell viability was estimated with CCK-8 assay. Apoptosis was detected by the Annexin V-FITC and PI assay. The reactive oxygen species (ROS) level was determined by MitoSOX-Red and CellROX-Green probes. Mitochondrial membrane potential was analyzed with TMRM staining. Immunofluorescence was utilized for hair cells and spiral ganglion neuron detection. Apoptosis-associated proteins were assessed by cleaved caspase-3 and TUNEL staining. These results showed that D4M pretreatment protected hair cells from cisplatin-induced damage, increased cell viability, and decreased apoptosis in House Ear Institute-Organ of Corti1 (HEI-OC1) cells and neonatal mouse cochlear explants. D4M significantly inhibited cisplatin-induced mitochondrial apoptosis and reduced ROS accumulation. In addition, the protective effect of D4M on cisplatin-induced ototoxicity was also confirmed in cochlear hair cells and spiral ganglion neurons in neonatal mice. Mechanistic studies showed that D4M markedly downregulated p-JNK and elevated the expression ratio of p-FoxO1/FoxO1, thereby reducing cisplatin-induced caspase-dependent apoptosis. Meanwhile, D4M-related protection of HEI-OC1 cells was significantly blunted by JNK signaling induction with anisomycin. This study supports the possibility that D4M may be used as a new compound to prevent cisplatin-related hearing loss.

Evaluation of per- and polyfluoroalkyl substances (PFAS) in leachate, gas condensate, stormwater and groundwater at landfills.

Per- and polyfluoroalkyl substances (PFAS), found in many consumer products, are commonly disposed of in landfills at the end of their service lives. To identify landfill liquids that should be prioritized for treatment, this study aimed to evaluate PFAS levels in different aqueous samples from landfills and identify relationships between PFAS and landfill characteristics. Twenty-six PFAS including 11 perfluoroalkyl carboxylic acids (PFCAs), 7 perfluoroalkyl sulfonates (PFSAs), and 8 perfluoroalkyl acid precursors (PFAA-precursors) were measured in municipal solid waste (MSW) leachate, construction and demolition debris (CDD) leachate, municipal solid waste incineration (MSWI) ash leachate, gas condensate, stormwater, and groundwater from landfills. Based on the median, results show that PFAS levels in MSW leachate were the highest (10,000 ng L-1), CDD leachate were intermediate (6200 ng L-1), and MSWI ash leachate were the lowest (1300 ng L-1) among the leachates evaluated. PFAS levels in gas condensate (7000 ng L-1) were similar to MSW leachate. PFAS in stormwater and groundwater were low (medians were less than 500 ng L-1). Dominant subgroups included PFCAs and PFAA-precursors in all leachates. PFSAs were also found in CDD leachate, PFAA-precursors in gas condensate, and PFCAs in stormwater and groundwater. Landfill characteristics significantly correlated with ∑26PFAS included waste proportions (percentage of MSWI ash in landfill, |rs| = 0.22), operational status (active or not, |rs| = 0.27) and rainfall (30-d cumulative rainfall, |rs| = 0.39). The results from this study can be used to prioritize which landfills and which reservoir of liquids (and corresponding subgroup of PFAS) to target for PFAS management.

CSMD1 Mutation Related to Immunity Can Be Used as a Marker to Evaluate the Clinical Therapeutic Effect and Prognosis of Patients with Esophageal Cancer.

INTRODUCTION: As a highly aggressive tumor with a poor prognosis, esophageal cancer (ESCA)'s relationship with gene mutations is unclear. Therefore, we tried to explore the role of gene mutation in ESCA progression and its relationship with immune response, clinical treatment, and prognosis. METHODS: In addition to copy number variation (CNV) situations of common genes obtained from 2 public databases, the relationship between mutations and prognosis/tumor mutational burden (TMB) was also analyzed. Kaplan-Meier survival and Cox regression analysis were used to identify the CSMD1 mutation status as an independent predictor of prognosis. We also enriched related functions and pathways. Next, the relationship between 22 immune cells and CSMD1 mutation status was analyzed. In addition to the differences in the expression levels of immune checkpoint inhibitors (ICIs)-related genes between the high TMB and low TMB groups, the differences in the expression levels of ICIs/m6a/multi-drug resistance-related genes and the sensitivity of three chemotherapeutic drugs between CSMD1 mutant and the wild group were also compared. In addition to differences and prognostic analysis of CSMD1 expression, the correlation analysis between the expression of these genes/immune cells and the expression of CSMD1 was also performed. Finally, a nomogram that could efficiently and conveniently predict the survival probability of ESCA patients was constructed and verified. RESULTS: We obtained 17 frequently mutated genes distribution. Mutation and loss of CSMD1 are frequent in ESCA. Only CSMD1 mutation can be used as an independent predictor of poor prognosis. Patients in the high TMB group have a lower survival probability. Wild CSMD1 may be involved in immune-related pathways. More helper T cells and fewer resting state dendritic cells were found in the CSMD1 mutant group. The PD-1 expression in the high TMB group showed higher. Paclitaxel sensitivity and ABCC1 expression were higher in the wild CSMD1 group. Most cancers show differential expression of CSMD1. Except for the prognosis of ESCA, the expression of CSMD1 is related to immune cell content and the expression of ICIs/m6a/multi-drug resistance related genes. DISCUSSION: CSMD1 mutation could be used as an immune-related biomarker to predict prognosis and treatment effect of paclitaxel. Mutation and loss of CSMD1 may promote the progression of ESCA.

Evaluation of the antioxidant effects of acid hydrolysates from Auricularia auricular polysaccharides using a Caenorhabditis elegans model.

Caenorhabditis elegans is an important model organism for studying stress response mechanisms and identifying genetic pathways that influence longevity. The present study was designed to explore the in vivo-antioxidant potential and the probable mechanism of acid hydrolysates prepared from A. auricula polysaccharides (AAPHs-F) with the optimal acid hydrolysis conditions using Box-Behnken design, and C. elegans was used as a model organism. The effects of AAPHs-F on the locomotory behavior, lifespan, activities of antioxidant-related enzymes and levels of antioxidants in C. elegans were studied. In addition, the potential of AAPHs-F in up-regulating the expression of antioxidant-related genes in C. elegans, such as daf-16, skn-1, sod-1, sod-2 and sir-2.1, and the inhibition of cell apoptosis of C. elegans were also discussed. The results indicated that AAPHs-F could significantly increase the U-Turn frequency of nematodes, extend their lifespan, enhance antioxidant systems including superoxide dismutase (SOD) by 70.60%, catalase (CAT) by 73.45% and glutathione reductase (GR) by 258.68% (p < 0.01), increase the level of glutathione (GSH) by 110.22% (p < 0.01), and decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA) by 31.86% and 46.16% (p < 0.01), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that AAPHs-F could up-regulate mRNA expression levels of daf-16, skn-1, sir, sod-1 and sod-2 in wild-type C. elegans (>1.3 fold) when treated at a concentration of 0.1 mg mL-1 (p < 0.05 or p < 0.01). AAPHs-F was concluded to be heteropolysaccharides composed of mannose, glucose and galactose with a molar ratio of 12.7 : 3.25 : 1. The molecular weight of AAPHs-F was determined to be 885.37 Da. Furthermore, AAPHs-F is mainly formed of (1 → 3)-linked-α-d-glucopyranose, and carboxyl or acetamide is present in the molecule. In summary, our studies provide evidence that AAPHs-F helps improve the antioxidant defense system, and up-regulation of stress and longevity related genes suggests the possible involvement of these genes in the prevention of stress damage in C. elegans.

Acute Changes of Bile Acids and FGF19 After Sleeve Gastrectomy and Roux-en-Y Gastric Bypass.

CONTEXT: Gastric bypass (GBP) and sleeve gastrectomy (SG) are both effective bariatric treatments that cause sustained weight loss as well as improvement of type 2 diabetes mellitus (T2DM). The underlying mechanisms are under investigation, including the contribution of alterations in bile acids (BAs) in achieving or maintaining the beneficial metabolic effects after bariatric surgery. AIMS: The aim of this study is to investigate the acute and short-term effects of GBP and SG on BA compositions and fibroblast growth factor 19 (FGF19) in obese individuals with T2DM and to evaluate any correlations between changes in these measures with glucose metabolic improvements. METHODS: The levels of both fasting and postprandial plasma BA compositions after oral glucose tolerance test (OGTT), fasting FGF19 and various metabolic indices were measured 1 day before and at 3 days and 3 months after GBP and SG in 19 obese patients (GBP = 8, SG = 11) with T2DM. RESULTS: Body weight loss was observed after both GBP and SG 3 months post-operatively, with no significant difference between the two intervention groups (15.0 ± 3.1% vs. 13.9 ± 5.2%, P = 0.761). At 3 days post-operation, FGF19 levels increased significantly in both surgery groups (GBP, 118.3 ± 57.3 vs. 363.6 ± 131.0 pg mL-1, post-operation P = 0.008; SG, 173.2 ± 127.8 vs. 422.0 ± 243.6 pg mL-1, post-operation P = 0.001). Fasting and postprandial increases from pre-operative values in secondary (r = 0.57, P = 0.02; r = 0.58, P = 0.01), conjugated (r = 0.50, P = 0.01; r = 0.48, P = 0.04), glycine-conjugated (r = 0.52, P = 0.05; r = 0.46, P = 0.05) and secondary-conjugated (r = 0.53, P = 0.02; r = 0.60, P = 0.01) BAs correlated with decreases in the postprandial states of glucose (defined by area under the curve (AUC) over 120 min (AUC0-120min)). Increases in postprandial primary-conjugated BAs were found to be associated with decreases in HOMA-IR (r = 0.45, P = 0.05). However, increases in fasting and postprandial taurine-conjugated BA correlated with decreases in both basal insulin secretion rate (r = 0.47, P = 0.04; r = 0.48, P = 0.04) and C-peptide level (r = 0.45, P = 0.05; r = 0.47, P = 0.04). After 3 months, fasting and postprandial increases in secondary (r = 0.51, P = 0.03; r = 0.48, P = 0.04), secondary-conjugated (r = 0.52, P = 0.02; r = 0.51, P = 0.03) and non-12α-OH (r = 0.51, P = 0.02; r = 0.58, P = 0.01) BAs were found to correlate with increases in Stumvoll Insulin Sensitivity Index. Increases in both fasting and postprandial 12α-OH BAs were correlated with the decreases in glucose AUC (r = 0.46, P = 0.05; r = 0.41, P = 0.04). CONCLUSIONS: Both GBP and SG achieve increases in many BA species as early as 3 days post-operation, which are sustained at 3 months post-operation. Rises in secondary BA and conjugated forms are correlated with early improvements in glucose metabolism at 3 days post-operation. These along with 12α-OH BA correlated with improved glucose metabolism at 3 months post-operation, suggesting they may contribute to the observed T2DM remission after bariatric surgery.

Hypoglycemic Effect of the Degraded Polysaccharides from the Wood Ear Medicinal Mushroom Auricularia auricula-judae (Agaricomycetes).

Auricularia auricula-judae is an important culinary-medicinal mushroom. The A. auricula-judae polysaccharides (AAPs) were prepared from A. auricula-judae in the early stage through alkali extraction and deproteination with the Sevag method, and optimal acid hydrolysis conditions were established by Box-Behnken to prepare the degraded polysaccharides (AAPs-F) from AAPs. In this study, a nonenzymatic glycosylation reaction system was used for the evaluation of the inhibitory effects on the formation of advanced glycation end products (AGEs). In addition, high glucose resistance was assessed by glucose consumption of HepG2 cells and the lifespan of Caenorhabditis elegans under high sugar stress. It was found that both 0.5 mg·mL-1 AAPs and 0.2 mg·mL-1 AAPs-F could significantly inhibit AGE formation in short- and long-term glycosylation (P < .05) in a dose-dependent manner, determined by ultraviolet and fluorospectrophotometry. It indicated activity against AGE formation for different concentrations of AAPs and AAPs-F. AAPs-F at 0.5 mg·mL-1 significantly enhanced the glucose absorption of HepG2 cells by 24.4% (P < .05) in a dose-dependent manner at 24 h, and markedly extended the lifespan of C. elegans by 32.9% (P < 0.05) under high sugar stress conditions. This study demonstrated that the derived hydrolysates produced by the hydrolysis of acid had a prominent effect on the inhibition of AGE formation and relieved the stress state caused by high sugar levels.

Preparation of iron oxide silica particles for Zika viral RNA extraction.

In this work, a robust synthetic pathway for magnetic core preparation and silica surface coating of magnetic microparticles is presented. Silica-coated magnetic particles are widely used to extract DNA and RNA from various biological samples. We present a novel route for the synthesis of iron oxide silica particles (Fe3O4@Silica) and demonstrate their performance for extracting ZIKA viral RNA from serum. The iron (II, III) oxide (Fe3O4), magnetite core is first prepared by ammonia neutralization of ferrous and ferric chloride aqueous solution under argon, followed by the addition of citrate salt to stabilize the surface of the resultant magnetic nanospheres. After this one-pot, two-step synthesis, the magnetic nanospheres are consumed during silica coating by hydrolysis of tetraethoxysilane (TEOS) under alkaline condition. The final product is a sphere-like magnetic aggregate with a size range of 1-2 micron. By simply suspending the magnetic aggregates in guanidinium chloride solution, the silica surface can be prepared for RNA binding. The RNA extraction efficiency was evaluated by extracting ZIKA viral RNA from serum followed by a PCR-based assay. The data indicate excellent recovery of target RNA and removal of PCR inhibitors. This manufacturing procedure for the silica coated microparticles provides a low-cost, effective and ready for scale-up method whose performance is equivalent to commercial alternatives such as magnetic silica surface particles for DNA and RNA sample preparations. The cost of the clinical assays could be largely decreased due to the 100 fold reduction in cost by replacing the commercially available magnetic particles with the developed material for RNA extraction.

Inhibition of sortase A by chalcone prevents Listeria monocytogenes infection.

The critical role of sortase A in gram-positive bacterial pathogenicity makes this protein a good potential target for antimicrobial therapy. In this study, we report for the first time the crystal structure of Listeria monocytogenes sortase A and identify the active sites that mediate its transpeptidase activity. We also used a sortase A (SrtA) enzyme activity inhibition assay, simulation, and isothermal titration calorimetry analysis to discover that chalcone, an agent with little anti-L. monocytogenes activity, could significantly inhibit sortase A activity with an IC50 of 28.41 ± 5.34 µM by occupying the active site of SrtA. The addition of chalcone to a co-culture of L. monocytogenes and Caco-2 cells significantly inhibited bacterial entry into the cells and L. monocytogenes-mediated cytotoxicity. Additionally, chalcone treatment decreased the mortality of infected mice, the bacterial burden in target organs, and the pathological damage to L. monocytogenes-infected mice. In conclusion, these findings suggest that chalcone is a promising candidate for the development of treatment against L. monocytogenes infection.

TIM-1 acts a dual-attachment receptor for Ebolavirus by interacting directly with viral GP and the PS on the viral envelope.

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%-90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.

[Comparative study of less invasive stabilization system (LISS) and the condylar support plates for the treatment of AO type C distal femoral fractures in adults].

OBJECTIVE: To summarize the complications and the early clinical effect of less invasive stabilization system and the femoral condylar support plates in the treatment of AO type C distal femoral fractures. METHODS: From September 2007 to February 2012, 46 patients with AO type C distal femoral fractures were retrospectively studied. Of all patients 25 were treated with less invasive stabilization system including 14 males and 11 females with a mean age of (56.3±4.2) years old; according to AO classification, there were 14 cases of C1, 8 cases of C2 and 3 cases of C3 with a mean hospital stay of (15.6±1.7) days. While 21 cases were treated with femoral condylar support plates fixation including 12 males and 9 females with a mean age of (53.8±5.1) years old;there were 13 cases of C1, 6 cases of C2 and 2 cases of C3 with a mean hospital stay of (17.8±2.2) days. Comparative analysis was performed from the operation related index,postoperative complications and Evanich score of the knee joint function between the two groups at follow-up. RESULTS: All 46 patients were followed up from 13 to 38 months with a mean time of 19.6 months after surgery. Complications included 1 case with infection,3 cases with internal fixation failure, 1 case with nonunion and 1 case with activity limitation of the affected knee. The differences in the incision length, blood loss, fracture healing time were significant between two groups (P<0.05), while there was no significant difference in the duration of operations, hospital stays and the incidence of postoperative complications between two groups (P>0.05). The statistical significance was also found in the Evanich score at last follow-up between two groups (P<0.05). CONCLUSION: Patients with less invasive stabilization system fixation had the characteristics of less trauma, shorter fracture healing time and better functional recovery. Less invasive stabilization system had became one of the ideal internal fixations in the treatment of AO type C distal femoral fractures.

Crystal structures of GI.8 Boxer virus P dimers in complex with HBGAs, a novel evolutionary path selected by the Lewis epitope.

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the ß-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.

Ins-4 and daf-28 function redundantly to regulate C. elegans L1 arrest.

Caenorhabditis elegans larvae reversibly arrest development in the first larval stage in response to starvation (L1 arrest or L1 diapause). Insulin-like signaling is a critical regulator of L1 arrest. However, the C. elegans genome encodes 40 insulin-like peptides, and it is unknown which peptides participate in nutritional control of L1 development. Work in other contexts has revealed that insulin-like genes can promote development ("agonists") or developmental arrest ("antagonists"), suggesting that such agonists promote L1 development in response to feeding. We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified thirteen candidate agonists and eight candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists is largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in control of L1 development. Transcriptional regulation of candidate agonists is most significant in the intestine, as if internal nutrient status is a more important influence on transcription than sensory perception. Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 developmental dynamics, though simultaneous disruption of ins-4 and daf-28 increases survival of L1 arrest. Furthermore, overexpression of ins-4, ins-6 or daf-28 alone decreases survival and promotes cell division during starvation. These results suggest extensive functional overlap among insulin-like genes in nutritional control of L1 development while highlighting the role of ins-4, daf-28 and to a lesser extent ins-6.

Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens.

Noroviruses, an important cause of acute gastroenteritis in humans, recognize the histo-blood group antigens (HBGAs) as host susceptible factors in a strain-specific manner. The crystal structures of the HBGA-binding interfaces of two A/B/H-binding noroviruses, the prototype Norwalk virus (GI.1) and a predominant GII.4 strain (VA387), have been elucidated. In this study we determined the crystal structures of the P domain protein of the first Lewis-binding norovirus (VA207, GII.9) that has a distinct binding property from those of Norwalk virus and VA387. Co-crystallization of the VA207 P dimer with Le(y) or sialyl Le(x) tetrasaccharides showed that VA207 interacts with these antigens through a common site found on the VA387 P protein which is highly conserved among most GII noroviruses. However, the HBGA-binding site of VA207 targeted at the Lewis antigens through the α-1, 3 fucose (the Lewis epitope) as major and the ß-N-acetyl glucosamine of the precursor as minor interacting sites. This completely differs from the binding mode of VA387 and Norwalk virus that target at the secretor epitopes. Binding pocket of VA207 is formed by seven amino acids, of which five residues build up the core structure that is essential for the basic binding function, while the other two are involved in strain-specificity. Our results elucidate for the first time the genetic and structural basis of strain-specificity by a direct comparison of two genetically related noroviruses in their interaction with different HBGAs. The results provide insight into the complex interaction between the diverse noroviruses and the polymorphic HBGAs and highlight the role of human HBGA as a critical factor in norovirus evolution.

Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center.

Highly pathogenic influenza virus strains currently in circulation pose a significant risk of a global pandemic. Following the reported crystal structure of the endonuclease domain from the avian influenza virus polymerase PA subunit, here we report the results of a systematic X-ray crystallographic analysis of its complex with adenosine, uridine, and thymidine nucleoside monophosphates (NMPs). Electron density corresponding to the monophosphate moiety of each nucleotide was apparent in each NMP complex and bound to the catalytic metal. A hydrophobic site was found to contribute to nucleoside binding. The NMP complex structures should represent the conformation of the bound product after nuclease cleavage. Moreover, one solvent molecule was found to occupy an equivalent position to the second reported Mn(2+) ion, where it mediates the interaction between bound NMPs and the N-terminal PA domain in the presence of the Mg(2+) ion. The results presented here indicate a possible cleavage mechanism and identify a distinct nucleotide binding pocket. The identification of this binding pocket opens a new avenue for anti-influenza drug discovery, targeting the cap-dependent endonuclease, in response to the worldwide threat of influenza.