Effects of probiotic supplementation on 12 min run performance, mood management, body composition and gut microbiota in amateur marathon runners: A double-blind controlled trial.

Background: Probiotic supplementation has a positive effect on endurance exercise performance and body composition in athletes, but the underlying mechanisms remain unclear. Gut microbiota can provide measurable markers of immune function in athletes, and microbial composition analysis may be sensitive enough to detect stress and metabolic disorders caused by exercise. Methods: Nineteen healthy active amateur marathon runners (15 male and 4 female) with a mean age of 29.11 years volunteered to participate in this double-blind controlled study. Based on the performance of the Cooper 12-min running test (CRT), the participants were allocated into two groups to receive either a probiotic formulation comprising lactobacillus acidophilus and bifidobacterium longum (n = 10) or placebo containing maltodextrin (n = 9) for five weeks. Consistency of diet and exercise was ensured throughout the experimental period. Before and after the intervention, all participants were assessed for CRT, emotional stability and gastrointestinal symptoms, gut microbiota composition, body composition and magnetic resonance imaging (MRI) indicators of skeletal muscle microcirculation. Results: Compared to before the intervention, the probiotics group showed an increase in CRT score (2.88 ± 0.57 vs 3.01 ± 0.60 km, P＜0.05), significant improvement in GSRS and GIQLI (9.20 ± 4.64 vs 7.40 ± 3.24, 118.90 ± 12.30 vs 127.50 ± 9.85, P＜0.05), while these indicators remained unchanged in the control group, with a significant time-group interaction effect on gastrointestinal symptoms. Additionally, some MRI metabolic cycling indicators of the thigh skeletal muscle also changed in the probiotics group (P＜0.05). Regarding microbiota abundance, the probiotics group exhibited a significant increase in the abundance of beneficial bacteria and a significant decrease in the abundance of harmful bacteria post-intervention (P＜0.05). Conclusion: As a sports nutritional supplement, probiotics have the potential to improve athletic performance by optimizing the balance of gut microbiota, alleviating gastrointestinal symptoms.

Rapamycin recruits SIRT2 for FKBP12 deacetylation during mTOR activity modulation in innate immunity.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation-deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells.

Aberrant Intraregional Brain Activity and Functional Connectivity in Patients With Diarrhea-Predominant Irritable Bowel Syndrome.

BACKGROUND AND PURPOSE: The appearance and aggravation of diarrhea-predominant irritable bowel syndrome (IBS-D) have proven to be closely related to psychosocial factors. We aimed to measure altered spontaneous brain activity and functional connectivity (FC) in patients with IBS-D using resting-state functional magnetic resonance imaging (RS-fMRI) and to analyze the relationship between these parameters and emotional symptoms. METHODS: Thirty-six adult IBS-D patients and thirty-six demographic-matched healthy controls (HCs) underwent RS-fMRI scans. After processing RS-fMRI data, the values of the amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (ReHo) of the two groups were compared. The abnormal regions were selected as the regions of interest to compare whole-brain seed-based FC between the groups. The relationships between RS-fMRI data and mood and gastrointestinal symptoms were analyzed using correlation and mediation analyses. RESULTS: Compared with HCs, IBS-D patients showed increased ALFF in the right cerebellum posterior lobe, the right lingual gyrus/calcarine, the right postcentral gyrus, the right superior frontal gyrus (SFG), and middle frontal gyrus (MFG), with decreased ALFF in the right inferior parietal lobule, the right striatum, the right anterior cingulated cortex, the right insula, the right hippocampus, the right thalamus, the right midbrain, and the left precuneus. IBS-D patients showed increased ReHo in the bilateral lingual gyrus/calcarine, the bilateral SFG, the right MFG, and the right postcentral gyrus, with decreased ReHo in the orbital part of the left inferior frontal gyrus and the right supplementary motor area. Patients showed enhanced FC between the left precuneus and the bilateral orbitofrontal cortex (OFC). There was a positive correlation between increased ALFF values in the right midbrain and anxiety-depression symptoms in IBS-D patients, and the mediating effect of gastrointestinal symptoms indirectly caused this correlation. CONCLUSION: IBS-D patients had dysregulated spontaneous activity and FC in regions related to pain regulation and emotional arousal involved in prefrontal-limbic-midbrain circuit and somatosensory processing. The development of mood disorders in IBS-D patients may be partly related to the dysfunction of components in the dopamine pathway (especially the midbrain, OFC) due to visceral pain.

[Quantitative evaluation of lumbar spine osteoporosis by apparent diffusion coefficient and signal intensity ratio of magnetic resonance diffusion-weighted magnetic resonance imaging].

OBJECTIVE: To investigate the application value of apparent diffusion coefficient (ADC) and signal intensity ratio (SIR) of MR diffusion-weighted imaging (DWI) in quantitative evaluation of lumbar spine osteoporosis. METHODS: A total of 175 patients with lumbar spine diseases who received dualenergy X-ray absorption (DXA) bone mineral density (BMD), routine MRI and DWI of the lumbar spine from May 2017 to October 2019 were selected. According to the T-value of DXA, the patients were divided into osteoporosis group (64 cases), osteopenia group (53 cases) and normal bone mass group (58 cases). The ADC and SIR values of L2-L4 were measured and compared among the three groups and the correlation between ADC, SIR and BMD was analyzed. The ROC curve was used to evaluate the differential diagnosis value of ADC and SIR for osteoporosis, osteopenia and normal bone mass. RESULTS: There were statistically significant differences in ADC and SIR values among three groups (F=41.386, 37.114, all P=0.000). The ADC value of the osteoporosis group was lower than that of the osteopenia group and the normal bone mass group, and the difference was statistically significant (t=3.540, 9.069, P=0.001, 0.000);the SIR value of the osteoporosis group was higher than that of the osteopenia group and the normal bone mass group, and the difference was statistically significant (t=5.083, 8.523, all P=0.000). Spearman correlation analysis showed that ADC value was positively correlated with BMD (r=0.313, P=0.004);SIR value was negatively correlated with BMD (r=- 0.589, P=0.000). Receiver operator characteristic (ROC) curve analysis showed that the area under curve (AUC), sensitivity andspecificity of ADC and SIR in the diagnosis of lumbar osteoporosis and osteopenia were 0.742, 89.1% , 52.8% and 0.729, 89.1%, 50.9% respectively (P=0.000);the AUC, sensitivity and specificity of ADC and SIR in the diagnosis of lumbar osteoporosis and normal bone mass were 0.815, 100.0%, 50.0% and 0.856, 65.6%, 93.1%, respectively (P=0.000);the AUC, sensitivity and specificity of ADC and SIR in the diagnosis of lumbar osteoporosis were 0.78, 89.1%, 51.4% and 0.795, 50.0% and 94.6% respectively (P=0.000);All have a certain diagnostic value. CONCLUSION: ADC and SIR can better reflect the BMD of patients with lumbar diseases, and can quantitatively evaluate the vertebral body of osteoporosis, which play an important role in the diagnosis of lumbar osteoporosis.

[CT Characteristics of Consolidation Type of Pulmonary Cryptococcosis in Immunocompetent Patients].

Objective To analyze the CT characteristics of consolidation type of pulmonary cryptococcosis in immunocompetent patients,and thus improve the diagnosis of this disease. Methods A total of 20 cases with consolidation-type pulmonary cryptococcosis confirmed by pathological examinations were studied.Each patient underwent breath-hold multislice spiral CT,and 10 patients underwent contrast enhanced CT.The data including lesion number,lesion distribution,lesion density,performance of enhanced CT scan,accompanying signs,and prognosis were analyzed. Results The occurrence rates of single and multiple lesions were 80.0%(n=16)and 20.0%(n=4),respectively.In all the 16 multiple-lesion patients,the occurrence rate of unilateral lobar distribution was 56.0%(n=9).The 76 measurable lesions mainly presented subpleural distribution(71.1%,n=54)and lower pulmonary distribution(75.0%,n=57).A total of 39 lesions were detected in the 10 patients received contrast enhanced CT,in which 31 lesions(79.5%)showed homogeneous enhancement,34 lesions(87.2%)showed moderate enhancement,and all the lesions manifested angiogram sign.Consolidation lesions were accompanied by many CT signs,of which air bronchogram sign had the occurrence rate of 63.2%(n=48),including types Ⅲ(n =37)and Ⅳ(n=11).Other signs included halo signs(43/76,56.6%),vacuoles or cavities(9/76,11.8%),pleural thickening(14/20,70.0%),and pleural effusion(2/20,10.0%).After treatment,the lesions of 7 patients were basically absorbed and eventually existed in the form of fibrosis. Conclusions The lesions in the immunocompetent patients with consolidation type of pulmonary cryptococcosis usually occur in the lower lobe and close to the pleura,mainly presenting unilateral distribution.The CT angiogram signs,proximal air bronchogram signs,and halo signs are the main features of this disease,which contribute to the diagnosis.

Associations between low-density lipoprotein cholesterol and haemorrhagic stroke.

OBJECTIVE: To investigate the associations between the blood concentrations of low-density lipoprotein cholesterol (LDL-C) and the clinical features of haemorrhagic stroke. METHODS: This study analysed the data from patients with acute haemorrhagic stroke at a comprehensive stroke centre from 2013 to 2018. Patients were stratified into three groups according to their baseline LDL-C levels: < 70, 70 to < 100 and ≥ 100 mg/dL. We used multivariate logistic regression models to analyse the associations between LDL-C and the risks of having severe neurological deficits (National Institute Health Stroke Scale [NIHSS] scores ≥ 15) and unfavourable outcomes (modified Rankin Scale [mRS] scores>2) at discharge. RESULTS: Six-hundred and six patients were analysed. Their median age was 58 years. Among the patients, 75 (12%) patients had LDL-C levels < 70 mg/dL, 194 (32%) patients had LDL-C levels between 70 to < 100 mg/dL and the other 337 (56%) patients had LDL-C levels ≥ 100 mg/dL. Patients with higher LDL-C levels were less likely to suffer severe neurological deficits (LDL-C: 70 to < 100 vs. < 70 mg/dL, adjusted odds ratio [OR]: 0.29, 95% CI: 0.15-0.57; LDL-C: ≥ 100 vs. < 70 mg/dL, adjusted OR = 0.27, 95% CI: 0.15-0.51) and to have unfavourable outcomes at discharge (LDL-C: 70 to < 100 vs. < 70 mg/dL, adjusted OR = 0.50, 95% CI: 0.29-0.87 and LDL-C: ≥ 100 vs. < 70 mg/dL, adjusted OR = 0.46, 95% CI: 0.28-0.78). CONCLUSIONS: An LDL-C level < 70 mg/dL was independently associated with severe neurological deficits of haemorrhagic stroke and may increase the risks of unfavourable outcomes at discharge.

The current status of syphilis prevention and control in Jiangsu province, China: A cross-sectional study.

OBJECTIVE: To analyze the midterm evaluation data from the National Syphilis Prevention and Control Plan (2010-2020) and evaluate the current status of syphilis prevention and control in Jiangsu province, China. METHODS: We collected data via (1) field surveys conducted in 2015 and (2) data recorded in existing syphilis surveillance systems. We conducted descriptive statistical analysis to evaluate the current landscape of syphilis control initiatives and their potential effect in syphilis control. RESULTS: The incidence of all cases of syphilis decreased from 2010 (32.3 per 100,000) to 2015 (30.1 per 100,000), with an annual growth of -1.17% (x2trend = -7.52, P<0.001) in Jiangsu province. The incidence of primary and secondary syphilis and congenital syphilis both decreased significantly from 2010 to 2015. The average awareness rate of syphilis knowledge among professional personnel was 95.4% (3781/3963). Rural residents had the lowest awareness rate (83.5%, 1875/2245) and commercial sex workers had the highest awareness rate (92.1%, 7804/8474) in 2015. Only 47.8% (33908/70894) of patients received provider-initiated syphilis counseling and testing (PISTC) services in sexually transmitted disease (STD) clinics, but 94.5% (87927/93020) of all syphilis patients received free testing for syphilis. Overall, 97.2% (9378/9648) of syphilis reported cases of syphilis at medical institutions were confirmed to be accurate, and 92.2% (5850/6345) of patients diagnosed with syphilis at medical institutions received treatment with penicillin. CONCLUSION: The syphilis incidence rate in Jiangsu has decreased in recent years, but remains at a high level. It is essential to promote PISTC services to improve knowledge of syphilis and rates of testing and treatment in Jiangsu province.

Functional Role of Histidine in the Conserved His-x-Asp Motif in the Catalytic Core of Protein Kinases.

The His-x-Asp (HxD) motif is one of the most conserved structural components of the catalytic core of protein kinases; however, the functional role of the conserved histidine is unclear. Here we report that replacement of the HxD-histidine with Arginine or Phenylalanine in Aurora A abolishes both the catalytic activity and auto-phosphorylation, whereas the Histidine-to-tyrosine impairs the catalytic activity without affecting its auto-phosphorylation. Comparisons of the crystal structures of wild-type (WT) and mutant Aurora A demonstrate that the impairment of the kinase activity is accounted for by (1) disruption of the regulatory spine in the His-to-Arg mutant, and (2) change in the geometry of backbones of the Asp-Phe-Gly (DFG) motif and the DFG-1 residue in the His-to-Tyr mutant. In addition, bioinformatics analyses show that the HxD-histidine is a mutational hotspot in tumor tissues. Moreover, the H174R mutation of the HxD-histidine, in the tumor suppressor LKB1 abrogates the inhibition of anchorage-independent growth of A549 cells by WT LKB1. Based on these data, we propose that the HxD-histidine is involved in a conserved inflexible organization of the catalytic core that is required for the kinase activity. Mutation of the HxD-histidine may also be involved in the pathogenesis of some diseases including cancer.

Exploring differentially expressed genes in the ovaries of uniparous and multiparous goats using the RNA-Seq (Quantification) method.

Fecundity improvement is one of the most important objectives for goat breeders as it greatly increases production efficiency. To investigate the genes associated with litter sizes in the Anhui White goat (AWG), gene expression differences in the ovaries of uniparous and multiparous AWG were assessed using the RNA-Seq (Quantification) method. This analysis generated 6,027,714 and 5,884,062 clean reads in uniparous and multiparous libraries, respectively. A total of 2201 differentially expressed genes (DEGs) were thereby identified (FDR≤0.001, |log2Ratio|≥1). There were 1583 up-regulated and 618 down-regulated genes in the multiparous samples compared with the uniparous samples. A large number of these DEGs were related to the terms cellular process, cell & cell part and binding. Twelve genes which may be associated with the high prolificacy of AWG were identified using a bioinformatic screen. In addition, pathway analysis revealed that these DEGs were significantly enriched in 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including ECM-receptor interactions, focal adhesion, and regulation of the actin cytoskeleton among others. This suggested a role for these pathways in the prolificacy of AWG. These results provide a list of new candidate genes for goat prolificacy.

[CT findings of tuberous sclerosis associated with hepatic and renal angiomyolipomas].

OBJECTIVE: To summarize the clinical and computed tomographic(CT) findings of tuberous sclerosis(TS) associated with hepatic and renal angiomyolipomas. METHOD: We retrospectively analyzed the clinical and CT findings of 7 TS patients with hepatic and renal angiomyolipomas. RESULTS: Brain CT showed calcified nodules and/or the uncalcified nodule in the lateral ventricle subependymal in 7 patients. Abdominal CT showed bilateral renal angiomyolipomas in 7 cases and hepatic angiomyolipomas in 2 cases. Chest CT showed lymphangioleiomyomatosis in one case. CONCLUSIONS: TS associated with merger hepatic and/or renal angiomyolipomas have typical CT findings. Liver angiomyolipomas shows certain correlation with bilateral renal angiomyolipomas.

Characterization and differential expression of microRNAs in the ovaries of pregnant and non-pregnant goats (Capra hircus).

BACKGROUND: Ovarian follicular development and hormone secretion are complex and coordinated biological processes which will usually be altered during pregnancy. Ovarian function is tightly regulated by a multitude of genes, and also by some specific miRNAs. It is necessary to identify the differentially expressed miRNAs in the ovaries of pregnant and non-pregnant mammals, in order to further understand the role of miRNA-mediated post-transcriptional regulation in mammalian reproduction. Here, we performed a comprehensive search for hircine miRNAs using two small RNA sequencing libraries prepared from the ovaries of pregnant and non-pregnant goats. RESULTS: 617 conserved and 7 putative novel miRNAs were identified in the hircine ovaries. A total of 471 conserved miRNAs (76.34%) were co-expressed in both pregnant and non-pregnant libraries, and 90 pregnancy-specific and 56 non-pregnancy-specific conserved miRNAs were identified. Additionally, 407 unique miRNAs (65.96%) were significantly differentially expressed in the pregnant and non-pregnant libraries, of which 294 were upregulated and 113 were downregulated in the pregnant library compared to the non-pregnant library. Further analysis showed that miR-143 was predicted to bind to the target sequences of Frizzled-6 and -3 receptor genes in the Wnt/beta-catenin signaling pathway, and let-7b may target the Activin receptor I and Smad 2/3 genes in the TGF-beta signaling pathway. The expression level of 5 randomly selected miRNAs were analyzed by quantitative real-time PCR (q-PCR), and the results demonstrated that the expression patterns were consistent with the Solexa sequencing results. CONCLUSIONS: The identification and characterization of differentially expressed miRNAs in the ovaries of pregnant and non-pregnant goats provides important information on the role of miRNA in the regulation of the ovarian development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during mammalian reproduction.

Reference gene screening for analyzing gene expression across goat tissue.

Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

Parameters and grading of evoked potentials: prediction of unfavorable outcome in patients with severe stroke.

To explore the prognostic accuracy for unfavorable outcome with short-latency somatosensory evoked potential (SLSEP) and brainstem auditory evoked potential (BAEP) in patients with severe stroke, 100 acute severe supratentorial stroke patients [Glasgow Coma Scale, (GCS) 0.4). Prognostic accuracy of SLSEP, BAEP, and GCS for unfavorable outcome was very high (94.3%-98.7%), among which BAEP is the highest (97.5%-98.7%). The overall prognostic accuracy of SLSEP (90.5%-93.7%) was higher than BAEP (83.0%-89.4%) and GCS (82%). The prognostic accuracy of SLSEP and BAEP for unfavorable outcome in patients with severe supratentorial stroke was high, whereas for favorable outcome, it was low. The overall prognostic accuracy was higher than GCS.

Predicting hospital mortality using APACHE II scores in neurocritically ill patients: a prospective study.

Four versions of Acute Physiology and Chronic Health Evaluation are limited in predicting hospital mortality for neurocritically ill patients. This prospective study aimed to develop and assess the accuracy of a modified APACHE II model in predicting mortality in neurologic intensive care unit (N-ICU). A total of 653 patients entered the study. APACHE II scores on admission, and worst 24-, 48-, and 72-h scores were obtained. Neurologic diagnoses on admission were classified into five categories: cerebral infarction, intracranial hemorrhage, neurologic infection, neuromuscular disease, and other neurologic diseases. We developed a modified APACHE II model based on the variables of the 72-h APACHE II score and disease category using a multivariate logistic regression procedure to estimate probability of death. We assessed the calibration and discrimination of the modified APACHE II model using the Hosmer-Lemeshow goodness-of-fit chi-squared statistic and area under the receiver operating characteristic curve (AU-ROC). The modified APACHE II model had good discrimination (AU-ROC = 0.88) and calibration (Hosmer-Lemeshow statistic: chi (2) = 3.707, P = 0.834). The discrimination of the 72-h APACHE II score for cerebral infarction, intracerebral hemorrhage, and neurologic infection was satisfactory, with AU-ROC of 0.858, 0.863, and 1.000, respectively, but it was poor in discriminating for the categories of other neurologic diseases and neuromuscular disease. The results showed that our modified APACHE II model can accurately predict hospital mortality for patients in N-ICU. It is more applicable to clinical practice than the previous model because of its simplicity and ease of use.

Comparison of the crystal structure and function to wild-type and His25Ala mutant human heme oxygenase-1.

Human heme oxygenase-1 (hHO-1) is a rate-limiting enzyme in heme metabolism. It regulates serum bilirubin level. Site-directed mutagenesis studies indicate that the proximal residue histidine 25 (His25) plays a key role in hHO-1 activity. A highly purified hHO-1 His25Ala mutant was generated and crystallized with a new expression system. The crystal structure of the mutant was determined by X-ray diffraction technology and molecular replacement at the resolution of 2.8 A, and the model of hHO-1 His25Ala mutant was refined. The final crystallographic and free R factors were 0.245 and 0.283, respectively. The standard bond length deviation was 0.007 A, and the standard bond angle deviation was 1.3 degrees . The mutation of His25 to Ala led to an empty pocket underneath the ferric ion in the heme, leading to loss of binding iron ligand. Although this did not cause an overall structural change, the enzymatic activity of the mutant hHO-1 was reduced by 90%. By supplementing imidazole, the HO-1 activity was restored approximately 90% to its normal level. These data suggest that Ala25 remains unchanged in the structure compared to His25, but the important catalytic function of hHO-1 is lost. Thus, it appears that His25 is a crucial residue for proper hHO-1 catalysis.

Isoquinoline-1,3,4-trione derivatives inactivate caspase-3 by generation of reactive oxygen species.

Caspase-3 is an attractive therapeutic target for treatment of diseases involving disregulated apoptosis. We report here the mechanism of caspase-3 inactivation by isoquinoline-1,3,4-trione derivatives. Kinetic analysis indicates the compounds can irreversibly inactivate caspase-3 in a 1,4-dithiothreitol (DTT)- and oxygen-dependent manner, implying that a redox cycle might take place in the inactivation process. Reactive oxygen species detection experiments using a chemical indicator, together with electron spin resonance measurement, suggest that ROS can be generated by reaction of isoquinoline-1,3,4-trione derivatives with DTT. Oxygen-free radical scavenger catalase and superoxide dismutase eliciting the inactivation of caspase-3 by the inhibitors confirm that ROS mediates the inactivation process. Crystal structures of caspase-3 in complexes with isoquinoline-1,3,4-trione derivatives show that the catalytic cysteine is oxidized to sulfonic acid (-SO(3)H) and isoquinoline-1,3,4-trione derivatives are bound at the dimer interface of caspase-3. Further mutagenesis study shows that the binding of the inhibitors with caspase-3 appears to be nonspecific. Isoquinoline-1,3,4-trione derivative-catalyzed caspase-3 inactivation could also be observed when DTT is substituted with dihydrolipoic acid, which exists widely in cells and might play an important role in the in vivo inactivation process in which the inhibitors inactivate caspase-3 in cells and then prevent the cells from apoptosis. These results provide valuable information for further development of small molecular inhibitors against caspase-3 or other oxidation-sensitive proteins.

Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion.

CD147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC)-associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8A resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected by a 5-residue flexible linker. This unique C2-I domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/hetero-oligomerizations and should represent a general architecture of other CD147 family members.

A putative new domain target for anti-hepatitis B virus: residues flanking hepatitis B virus reverse transcriptase residue 306 (rtP306).

Previous work showed that conservation of proline at residue 306 (rtP306) of hepatitis B virus (HBV) reverse transcriptase (RT) is crucial for virus replication and encapsidation of pregenomic RNA (pgRNA). In this study, the functions of residues flanking rtP306 in HBV RT (rtG304, rtY305, rtA307, rtL308 and rtL311) are presented. Alanine or phenylalanine was used to substitute these residues by constructing site-directed mutants which were used to transfect Huh-7 cells. Replication competencies and encapsidation efficiencies were compared between the mutants and the parental viral strain. Substitutions at these residues resulted in marked decrease of replication competency, which was confirmed by Southern blot hybridization of HBV DNA isolated from intracytoplasmic core particles, and trans-complementation between a non-replicative defective mutant and corresponding RT mutants. Impaired pgRNA encapsidation efficiency of these mutants was shown as the major mechanism for decreased replication efficiency. Since residues from rt304 to rt311 are highly conserved among genotypes A-H HBV strains, results suggest that rt304 to rt311 in HBV RT may serve as a putative anti-HBV new target domain.

Mutational analysis revealed that conservation of hepatitis B virus reverse transcriptase residue 306 (rtP306) is crucial for encapsidation of pregenomic RNA.

Hepatitis B virus (HBV) is a DNA virus which replicates via reverse transcription. The structure and function of the reverse transcriptase play important roles in HBV replication. We have previously reported that when proline at residue 306 in HBV reverse transcriptase was substituted by other amino acids, most of the mutants showed decreased replicative competency. To explore the mechanisms for this decrease in replicative competency, constructs with substituted amino acid residues at rtP306 were used to transfect Huh-7 cells, and replication competencies, transcription levels and encapsidation efficiencies of the mutants and the parental viral strain were compared. Decreased replication competency was found with many of the mutants and confirmed by trans-complementation between each mutant and a replication-defective replicon. No change in transcriptional level was detected between all mutated constructs. The encapsidation competencies of these constructs were studied by assaying pregenomic RNAs in intracytoplamic core particles from transfected cells, which were normalized for the amount of HBV core protein by Western blotting using anti-core antibodies. Impaired encapsidation was found in several mutants substituted at residue 306, thereby demonstrating for the first time that conservation of proline at this residue is crucial for efficient encapsidation of pregenomic RNA.