MetaGeneHunt for protein domain annotation in short-read metagenomes.

The annotation of short-reads metagenomes is an essential process to understand the functional potential of sequenced microbial communities. Annotation techniques based solely on the identification of local matches tend to confound local sequence similarity and overall protein homology and thus don't mirror the complex multidomain architecture and the shuffling of functional domains in many protein families. Here, we present MetaGeneHunt to identify specific protein domains and to normalize the hit-counts based on the domain length. We used MetaGeneHunt to investigate the potential for carbohydrate processing in the mouse gastrointestinal tract. We sampled, sequenced, and analyzed the microbial communities associated with the bolus in the stomach, intestine, cecum, and colon of five captive mice. Focusing on Glycoside Hydrolases (GHs) we found that, across samples, 58.3% of the 4,726,023 short-read sequences matching with a GH domain-containing protein were located outside the domain of interest. Next, before comparing the samples, the counts of localized hits matching the domains of interest were normalized to account for the corresponding domain length. Microbial communities in the intestine and cecum displayed characteristic GH profiles matching distinct microbial assemblages. Conversely, the stomach and colon were associated with structurally and functionally more diverse and variable microbial communities. Across samples, despite fluctuations, changes in the functional potential for carbohydrate processing correlated with changes in community composition. Overall MetaGeneHunt is a new way to quickly and precisely identify discrete protein domains in sequenced metagenomes processed with MG-RAST. In addition, using the sister program "GeneHunt" to create custom Reference Annotation Table, MetaGeneHunt provides an unprecedented way to (re)investigate the precise distribution of any protein domain in short-reads metagenomes.

Systemic lupus erythematosus-associated diffuse alveolar haemorrhage: a single-centre experience in Han Chinese patients.

OBJECTIVE: Despite aggressive therapeutic regimens, diffuse alveolar haemorrhage (DAH) is still associated with a high mortality rate in systemic lupus erythematosus (SLE). This study was carried out in patients with SLE-associated DAH with a focus on their therapeutic modality. METHOD: A retrospective review was performed in 839 Han Chinese lupus patients hospitalized for their DAH manifestation from May 2006 to December 2016. RESULTS: There were 24 episodes in 17 cases (2.0% incidence), 15 females and two males aged 19-67 years (mean ± sd 38.2 ± 15.1 years). High disease activity [Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) 12-31, 19.8 ± 5.6] was found at the onset of DAH. All patients were treated with high-dose corticosteroid, followed by pulse methylprednisolone (70.6%), plasmapheresis (41.2%), pulse cyclophosphamide (35.3%), and rituximab (23.5%). Six patients (35.3%), including three with extracorporeal membrane oxygenation, died owing to acute respiratory failure. All patients receiving rituximab treatment survived with a follow-up period of 12-58 months (40.8 ± 21.1 months), and no further relapse was noted in three cases with a history of recurrent DAH episodes. In addition, there was a significant decrease in their lupus activity (SLEDAI-2K 21.5 ± 6.0 to 6.3 ± 1.7, p = 0.0286). CONCLUSION: In this single-centre series with SLE-associated DAH in Han Chinese patients, a beneficial effect of rituximab therapy was observed.

Potent antitumor activity of Oct4 and hypoxia dual-regulated oncolytic adenovirus against bladder cancer.

Most solid tumors undergo hypoxia, leading to rapid cell division, metastasis and expansion of a cell population with hallmarks of cancer stem cells (CSCs). Tumor-selective replication of oncolytic adenoviruses may be hindered by oxygen deprivation in tumors. It is desirable to develop a potent oncolytic adenovirus, retaining its antitumor activity even in a hypoxic environment. We have previously generated an Oct4-dependent oncolytic adenovirus, namely Ad9OC, driven by nine copies of the Oct4 response element (ORE) for specifically killing Oct4-overexpressing bladder tumors. Here, we developed a novel Oct4 and hypoxia dual-regulated oncolytic adenovirus, designated AdLCY, driven by both hypoxia response element (HRE) and ORE. We showed that hypoxia-inducible factor (HIF)-2α and Oct4 were frequently overexpressed in hypoxic bladder cancer cells, and HIF-2α was involved in HRE-dependent and Oct4 transactivation. AdLCY exhibited higher cytolytic activities than Ad9OC against hypoxic bladder cancer cells, while sparing normal cells. AdLCY exerted potent antitumor effects in mice bearing human bladder tumor xenografts and syngeneic bladder tumors. It could target hypoxic CD44- and CD133-positive bladder tumor cells. Therefore, AdLCY may have therapeutic potential for targeting hypoxic bladder tumors and CSCs. As Oct4 is expressed in various cancers, AdLCY may be further explored as a broad-spectrum anticancer agent.

Electromagnetic thermotherapy using fine needles for hepatoma treatment.

BACKGROUND AND AIMS: Hepatocellular carcinoma can be treated with heat-based therapies, especially radiofrequency ablation (RFA). However, RFA has limited efficacy and is quite expensive. We designed a new system using fine needles combined with an alternating magnetic field to generate hyperthermia for the treatment of hepatocellular carcinoma in a rat hepatoma model. Our aims are to assess the efficacy of our method and determine survival up to 30 days. METHODS: An N1-S1 cell line was inoculated into the livers of Sprague-Dawley rats, generating tumors after 14 days. The animals were randomized into 5 groups and treated after laparotomy either with normal saline (group I), iron oxide nanoparticles (group II), fine needles (group III), fine needles and iron oxide nanoparticles combined (group IV) or self-designed two-part needles placed under ultrasonographic guidance percutaneously (group V). Every rat was placed in an alternating magnetic field. The temperature in the treatment area was maintained between 55 and 60 °C. At day 30 after treatment, tumor volumes and mortality were assessed and histology samples were studied. RESULTS: Tumor volumes were significantly reduced and survival rate was prolonged in groups III, IV and V versus groups I and II (P < 0.05). On pathological examination, groups III, IV and V presented obvious necrosis, apoptosis, calcifications and inflammatory changes in the treatment area. CONCLUSION: Our study demonstrates that hyperthermia generated by fine stainless-steel needles combined with an alternating magnetic field effectively inhibits hepatoma growth in rats and prolongs their survival. Further, this method can be applied percutaneously under ultrasonographic guidance.

Identifying LRRC16B as an oncofetal gene with transforming enhancing capability using a combined bioinformatics and experimental approach.

Oncofetal genes are expressed in embryos or fetuses, are downregulated or undetectable in adult tissues, and then re-expressed in tumors. Known oncofetal genes, such as AFP, GCB, FGF18, IMP-1 and SOX1, often have important clinical applications or pivotal biological functions. To find new oncofetal-like genes, we used the public information of expressed sequence tags to systematically analyze gene expression patterns and identified a novel oncofetal-like gene, LRRC16B. It increased the proliferation, anchorage-independent growth and tumorigenesis of transformed cells in xenografts, possibly through its effects on cyclin B1 protein levels. These findings exemplify the feasibility of using bioinformatics to find new oncofetal-like genes and suggest that more genes with important functional roles will be uncovered in the candidate gene list.

Association of tryptophan hydroxylase gene polymorphism with depression, anxiety and comorbid depression and anxiety in a population-based sample of postpartum Taiwanese women.

Depression and anxiety disorders often coexist clinically and both are known to have a genetic basis, but the mode of inheritance is too complicated to be determined so far. Serotonin is the biogenic amine neurotransmitter most commonly associated with depression and anxiety. Since tryptophan hydroxylase (TPH1) is the rate-limiting enzyme in serotonin biosynthesis, its role in the pathophysiology of these psychiatric diseases has been intensively studied. In this study, we examined whether polymorphism of the TPH1 gene is related to the etiology of major depression, anxiety and comorbid depression and anxiety. Five single nucleoside polymorphisms of the TPH1 gene were studied in a population-based sample of postpartum Taiwanese women consisting of 120 subjects with depression or/and anxiety and 86 matched normal controls. A significant difference (P = 0.0107) in genotype frequency for the T27224C polymorphism was found between the comorbid and normal groups, and risk analysis showed that the C allele conferred a strong protective effect (odds ratio = 0.27; 95% confident interval = 0.11-0.7). Three-allele haplotypes involving T27224C polymorphism were constructed and haplotype associations between particular haplotype combinations and various diseases identified. However, the associations were weak and the overall haplotype frequency profiles in all groups were similar. The results suggest that depression, anxiety, and comorbid depression and anxiety disorders may have related etiologies. In addition, this study suggests that the TPH1 gene might play a role in the pathogenesis of these closely related disorders.

Oestrogen receptor-alpha and -beta immunoreactivity in gonadotropin-releasing hormone neurones after ovariectomy and chronic exposure to oestradiol.

Oestradiol exerts negative- and positive-feedback actions on luteinizing hormone (LH) secretion by modulating gonadotropin-releasing hormone (GnRH) release. Furthermore, a chronic increase in circulating oestradiol in either young ovariectomized (OVX) rats, or in middle-aged persistent oestrous (PE) rats, causes a gradual attenuation of LH surges until the positive-feedback action of oestradiol disappears. Based on these findings, and on the equivocal evidence regarding a direct action of oestradiol on GnRH neurones, we tested the hypothesis that chronic oestradiol abolishes LH surges by decreasing the proportion of GnRH neurones containing oestrogen receptor (ER)alpha or beta. Regularly cycling rats were ovariectomized, and half immediately received oestradiol. Three days, or 2 or 4 weeks later, rats were perfused at 18.00 h, and GnRH was colocalized with ERalpha or ERbeta by immunocytochemistry. ERbeta was expressed in 76% of GnRH neurones, whereas virtually no GnRH cells were immunopositive for ERalpha. The proportion of GnRH cells expressing ERalpha or beta in OVX rats was not altered by oestradiol or time after OVX, and this was the case regardless of their medial to lateral, or rostral to caudal location. The results indicate that the mechanisms for the positive-feedback action of oestradiol, and the loss of LH surges in OVX rats after chronic oestradiol, are not mediated by changes in the proportion of oestrogen-receptor containing GnRH neurones.

Chronic elevation of estradiol in young ovariectomized rats causes aging-like loss of steroid-induced luteinizing hormone surges.

This study was designed to test the hypothesis that the loss of LH surges in response to the stimulatory actions of estradiol and progesterone in middle-aged, persistent-estrous (PE) rats may be caused by chronic elevations in circulating estradiol. Five groups of regularly cycling young rats received an s.c. estradiol implant immediately after ovariectomy (Day 0). For determination of LH surges, blood samples were collected hourly between 1200-1900 h from each of the five groups at one of the following times: 3 days, or 1, 2, 4, or 8 wk later. On the next day, either progesterone (0.5 mg/100 g BW) or corn oil was injected s.c. at 1200 h, and samples were obtained as before. Incidence and amplitude of estradiol-induced LH surges decreased during the first 2 wk of estradiol treatment, after which no surges occurred. Progesterone enhanced the incidence and amplitude of estradiol-induced LH surges thus delaying their disappearance. These results support our hypothesis and demonstrate that the stimulatory actions of estradiol and progesterone on the LH surge sequentially diminish with time after exposure to estradiol in young rats. Thus, young rats chronically treated with estradiol may be a useful model for studying the mechanisms whereby LH surges are abolished in middle age during the hyperestrogenic state of PE.

Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA.

We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in E. coli DH5alpha reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E. coli or V. vulnificus. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of V. vulnificus mice.

Neoplasia in zebrafish (Danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two exposure routes at different developmental stages.

Using zebrafish, Danio rerio, initial pioneering work in the 1960s revealed carcinogen responsiveness of fish, yet very few subsequent tumorigenesis investigations have utilized this species. We exposed embryos (60 hours postfertilization) and fry (3 week posthatch) to 7,12-dimethylbenz[a]anthracene (DMBA) by immersion in aqueous solutions for 24 hours, at concentrations of 0-1 or 0-5 ppm (mg/L), respectively. Juvenile zebrafish 2 months posthatch were fed a diet containing 0-1,000 ppm DMBA for 4 months. Fish were sampled for histologic evaluation at 7-12 months after the onset of carcinogen treatment. Fry were most responsive to DMBA and showed the widest diversity of target tissues and histologic types of neoplasia, having several types of epithelial, mesenchymal, and neural neoplasia. The principal target tissues for carcinogenic response were liver following embryo or fry exposure, with gill and blood vessel the second and third most responsive tissues in fry. Intestine was the primary target and gill a secondary target in fish that received dietary DMBA as juveniles. These studies indicate that young zebrafish are most responsive to DMBA, showing a greater diversity of neoplasm types than rainbow trout. Thus, zebrafish are a valuable model system in which to study mechanistic aspects of the carcinogenesis process.

Neoplasia in zebrafish (Danio rerio) treated with N-methyl-N'-nitro-N-nitrosoguanidine by three exposure routes at different developmental stages.

We exposed embryos (83 hours postfertilizaton) and fry (3 weeks posthatch) to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by immersion in aqueous solutions of 0-10 ppm for 1 hour (embryo) or 0-2 ppm for 24 hours (fry). Zebrafish embryos were microinjected with MNNG at levels of 0 or 96 ng/egg. Diets containing 0-2,000 ppm MNNG were fed to juvenile zebrafish for 3 months beginning at 2 months posthatch. Fish were sampled for histopathologic study at 6-12 months after initiation of carcinogen exposure. Embryos and fry were both quite responsive to MNNG; however, juvenile zebrafish were remarkably refractory to MNNG-induced neoplasia. Principal target organs in zebrafish treated as embryos with MNNG were liver and testis, with hepatocellular adenoma the most prevalent hepatic neoplasm. A variety of mesenchymal neoplasms occurred in zebrafish following embryo exposure to MNNG, including chondroma, hemangioma, hemangiosarcoma, leiomyosarcoma, and rhabdomyosarcoma. Testis and blood vessels were primary target organs for MNNG following fry exposure, with seminoma, hemangioma, hemangiosarcoma, and various other epithelial and mesenchymal neoplasms occurring. The zebrafish is a responsive, cost-effective lower vertebrate model system in which to study mechanisms of carcinogenesis.

Malignant fibrous histocytoma of the heart presenting as right ventricular outflow tract obstruction: a case report.

Primary malignant fibrous histocytoma (MFH) of the heart is extremely rare. Herein, we report the case of a 17-year-old woman with a primary MFH in the right ventricular outflow tract, presenting with increasing dyspnea on exertion. Wide excision of the tumor, including part of the pulmonary artery and pulmonary valve, was performed under cardiopulmonary bypass. Her postoperative recovery was uneventful.

Male sexual behavior is associated with LHRH neuron number in middle-aged rats.

LHRH administration is reported to facilitate male sexual behavior. The aim of the present study was to investigate whether male sexual behavior is associated with the number of LHRH neurons in the forebrain in middle-aged rats. Male Long-Evans rats (18-19 months) were assigned to three groups on the basis of sexual performance: (1) group MEI consisted of rats showing complete copulatory patterns, including mounts, intromissions and ejaculations, (2) group MI was composed of rats showing mounts and intromissions, but no ejaculation and (3) group NC were non-copulators, i.e. they did not show any copulatory behavior. Young adult rats (4-5 months), displaying sexual behavior, were used as controls. Following the sexual behavior tests, the number of LHRH neurons in the medial septum (MS), organum vasculosum of the lamina terminalis (OVLT), preoptic area (POA) and anterior hypothalamus (AH) was determined by immunocytochemistry. No difference was seen in the total number of LHRH neurons in these combined brain areas between group MIE and young controls. In the three middle-aged groups, the total number of LHRH neurons was greatest in group MIE, less in group MI, and lowest in group NC. In general, a similar trend was seen separately in the MS, OVLT and POA. These results suggest that changes in the number of LHRH neurons in the forebrain, in most cases, are age-related, at least in the middle-aged rats, but they also seem to be associated with male sexual performance.