Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain

It has been shown that induction of HSP70 by administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury. The present study was performed to determine the effect of GGA on the survival of mice and on brain damage under acute hypobaric hypoxia. The data showed that the mice injected with GGA survived significantly longer than control animals (survival time of 9.55 ± 3.12 min, n = 16 vs. controls at 4.28 ± 4.29 min, n = 15, P < 0.005). Accordingly, the cellular necrosis or degeneration of the hippocampus and the cortex induced by sublethal hypoxia for 6 h could be attenuated by preinjection with GGA, especially in the CA2 and CA3 regions of the hippocampus. In addition, the activity of nitric oxide synthase (NOS) of the hippocampus and the cortex was increased after exposure to sublethal hypoxia for 6 h but could be inhibited by the preinjection of GGA. Furthermore, the expression of HSP70 was significantly increased at 1 h after GGA injection. These results suggest that administration of GGA improved survival rate and prevented acute hypoxic damage to the brain and that the underlying mechanism involved induction of HSP70 and inhibition of NOS activity.     

A Genetic Survey of Fluoxetine Action on Synaptic Transmission in Caenorhabditis elegans

Fluoxetine is one of the most commonly prescribed medications for many behavioral and neurological disorders. Fluoxetine acts primarily as an inhibitor of the serotonin reuptake transporter (SERT) to block the removal of serotonin from the synaptic cleft, thereby enhancing serotonin signals. While the effects of fluoxetine on behavior are firmly established, debate is ongoing whether inhibition of serotonin reuptake is a sufficient explanation for its therapeutic action. Here, we provide evidence of two additional aspects of fluoxetine action through genetic analyses in Caenorhabditis elegans. We show that fluoxetine treatment and null mutation in the sole SERT gene mod-5 eliminate serotonin in specific neurons. These neurons do not synthesize serotonin but import extracellular serotonin via MOD-5/SERT. Furthermore, we show that fluoxetine acts independently of MOD-5/SERT to regulate discrete properties of acetylcholine (Ach), gamma-aminobutyric acid (GABA), and glutamate neurotransmission in the locomotory circuit. We identified that two G-protein–coupled 5-HT receptors, SER-7 and SER-5, antagonistically regulate the effects of fluoxetine and that fluoxetine binds to SER-7. Epistatic analyses suggest that SER-7 and SER-5 act upstream of AMPA receptor GLR-1 signaling. Our work provides genetic evidence that fluoxetine may influence neuronal functions and behavior by directly targeting serotonin receptors.FLUOXETINE is a selective serotonin reuptake inhibitor (SSRI) and has made a major impact on the treatment of many behavioral disorders. The empirical action of SSRIs is blocking the serotonin reuptake transporter (SERT). SERT is localized in the plasma membrane and transports extracellular serotonin (5-HT) into the cytoplasm (Blakely et al. 1991; Hoffman et al. 1991), this being the major mechanism of terminating 5-HT signaling. Consequently, SSRIs are thought to exert therapeutic effects by blocking SERT from removal of 5-HT in the synaptic clef, thereby increasing the level of 5-HT signals (Schatzberg and Nemeroff 2004). However, several observations point to additional actions of SSRIs on the 5-HT system and neuronal functions. First, knockout of SERT in mouse caused a marked reduction of 5-HT in the brain (Bengel et al. 1998). Second, a variety of studies with cultured mammalian cells and mouse brain slices showed that SSRIs and tricyclic antidepressant agents (TCAs) have high affinities to many 5-HT receptor subtypes and act as agonists or antagonists depending on particular receptors being tested (Ni and Miledi 1997; Kroeze and Roth 1998; Eisensamer et al. 2003). Third, genetic analyses of the nematode Caenorhabditis elegans in our laboratory and others showed that fluoxetine and the TCA imipramine (Tofrani) could influence behavior independent of SERT function (Weinshenker et al. 1995; Ranganathan et al. 2001; Dempsey et al. 2005). In this study, we carried out a systematic survey of SSRIs treatment in C. elegans to gain new insights into actions of SSRIs on the 5-HT system and other neurotransmitter systems.In both vertebrates and invertebrates, 5-HT functions as a neuromodulator to either facilitate or inhibit synaptic transmission of other neurotransmitters (Fink and Gothert 2007). Modulation of synaptic activity by 5-HT signaling underscores the synaptic plasticity involved in stress responses, learning, adaptation, and memory (Kandel 2001; Zhang et al. 2005). The role of 5-HT in C. elegans was initially identified through pharmacological experiments showing that exogenous 5-HT can promptly induce changes in a variety of behaviors, including feeding, egg laying, and locomotion (Avery and Horvitz 1990; Weinshenker et al. 1995; Nurrish et al. 1999). The relevance of these behaviors to endogenous 5-HT has since been validated through studies of mutants of 5-HT signaling. Importantly, multiple 5-HT receptors may function in distinct cells synergistically or antagonistically to regulate a specific behavior (Carnell et al. 2005; Dernovici et al. 2007; Murakami and Murakami 2007; Hapiak et al. 2009). In nearly all tested paradigms, fluoxetine and imipramine induce behavioral changes similarly to exogenous 5-HT (Weinshenker et al. 1995; Nurrish et al. 1999), implying that fluoxetine regulates 5-HT inputs to these neural circuits. However, the tryptophan hydroxylase gene tph-1 is required for 5-HT biosynthesis in C. elegans (Sze et al. 2000), mod-5 encodes its sole SERT (Ranganathan et al. 2001), and yet fluoxetine could stimulate egg laying and inhibit locomotion in mod-5 and tph-1 mutants (Weinshenker et al. 1995; Choy and Thomas 1999; Ranganathan et al. 2001; Dempsey et al. 2005). These findings provided a basis for further investigation into genes and synaptic functions regulated by 5-HT and the impact of fluoxetine on 5-HT signaling.Here we present genetic evidence of multifaceted effects of fluoxetine on the 5-HT system and its downstream targets in C. elegans. We show that fluoxetine treatment and loss of MOD-5/SERT function do not simply increase presynaptic 5-HT signals. Rather, they may eliminate 5-HT in specific neurons. Furthermore, fluoxetine acts independently of SERT to regulate 5-HT serotonin receptors and their downstream targets involved in acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glutamate neurotransmission.     

Bioturbation of Burrowing Crabs Promotes Sediment Turnover and Carbon and Nitrogen Movements in an Estuarine Salt Marsh

Ecological functions of bioturbation in ecosystems have received increasing attention over the recent decades, and crab burrowing has been considered as one of the major bioturbations affecting the physical and chemical processes in salt marshes. This study assessed the integrated effects of crab excavating and burrow mimic trapping on sediment turnover and vertical C and N distributions in a Chinese salt marsh in the Yangtze River estuary. Crab burrowing increased soil water content and the turnover of carbon and nitrogen and decreased bulk soil density. Vertical movement of materials, nutrient cycling and reuse driven by crab burrowing might be obstructed by vegetation (Phragmites australis and Spartina alterniflora communities). The amount of soil excavated by crab burrowing was higher than that deposited into burrow mimics. In Phragmites marshes, Spartina marshes and unvegetated mudflats, net transport of soil to the marsh surface was 171.73, 109.54, and 374.95 g m⁻² d⁻¹, respectively; and the corresponding estimated soil turnover time was 2.89, 4.07 and 1.83 years, respectively. Crab burrowing in salt marshes can mix surface and deeper soil over a period of years, accelerating litter decomposition and promoting the efficient reuse of nutrients by plants. Therefore, bioturbation affects soil physical processes and functioning of ecosystems, and needs to be addressed in ecosystem management.     

Upregulation of plasma C9 protein in gastric cancer patients

Gastric cancer is one of the leading causes of cancer‐related deaths worldwide. Current biomarkers used in the clinic do not have sufficient sensitivity for gastric cancer detection. To discover new and better biomarkers, protein profiling on plasma samples from 25 normal, 15 early‐stage and 21 late‐stage cancer was performed using an iTRAQ‐LC‐MS/MS approach. The level of C9 protein was found to be significantly higher in gastric cancer compared with normal subjects. Immunoblotting data revealed a congruent trend with iTRAQ results. The discriminatory power of C9 between normal and cancer states was not due to inter‐patient variations and was independent from gastritis and Helicobacter pylori status of the patients. C9 overexpression could also be detected in a panel of gastric cancer cell lines and their conditioned media compared with normal cells, implying that higher C9 levels in plasma of cancer patients could be attributed to the presence of gastric tumor. A subsequent blind test study on a total of 119 plasma samples showed that the sensitivity of C9 could be as high as 90% at a specificity of 74%. Hence, C9 is a potentially useful biomarker for gastric cancer detection.     

Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target.     

High Dengue Case Capture Rate in Four Years of a Cohort Study in Nicaragua Compared to National Surveillance Data

Dengue is a major public health problem in tropical and subtropical regions; however, under-reporting of cases to national surveillance systems hinders accurate knowledge of disease burden and costs. Laboratory-confirmed dengue cases identified through the Nicaraguan Pediatric Dengue Cohort Study (PDCS) were compared to those reported from other health facilities in Managua to the National Epidemiologic Surveillance (NES) program of the Nicaraguan Ministry of Health. Compared to reporting among similar pediatric populations in Managua, the PDCS identified 14 to 28 (average 21.3) times more dengue cases each year per 100,000 persons than were reported to the NES. Applying these annual expansion factors to national-level data, we estimate that the incidence of confirmed pediatric dengue throughout Nicaragua ranged from 300 to 1000 cases per 100,000 persons. We have estimated a much higher incidence of dengue than reported by the Ministry of Health. A country-specific expansion factor for dengue that allows for a more accurate estimate of incidence may aid governments and other institutions calculating disease burden, costs, resource needs for prevention and treatment, and the economic benefits of drug and vaccine development.     

Nuclear Magnetic Resonance Studies on Huwentoxin-Ⅺ from the Chinese Bird Spider Ornithoctonus huwena:~(15)N Labeling and Sequence-specific ~1H,~(15)N Nuclear Magnetic Resonance Assignments

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin withboth antiprotease activity and potassium channel blocking activity.To determine its solution structure,huwentoxin-Ⅺ was expressed in a yeast eukaryotic expression system and studied by NMR.The ~(15)N labelingstrategy was used to facilitate the process of resonance assignments.The nearly complete sequence-specificassignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional(2D)and three-dimensional(3D)spectra,including DQF-COSY,TOCSY,NOESY,~(15)N-~1H HSQC,~(15)N-~1H HNHA,~(15)N-~1H HNHB,~(15)N-~1H TOCSY-HSQC and ~(15)N-~1H NOESY-HSQC spectra.Secondary structure analysis ofhuwentoxin-Ⅺ showed that it mainly contains an N-terminal 3_(10)-helix from Thr3 to Arg5 and a C-terminalα-helix from Gln45 to Cys52,plus a triple-stranded antiparallel β-sheet of Glul 8-Asn23,Thr26-Ile31 andAsn40-Lys41.These studies provide a solid basis for the final structure determination of huwentoxin-Ⅺ.     

Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model

We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitro phenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis.