Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (⌬ssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ⌬ssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.Salmonella enterica serovars are intracellular pathogens that can cause gastroenteritis and typhoid fever. In the developing world, they are a leading cause of morbidity and mortality resulting from dehydration and untreated sepsis (21)(22)43). Salmonella actively secretes effector proteins into the host cell cytoplasm to create a replicative niche and inhibit the immune system. Many of these effectors are delivered by one of two type III secretion systems (TTSS), which are encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2, respectively) (24). The SPI-1 TTSS facilitates host cell entry and inflammation, whereas SPI-2 mediates intracellular survival (19,51). Both SPI-1 and SPI-2 are required in a mouse model of persistent infection (35). While over 30 TTSS effectors have been identified to date (5,25, 42,(58)(59), the list is thought to be an underestimate of the true effector repertoire because several virulence phenotypes are dependent on TTS but are not linked to any known effectors (34,57).A proteomic study of Escherichia coli O157:H7 identified over 31 new type III effectors. This analysis took advantage of a sepL mutant that secreted effector proteins into culture medium in vitro (60). E. coli SepL interacts with Tir (63), a type III effector that inserts into the host plasma membrane and functi...

We investigated the capability of Microcystis aeruginosa to cause apoptosis by pursuing morphological, molecular and physiological characteristics after exposure to H 2 O 2 . Microcystis proliferation was only weakly affected after exposure to 150 mM H 2 O 2 but cell numbers decreased dramatically after exposures of 250 and 325 mM H 2 O 2 . Cells exposed to 250 and 325 mM H 2 O 2 were examined using transmission electron microscopy, and they exhibited membrane deformation and partial disintegration of thylakoids. Correspondingly, fluorescence imaging of DNA by Hoechst 33342 staining revealed the condensation of nucleoid chromatin. Moreover, cellular injury was concomitant with dramatic decreases in photosynthetic efficiency (ratio of variable fluorescence to maximum fluorescence [Fv/Fm], maximum electron transport rate [ETRmax]) and elevated caspase-3-like activity after exposure of 250 and 325 mM H 2 O 2 . Terminal deoxynucleotidyl transferase Deoxyuridine 5-triphosphate nick end labelling (TUNEL) positive staining appeared in cells exposed to 250 mM and 325 mM H 2 O 2 , and the percentage staining increased with increasing H 2 O 2 concentration. These data suggested that M. aeruginosa exposed to H 2 O 2 underwent an apoptotic event. Additionally, cells exposed to H 2 O 2 had increased cytoplasmic vacuolation and nontypical DNA laddering. Increased caspase-3-like activity was not inhibited in the presence of the synthetic caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone. Therefore, H 2 O 2 induced apoptotic-like cell death in a dose-dependent manner. Taken together, our results provided a novel mechanism for explaining cyanobacterial bloom dynamics in response to environmental stress. The results also contributed to the understanding of the origin and evolution of programmed cell death.

Aquatic origin collagen (Aqua-collagen) has been pursued as an alternative to mammalian origins. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were successfully extracted from the skin of tilapia with the yield about 19-20% (basis of lyophilized dry weight), and examined for their physico-functional and structural properties. Both ASC and PSC containing α 1 , α 2 chains were characterized to be type I collagen and had lower denaturation temperature compared to mammalian origins. PSC (low telopeptides collagen) was selected for biomedical scaffolds construction due to its low immunogenicity. SEM analysis of fish collagen scaffolds showed a wide range of pore size distribution, high porosity, and high surface area-to-volume ratios. The tilapia collagen microfiber matrix scaffolds were grafted beneath the dorsal skin in 96 mice towards tissue regeneration, with bovine collagen microfiber matrix scaffolds (Avitene™ UltraFoam™ sponge) serving as control. Biocompatibility evaluation in the dorsal tissue showed that implanted scaffolds degraded completely after 20 days with no pathological inflammatory tissue responses. These findings indicated that aqua-collagens microfiber matrix scaffolds were highly biocompatible in nature, exploring its feasibility for the development of scaffolds in tissue engineering. 1. Introduction Collagen is one of non-branched chain fibrous proteins in the extracellular matrix (ECM) component, plays a pivotal role in maintaining tissue homeostasis, biological integrity and structural mechanics [1]. Collagen, up to 25% of the total proteins, being presence in skin, bone, muscle sheath, ligaments, cartilage and other connective tissues of animals [2], is contributing to unique physiological functions [3]. Generally, collagens are formed by polypeptide chains constituted by repeating triplets Gly-X-Y of Glycine and two other amino acids, where proline (Pro) and hydroxyproline (Hyp) are the most common ones among about 1000 total amino acids [4]. Currently, researchers have identified at least 27 variants of collagens on the basis of sequence homology and the molecular structure [5]. Type I was the most abundant and important collagen and was a natural macromolecular hydrocolloid with widespread use in the food, cosmetic, biomedical, and pharmaceutical industries because of its excellent biocompatibility and biodegradability, and weak antigenicity [6]. Type I collagen was most prevalent and found in connective tissues such as bone, skin, and cornea of the eyes [7], contains three α chains such as [α 1 (I)] 2 α 2 (I) and [α 1 (I)] 3. Every chain contains more than 1000 amino acids with minor difference in amino acid sequences of α chains between different vertebrates. There has a repetition of glycine in every three amino acid sequence, such sequences make the distance of α chain shortened and the space minimized, so the chains close together to form a more stable helical structure since glycine has the smallest side groups. Marine and aquaculture capture fisheries are widely dist...

Several assay methods were screened for viability assessment in cyanobacteria using Microcystis aeruginosa FACHB 905. Compared with fluorescent diacetate (FDA), Evan's Blue and autofluorescence, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell by counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. Half-life of oxidoreductase in dead cell was calculated to be 3 h. The MTT assay was also found to be applicable to other cyanobacteria and diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. Based on the above results, we proposed an optimized procedure for the MTT method on Microcystis strains. The use of this assay may be of importance to better understand the dynamics of bloom and the fate of Microcystis under natural or disturbed conditions.

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We studied daily periodicity, taxonomic composition and annual variation of fish larvae abundance from a survey of the Zhaoqing section of the Pearl River over a 3-year period, 2006-2008, and collected 52 species, in eight families and 16 orders. The fish larval samples were numerically dominated by six species, Barbel Chub (Squaliobarbus curriculus) 33.1%, Black Guangdong Bream (Megalobrama hoffmanni) 20.6%, Yellowfin (Xenocypris argentea) 15.4%, Mud Carp (Cirrhina molitorella) 10%, Common Sawbelly (Hemiculter leucisculus) 4.3%, and Freshwater Bream (Parabramis pekinensis) 3.1%. These six species accounted for 86.5% of total numbers. Black Carp (Mylopharyngodon piceus), Grass Carp (Ctenopharyngodon idellus), Silver Carp (Hypophthalmichthys molitrix) and Bighead Carp (Aristichthys nobilis) are four of the most economically important fish species in China, and accounted for approximately 4.6% of the total. When compared with historical records from 1931, there are obvious changes in the fish larvae community. Chinese Sturgeon (Acipenser sinensis), Seasonal Shad (Macrura reevesi) and Long Spiky-head Carp (Luciobrama macrocephalus) populations have disappeared and there are also decreases in several other economically important fish populations. We observed a delay in spawning time of Black Carp, Grass Carp, Silver Carp and Bighead Carp, which may be the result of the hydrological regime change in the Pearl River. We used canonical correspondence analysis (CCA) and cross-covariance function (CCF) to analyze the relationship between fish larvae and environmental variables. Our results clearly show that discharge affects the majority of fish larvae, with the various species having different responses. Larval abundances of 15 species such as Black Carp, Grass Carp, Silver Carp, Bighead Carp, Mud Carp and Black Guangdong Bream are significantly and positively related to the discharge. Other species such as Chinese Noodlefish (Leucosoma chinensis) and Rhinogobius olivaceus are negatively related to the discharge. Abundances of larval Mosquitofish (Gambusia affinis), Common Carp (Cyprinus carpio) and Eleotris oxycephala were not related to the discharge. As most economic fish species are affected by discharge, we also conclude that changes of hydrological regime, mainly caused by river damming, will result in the decline of fisheries resources in the Pearl River. The findings of this study have important implications for our ability to model the responses of fish larvae to environmental flows for the purpose of river regulation.

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