Toxicity of the dinoflagellate Gambierdiscus sp. toward the marine copepod Tigriopus japonicus

The toxic dinoflagellate Gambierdiscus sp. has recently been observed in temperate areas in the Southern Sea of the Republic of Korea. The purpose of this study was to examine the toxicity of Gambierdiscus sp. toward the marine copepod Tigriopus japonicus. T. japonicus adult females and nauplii were exposed to various concentrations of algae or culture medium to assess toxicity and analyze gene expression patterns in the copepod. Based on the toxicity tests, Gambierdiscus sp. increased the mortality of nauplii and the immobility of adult females. The survival and mobility of T. japonicus were not affected by culture medium lacking Gambierdiscus sp. cells in the toxicity test. However, based on the analysis of gene expression in the copepod, exposure of the copepod to culture medium affected the expression of stress or detoxification-related genes. Further studies to identify toxins in Gambierdiscus sp. are required to increase our understanding of dinoflagellate toxicity.     

Chemical Composition and Cytotoxic Evaluation of the Essential Oil of Phyllogonium viride (Phyllogoniaceae,Bryophyta)

The present study aimed to determine the chemical composition and biological activity of the essential oil obtained from Phyllogonium viride Brid. (Phyllogoniaceae, Bryophyta), whose samples were collected in southern Brazil. For the first time, the cytotoxic activity of the essential oil of P. viride in breast and colorectal tumor cells (MCF-7 and HCT-116) was evaluated, as well as the cytotoxic potential of this oil in non-tumoral cells of human immortalized keratinocytes (HaCaT) via MTT assay. The compounds majorly found in P. viride essential oil were β-bazzanene (20.30 %), β-caryophyllene (17.06 %), β-chamigrene (14.02), and germacrene B (11.72 %). Treatment with P. viride essential oil in the different tested cell lines did not induce any toxicity in most of the tested concentrations. These data contribute to generating new scientific information about this understudied plant species. Furthermore, the chemical characterization of the compounds present in the essential oil of P. viride can lead to greater elucidation of its biotechnological potential.     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.     

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt,p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

In silico synteny based comparative genomics approach for identification and characterization of novel therapeutic targets in Chlamydophila pneumoniae

Chlamydophila pneumoniae is one of the most important and well studied gram negative bacterial strain with respect to community acquired pneumonia and other respiratory diseases like Chronic obstructive pulmonary disease (COPD), Chronic asthma, Alzheimer''s disease, Atherosclerosis and Multisclerosis which have a great potential to infect humans and many other mammals. According to WHO prediction, COPD is to become the third leading cause of death by 2030. Unfortunately, the molecular mechanisms leading to chronic infections are poorly understood and the difficulty in culturing C pneumoniae in experimental conditions and lack of entirely satisfactory serological methods for diagnosis is also a hurdle for drug discovery and development. We have performed an insilico synteny based comparative genomics analysis of C pneumoniae and other eight Chlamydial organisms to know the potential of C pneumoniae which cause COPD but other Chlamydial organisms lack in potential to cause COPD though some are involved in human pathogenesis. We have identified total 354 protein sequences as non-orthologous to other Chlamydial organisms, except hypothetical proteins 70 were found functional out of which 60 are non homologous to Homo sapiens proteome and among them 18 protein sequences are found to be essential for survival of the C pneumoniae based on BLASTP search against DEG database of essential genes. CELLO analysis results showed that about 80% proteins are found to be cytoplasmic, Among which 5 were found as bacterial exotoxins and 2 as bacterial endotoxins, remaining 11 proteins were found to be involved in DNA binding, RNA binding, catalytic activity, ATP binding, oxidoreductase activity, hydrolase activity and proteolysis activity. It is expected that our data will facilitate selection of C pneumoniae proteins for successful entry into drug design pipelines.     

A Survey Investigating the Infection of Fusarium langsethiae and Production of HT‐2 and T‐2 Mycotoxins in UK Oat Fields

Fusarium langsethiae is a toxigenic fungal species that has been reported in European small‐grain cereal crops such as oats, wheat and barley. Although its relative contribution to fusarium head blight (FHB) symptoms is not well understood, it is reported to contaminate these cereals with high levels of HT‐2 and T‐2 trichothecenes mycotoxins that are currently under consideration for legislation by the European Commission. Ten commercial oat fields in Shropshire and Staffordshire (two adjacent counties in the Midlands) in the UK were surveyed in the 2006/2007 growing season. Samples were taken from predetermined field locations at Zadoks growth stages 32/33, 69, 77‐85 and 90‐92 for F. langsethiae biomass and HT‐2 and T‐2 toxins quantification. The results from this study showed that oats can be heavily infected with F. langsethiae and have high concentrations of HT‐2 and T‐2 toxins with no apparent FHB symptoms. The regression of HT‐2 + T‐2 toxins on F. langsethiae DNA concentration was highly significant (P < 0.001, r² = 0.55). The results indicated that although F. langsethiae had no direct effect on crop yield, it may result in indirect economic losses where the grain can be rejected or downgraded as a result of intolerable levels of HT‐2 and T‐2 toxins, which are of human food and animal feed safety concern. The influence of cultural field practices on the infection and HT‐2 and T‐2 toxins accumulation in oats was not clear and warrants further studies to identify the sources of F. langsethiae inoculum and conditions favourable for infection and mycotoxin production.     

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.