Isolation and enumeration of marine actinomycetes from seawater and sediments in Alexandria

Actinomycetes were isolated from near-shore marine sediments and water at four different sites in Alexandria. Statistical analysis revealed that variation in temperature, pH, and dissolved phosphate were of insignificant values, but that variation in total nitrogen and organic matter were significant. The treatment of sediments and water samples by heat resulted in a selective reduction of the nonactinomycetal heterotrophic microflora. Four selective culture media were used for counting actinomycetes in marine water and sediments. The starch nitrate medium favored the growth of these microorganisms. The diversity and counts of actinomycetes varied with the seasonal variation, and the highest counts were detected in dry warm seasons. The numbers of this bacterial group in sediments exceeded by far their numbers in seawater. A positive correlation was found between population size and location. Actinomycetes were found in the highest numbers in the upper layers (0-20 cm depth). In a few cases, the counts of actinomycetes showed bimodal maxima 0-20 and 60-100 cm deep. Sediments were the best source of marine actinomycetes, and their distribution varied depending on the depth from which samples were collected. The ratio of actinomycetes to the total microflora ranged from 0.48 to 2.29, depending on location.     

Analyzing of expression of novel polypeptide complexes consisting of Shiga toxin B subunit and Adherence Fimbriae of Escherichia coli based on in silico modeling

Enterohemorrhagic (EHEC) and enteroaggregative (EAEC) are two pathotypes of diarrheagenic Escherichia coli. EAEC strains express adhesins called aggregate adherence fimbriae (AAFs) which the bacteria use to adhere to intestinal mucosa. EHEC virulence factor is Shiga toxin which belongs to the AB5 toxin family. B subunit, the nontoxic part of Shiga toxin (StxB), forms a homo pentamer and is responsible for binding to target cells. StxB has recently been proven to have adjuvant activity. In the current study we fused StxB encoding gene to 3' end of genes encoding two variants of AAFs, i.e., AAF/I and AAF/II. The in silico studies on tertiary structure and biochemical characteristics of Shiga toxin A subunit (StxA) revealed more resemblance to AAF/II than AAF/I. The constructs were prepared in a way that StxB could imitate its natural structure (pentamer formation) and its position (C-terminus) in the native toxin complex. The expression of these constructs showed the formation of AAF/II-B as a protein complex but with lower molecular mass than its expected size. In contrast, the AAF/I-B complex was not formed. Overall, the results of in silico studies and expression experiments together revealed that despite AAF/II-B expression, StxB failed to form pentamer. Therefore the observed protein complex has lower molecular mass. Since StxB is bound to AAF/II through disulfide bond, this bond prevents pentamer formation of StxB. However, due to the lack of disulfide bond between AAF/I and StxB, no protein complex is formed, thus StxB maintains its pentamer structure.     

Impact of Response Shift on Time to Deterioration in Quality of Life Scores in Breast Cancer Patients

Background

This prospective multicenter study aimed to study the impact of the recalibration component of response-shift (RS) on time to deterioration (TTD) in health related quality of life (QoL) scores in breast cancer (BC) patients and the influence of baseline QoL expectations on TTD.

Methods

The EORTC-QLQ-C30 and BR-23 questionnaires were used to assess the QoL in a prospective multicenter study at inclusion (T0), at the end of the first hospitalization (T1) and, three (T2) and 6 months after the first hospitalization (T3). Recalibration was investigated by the then-test method. QoL expectancy was assessed at diagnosis. Deterioration was defined as a 5-point decrease in QoL scores, considered a minimal clinically important difference (MCID). TTD was estimated using the Kaplan-Meier method. Cox regression analyses were used to identify factors influencing TTD.

Results

From February 2006 to February 2008, 381 women were included. Recalibration of breast cancer patients'' internal standards in the assessment of their QoL had an impact on TTD. Median TTD were significantly shorter when recalibration was not taken into account than when recalibration was taken into account for global health, role-functioning, social-functioning, body-image and side effects of systemic therapy. Cox multivariate analyses showed that for body image, when recalibration was taken into account, radiotherapy was associated with a shorter TTD (HR: 0.60[0.38–0.94], whereas, no significant impact of surgery type on TTD was observed. For global health, cognitive and social functioning dimensions, patients expecting a deterioration in their QoL at baseline had a significantly shorter TTD.

Conclusions

Our results showed that RS and baseline QoL expectations were associated with time to deterioration in breast cancer patients.     

Downregulation of Stemness Genes and Induction of Necrosis in Rat LA7 Cancer Stem Cells Induced Tumors Treated with Starved Fibroblasts Culture Supernatant

Background:Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy. Methods:The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining.Results:All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67⁺, cytokeratin⁺, vimentine⁺, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium.Conclusion:This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.Key Words: Breast cancer, Cancer Stem cells, Cell differentiation, Fibroblasts, Gene expression     

Molecular phylogeny and ecogeography of Onobrychis viciifolia Scop. (Fabaceae) based on nrDNA ITS sequences and genomic ISSR fingerprinting

In this study 40 specimens from 8 representative Iranian populations of Onobrychis viciifolia SCOP. were collected from their natural habitats. The specimens were biometrically assessed using 43 quantitative and 15 qualitative morphological characters. At each sample site we recorded ecogeographical variables. In order to evaluate the difference between the variation due to phenotypic responses and genetic adaptation, we generated nucleotide sequence data from the internal transcribed spacer of the nuclear rDNA genes (ITS) and carried out genomic fingerprints using inter‐simple sequence repeat PCR (ISSR). Cluster analysis of morphological characters divided the eight populations into three major groups. Leaf length, leaflet length and width, calyx length, plant height, and stem length were introduced as diagnostic characters for three different morphological groups. Furthermore, canonical correspondence analysis (CCA) of ecogeographical data showed correlations between morphological variations and ecogeographical factors. Clay%, saturation percentage (SP), total neutralizing value (TNV%), texture, minimum temperature, and geographic separation were the main environmental variables associated with morphological groups of O. viciifolia. ISSR analysis revealed three main groups which are in correspondence with morphological groups, indicating that the three morphological groups have been shaped by genetic and phenotypic responses to environmental conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Design and synthesis of 4-methoxyphenylacetic acid esters as 15-lipoxygenase inhibitors and SAR comparative studies of them

A group of 4-methoxyphenylacetic acid esters were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compounds 7d–e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 3.8 and 1.9 μM, respectively). All compounds were docked in SLO active site and showed that carbonyl group of compounds is oriented toward the Fe^III–OH moiety in the active site of enzyme and fixed by hydrogen bonding with hydroxyl group. It is assumed that lipophilic interaction of ligand–enzyme would be in charge of inhibiting the enzyme activity. The selectivity of the synthetic esters in inhibiting of 15-HLOb was also compared with 15-HLOa by molecular modeling and multiple alignment techniques.     

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

A comprehensive study reveals that kinesins in the malaria parasite Plasmodium have diverse cellular roles and locations, including functions in spindle assembly during proliferation, axoneme formation in flagellum biogenesis, and determining the apical morphology of the cell.     

Regulation of mitochondrial proteostasis by the proton gradient

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m‐AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca²⁺/H⁺ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m‐AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca²⁺ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m‐AAA protease. The m‐AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca²⁺ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.     

An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in Candida albicans

A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.2500 is a conserved mitochondrial protein, structurally similar to, but functionally diverged from, the squalene/phytoene synthases family. The C. albicans orf19.2500 is distinguished by 3 evolutionarily acquired stretches of amino acid inserts, absent from all other eukaryotes except a small number of ascomycete fungi. Biochemical assays showed that orf19.2500 is required for the assembly and activity of the NADH ubiquinone oxidoreductase Complex I (CI) of the respiratory electron transport chain (ETC) and was thereby named NDU1. NDU1 is essential for respiration and growth on alternative carbon sources, important for immune evasion, required for virulence in a mouse model of hematogenously disseminated candidiasis, and for potentiating resistance to antifungal drugs. Our study is the first report on a protein that sets the Candida-like fungi phylogenetically apart from all other eukaryotes, based solely on evolutionary “gain” of new amino acid inserts that are also the functional hub of the protein.