Survey of sediment quality in Sabine Lake, Texas and vicinity

The toxicity of sediments in Sabine Lake, Texas, and adjoining Intracoastal Waterway canals was determined as part of bioeffects assessment studies managed by NOAA’s National Status and Trends Program. The objectives of the survey were to determine: (1) the incidence and degree of toxicity of sediments throughout the study area; (2) the spatial patterns (or gradients) in chemical contamination and toxicity, if any, throughout the study area; (3) the spatial extent of chemical contamination and toxicity; and (4) the statistical relationships between measures of toxicity and concentrations of chemicals in the sediments. Surficial sediment samples were collected during August, 1995 from 66 randomly-chosen locations. Laboratory toxicity tests were performed as indicators of potential ecotoxicological effects in sediments. A battery of tests was performed to generate information from different phases (components) of the sediments. Tests were selected to represent a range in toxicological endpoints from acute to chronic sublethal responses. Toxicological tests were conducted to measure: reduced survival of adult amphipods exposed to solid-phase sediments; impaired fertilization success and abnormal morphological development in gametes and embryos, respectively, of sea urchins exposed to pore waters; reduced metabolic activity of a marine bioluminescent bacteria exposed to organic solvent extracts; and induction of a cytochrome P-450 reporter gene system in exposures to solvent extracts of the sediments. Chemical analyses were performed on portions of each sample to quantify the concentrations of trace metals, polynuclear aromatic hydrocarbons, and chlorinated organic compounds. Correlation analyses were conducted to determine the relationships between measures of toxicity and concentrations of potentially toxic substances in the samples. Based upon the compilation of results from chemical analyses and toxicity tests, the quality of sediments in Sabine Lake and vicinity did not appear to be severely degraded. Chemical concentrations rarely exceeded effects-based numerical guidelines, suggesting that toxicant-induced effects would not be expected in most areas. None of the samples was highly toxic in acute amphipod survival tests and a minority (23%) of samples were highly toxic in sublethal urchin fertilization tests. Although toxic responses occurred frequently (94% of samples) in urchin embryo development tests performed with 100% pore waters, toxicity diminished markedly in tests done with diluted pore waters. Microbial bioluminescent activity was not reduced to a great degree (no EC50 <0.06 mg/ml) and cytochrome P-450 activity was not highly induced (6 samples exceeded 37.1 ug/g benzo[a]pyrene equivalents) in tests done with organic solvent extracts. Urchin embryological development was highly correlated with concentrations of ammonia and many trace metals. Cytochrome P450 induction was highly correlated with concentrations of a number of classes of organic compounds (including the polynuclear aromatic hydrocarbons and chlorinated compounds). (PDF contains 51 pages)

Desenvolvimento de metodologias para a determinação da atividade biogênica de bactérias redutoras de sulfato

A corrosão causada por H2S biogênico frequentemente resulta em danos extensos na indústria do petróleo. O presente trabalho avaliou parâmetros de crescimento microbiano e aplicou metodologias de determinação de sulfetos por técnicas espectrofotométrica na região da luz visível e radiorespirométrica para avaliação da atividade metabólica, correlacionando com a população de bactérias redutoras de sulfato, determinada através da técnica do Número Mais Provável (NMP). Amostras de água de formação e consórcio de BRS foram avaliadas através do arraste de sulfetos estáveis produzidos biogenicamente e quantificados por espectrofotometria. O cálculo das velocidades instantâneas e específicas de produção de sulfetos permitiu avaliar de que maneira alguns parâmetros de crescimento microbiano podem afetar o metabolismo das BRS. A detecção de concentrações traço de sulfetos biogênicos pode ser realizada através de ensaios radiorespirométricos. Para isto, diluições em série de água do mar sintética com três amostras distintas foram avaliadas. Os testes realizados indicam que o acréscimo do tempo de incubação de cultura microbiana anaeróbia mista contribuiu para o aumento das capacidades de redução de sulfato, assim como o aumento das fontes de carbono. Ambas as técnicas provaram ser um rápido teste para a detecção de sulfetos biogênicos, particularmente aqueles associados aos produtos de corrosão, sendo uma ferramenta muito útil para monitoração e controle de tanques de armazenamento de água e óleo, plataformas continentais de petróleo e diversos tipos de reservatórios. O presente trabalho prevê a continuidade dos experimentos, através de avaliação de um maior universo de amostras da indústria do petróleo e medições menos espaçadas da técnica espectrofotométrica, além da avaliação radiorespirométrica em modo contínuo, evitando os efeitos inibitórios do H2S

Award Winner in the Young Investigator Category, 2014 Society for Biomaterials Annual Meeting and Exposition, Denver, Colorado, April 16-19, 2014: Periodically perforated core-shell collagen biomaterials balance cell infiltration, bioactivity, and mechanical properties.

Orthopedic tissue engineering requires biomaterials with robust mechanics as well as adequate porosity and permeability to support cell motility, proliferation, and new extracellular matrix (ECM) synthesis. While collagen-glycosaminoglycan (CG) scaffolds have been developed for a range of tissue engineering applications, they exhibit poor mechanical properties. Building on previous work in our lab that described composite CG biomaterials containing a porous scaffold core and nonporous CG membrane shell inspired by mechanically efficient core-shell composites in nature, this study explores an approach to improve cellular infiltration and metabolic health within these core-shell composites. We use indentation analyses to demonstrate that CG membranes, while less permeable than porous CG scaffolds, show similar permeability to dense materials such as small intestine submucosa (SIS). We also describe a simple method to fabricate CG membranes with organized arrays of microscale perforations. We demonstrate that perforated membranes support improved tenocyte migration into CG scaffolds, and that migration is enhanced by platelet-derived growth factor BB-mediated chemotaxis. CG core-shell composites fabricated with perforated membranes display scaffold-membrane integration with significantly improved tensile properties compared to scaffolds without membrane shells. Finally, we show that perforated membrane-scaffold composites support sustained tenocyte metabolic activity as well as improved cell infiltration and reduced expression of hypoxia-inducible factor 1α compared to composites with nonperforated membranes. These results will guide the design of improved biomaterials for tendon repair that are mechanically competent while also supporting infiltration of exogenous cells and other extrinsic mediators of wound healing.

Human mesenchymal stem cell response to 444 ferritic stainless steel networks

The aim of this work is to improve bone-implant bonding. This can, potentially, be achieved through the use of an implant coating composed of fibre networks. It is hypothesised that such an implant can achieve strong peri-prosthetic bone anchorage, when seeded with human mesenchymal stem cells (hMSCs). The materials employed were 444 and 316L stainless steel fibre networks of the same fibre volume fraction. The present work confirms that hMSCs are able to proliferate and differentiate towards the osteogenic lineage when seeded onto the fibre networks. Cellular viability, proliferation and metabolic activity were assessed and the results suggest higher proliferation rates when hMSC are seeded onto the 444 networks as compared to 316L. Cell distribution was found uniform across the seeded surfaces with 444 showing a somewhat higher infiltration depth. Copyright © Materials Research Society 2013.

不同抗旱性青稞LEA2/LEA3蛋白基因的克隆与表达

作物的抗旱性是一个多基因控制的、极为复杂的数量性状，植物对干旱在分子水平上的差异反应通过植物组织生理和细胞生物学水平，最终表现为植物抗旱性的不同。在我国，旱地农业超过耕地面积的50%，但水资源短缺，因此培育和选育抗旱高产作物是发展节水型农业最有效的途径。 青藏高原气候恶劣、年均降雨量少，也是世界大麦初生起源中心，因而蕴藏了十分丰富的与抗逆相关的种质资源材料，从这些特殊的资源材料克隆抗旱基因，不仅对培育抗旱、优质、高产大麦新品种具有重要理论意义和经济价值，而且对整个作物抗旱基础和育种应用研究都具重大促进作用。 为了筛选青稞（裸大麦，Hordeum vulgare ssp. vulgare）抗旱性材料，本研究选用来自青藏高原不同地区的84份青稞为材料，在叶片失水率(water loss rate, WLR)检测分析的基础上，选择失水率值差异显著的12个品种，通过相对含水量（relative water content, RWC）和反复干旱法评价其抗旱性，并通过植株对干旱胁迫下的丙二醛（MDA）含量和游离脯氨酸（free-proline）含量变化，了解不同抗旱性材料的生理反应特性。选择抗旱性强弱不同的品种各两份进行LEA2蛋白基因(Dhn6基因)、LEA3蛋白基因(HVA1基因)的克隆，比较LEA蛋白结构差异与作物抗旱性之间的关系。同时，对抗旱性不同的青稞品种受到干旱时间不同的失水变化率（dynamics water loss rate, DWLR）进行了检测；对抗旱性不同的青稞对照材料进行2 h、4 h、8 h和12 h的快速干旱处理，通过SYBR Green实时荧光定量RT-PCR技术对Dhn6基因、Dhn11基因、Dhn13基因和HVA1基因在不同抗旱性材料受到不同干旱时间处理后的相对表达水平进行了检测。本研究对LEA蛋白基因在抗旱性不同的青稞材料中的干旱胁迫分子水平上的差异反应进行了研究，也对植物的抗旱机理进行了初步探讨。主要研究结果如下： 1. 青稞苗期进行离体叶片失水率测定结果表明，来自青藏高原的84份青稞材料的WLR在0.086~0.205gh-1g-1DW之间。选择WLR低于0.1gh-1g-1DW和WLR高于0.18gh-1g-1DW的品种各6份，并对苗期分别进行未干旱及干旱12小时的处理。相对含水量检测结果表明，低失水率青稞材料干旱后的具有更高的相对含水量，盆栽缺水试验也显示叶片失水率低的材料耐旱能力强于失水率高的材料。通过水合茚三酮法测定离体叶片游离脯氨酸的含量，结果表明，所有品种未干旱处理时，游离脯氨酸含量差异不大（17.10~25.74 µgg-1FW）；干旱12小时后，低失水率的品种游离脯氨酸含量明显增高（32.99~53.45µgg-1FW），高失水率品种的游离脯氨酸含量与干旱前变化不明显(P<0.05)。硫代巴比妥酸法测定离体叶片丙二醛(MDA)含量，结果显示，12份所选对照品种中，丙二醛的含量在0.97~2.74nmolg-1FW，干旱12小时后丙二醛的含量显著上升（1.46~4.74nmolg-1FW），高失水率的6个品种的丙二醛含量在未干旱和干旱处理时都明显高于低WLR品种。本研究结果表明青稞的低失水率、低丙二醛含量、高相对含水量和高脯氨酸含量具相关性（P<0.05）。综上研究，我们认为作物失水率的测定可以作为快速检测作物抗旱性的指标之一，因此，强抗旱品种喜玛拉10号(TR1)、品比14号(TR2)和弱抗旱品种冬青8号(TS1)、QB24 (TS2)被选作抗旱基因克隆和表达分析的研究材料。 2. 高等植物胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins, LEA proteins)与植物耐脱水性密切相关，为了探讨青稞LEA蛋白结构差异性与植物抗旱性的关系，本研究以强抗旱品种（喜玛拉10号、品比14号）和弱抗旱品种（冬青8号、QB24）为材料，利用同源克隆法，通过RT-PCR，分别克隆了与抗旱性密切相关的Dhn6基因和HVA1基因。Dhn6基因序列分析结果表明，强抗旱品种品比14号和弱抗旱品种冬青8号Dhn6基因所克隆到的序列为1026bp，它们之间只有5个碱基的差异；喜玛拉10号和QB24克隆到的序列长963bp。在强弱不同的抗旱品种中有22个核苷酸易突变位点，相应的脱水素氨基酸序列推导结果表明，22个核苷酸突变位点中，仅有8个位点导致相应的氨基酸残基的改变，其余的位点系同义突变，另外，21个富含甘氨酸序列的缺失并没有联系作物抗旱性特征。推测这些同义突变位点的氨基酸残基对维持青稞DHN6蛋白的正常结构和功能起着非常重要的作用，也可能DHN6蛋白对青稞长期适应逆境胁迫和遗传进化的结果。对HVA1基因的序列分析结果表明，冬青8号、QB24、品比14号和喜玛拉10号的目的基因核苷酸序列全长分别为661bp、697bp、694bp和691bp，它们都包含1个完整的开放阅读框。相应的LEA3蛋白氨基酸序列结果表明，11个高度保守的氨基酸残基组成基元重复序列的拷贝数与青稞抗旱性之间没有必然关系，在强抗旱品种（喜玛拉10号、品比14号）中三个共同的氨基酸突变位点Gln32、Arg33和Ala195可能对抗旱蛋白的结构和功能有影响；另外，强抗旱青稞品种LEA3蛋白质中11-氨基酸保守基元序列拷贝数和极性氨基酸占蛋白的比例更高，推测LEA3蛋白中基元序列拷贝数和极性氨基酸占蛋白的比例对该蛋白的结构和功能影响更大。 3. LEA蛋白基因的表达水平的上调与植物的耐脱水性密切相关，我们对强抗旱性材料（喜玛拉10号、品比14号）和弱抗旱材料（冬青8号、QB24）进行干旱处理2 h、4 h、6 h、8 h和10 h的失水变化率进行测定，结果表明弱抗旱品种在2~4小时之间失水率变化最明显，而四个对照品种的失水率在8小时后和24小时的失水率值变化不大。进一步提取青稞苗期进行2 h、4 h、8 h和12 h的干旱处理后的总RNA，通过SYBR Green实时荧光定量RT-PCR技术对青稞脱水素基因(Dhn6、Dhn11和Dhn13)和LEA3蛋白基因(HVA1)的相对表达水平受干旱时间和作物抗旱性的影响进行了检测。研究发现，抗旱性不同的青稞品种随干旱处理的时间延长，Dhn6、Dhn11、Dhn13和HVA1基因的相对表达水平不同。 Dhn6基因的相对表达水平在强抗旱青稞品种干旱8小时后快速上升，但在弱抗旱青稞品种干旱处理12小时后检测到更高表达量；Dhn11基因在对照青稞抗旱品种的表达累积水平随干旱时间的延长持续下降；整个干旱过程中，Dhn13基因的相对表达水平在弱抗旱品种持续上升，在强抗旱品种中干旱处理8小时快速上升并达到最高，干旱12小时后降低。与脱水素基因相比较，强抗旱青稞品种在干旱2小时后HVA1基因的相对表达水平显著升高，相对表达量随干旱处理的时间持续上升，在干旱12小时后达到最高；与之相比较，在整个干旱过程中，弱抗旱品种的相对表达水平显著低于强抗旱品种，在干旱8小时之前弱抗旱品种的相对表达水平变化不明显；在干旱8~12小时后却显著上升。上述结果表明，不同的LEA蛋白在植物耐脱水过程中的干旱表达累积水平不同；干旱不是诱导高等植物Dhn11基因表达的主要因素；植物的抗旱性不同，不同LEA蛋白基因对干旱的反应有差异。推测某些LEA蛋白基因的干旱胁迫早期表达累积程度与植物的抗旱性直接相关；其中，Dhn11基因和Dhn12基因不同的表达模式可能与干旱调控表达顺式作用成分(dehydration responsive element, DRE)的有无或结构上的差异有关。 本研究结果认为，(1)失水率和相对含水量可作为植物抗旱性检测的指标之一；(2) DHN6同义突变位点的氨基酸残基对维持该蛋白的正常结构和功能起着重要作用；(3) 11-氨基酸保守基元序列拷贝数和极性氨基酸的比例对LEA3蛋白结构和功能有重要影响；(4)LEA蛋白表达随着干旱胁迫程度而增加，但Dhn11基因并不受干旱诱导表达；(5)作物的抗旱性不同，LEA蛋白对干旱的累积反应并不相同，干旱早期LEA蛋白的累积程度可能会影响植物的抗旱性。 Drought resistance was a complex trait which involved multiple physiological and biochemical mechanisms and regulation of numerous genes. Because its complex traits, it is difficult to understand the mechanisms of drought resistance in plants. Plants respond to water stress through multiple physiological mechanisms at the cellular, tissue, and whole-plant levels. Tibetan hulless barley, a pure line, is a selfing annual plant that has predominantly penetrated into the Qinghai-Tibetan Plateau and remains stable populations there. The wide ecological range of Tibetan hulless barley differs in water availability, temperature, soil type and vegetation, which makes it possess a high potential of adaptive diversity to abiotic stresses. This adaptive genetic diversity indicates that the potential of Tibetan hulless barley serves as a good source for drought resistance alleles for breeding purposes. 12 contrasting drought-tolerant genotypes were selected to measure relative water content (RWC), maldondialdehyde (MDA) and proline content, based on values of water loss rate (WLR) and repeated drought methods from Tibetan populations of cultivated hulless barley. As a result of the screening, sensitive and tolerant genotypes were identified to clarify relationships between characteristics of LEA2/LEA3 genes sequences and expression and drought-tolerant genotypes, associated with resistance to water deficit. In addition, dynamics water loss rate (DWLR) was measured to observe the changes on diffrential drought-tolerant genotypes. Real-time quantitative RT-PCR was applied to detect relative expression levels of Dhn6, Dhn11, Dhn13 and HVA1 genes in sensitive and tolerant genotypes with 2 h, 4 h, 8h and 12 h of dehydration. In the present study, differential sequences and expression of LEA2/LEA3 genes were explored in Tibetan hulless barley, associated with phenotypically diverse drought-tolerant genotypes. 1. The assessments of WLR and RWC were considered as an alternative measure of plant water statues reflecting the metabolic activity in plants, and the parameters of MDA and proline contents were usually consistent with the resistance to water stress. The values of detached leaf WLR of the tested genotypes were highly variable among 84 genotypes, ranging from 0.086 to 0.205 g/h.g DW. The 12 most contrasting genotypes (6 genotypes with the lowest values of WLR and 6 genotypes with the highest values of WLR) were further validated by measuring RWC, MDA and free-proline contents, which were well watered and dehydrated for 12 h. Results of RWC indicated that the values of 12 contrasting genotypes RWC ranged from 89.94% to 93.38% under condition of well water, without significant differences, but 6 genotypes with lower WLR had higher RWC suffered from 12 h dehydration. The results indicated that lower MDA contents, lower scores of WLR and higher proline contents were associated with drought-tolerant genotypes in hulless barley. Remarkably, proline amounts were increased more notable in 6 tolerant genotypes than 6 sensitive genotypes after excised leaves were dehydrated for 12 h, with control to slight changes under condition of well water. Results of MDA contents showed that six 6 tolerant genotypes had lower MDA contents than the 6 sensitive genotypes under both stressed and non-stressed conditions. As a result of that screening, drought- resistant genotypes (Ximala 10 and Pinbi 14) and drought-sensitive genotypes (Dongqing 8 and QB 24) were chosen for comparing the differential characteristics of LEA2/LEA3 genes and their expression analysis. It was conclusion that measurements of WLR could be considered an alternative index as screening of drought-tolerant genotypes in crops. 2. Late embryogenesis abundant (LEA) proteins were thought to protect against water stress in plants. To explore the relationships between configuration of LEA proteins and phenotypically diverse drought-tolerant genotypes, sequences of LEA genes and their deduced proteins were compared in Tibetan hulless barley. Results of comparing Dhn6 gene in Ximala 10 and QB24 indicated that absence of 63bp was found, except that only 5 mutant nucleotides were found. While 22 mutant sites were taken place in Dhn6 gene between sensitive and tolerant lines, 14 synonymous mutation sites appeared in the contrasting genotypes. The additional/absent polypeptide of 21 polar amino acid residues was not consistent with phenotypically drought-tolerant genotypes in hulless barley. It was deduced that synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein. The sequencing analysis results indicated that each cloned HVA1 gene from four selected genotypes contained an entire open reading frame. The whole sequence of HVA1 gene from Dongqing 8, QB24, Pinbi 14 and Ximala 10 was respectively 661bp, 697bp, 694bp and 691bp. Results of DNA sequence analyses showed that the differences in nucleotides of HVA1 gene in sensitive genotypes were not consistent with that of tolerant genotypes, except for absence of 33 nucleotides from +154 to +186 (numbering from ATG) in QB24. Database searches using deduced amino acid sequences showed a high homology in LEA3 proteins in the selected genotypes. Multiple sequence alignments revealed that LEA3 protein from Dongqing 8 was composed of 8 repeats of an 11 amino acid motif, less the fourth motif than Pinbi 14, Ximala 10 and QB24. Consistent mutant amino acid residues appeared in contrasting genotypes by aligning and comparing the coding sequence region, including Gln32, Arg33 and Ala195 in tolerant genotypes as compared to Asp32, Glu33 and Thr195 (Thr184 in Dongqing 8) in sensitive lines. It was concluded that consistent appearance of Gln32, Arg33 and Ala195 would contributed to functions of LEA3 protein in crops, as well as higher proportion of 11-amino-repeating motifs and polar amino acid residues. 3. Most of the LEA genes are up-regulated by dehydration, salinity, or low temperature, are also induced by application of exogenous ABA, which increases in concentration in plants under various stress conditions and acts as a mobile stress signal. Higher levels of proteins of LEA group 3 accumulated was correlated well with high level of desiccation tolerance in severely dehydrated plant seedlings. Dehydrins (DHNs), members of LEA2 protein, are an immunologically distinct protein family, and Dhn genes expression is associated with plant response to dehydration. Dynamic water loss rate was measured between sensitive genotypes and tolerant genotypes after they were dehydrated for 2 h, 4 h, 6h and 8 h. Detailed measurements of WLR at the early stage of dehydration (2, 4, 6, and 8 h) showed that WLR was stabilizing after 8 h, and there were no significant changes between these values and WLR after 24 h. Drought stress was applied to 10-day-old seedlings by draining the solution from the container for defined dehydration periods. Leaf tissues of the selected genotypes were harvested from control plants (time 0); and after 2, 4, 8, and 12 h of dehydration. Differential expression trends of Dhn6, Dhn11, Dhn13 and HVA1 genes were detected in phenotypically diverse drought-tolerant hulless barleys, related to different time of dehydration. Results of quantitative real-time PCR indicated that relative level of HVA1 expression was always higher in tolerant genotypes, rapidly increasing at the earlier stages (after 2-4 h of dehydration). However, HVA1 expressions of sensitive genotypes had a fast increase from 8 h to 12 h of stress. Significant differences in expression trends of dehydrin genes between tolerant genotypes and sensitive lines were detected, mainly in Dhn6 and Dhn13 gene, depending on the duration of the dehydration stress. The relative expression levels of Dhn6 gene were significantly higher in tolerant genotypes after 8 h dehydration, by control with notable higher expression levels after 12 h water stress in sensitive ones. The relative expression levels of Dhn13 gene tended to ascend during exposure to dehydration in drought-sensitive genotypes. However, fluctuate trends of Dhn13 expression level were detected in drought-resistant lines, including in lower expression levels of 12 h dehydration as compared to 8 h water stress. It was conclusion that (1) diverse LEA proteins would play variable roles in resisting water stress in plants; (2) expression of Dhn11 gene was not induced by dehydrated signals because of the trends of expression descended in contrasting genotypes suffered from water deficit and (3) variable accumulations on LEA proteins would be appear in diverse drought-tolerant genotypes during dehydrations. It is deduced that higher accumulations of Dhn6 and Dhn13 expression in 8 h dehydration are related to diverse drought-tolerant lines in crops. The present results indicated that different dehydrin genes would play variable functional roles in resisting water stress when plants were suffered from water deficit. The authors suggest physiologically different reactions between resistant and sensitive genotypes may be the results of differential expression of drought-resistant genes and related signal genes in plants. In addition, contrarily induced expression of Dhn11 and Dhn12 was related to dehydration responsive element (DRE) in barleys. The present study indicated that (1) measurements of WLR and RWC could be considered as one index of drought-tolerant screenings; (2) synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein, (3) higher proportion of 11-amino-repeating motifs and polar amino acid residues would contribute to functions on LEA3 protein, (4) the longer drought, the more accumulation on LEA proteins, except for Dhn11 gene in crops and (5) differential responses on expression of LEA protein genes would result in physiological traits of drought tolerance in plants.

Primmorphs from archaeocytes-dominant cell population of the sponge Hymeniacidon perleve: Improved cell proliferation and spiculogenesis

Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs. (C) 2003 Wiley Periodicals, Inc.

Effect of sulfate-reducing bacteria on corrosion behavior of mild steel in sea mud

Microbiologically influenced corrosion (MIC) is very severe corrosion for constructions buried under sea mud environment. Therefore it is of great importance to carry out the investigation of the corrosion behavior of marine steel in sea mud. In this paper, the effect of sulfate-reducing bacteria (SRB) on corrosion behavior of mild steel in sea mud was studied by weight loss, dual-compartment cell, electronic probe microanalysis (EPMA), transmission electron microscopy (TEM).combined with energy dispersive X-ray analysis (EDX) and electrochemical impedance spectroscopy (EIS). The results showed that corrosion rate and galvanic current were influenced by the metabolic activity of SRB. In the environment of sea mud containing SRB, the original corrosion products, ferric (oxyhydr) oxide, transformed to iron sulfide. With the excess of the dissolved H2S, the composition of the protective layer formed of FeS transformed to FeS2 or other non-stoichiometric polysulphide, which changed the state of the former layer and accelerated the corrosion process.

Shining light on food microbiology; applications of luciferase-tagged microorganisms in the food industry

Bioluminescence is the production of light by living organisms as a result of a number of enzyme catalysed reactions caused by enzymes termed luciferases. The lux genes responsible for the emission of light can be cloned from one bioluminescent microorganism into one that is not bioluminescent. The light emitted can be monitored and quantified and will provide information on the metabolic activity, quantity and location of cells in a particular environment, in real-time. The primary aim of this thesis was to investigate and identify several food industry related applications of lux-tagged microorganisms. The first aim was to monitor a lux-tagged Cronobacter sakazakii in reconstituted infant milk formula, in realtime. The second aim was to investigate a bioluminescent-based early warning system for starter culture disruption by bacteriophages and antibiotic residues. The third of this thesis was to examine the use of a bioluminescent-based assay to test the activity of bioengineered Nisin derivatives M21V and S29A against foodborne pathogens in laboratory media and selected foods.

Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

The effects of osmotic stress on the structure and function of the cell nucleus.

Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis.

Changes in the properties of Bacillus thuringiensis after prolonged culture in a rich medium

AIM: To investigate the effect of repeated culture in a rich medium on certain genetic, metabolic, pathogenic and structural characteristics of fresh isolates of Bacillus thuringiensis. METHODS AND RESULTS: Four strains of B. thuringiensis, which had been isolated in vegetative form from leaf surfaces, were grown for 500 generations in batch culture in a rich medium. One of the strains, S4g, differed from the parent in the following respects: greater cell width; changed plasmid profile; complete loss of ability to produce delta-endotoxins; loss of ability to produce beta-exotoxin and disruption of vip3 gene; radically different fatty acid composition; and altered metabolic activity. Two of the other evolved strains (S1g and S6g) showed differences in fatty acid profiles compared with the parents. Genetic finger-printing showed that there were also mutations in the cry genes of two of the evolved strains (S1g and S2g). The delta-endotoxins of strain S6g were significantly less toxic to the larvae of Pieris brassica compared with those of the parent and it also differed in the plasmid content. CONCLUSION: Radical and unpredictable changes can occur in fresh isolates of B. thuringiensis when subjected to growth in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first analysis of a Gram positive and biotechnologically significant bacterium after repeated laboratory culture. It is of great relevance to the biotechnological exploitation of B. thuringiensis that prolonged growth of environmental isolates on laboratory culture media can have profound effects on their structure, genome and virulence determinants.

Changes in pH at the exterior surface of plankton with ocean acidification

Anthropogenically released CO2 is dissolving in the ocean, causing a decrease in bulk-seawater pH (ocean acidification). Projections indicate that the pH will drop 0.3 units from its present value by 2100 (ref. 1). However, it is unclear how the growth of plankton is likely to respond. Using simulations we demonstrate how pH and carbonate chemistry at the exterior surface of marine organisms deviates increasingly from those of the bulk sea water as organism metabolic activity and size increases. These deviations will increase in the future as the buffering capacity of sea water decreases with decreased pH and as metabolic activity increases with raised seawater temperatures. We show that many marine plankton will experience pH conditions completely outside their recent historical range. However, ocean acidification is likely to have differing impacts on plankton physiology as taxon-specific differences in organism size, metabolic activity and growth rates during blooms result in very different microenvironments around the organism. This is an important consideration for future studies in ocean acidification as the carbonate chemistry experienced by most planktonic organisms will probably be considerably different from that measured in bulk-seawater samples. An understanding of these deviations will assist interpretation of the impacts of ocean acidification on plankton of different size and metabolic activity.

Selection of yeast strains for bioethanol production from UK seaweeds

Macroalgae (seaweeds) are a promising feedstock for the production of third generation bioethanol, since they have high carbohydrate contents, contain little or no lignin and are available in abundance. However, seaweeds typically contain a more diverse array of monomeric sugars than are commonly present in feedstocks derived from lignocellulosic material which are currently used for bioethanol production. Hence, identification of a suitable fermentative microorganism that can utilise the principal sugars released from the hydrolysis of macroalgae remains a major objective. The present study used a phenotypic microarray technique to screen 24 different yeast strains for their ability to metabolise individual monosaccharides commonly found in seaweeds, as well as hydrolysates following an acid pre-treatment of five native UK seaweed species (Laminaria digitata, Fucus serratus, Chondrus crispus, Palmaria palmata and Ulva lactuca). Five strains of yeast (three Saccharomyces spp, one Pichia sp and one Candida sp) were selected and subsequently evaluated for bioethanol production during fermentation of the hydrolysates. Four out of the five selected strains converted these monomeric sugars into bioethanol, with the highest ethanol yield (13 g L−1) resulting from a fermentation using C. crispus hydrolysate with Saccharomyces cerevisiae YPS128. This study demonstrated the novel application of a phenotypic microarray technique to screen for yeast capable of metabolising sugars present in seaweed hydrolysates; however, metabolic activity did not always imply fermentative production of ethanol.

Oncogenic Kras Promotes Chemotherapy-Induced Growth Factor Shedding via ADAM17.

Oncogenic mutations in Kras occur in 40% to 45% of patients with advanced colorectal cancer (CRC). We have previously shown that chemotherapy acutely activates ADAM17, resulting in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. In this study, we examined the role of mutant Kras in regulating growth factor shedding and ADAM17 activity, using isogenic Kras mutant (MT) and wild-type (WT) HCT116 CRC cells. Significantly higher levels of TGF-a and VEGF were shed from KrasMT HCT116 cells, both basally and following chemotherapy treatment, and this correlated with increased pErk (phosphorylated extracellular signal regulated kinase)1/2 levels and ADAM17 activity. Inhibition of Kras, MEK (MAP/ERK kinase)1/2, or Erk1/2 inhibition abrogated chemotherapy-induced ADAM17 activity and TGF-a shedding. Moreover, we found that these effects were not drug or cell line specific. In addition, MEK1/2 inhibition in KrasMT xenografts resulted in significant decreases in ADAM17 activity and growth factor shedding in vivo, which correlated with dramatically attenuated tumor growth. Furthermore, we found that MEK1/2 inhibition significantly induced apoptosis both alone and when combined with chemotherapy in KrasMT cells. Importantly, we found that sensitivity to MEK1/2 inhibition was ADAM17 dependent in vitro and in vivo. Collectively, our findings indicate that oncogenic Kras regulates ADAM17 activity and thereby growth factor ligand shedding in a MEK1/2/Erk1/2-dependent manner and that KrasMT CRC tumors are vulnerable to MEK1/2 inhibitors, at least in part, due to their dependency on ADAM17 activity.