Low Oxygen Availability Research Articles

Abstract 2922: Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

Abstract Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumour that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22rV1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that promote cell invasion and neuroendocrine differentiation. Gene ontology analysis of the hypoxia-inducible genes sensitive to loss of Rb revealed that a significant portion of these genes are involved in neuroendocrine differentiation (NED), specifically ENO2, KISS1R and HTR5A. ENO2 is the bonafide marker of neuroendocrine differentiation and it's presence is a signature of late stage castrate resistant prostate cancer. Furthermore, we have functional evidence KISS1R is linked to intracellular calcium mobilization in 22RV1 cells. We have demonstrated that increased expression of HIF-regulated genes in response to loss of Rb activates Akt and ERK signaling pathways and promotes neuroendocrine differentiation and invasion. Inhibition of these signaling pathways significantly decreased actin polymerization in LNCaP cells. For the first time, we have established a direct link between hypoxic tumour environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumours to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. Citation Format: Mark Labrecque, Mandeep Takhar, Rebecca J. Nason, Stephanie Santacruz, Kevin Tam, Shabnam Massah, Anne Haegert, Robert Bell, Manuel Altamirano-Dimas, Colin Collins, Frank Lee, Gratien Prefontaine, Michael Cox, Timothy Beischlag. Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2922.

Quality of minimally processed carrots in perforation-mediated modified-atmosphere packaging (PM-MAP)

Studies were carried out to investigate the effect of PM-MAP on minimally processed carrots with different number of perforations (0, 4, 12 and control) on flexible package, stored at 2 °C temperature. The effect of PM-MAP on headspace oxygen and carbon dioxide concentration, weight loss, surface color, firmness, pH, total soluble solid, β-carotene and microbial load were evaluated. It was observed that equilibrium modified atmospheric condition of 5–7 % oxygen and 7.8 % carbon dioxide was achieved in 4 perforation pack. Whiteness index (WI) increased in all the samples from the initial value of 22.75 to a range of 30–35 within 8 days of storage. After that, it remained almost constant for all samples except for 0 perforation packs where a decreasing trend was observed. Chroma value (CV) of samples also followed similar trends like WI and reduced from initial value of 63.08 to a range of 50–55 within 8 days of storage and remained in a constant range during further storage. Firmness of carrot slices decreased to 22–24 N and 25–28 N for 4 and 12 perforation pack respectively from initial value of 30 N after 40 days of storage. Lower oxygen availability in 4 perforation pack was main reason behind lower microbial count (5.2 log10 cfu/g) after 30 days of storage. Reduction of 5.52 % and 10.10 % in β-carotene content was observed in 4 and 12 perforation pack samples. Thus for PM-MAP, 150 g of carrot samples in 4 perforation packs proved to be better than other conditions.

Treatment of fish farm sludge supernatant by aerated filter beds and steel slag filters—effect of organic loading rate

The goal of our study was to develop an on-site cost-effective and extensive method for the treatment of supernatant of the sludge settling silo of fresh water fish farms. The main objectives of this study were 1) to determine the effect of the organic loading rate (OLR) on organic matter and nutrient removal efficiency of a pilot treatment system consisting of a series of aerated filter beds (AFBs) and electric arc furnace steel slag filters operated at different void hydraulic retention times (HRTV), 2) to validate the P-Hydroslag model as a design tool for slag filters and 3) to propose preliminary design options for fish farm sludge treatment. The 11.5 month experiment was divided into two phases: Phase 1 (P1) of 8.5 months with a low OLR to the AFBs of 0.015kg BOD5m−3 d−1, and Phase 2 (P2) of 3 months with a high OLR of 0.5kg BOD5m−3 d−1. The results showed that the OLR affected the organic matter mineralization and nitrification efficiency of AFBs. With a low OLR and an influent COD concentration of 320mgL−1, an average COD removal efficiency of 95% was observed while with a high OLR and an influent COD of 5400mgL−1, an average COD reduction of 65% was achieved. Due to the high OLR during P2, no nitrification took place probably due to the low availability of oxygen in the AFBs. The TP removal efficiency of the AFBs was 50% during P1 (influent of 5.2mgL−1), and 88% during P2 (influent of 110mgL−1). All SCs showed a high o-PO4 removal efficiency of >98% during P1 (average influent of 1.8mg P L−1, effluent of 0.04mg P L−1) and >85% during P2 (average influent of 8.7mg P L−1, effluent of 0.65mg P L−1) with a decrease in efficiency only observed over time in the SC that received the highest organic loading rate. It was concluded that with optimal loading rates, this compact biological and physicochemical semi-extensive treatment system offers a promising alternative to the high energy demand and maintenance treatment systems for organic matter and phosphorus removal, and that this treatment system could be applicable to other agro-environmental, municipal or residential effluents.

The effect of soil type on the bioremediation of petroleum contaminated soils

In this research the bioremediation of four different types of contaminated soils was monitored as a function of time and moisture content. The soils were categorized as sandy soil containing 100% sand (type I), clay soil containing more than 95% clay (type II), coarse grained soil containing 68% gravel and 32% sand (type III), and coarse grained with high clay content containing 40% gravel, 20% sand, and 40% clay (type IV). The initially clean soils were contaminated with gasoil to the concentration of 100 g/kg, and left on the floor for the evaporation of light hydrocarbons. A full factorial experimental design with soil type (four levels), and moisture content (10 and 20%) as the factors was employed. The soils were inoculated with petroleum degrading microorganisms. Soil samples were taken on days 90, 180, and 270, and the residual total petroleum hydrocarbon (TPH) was extracted using soxhlet apparatus. The moisture content of the soils was kept almost constant during the process by intermittent addition of water. The results showed that the efficiency of bioremediation was affected significantly by the soil type (Pvalue < 0.05). The removal percentage was the highest (70%) for the sandy soil with the initial TPH content of 69.62 g/kg, and the lowest for the clay soil (23.5%) with the initial TPH content of 69.70 g/kg. The effect of moisture content on bioremediation was not statistically significant for the investigated levels. The removal percentage in the clay soil was improved to 57% (within a month) in a separate experiment by more frequent mixing of the soil, indicating low availability of oxygen as a reason for low degradation of hydrocarbons in the clay soil.

Clam mitochondria respond better to hypoxia than scallop mitochondria

Few terrestrial animals ever experience hypoxia (when oxygen is scarce), but spells of low oxygen availability are routine for creatures clinging to life on the seashore: ‘Diurnal cycles of respiration and photosynthesis in the coastal zones commonly lead to O2 swings from near-anoxia during the night to hyperoxia during the day’, says Inna Sokolova from the University of North Carolina at Charlotte, USA. Yet, many of these creatures can withstand periods of oxygen deprivation lasting weeks and even months. Sokolova explains that several of the mechanisms that underpin the coast dwellers’ survival are already known, including the use of alternative mechanisms to generate ATP and the animals’ ability to drop their metabolic rate to conserve energy. However, Sokolova explains that little was known about how these resilient species protected the essential mitochondria – which consume oxygen to generate ATP – from the damaging effects of oxygen deprivation.Sokolova and a team of colleagues investigated the function of mitochondria in two shoreline species: the hypoxia-tolerant hard clam (Mercenaria mercenaria), which can survive for 2 weeks with no oxygen at all, and the less robust bay scallop (Argopecten irradians), which can only survive a few hours in anoxia. Keeping the bivalves in deoxygenated water (less than 0.1% oxygen) for 18 h, the team then collected samples from the gills, hepatopancreas tissue and adductor muscles to find out how the mitochondria had responded to oxygen deprivation. In addition, the team collected tissues from molluscs that had not experienced hypoxia, and from molluscs that had been transferred back into water with normal oxygen concentrations after hypoxia to find out how they responded as damaging oxygen flooded back into their bodies.Analysing the animals’ responses, the team saw that when the oxygen returned after an extended period of hypoxia, the scallops suffered similar experiences to those of other hypoxia-sensitive animals: the mitochondria were impaired, reducing their oxidation and phosphorylation capacities as they partially depolarized and the potential difference across the membrane reduced. In parallel, the vulnerable scallops ramped up production of heat shock proteins – which mop up proteins that have been damaged by the oxygen influx – in order to reduce the damage incurred by the mitochondria during reoxygenation. In contrast, the robust clams increased the oxidative capacity of their mitochondria during hypoxia and this also continued rising when the oxygen returned. Meanwhile, both of the molluscs reduced phosphorylation activity (which is normally responsible for ATP production via the Fo,F1-ATPase) during the extended period of hypoxia, ‘Likely to prevent ATP wastage by the reverse action of the ATPase’, say Sokolova and colleagues, although the clams regained this activity during the 1 h period of reoxygenation, when the scallops’ phosphorylation activity failed to improve. And when the team tested the bivalves’ ability to conserve energy by downregulating non-essential metabolic processes, it was clear that the clam was better able to reduce its energy expenditure than the more vulnerable scallop, although neither animal was severely energy deprived.So, a period of hypoxia dramatically affects both the hypoxia-tolerant hard clam and the more susceptible bay scallop, but alterations in the physiology of the clam leave it better prepared to tackle extended periods of hypoxia than its scallop shore-mate.

Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH).

In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants’ growth and yield—even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD+ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function.

MiR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga.

Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.

Improved shelf life of dried Beauveria bassiana blastospores using convective drying and active packaging processes.

The yeast form (blastospore) of the dimorphic insect-pathogenic fungus Beauveria bassiana can be rapidly produced using liquid fermentation methods but is generally unable to survive rapid dehydration processes or storage under non-refrigerated conditions. In this study, we evaluated the influence of two convective drying methods, various modified atmosphere packaging systems, and storage temperatures on the desiccation tolerance, storage stability, and virulence of blastospores of B. bassiana ESALQ 1432. All blastospore formulations were dried to <5% water content equivalent to aw<0.3. The viability of B. bassiana blastospores after air drying and spray drying was greater than 80%. Vacuum-packaged blastospores remained viable longer when stored at 4°C compared with 28°C with virtually no loss in viability over 9months regardless the drying method. When both oxygen and moisture scavengers were added to sealed packages of dried blastospore formulations stored at 28°C, viability was significantly prolonged for both air- and spray-dried blastospores. The addition of ascorbic acid during spray drying did not improve desiccation tolerance but enhanced cell stability (∼twofold higher half-life) when stored at 28°C. After storage for 4months at 28°C, air-dried blastospores produced a lower LC80 and resulted in higher mortality to whitefly nymphs (Bemisia tabaci) when compared with spray-dried blastospores. These studies identified key storage conditions (low aw and oxygen availability) that improved blastospore storage stability at 28°C and will facilitate the commercial development of blastospores-based bioinsecticides.

New insights into cell non-autonomous mechanisms of theC. eleganshypoxic response

The hypoxic response is a well-studied and highly conserved biological response to low oxygen availability. First described more than 20 y ago, the traditional model for this response is that declining oxygen levels lead to stabilization of hypoxia-inducible transcription factors (HIFs), which then bind to hypoxia responsive elements (HREs) in target genes to mediate the transcriptional changes collectively known as the hypoxic response.(1,2) Recent work in C. elegans has forced a re-evaluation of this model by indicating that the worm HIF (HIF-1) can mediate effects in a cell non-autonomous fashion and, in at least one case, increase expression of an intestinal hypoxic response target gene in cells lacking HIF-1.

The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells.

Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.

Hydrographic conditions driving sardine and anchovy populations in a land-locked sea

The aim of this paper is to establish a relationship between long-term variability in sardine and anchovy populations in the Adriatic Sea and ocean dynamics and processes that occur over interannual and decadal timescales in the Adriatic-Ionian basin. Basis for such analysis are annual time series of sardine and anchovy landings and recruits at age 0 and annual time series of environmental parameters observed at a representative Adriatic station between 1975 and 2010. Pearson correlations and robust Dynamic Factor Analysis (DFA) were applied to quantify the connections between fisheries and environmental parameters. Variations and trends in fisheries series were best explained by changes in near-bottom temperature and salinity, being an appropriate proxy for tracking changes in water masses' dynamics and hydrographic conditions in the basin. It seems that a prolonged period of decreasing sardine population was characterized by low oxygen availability and environmental conditions in the deep Adriatic waters, triggered by an extraordinary basin-wide event called the Eastern Mediterranean Transient. A collapse in anchovy population has been observed after an exceptional cooling event followed by dense water formation.

PLC-β2 is modulated by low oxygen availability in breast tumor cells and plays a phenotype dependent role in their hypoxia-related malignant potential.

Limited oxygen availability plays a critical role in the malignant progression of breast cancer by orchestrating a complex modulation of the gene transcription largely dependent on the tumor phenotype. Invasive breast tumors belonging to different molecular subtypes are characterized by over-expression of PLC-β2, whose amount positively correlates with the malignant evolution of breast neoplasia and supports the invasive potential of breast tumor cells. Here we report that hypoxia modulates the expression of PLC-β2 in breast tumor cells in a phenotype-related manner, since a decrease of the protein was observed in the BT-474 and MCF7 cell lines while an increase was revealed in MDA-MB-231 cells as a consequence of low oxygen availability. Under hypoxia, the down-modulation of PLC-β2 was mainly correlated with the decrease of the EMT marker E-cadherin in the BT-474 cells and with the up-regulation of the stem cell marker CD133 in MCF7 cells. The increase of PLC-β2 induced by low oxygen in MDA-MB-231 cells supports the hypoxia-related reorganization of actin cytoskeleton and sustains invasion capability. In all examined cell lines, but with an opposite role in the ER-positive and ER-negative cells, PLC-β2 was involved in the hypoxia-induced increase of HIF-1α, known to affect both EMT and CD133 expression. Our data include PLC-β2 in the complex and interconnected signaling pathways induced by low oxygen availability in breast tumor cells and suggest that the forced modulation of PLC-β2 programmed on the basis of tumor phenotype may prevent the malignant progression of breast neoplasia as a consequence of intra-tumoral hypoxia. © 2016 Wiley Periodicals, Inc.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution.

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [(13)C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [(13)C]glutamate and [(15)N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the (13)C/(15)N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

Skeletal muscle hypoxia-inducible factor-1 and exercise.

Reduced oxygen levels in skeletal muscle during exercise are a consequence of increased oxygen consumption. The cellular response to hypoxia is conferred to a large extent by activation of the hypoxia-sensitive transcription factor hypoxia-inducible factor-1 (HIF-1). The target genes of HIF-1 increase oxygen transport through mechanisms such as erythropoietin-mediated erythropoiesis and vascular endothelial growth factor-induced angiogenesis and improve tissue function during low oxygen availability through increased expression of glucose transporters and glycolytic enzymes, which makes HIF-1 an interesting candidate as a mediator of skeletal muscle adaptation to endurance training. However, HIF-1 may also inhibit cellular oxygen consumption and mitochondrial oxidative metabolism, features discordant with the phenotype of a trained muscle. Skeletal muscle readily adjusts to altered functional demands. Adaptation of skeletal muscle to long-term aerobic training enables better aerobic performance at higher intensities through improved metabolic capacity and oxygen supply. The components of acute exercise that act as triggers for adaptation are still largely unknown; however, an early hypothesis was that local hypoxia acts as a possible stimulus for exercise adaptation. The hypoxia-sensitive subunit, HIF-1α, is stabilized in skeletal muscle in response to an acute bout of endurance exercise. However, long-term endurance exercise seems to attenuate the acute HIF-1α response. This attenuation is concurrent with an increase in expression of several negative regulators of the HIF system. We propose that the HIF-1α response is blunted in response to long-term exercise training through induction of its negative regulators and that this inhibition enables the enhanced oxidative metabolism that is part of a local physiological response to exercise.

Hypoxia-inducible factors regulate T cell metabolism and function.

Resolution of infection requires the coordinated response of heterogeneous cell types to a range of physiological and pathological signals to regulate their proliferation, migration, differentiation, and effector functions. One mechanism by which immune cells integrate these signals is through modulating metabolic activity. A well-studied regulator of cellular metabolism is the hypoxia-inducible factor (HIF) family, the highly conserved central regulators of adaptation to limiting oxygen tension. HIF's regulation of cellular metabolism and a variety of effector, signaling, and trafficking molecules has made these transcription factors a recent topic of interest in T cell biology. Low oxygen availability, or hypoxia, increases expression and stabilization of HIF in immune cells, activating molecular programs both unique and common among cell types, including glycolytic metabolism. Notably, numerous oxygen-independent signals, many of which are active in T cells, also result in enhanced HIF activity. Here, we discuss both oxygen-dependent and -independent regulation of HIF activity in T cells and the resulting impacts on metabolism, differentiation, function, and immunity.

Parasite‐induced inversion of geotaxis in a freshwater amphipod: a role for anaerobic metabolism?

SummaryMany parasites with complex life cycles alter the phenotype of their intermediate hosts in ways that seem to favour transmission to a final host. Although there is a large literature on host manipulation, how parasites alter the phenotype of their hosts remains poorly known.The bird acanthocephalanPolymorphus minutusis known to alter geotaxis in its amphipod host,Gammarus roeseli. Here, we examine the potential roles of low oxygen availability and the excretion, by the parasite, of two products from its own anaerobic metabolism (lactate and succinate) in altered geotaxis.Under hypoxia, uninfectedG. roeselishowed negative geotaxis and lower metabolic rate, two traits also altered by infection withP. minutus, albeit with different intensities. The injection of a mixture of lactate and succinate in uninfected amphipods mimicked the parasite‐induced reversion of geotaxis, without affecting the metabolic rate.In addition, bothP. minutus‐infected gammarids and uninfected ones conditioned to hypoxia for 2 days showed elevated levels of lactate in the brain, but not in the haemolymph.Overall, our results indicate that the pathways involved in anaerobic metabolism and hypoxia signalling might be responsible for the changes in geotaxis and metabolic rate induced byP. minutusinfection.Our study emphasizes the need to consider the tight and complex connections between physiological processes and behavioural adjustments, in particular at the brain level, in the understanding of parasitic manipulation, and more broadly of behavioural changes in infected hosts.

Quantitative Trait Loci for Salinity Tolerance Identified under Drained and Waterlogged Conditions and Their Association with Flowering Time in Barley (Hordeum vulgare. L).

IntroductionSalinity is one of the major abiotic stresses affecting crop production via adverse effects of osmotic stress, specific ion toxicity, and stress-related nutritional disorders. Detrimental effects of salinity are also often exacerbated by low oxygen availability when plants are grown under waterlogged conditions. Developing salinity-tolerant varieties is critical to overcome these problems, and molecular marker assisted selection can make breeding programs more effective.MethodsIn this study, a double haploid (DH) population consisting of 175 lines, derived from a cross between a Chinese barley variety Yangsimai 1 (YSM1) and an Australian malting barley variety Gairdner, was used to construct a high density molecular map which contained more than 8,000 Diversity Arrays Technology (DArT) markers and single nucleotide polymorphism (SNP) markers. Salinity tolerance of parental and DH lines was evaluated under drained (SalinityD) and waterlogged (SalinityW) conditions at two different sowing times.ResultsThree quantitative trait loci (QTL) located on chromosome 1H, single QTL located on chromosomes 1H, 2H, 4H, 5H and 7H, were identified to be responsible for salinity tolerance under different environments. Waterlogging stress, daylight length and temperature showed significant effects on barley salinity tolerance. The QTL for salinity tolerance mapped on chromosomes 4H and 7H, QSlwd.YG.4H, QSlwd.YG.7H and QSlww.YG.7H were only identified in winter trials, while the QTL on chromosome 2H QSlsd.YG.2H and QSlsw.YG.2H were only detected in summer trials. Genes associated with flowering time were found to pose significant effects on the salinity QTL mapped on chromosomes 2H and 5H in summer trials. Given the fact that the QTL for salinity tolerance QSlsd.YG.1H and QSlww.YG.1H-1 reported here have never been considered in the literature, this warrants further investigation and evaluation for suitability to be used in breeding programs.

Left-Biased Spermatogenic Failure in 129/SvJ Dnd1Ter/+ Mice Correlates with Differences in Vascular Architecture, Oxygen Availability, and Metabolites.

Homozygosity for the Ter mutation in the RNA-binding protein Dead end 1 (Dnd1(Ter/Ter)) sensitizes germ cells to degeneration in all mouse strains. In 129/SvJ mice, approximately 10% of Dnd1(Ter/+) heterozygotes develop spermatogenic failure, and 95% of unilateral cases occur in the left testis. The first differences between right and left testes were detected at Postnatal Day 15 when many more spermatogonial stem cells (SSCs) were undergoing apoptosis in the left testis compared to the right. As we detected no significant left/right differences in the molecular pathway associated with body axis asymmetry or in the expression of signals known to promote proliferation, differentiation, and survival of germ cells, we investigated whether physiological differences might account for asymmetry of the degeneration phenotype. We show that left/right differences in vascular architecture are associated with a decrease in hemoglobin saturation and increased levels of HIF-1alpha in the left testis compared to the right. In Dnd1 heterozygotes, lower oxygen availability was associated with metabolic differences, including lower levels of ATP and NADH in the left testis. These experiments suggest a dependence on oxygen availability and metabolic substrates for SSC survival and suggest that Dnd1(Ter/+) SSCs may act as efficient sensors to detect subtle environmental changes that alter SSC fate.

Effect of CoCl₂ on fracture repair in a rat model of bone fracture.

Low oxygen availability is known to activate the hypoxia-inducible factor-1α (HIF-1α) pathway, which is involved in the impairment of fracture healing. However, the role of low oxygen in fracture healing remains to be fully elucidated. In the present study, rats were divided into two groups and treated with CoCl2 or saline, respectively. Mice with tibial fractures were sacrificed at 14, 28 and 42 days subsequent to fracture. Autoradiography was performed to measure healing of the bone tissue. In addition, the effects of cobalt chloride (CoCl2) on the expression of two major angiogenic mediators, HIF‑1α and vascular endothelial growth factor (VEGF), as well as the osteoblast markers runt‑related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) were determined at mRNA and protein levels by reverse transcription‑quantitative polymerase chain reaction, western blot analysis and immunohistochemistry. Systemic administration of CoCl2 (15 mg/kg/day intraperitoneally) significantly promoted fracture healing and mechanical strength. The present study demonstrated that in rats treated with CoCl2, the expression of HIF‑1α, VEGF, Runx2, ALP and OC was significantly increased at mRNA and protein levels, and that CoCl2 treatment enhances fracture repair in vivo.

Corrigenda: Epigean and hypogean Palaemonetes sp. (Decapoda, Palaemonidae) from Edwards Aquifer: An examination of trophic structure and metabolism. Subterranean Biology 14: 79–102.

This study addresses the causes of the metabolic depression observed when examining the metabolism of hypogean versus epigean organisms. We examined the two current hypotheses regarding the cause of metabolic cave adaptation, a paucity of food and low oxygen availability, both necessary for ATP production, by first determining if the hypogean environment examined, Edwards Aquifer, was resource limited. Stable isotope analyses indicate that there is extensive microbial chemolithoautotrophic production providing resources for the hypogean organisms. &delta;13C values ( &le;30&permil; ) were well below that of terrestrial biome indicating that C in the aquifer originates from chemolithoautotrophic inorganic carbon fixation, not photosynthetically derived material resulting from terrigenous sources. Data suggest the artesian system is a complex geochemical ecosystem providing inorganic energy sources from both methane and sulfates. Metabolism, examined via key aerobic and anaerobic proxies, and organismal proximate composition indicated there was no difference between metabolic rates and energy storage of Palaemonetes antrorum (stygobitic) and Palaemonetes kadiakensis (epigean). This indicates that resources within the oxic aquifer are not limited. We demonstrate that it is necessary for one, or both, of these selective pressures to be present for metabolic cave adaptation to occur.