Low serum testosterone is associated with adverse outcome in men with cirrhosis independent of the model for end-stage liver disease score.

Low serum testosterone has been retrospectively associated with mortality in men on the liver transplant waiting list. The impact of testosterone deficiency on other outcomes has not previously been assessed. We conducted a single center prospective observational study of all men with cirrhosis seen between 2013 and 2014. Baseline data included sex hormone profile, Model for End-Stage Liver Disease (MELD) score, and standard biochemistry. Outcomes were recorded over 12 months including major infection, liver transplantation, and death. Of 268 cirrhotic men, the median MELD score was 10 (interquartile range [IQR], 8-15) and median serum testosterone was 17.4 nmol/L (IQR, 8.9-25.0 nmol/L). During the study period, 32 (12%) men died, 18 (7%) received a liver transplant, and 51 (19%) suffered a major infection. Mortality markedly increased when total testosterone fell below a threshold value of 8.3 nmol/L, and this cutoff was used for further analysis. Testosterone below 8.3 nmol/L was associated with the combined outcome of death or transplantation independently of the MELD score (hazard ratio [HR], 2.36; IQR, 1.16-4.81; P = 0.02) for testosterone (and HR, 1.22; IQR, 1.18-1.27; P < 0.001 for MELD). Low total testosterone was also an independent risk factor for major infection (HR, 3.61; IQR, 1.61-8.06; P < 0.001) and nearly significant for mortality alone (HR, 2.39; IQR, 0.97-5.88; P = 0.057). Low free testosterone (<139 pmol/L) was similarly independently associated with death or transplantation (HR, 2.43; IQR, 1.12-5.29; P = 0.03) and infection (HR, 3.3; IQR, 1.46-7.46; P = 0.004). In conclusion, low testosterone is a novel prognostic marker in men with cirrhosis that is numerically associated with increased mortality or need for transplantation, as well as risk for major infection. Interventional studies of testosterone therapy are required to investigate whether correcting low testosterone can reduce mortality and improve other clinical outcomes. Liver Transplantation 22 1482-1490 2016 AASLD.

Ecotoxicological impact of Zequanox®, a novel biocide, on selected non-target Irish aquatic species.

Effective, species-specific zebra mussel control is needed urgently for Ireland׳s freshwater bodies, which became infested with non-native zebra mussels in the 1990s. Zequanox®, a newly commercialized product for zebra and quagga mussel control, is composed of dead Pseudomonas fluorescens CL 145A cells. This paper describes ecotoxicology tests on three representative native Irish freshwater species: Anodonta (duck mussel), Chironomus plumosus (non-biting midge), and Austropotamobius pallipes (white-clawed crayfish). The species were exposed to Zequanox in a 72-h static renewal toxicity test at concentrations of 100-750mg active ingredient per liter (mga.i./L). Water quality parameters were measured every 12-24h before and after water and product renewal. After 72h, endpoints were reported as LC10, LC50, and LC100. The LC50 values derived were (1) Anodonta: ≥500mga.i./L (2) C. plumosus: 1075mga.i./L, and (3) A. pallipes: ≥750mga.i./L. These results demonstrate that Zequanox does not negatively affect these organisms at the concentration required for >80percent zebra mussel mortality (150mg a.i/L) and the maximum allowable treatment concentration in the United Sates (200mga.i./L). They also show the overall species-specificity of Zequanox, and support its use in commercial facilities and open waters.

Systemic RNAi mediated gene silencing in the anhydrobiotic nematode Panagrolaimus superbus.

BACKGROUND: Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in Caenorhabditis elegans, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms Pristionchus pacificus and Oscheius tipulae. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus Panagrolaimus contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many Panagrolaimus species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether Panagrolaimus is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics. RESULTS: We studied two species: Panagrolaimus sp. PS1159 and Panagrolaimus superbus. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of Escherichia coli expressing dsRNA for the C. elegans embryonic lethal genes Ce-lmn-1 and Ce-ran-4. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the Panagrolaimus genes ef1b and rps-2. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target ef1b and rps-2 genes in RNAi treated Panagrolaimus sp. 1159 nematodes. Visible RNAi phenotypes were also observed when P. superbus was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in P. superbus. CONCLUSION: This demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes.

Anhydrobiosis and freezing-tolerance: adaptations that facilitate the establishment of Panagrolaimus nematodes in polar habitats.

Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. Panagrolaimus davidi, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent "ecological fitting" of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.

The anhydrobiotic potential and molecular phylogenetics of species and strains of Panagrolaimus (Nematoda, Panagrolaimidae).

Members of the genus Panagrolaimus are bacterial-feeding nematodes that occupy a diversity of niches ranging from Antarctic and temperate soils to terrestrial mosses. Some members of this genus are able to survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. We have assembled a collection of Panagrolaimus species and strains and have investigated their anhydrobiotic phenotypes. Our data show that within the genus Panagrolaimus there is a continuum of strains ranging from those unable to survive exposure to low relative humidity (RH) without prior preconditioning at high RH (slow desiccation strategists), through strains that have limited ability to survive rapid desiccation but whose anhydrobiotic ability improves upon preconditioning, to strains such as P. superbus that can readily survive immediate exposure to severe desiccation (fast desiccation strategists). Using this panel of nematodes we investigated the effect of preincubation at high RH on the accumulation of trehalose and on the nematodes' anhydrobiotic potential. We found that there is a strong correlation between trehalose induction and anhydrobiotic survival in Panagrolaimus. Furthermore, the high trehalose levels observed in fully hydrated P. superbus (10% dry mass) suggest that constitutive expression of trehalose pre-adapts this fast dehydration strategist to combat desiccation. All the strains observed, regardless of survival rates, undertook both coiling and clumping, which has the effect of reducing surface area and slowing the rate of water loss during desiccation. Phylogenetic analyses were carried out to investigate whether the observed anhydrobiotic phenotypes were the result of convergent evolution or represented a single phylogenetic lineage. These analyses, derived from alignments of the rDNA ITS and D3 sequences, indicate that the strongly anhydrobiotic strains of Panagrolaimus form a single phylogenetic lineage, which is separate from the weakly anhydrobiotic strains. The weakly anhydrobiotic strains are also phylogenetically divergent from each other. Our data indicate that Panagrolaimus has the potential to be an excellent model system for the investigation of molecular aspects of nematode anhydrobiosis.