Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Pilot study to investigate the effect of long-term exposure to high pCO2 on adult cod (Gadus morhua) otolith morphology and calcium carbonate deposition.

To date the study of ocean acidification on fish otolith formation has been mainly focused on larval and juvenile stages. In the present pilot study, wild-captured adult Atlantic cod (Gadus morhua) were exposed to two different levels of pCO2, 422µatm (ambient, low pCO2) or 1091µatm (high pCO2), for a period of 30 weeks (from mid-October to early April 2014-2015) in order to study the effects on otolith size, shape and CaCO3 crystallization amongst other biological parameters. We found that otoliths from cod exposed to high pCO2 were slightly smaller (- 3.4% in length; - 3.3% in perimeter), rounder (- 2.9% circularity and + 4% roundness) but heavier (+ 5%) than the low pCO2 group. Interestingly, there were different effects in males and females; for instance, male cods exposed to high pCO2 exhibited significant changes in circularity (- 3%) and roundness (+ 4%) compared to the low pCO2 males, but without significant changes on otolith dimensions, while females exposed to high pCO2 had smaller otoliths as shown for length (- 5.6%), width (- 2%), perimeter (- 3.5%) and area (- 4.8%). Furthermore, while the majority of the otoliths analysed showed normal aragonite deposition, 10% of fish exposed to 1091µatm of pCO2 had an abnormal accretion of calcite, suggesting a shift on calcium carbonate polymorph crystallization in some individuals under high pCO2 conditions. Our preliminary results indicate that high levels of pCO2 in adult Atlantic cod might affect otolith growth in a gender-specific way. Our findings reveal that otoliths from adult cod are affected by ocean acidification, and we believe that the present study will prompt further research into this currently under-explored area.

Acid-base adjustments and first evidence of denticle corrosion caused by ocean acidification conditions in a demersal shark species.

Global ocean acidification is expected to chronically lower the pH to 7.3 (>2200 µatm seawater pCO2) by the year 2300. Acute hypercapnia already occurs along the South African west and south coasts due to upwelling- and low-oxygen events, with increasing frequency. In the present project we investigated the impact of hypercapnia on the endemic demersal shark species Haploblepharus edwardsii. Specifically, we experimentally analysed acid-base regulation during acute and chronic hypercapnia, the effects of chronic hypercapnia on growth rates and on denticle structure- and composition. While H. edwardsii are physiologically well adapted to acute and chronic hypercapnia, we observed, for the first time, denticle corrosion as a result of chronic exposure. We conclude that denticle corrosion could increase denticle turnover and compromise hydrodynamics and skin protection.

Establishing key criteria to define and compare models of specialist palliative care: A mixed-methods study using qualitative interviews and Delphi survey.

BACKGROUND: Specialist palliative care services have various configurations of staff, processes and interventions, which determine how care is delivered. Currently, there is no consistent way to define and distinguish these different models of care. AIM: To identify the core components that characterise and differentiate existing models of specialist palliative care in the United Kingdom. DESIGN: Mixed-methods study: (1) semi-structured interviews to identify criteria, (2) two-round Delphi study to rank/refine criteria, and (3) structured interviews to test/refine criteria. SETTING/PARTICIPANTS: Specialist palliative care stakeholders from hospice inpatient, hospital advisory, and community settings. RESULTS: (1) Semi-structured interviews with 14 clinical leads, from eight UK organisations (five hospice inpatient units, two hospital advisory teams, five community teams), provided 34 preliminary criteria. (2) Delphi study: Round 1 (54 participants): thirty-four criteria presented, seven removed and seven added. Round 2 (30 participants): these 34 criteria were ranked with the 15 highest ranked criteria, including setting, type of care, size of service, diagnoses, disciplines, mode of care, types of interventions, 'out-of-hours' components (referrals, times, disciplines, mode of care, type of care), external education, use of measures, bereavement follow-up and complex grief provision. (3) Structured interviews with 21 UK service leads (six hospice inpatients, four hospital advisory and nine community teams) refined the criteria from (1) and (2), and provided four further contextual criteria (team purpose, funding, self-referral acceptance and discharge). CONCLUSION: In this innovative study, we derive 20 criteria to characterise and differentiate models of specialist palliative care - a major paradigm shift to enable accurate reporting and comparison in practice and research.

Ocean acidification promotes otolith growth and calcite deposition in gilthead sea bream (Sparus aurata) larvae.

The effects of ocean acidification on otolith crystallization and growth rates were investigated in gilthead sea bream (Sparus aurata) larvae. Larvae were exposed to three different pH levels: pH8.2, pH7.7 and pH7.3 for a period of 18 days post-fertilization. For the first time, we demonstrate that pH has a significant impact on the carbonate polymorph composition, showing calcite in a significant percentage of individuals at low pH. Around 21% of the larvae exposed to pH7.3 showed irregular calcitic otoliths rather than commonly found round aragonitic otoliths. Calcitic otoliths showed a moderate level of heritability suggesting an important role of genetic factors. We also observed significantly larger otoliths in larvae reared at pH7.7 and pH7.3 compared to pH8.2 in both sagittae and lapilli. Our results demonstrate that otolith growth rates in gilthead sea bream larvae increase at low pH while a significant proportion of larvae are prone to the formation of calcitic otoliths at pH7.3.

Acid-base balance and changes in haemolymph properties of the South African rock lobsters, Jasus lalandii, a palinurid decapod, during chronic hypercapnia.

Few studies exist reporting on long-term exposure of crustaceans to hypercapnia. We exposed juvenile South African rock lobsters, Jasus lalandii, to hypercapnic conditions of pH 7.3 for 28 weeks and subsequently analysed changes in the extracellular fluid (haemolymph). Results revealed, for the first time, adjustments in the haemolymph of a palinurid crustacean during chronic hypercapnic exposure: 1) acid-base balance was adjusted and sustained by increased bicarbonate and 2) quantity and oxygen binding properties of haemocyanin changed. Compared with lobsters kept under normocapnic conditions (pH 8.0), during prolonged hypercapnia, juvenile lobsters increased bicarbonate buffering of haemolymph. This is necessary to provide optimum pH conditions for oxygen binding of haemocyanin and functioning of respiration in the presence of a strong Bohr Effect. Furthermore, modification of the intrinsic structure of the haemocyanin molecule, and not the presence of molecular modulators, seems to improve oxygen affinity under conditions of elevated pCO2.

Intra-pituitary relationship of follicle stimulating hormone and luteinizing hormone during pubertal development in Atlantic bluefin tuna (Thunnus thynnus).

As part of the endeavor aiming at the domestication of Atlantic bluefin tuna (BFT; Thunnus thynnus), first sexual maturity in captivity was studied by documenting its occurrence and by characterizing the key hormones of the reproductive axis: follicle stimulating hormone (FSH) and luteinizing hormone (LH). The full length sequence encoding for the related hormone ß-subunits, bftFSHß and bftLHß, were determined, revealing two bftFSHß mRNA variants, differing in their 5' untranslated region. A quantitative immuno-dot-blot assay to measure pituitary FSH content in BFT was developed and validated enabling, for the first time in this species, data sets for both LH and FSH to be compared. The expression and accumulation patterns of LH in the pituitary showed a steady increase of this hormone, concomitant with fish age, reaching higher levels in adult females compared to males of the same age class. Conversely, the pituitary FSH levels were elevated only in 2Y and adult fish. The pituitary FSH to LH ratio was consistently higher (>1) in immature than in maturing or pubertal fish, resembling the situation in mammals. Nevertheless, the results suggest that a rise in the LH storage level above a minimum threshold may be an indicator of the onset of puberty in BFT females. The higher pituitary LH levels in adult females over males may further support this notion. In contrast three year-old (3Y) males were pubertal while cognate females were still immature. However, it is not yet clear whether the advanced puberty in the 3Y males was a general feature typifying wild BFT populations or was induced by the culture conditions. Future studies testing the effects of captivity and hormonal treatments on precocious maturity may allow for improved handling of this species in a controlled environment which would lead to more cost-efficient farming.

Building local community commitment to wetlands restoration: a case study of the Cache River Wetlands in southern Illinois, USA.

Natural resource professionals are increasingly faced with the challenges of cultivating community-based support for wetland ecosystem restoration. While extensive research efforts have been directed toward understanding the biophysical dimensions of wetland conservation, the literature provides less guidance on how to successfully integrate community stakeholders into restoration planning. Therefore, this study explores the social construction of wetlands locally, and community members' perceptions of the wetland restoration project in the Cache River Watershed of southern Illinois, where public and private agencies have partnered together to implement a large-scale wetlands restoration project. Findings illustrate that the wetlands hold diverse and significant meanings to community members and that community members' criteria for project success may vary from those identified by project managers. The case study provides managers with strategies for building community commitment such as engaging local citizens in project planning, minimizing local burdens, maximizing local benefits, and reducing uncertainty.

Respiratory adaptations to the deep-sea hydrothermal vent environment: the case of Segonzacia mesatlantica, a crab from the Mid-Atlantic Ridge.

Segonzacia mesatlantica (Crustacea; Decapoda; Brachyura) is the only endemic crab species known from the Mid-Atlantic Ridge (MAR) hydrothermal vents. Known from all explored sites in the Atlantic, its wide distribution makes this species a model to study physiological adaptation, and specifically respiratory strategies. Native haemocyanin (Hc) comprises four non-covalent associations in equilibrium formed by monomers, hexamers, dodecamers and octadecamers made up of approximately 75 kDa polypeptide chains. Four different amino acid chains are observed with a molecular mass ranging from 75,234 to 75,972 Da. Experiments carried-out under pressure suggested that the percentage of monomer increased in the haemolymph under hypoxic condition. We have also observed a shift of the proportion of the two dodecamer series, suggesting a rapid modification of the Hc phenotype between hypoxic and hyperoxic conditions. Native Hc possesses a high oxygen affinity ( P50 = 2.2 Torr at 15 degrees C and pH 7.5), a large Bohr effect (Deltalog P50 / DeltapH approximately -2.7) and a slightly reverse temperature effect (DeltaH = +17.19 kJ mol(-1). The composition of Segonzacia haemolymph is similar to that of other littoral species except for the large enrichment in free copper and zinc. As for other species from hydrothermal vent sites, Segonzacia haemolymph possesses a higher buffer capacity than littoral species. Moreover, species from the hydrothermal vent decapods from Pacific hydrothermal vent that encounter higher CO2 content in their environment have a higher buffer capacity than Atlantic vent species. The results presented are discussed in relation with the physico-chemical characteristics of the hydrothermal vent environment.