Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5′-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 μM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1- W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55°C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des-(A1-K6)-SHMT (55°C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8 ± 0.1 M) and des-(A1-K6)-SHMT (1.7 ± 0.1 M). The apoenzyme of des-(A1- W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5′-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-W14)-SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT

VeSV- Value at the end of the Sanitation Value Chain: Final Report

Bangladesh is no stranger to composting projects using both green waste and faecal sludge (FS). There have been many initiatives over the years with varying degrees of success. Similarly there have been hundreds, if not thousands of projects to improve access to latrines, latrine use and latrine management. Again there has been a great deal of success, especially in increasing the number of latrines being built. However, a key gap regarding the safe collection and processing of the waste from the pit still remains. In cases where projects have attempted addressing this, the solution has rarely been viable on a large scale. That is where this project—VeSV—is different. The aim of this project is to provide scientific evidence to support the commercial viability of collecting and composting faecal sludge for use in agriculture and horticulture. The gap between a good idea and commercial success is bridged on this project by producing primary scientific data based on qualitative and quantitative research methods and by engaging a number of stakeholders across sectors. A rigorous research was conducted to characterize raw faecal sludge material from single pit latrines in rural Bangladesh, as the starting point to develop value across the sanitation chain from processing FS material, through adding value by recovering nutrient and finally by assessing the potential commercialization of the final product in the fertilizer market. Crucially academics, NGOs, business groups and existing fertilizer, composting and latrine management companies were involved as part of our Reference Group, which helped to develop practical engineering solutions in harmony with the right and relevant context in rural Bangladesh. Our research outcomes include the development of safe methodologies for pit emptying; the assessment of people's intentions to change current operation and maintenance practices of pit latrines at household level and their willingness to participate in commercially viable and sustainable methods for FS management; the assessment of optimised engineering process for FS stabilisation and the production of a safe, high quality fertilizer that is desirable to farmers; and the identification of potential hurdles that may obstruct the widespread adoption of business models for FS fertiliser

Towards sustainable sanitation management: Establishing the costs and willingness to pay for emptying and transporting sludge in rural districts with high rates of access to latrines

MOTIVATION: Proper management of fecal sludge has significant positive health and environmental externalities. Most research on managing onsite sanitation so far either simulates the costs of, or the welfare effects from, managing sludge in situ in pit latrines. Thus, designing management strategies for onsite rural sanitation is challenging, because the actual costs of transporting sludge for treatment, and sources for financing these transport costs, are not well understood. METHODS: In this paper we calculate the actual cost of sludge management from onsite latrines, and identify the contributions that latrine owners are willing to make to finance the costs. A spreadsheet-based model is used to identify a cost-effective transport option, and to calculate the cost per household. Then a double-bound contingent valuation method is used to elicit from pit-latrine owners their willingness-to-pay to have sludge transported away. This methodology is employed for the case of a rural subdistrict in Bangladesh called Bhaluka, a unit of administration at which sludge management services are being piloted by the Government of Bangladesh. RESULTS: The typical sludge accumulation rate in Bhaluka is calculated at 0.11 liters/person/day and a typical latrine will need to be emptied approximately once every 3 to 4 years. The costs of emptying and transport are high; approximately USD 13 per emptying event (circa 14% of average monthly income); household contributions could cover around 47% of this cost. However, if costs were spread over time, the service would cost USD 4 per year per household, or USD 0.31 per month per household-comparable to current expenditures of rural households on telecommunications. CONCLUSION: This is one of few research papers that brings the costs of waste management together with financing of that cost, to provide evidence for an implementable solution. This framework can be used to identify cost effective sludge management options and private contributions towards that cost in other (context-specific) administrative areas where onsite sanitation is widespread

Incisional hernia following colorectal cancer surgery according to suture technique: Hughes Abdominal Repair Randomized Trial (HART).

BACKGROUND: Incisional hernias cause morbidity and may require further surgery. HART (Hughes Abdominal Repair Trial) assessed the effect of an alternative suture method on the incidence of incisional hernia following colorectal cancer surgery. METHODS: A pragmatic multicentre single-blind RCT allocated patients undergoing midline incision for colorectal cancer to either Hughes closure (double far-near-near-far sutures of 1 nylon suture at 2-cm intervals along the fascia combined with conventional mass closure) or the surgeon's standard closure. The primary outcome was the incidence of incisional hernia at 1 year assessed by clinical examination. An intention-to-treat analysis was performed. RESULTS: Between August 2014 and February 2018, 802 patients were randomized to either Hughes closure (401) or the standard mass closure group (401). At 1 year after surgery, 672 patients (83.7 per cent) were included in the primary outcome analysis; 50 of 339 patients (14.8 per cent) in the Hughes group and 57 of 333 (17.1 per cent) in the standard closure group had incisional hernia (OR 0.84, 95 per cent c.i. 0.55 to 1.27; P = 0.402). At 2 years, 78 patients (28.7 per cent) in the Hughes repair group and 84 (31.8 per cent) in the standard closure group had incisional hernia (OR 0.86, 0.59 to 1.25; P = 0.429). Adverse events were similar in the two groups, apart from the rate of surgical-site infection, which was higher in the Hughes group (13.2 versus 7.7 per cent; OR 1.82, 1.14 to 2.91; P = 0.011). CONCLUSION: The incidence of incisional hernia after colorectal cancer surgery is high. There was no statistical difference in incidence between Hughes closure and mass closure at 1 or 2 years. REGISTRATION NUMBER: ISRCTN25616490 (http://www.controlled-trials.com)

Genome-wide association study of thyroid-stimulating hormone highlights new genes, pathways and associations with thyroid disease

Thyroid hormones play a critical role in regulation of multiple physiological functions and thyroid dysfunction is associated with substantial morbidity. Here, we use electronic health records to undertake a genome-wide association study of thyroid-stimulating hormone (TSH) levels, with a total sample size of 247,107. We identify 158 novel genetic associations, more than doubling the number of known associations with TSH, and implicate 112 putative causal genes, of which 76 are not previously implicated. A polygenic score for TSH is associated with TSH levels in African, South Asian, East Asian, Middle Eastern and admixed American ancestries, and associated with hypothyroidism and other thyroid disease in South Asians. In Europeans, the TSH polygenic score is associated with thyroid disease, including thyroid cancer and age-of-onset of hypothyroidism and hyperthyroidism. We develop pathway-specific genetic risk scores for TSH levels and use these in phenome-wide association studies to identify potential consequences of pathway perturbation. Together, these findings demonstrate the potential utility of genetic associations to inform future therapeutics and risk prediction for thyroid diseases

Intraperitoneal drain placement and outcomes after elective colorectal surgery: international matched, prospective, cohort study

Despite current guidelines, intraperitoneal drain placement after elective colorectal surgery remains widespread. Drains were not associated with earlier detection of intraperitoneal collections, but were associated with prolonged hospital stay and increased risk of surgical-site infections.Background Many surgeons routinely place intraperitoneal drains after elective colorectal surgery. However, enhanced recovery after surgery guidelines recommend against their routine use owing to a lack of clear clinical benefit. This study aimed to describe international variation in intraperitoneal drain placement and the safety of this practice. Methods COMPASS (COMPlicAted intra-abdominal collectionS after colorectal Surgery) was a prospective, international, cohort study which enrolled consecutive adults undergoing elective colorectal surgery (February to March 2020). The primary outcome was the rate of intraperitoneal drain placement. Secondary outcomes included: rate and time to diagnosis of postoperative intraperitoneal collections; rate of surgical site infections (SSIs); time to discharge; and 30-day major postoperative complications (Clavien-Dindo grade at least III). After propensity score matching, multivariable logistic regression and Cox proportional hazards regression were used to estimate the independent association of the secondary outcomes with drain placement. Results Overall, 1805 patients from 22 countries were included (798 women, 44.2 per cent; median age 67.0 years). The drain insertion rate was 51.9 per cent (937 patients). After matching, drains were not associated with reduced rates (odds ratio (OR) 1.33, 95 per cent c.i. 0.79 to 2.23; P = 0.287) or earlier detection (hazard ratio (HR) 0.87, 0.33 to 2.31; P = 0.780) of collections. Although not associated with worse major postoperative complications (OR 1.09, 0.68 to 1.75; P = 0.709), drains were associated with delayed hospital discharge (HR 0.58, 0.52 to 0.66; P < 0.001) and an increased risk of SSIs (OR 2.47, 1.50 to 4.05; P < 0.001). Conclusion Intraperitoneal drain placement after elective colorectal surgery is not associated with earlier detection of postoperative collections, but prolongs hospital stay and increases SSI risk

Outcome of corneal transplant rejection: a 10-year study

Purpose: To study the incidence of graft rejection and the predictive factors for its reversibility. Methods: It is a retrospective study of 1927 consecutive penetrating keratoplasties performed between January 1990 and January 2000 with more than 6 months follow up. A total of 224 rejection episodes were noted in 183 patients. Of these, 184 first rejection episodes were included in this study. Results: The incidence of first rejection episode was 9.55%. Of patients 87% were symptomatic during the episode with vision loss being the commonest. The average time of onset of rejection was 15.25 ± 14.4 months (median 11.7 months). In total, 53.3% of rejections occurred within 1 year after surgery. Of the patients who completed minimum 3 months follow up after the rejection episode, the rate of reversibility was 63.3%. Major predictive factors for a poor outcome after rejection were repeated grafting and associated anterior vitrectomy during surgery. The reversibility of the episode did not differ significantly with the modality of treatment used, but treatment with intravenous steroids within 7 days of onset of rejection may have a role in reducing the recurrences of rejection episodes, thus increasing the graft survival. Conclusion: Regrafts and associated anterior vitrectomy were significant risk factors for a poor outcome following rejection episode

DeepPheWAS : an R package for phenotype generation and association analysis for phenome-wide association studies

DeepPheWAS is an R package for phenome-wide association studies that creates clinically curated composite phenotypes and integrates quantitative phenotypes from primary care data, longitudinal trajectories of quantitative measures, disease progression and drug response phenotypes. Tools are provided for efficient analysis of association with any genetic input, under any genetic model, with optional sex-stratified analysis, and for developing novel phenotypes.Peer reviewe