Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide.

Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.

Microbial production of human milk oligosaccharide lactodifucotetraose.

Human milk oligosaccharides (HMOs) are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of chemical syntheses and in vitro enzymatic catalysis strategies. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have the potential to reduce the cost for enzymatic synthesis of HMOs. Whole cell biocatalysts for the production of short-chained, linear and small monofucosylated HMOs have been reported but those for fucosylated structures with higher complexity have not been explored. In this study, we established a strategy for producing a difucosylated HMO, lactodifucotetraose (LDFT), from lactose and L-fucose in Escherichia coli. We used two bacterial fucosyltransferases with narrow acceptor selectivity to drive the sequential fucosylation of lactose and intermediate 2'-fucosyllactose (2'-FL) to produce LDFT. Deletion of substrate degradation pathways that decoupled cellular growth from LDFT production, enhanced expression of native substrate transporters and modular induction of the genes in the LDFT biosynthetic pathway allowed complete conversion of lactose into LDFT and minor quantities of the side product 3-fucosyllactose (3-FL). Overall, 5.1 g/L of LDFT was produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. Our results demonstrate promising applications of engineered microbial biosystems for the production of multi-fucosylated HMOs for biochemical studies.

Catalytic Cycle of Neisseria meningitidis CMP-Sialic Acid Synthetase Illustrated by High-Resolution Protein Crystallography.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

L. pneumophila CMP-5,7-di-N-acetyllegionaminic acid synthetase (LpCLS)-involved chemoenzymatic synthesis of sialosides and analogues.

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac2) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac2-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac2 synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac2-glycosides from chemoenzymatically synthesized Leg5,7Ac2 using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac2 and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac2), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).

Classifying Sustainable Development Goal trajectories: A country-level methodology for identifying which issues and people are getting left behind.

How useful are the Sustainable Development Goals for conducting empirical analysis at the country level? We develop a methodological framework for answering this question, with special emphasis on the SDGs' normative ambition of "no one left behind." We first classify all 169 SDG targets and find that 78 incorporate an outcome-focus that is quantitatively assessable at the country level, including 43 through a systematic approach to establishing "proxy targets." We then present a framework for diagnosing the embedded diversity of absolute and relative indicator trajectories in a harmonized manner, based on a country's share of its starting gap on course to be closed by the relevant deadline. In turn, we present a method for estimating the human consequences of falling short on targets, measured by the number of lives at stake and people's basic needs at stake. As a case study, we apply the framework to Canada, an economy not commonly examined in the context of global goals. We are able to assess a total of 61 targets through the use of 70 indicators, including 28 indicators drawn from the United Nations' official database. Overall, we find Canada is on course to succeed on 18 indicators; to cover at least half but less than the full objective on 7 indicators; to cover less than half the required distance on 33 indicators; and to remain stagnant or move backwards on 12 indicators. Among indicators assessed, the country is only fully on track to achieve one SDG. Shortfalls suggest approximately 54,000 Canadian lives at stake and millions of people left behind on issues like poverty, education, intimate partner violence, and access to water and sanitation. Our diagnostic framework enables considerable, if only partial, quantification of a country's SDG challenges, recognizing the wide range of contexts for underlying data availability and societal problems.

Agriculture, Aid, and Economic Growth in Africa.

How can foreign aid to agriculture support economic growth in Africa? This paper constructs a geographically indexed applied general equilibrium model that considers pathways through which aid might affect growth and structural transformation of labor markets in the context of soil nutrient variation, minimum subsistence consumption requirements, domestic transport costs, labor mobility, and constraints to self-financing of agricultural inputs.Using plausible parameters, the model is presented for Uganda as an illustrative case.We present three stylized scenarios to demonstrate the potential economy-wide impacts of both soil nutrient loss and replenishment, and how foreign aid can be targeted to support agricultural inputs that boost rural productivity and shift labor to boost real wages. One simulation shows how a temporary program of targeted official development assistance (ODA) for agriculture could generate, contrary to traditional Dutch disease concerns, an expansion in the primary tradable sector and positive permanent productivity and welfare effects, leading to a steady decline in the need for complementary ODA for budget support.

Streamlined chemoenzymatic total synthesis of prioritized ganglioside cancer antigens.

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacßSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.

Triazole-linked transition state analogs as selective inhibitors against V. cholerae sialidase.

Sialidases or neuraminidases are enzymes that catalyze the cleavage of terminal sialic acids from oligosaccharides and glycoconjugates. They play important roles in bacterial and viral infection and have been attractive targets for drug development. Structure-based drug design has led to potent inhibitors against neuraminidases of influenza A viruses that have been used successfully as approved therapeutics. However, selective and effective inhibitors against bacterial and human sialidases are still being actively pursued. Guided by crystal structural analysis, several derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) were designed and synthesized as triazole-linked transition state analogs. Inhibition studies revealed that glycopeptide analog E-(TriazoleNeu5Ac2en)-AKE and compound (TriazoleNeu5Ac2en)-A were selective inhibitors against Vibrio cholerae sialidase, while glycopeptide analog (TriazoleNeu5Ac2en)-AdE selectively inhibited Vibrio cholerae and A. ureafaciens sialidases.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2 ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac2 -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

Converting Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-sialyltransferase by saturation mutagenesis and regioselective screening.

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

Fertilizing growth: Agricultural inputs and their effects in economic development.

This paper estimates the role of agronomic inputs in cereal yield improvements and the consequences for countries' processes of structural change. The results suggest a clear role for fertilizer, modern seeds and water in boosting yields. We then test for respective empirical links between agricultural yields and economic growth, labor share in agriculture and non-agricultural value added per worker. The identification strategy includes a novel instrumental variable that exploits the unique economic geography of fertilizer production and transport costs to countries' agricultural heartlands. We estimate that a half ton increase in staple yields generates a 14 to 19 percent higher GDP per capita and a 4.6 to 5.6 percentage point lower labor share in agriculture five years later. The results suggest a strong role for agricultural productivity as a driver of structural change.

Glycosyltransferase engineering for carbohydrate synthesis.

Glycosyltransferases (GTs) are powerful tools for the synthesis of complex and biologically-important carbohydrates. Wild-type GTs may not have all the properties and functions that are desired for large-scale production of carbohydrates that exist in nature and those with non-natural modifications. With the increasing availability of crystal structures of GTs, especially those in the presence of donor and acceptor analogues, crystal structure-guided rational design has been quite successful in obtaining mutants with desired functionalities. With current limited understanding of the structure-activity relationship of GTs, directed evolution continues to be a useful approach for generating additional mutants with functionality that can be screened for in a high-throughput format. Mutating the amino acid residues constituting or close to the substrate-binding sites of GTs by structure-guided directed evolution (SGDE) further explores the biotechnological potential of GTs that can only be realized through enzyme engineering. This mini-review discusses the progress made towards GT engineering and the lessons learned for future engineering efforts and assay development.

Isolation of an arsenate-respiring bacterium from a redox front in an arsenic-polluted aquifer in West Bengal, Bengal Basin.

Natural pollution of groundwater by arsenic adversely affects the health of tens of millions of people worldwide, with the deltaic aquifers of SE Asia being particularly polluted. The pollution is caused primarily by, or as a side reaction of, the microbial reduction of sedimentary Fe(III)-oxyhydroxides, but the organism(s) responsible for As release have not been isolated. Here we report the first isolation of a dissimilatory arsenate reducer from sediments of the Bengal Basin in West Bengal. The bacterium, here designated WB3, respires soluble arsenate and couples its reduction to the oxidation of acetate; WB3 is therefore implicated in the process of arsenic pollution of groundwater, which is largely by arsenite. The bacterium WB3 is also capable of reducing dissolved Fe(III) citrate, solid Fe(III)-oxyhydroxide, and elemental sulfur, using acetate as the electron donor. It is a member of the Desulfuromonas genus and possesses a dissimilatory arsenate reductase that was identified using degenerate polymerase chain reaction primers. The sediment from which WB3 was isolated was brown, Pleistocene sand at a depth of 35.2 m below ground level (mbgl). This level was some 3 cm below the boundary between the brown sands and overlying reduced, gray, Holocene aquifer sands. The color boundary is interpreted to be a reduction front that releases As for resorption downflow, yielding a high load of labile As sorbed to the sediment at a depth of 35.8 mbgl and concentrations of As in groundwater that reach >1000 µg/L.

Interventions for treating fractures of the distal femur in adults.

BACKGROUND: Fractures of the distal femur (the part of the thigh bone nearest the knee) are a considerable cause of morbidity. Various different surgical and non-surgical treatments have been used in the management of these injuries but the best treatment remains controversial. OBJECTIVES: To assess the effects (benefits and harms) of interventions for treating fractures of the distal femur in adults. SEARCH METHODS: We searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised Register (9 September 2014); the Cochrane Central Register of Controlled Trials (The Cochrane Library, 2014, Issue 8); MEDLINE (1946 to August week 4 2014); EMBASE (1980 to 2014 week 36); World Health Organization (WHO) International Clinical Trials Registry Platform (January 2015); conference proceedings and reference lists without language restrictions. SELECTION CRITERIA: Randomised and quasi-randomised controlled clinical trials comparing interventions for treating fractures of the distal femur in adults. Our primary outcomes were patient-reported outcome measures (PROMs) of knee function and adverse events, including re-operations. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies and performed data extraction and risk of bias assessment. We assessed treatment effects using risk ratios (RR) or mean differences (MD) and, where appropriate, we pooled data using a fixed-effect model. MAIN RESULTS: We included seven studies that involved a total of 444 adults with distal femur fractures. Each of the included studies was small and assessed to be at substantial risk of bias, with four studies being quasi-randomised and none of the studies using blinding in outcome assessment. All studies provided an incomplete picture of outcome. Based on GRADE criteria, we assessed the quality of the evidence as very low for all reported outcomes, which means we are very uncertain of the reliability of these results.One study compared surgical (dynamic condylar screw (DCS) fixation) and non-surgical (skeletal traction) treatment in 42 older adults (mean age 79 years) with displaced fractures of the distal femur. This study, which did not report on PROMs, provided very low quality evidence of little between-group differences in adverse events such as death (2/20 surgical versus 1/20 non-surgical), re-operation or repeat procedures (1/20 versus 3/20) and other adverse effects including delayed union. However, while none of the findings were statistically significant, there were more complications such as pressure sores (0/20 versus 4/20) associated with prolonged immobilisation in the non-surgical group, who stayed on average one month longer in hospital.The other six studies compared different surgical interventions. Three studies, including 159 participants, compared retrograde intramedullary nail (RIMN) fixation versus DCS or blade-plate fixation (fixed-angle devices). None of these studies reported PROMS relating to function. None of the results for the reported adverse events showed a difference between the two implants. Thus, although there was very low quality evidence of a higher risk of re-operation in the RIMN group, the 95% confidence interval (CI) also included the possibility of a higher risk of re-operation for the fixed-angle device (9/83 RIMN versus 4/96 fixed-angle device; 3 studies: RR 1.85, 95% CI 0.62 to 5.57). There was no clinically important difference between the two groups found in quality of life assessed using the 36-item Short Form in one study (23 fractures).One study (18 participants) provided very low quality evidence of there being little difference in adverse events between RIMN and non-locking plate fixation. One study (53 participants) provided very low quality evidence of a higher risk of re-operation after locking plate fixation compared with a single fixed-angle device (6/28 locking plate versus 1/25 fixed-angle device; RR 5.36, 95% CI 0.69 to 41.50); however, the 95% CI also included the possibility of a higher risk of re-operation for the fixed-angle device. Neither of these trials reported on PROMs.The largest included study, which reported outcomes in 126 participants at one-year follow-up, compared RIMN versus locking plate fixation; both implants are commonly used in current practice. None of the between-group differences in the reported outcomes were statistically significant; thus the CIs crossed the line of no effect. There was very low quality evidence of better patient-reported musculoskeletal function in the RIMN group based on Short Musculoskeletal Function Assessment (0 to 100: best function) scores (e.g. dysfunction index: MD -5.90 favouring RIMN, 95% CI -15.13 to 3.33) as well as quality of life using the EuroQoL-5D Index (0 to 1: best quality of life) (MD 0.10 favouring RIMN, 95% CI -0.01 to 0.21). The CIs for both results included a clinically important effect favouring RIMN but also a clinically insignificant effect in favour of locking plate fixation. AUTHORS' CONCLUSIONS: This review highlights the major limitations of the available evidence concerning current treatment interventions for fractures of the distal femur. The currently available evidence is incomplete and insufficient to inform current clinical practice. Priority should be given to a definitive, pragmatic, multicentre randomised controlled clinical trial comparing contemporary treatments such as locked plates and intramedullary nails. At minimum, these should report validated patient-reported functional and quality-of-life outcomes at one and two years. All trials should be reported in full using the CONSORT guidelines.

Multi-factorial influences on sex ratio: a spatio-temporal investigation of endocrine disruptor pollution and neighborhood stress.

BACKGROUND: It is suggested the declining male birth proportion in some industrialized countries is linked to ubiquitous endocrine disruptor exposure. Stress and advanced parental age are determinants which frequently present positive findings. Multi-factorial influences on population sex ratio are rarely explored or tested in research. OBJECTIVES: To test the hypothesis that dual factors of pollution and population stress affects sex proportion at birth through geographical analysis of Central Scotland. METHODS: The study incorporates the use of Geographical Information Systems (GIS) tools to overlay modeled point source endocrine disruptor air emissions with "small-area" data on multiple deprivation (a proxy measurement of stress) and birth sex. Historical review of regional sex ratio trends presents additional data on sex ratio in Scotland to consider. RESULTS: There was no overall concentration in Central Scotland of low sex ratio neighborhoods with areas where endocrine disruptor air pollution and deprivation or economic stress were high. Historical regional trends in Scotland (from 1973), however, do show significantly lower sex ratio values for populations where industrial air pollution is highest (i.e. Eastern Central Scotland). CONCLUSIONS: Use of small area data sets and pollution inventories is a potential new method of inquiry for reproductive environmental and health protection monitoring and has produced interesting findings.

The effect of an integrated multisector model for achieving the Millennium Development Goals and improving child survival in rural sub-Saharan Africa: a non-randomised controlled assessment.

BACKGROUND: Simultaneously addressing multiple Millennium Development Goals (MDGs) has the potential to complement essential health interventions to accelerate gains in child survival. The Millennium Villages project is an integrated multisector approach to rural development operating across diverse sub-Saharan African sites. Our aim was to assess the effects of the project on MDG-related outcomes including child mortality 3 years after implementation and compare these changes to local comparison data. METHODS: Village sites averaging 35,000 people were selected from rural areas across diverse agroecological zones with high baseline levels of poverty and undernutrition. Starting in 2006, simultaneous investments were made in agriculture, the environment, business development, education, infrastructure, and health in partnership with communities and local governments at an annual projected cost of US$120 per person. We assessed MDG-related progress by monitoring changes 3 years after implementation across Millenium Village sites in nine countries. The primary outcome was the mortality rate of children younger than 5 years of age. To assess plausibility and attribution, we compared changes to reference data gathered from matched randomly selected comparison sites for the mortality rate of children younger than 5 years of age. Analyses were done on a per-protocol basis. This trial is registered with ClinicalTrials.gov, number NCT01125618. FINDINGS: Baseline levels of MDG-related spending averaged $27 per head, increasing to $116 by year 3 of which $25 was spent on health. After 3 years, reductions in poverty, food insecurity, stunting, and malaria parasitaemia were reported across nine Millennium Village sites. Access to improved water and sanitation increased, along with coverage for many maternal-child health interventions. Mortality rates in children younger than 5 years of age decreased by 22% in Millennium Village sites relative to baseline (absolute decrease 25 deaths per 1000 livebirths, p=0·015) and 32% relative to matched comparison sites (30 deaths per 1000 livebirths, p=0·033). INTERPRETATION: An integrated multisector approach for addressing the MDGs can produce rapid declines in child mortality in the first 3 years of a long-term effort in rural sub-Saharan Africa. FUNDING: UN Human Security Trust Fund, the Lenfest Foundation, Bill & Melinda Gates Foundation, and Becton Dickinson.

Promiscuity of a modular polyketide synthase towards natural and non-natural extender units.

Combinatorial biosynthesis approaches that involve modular type I polyketide synthases (PKSs) are proven strategies for the synthesis of polyketides. In general however, such strategies are usually limited in scope and utility due to the restricted substrate specificity of polyketide biosynthetic machinery. Herein, a panel of chemo-enzymatically synthesized acyl-CoA's was used to probe the promiscuity of a polyketide synthase. Promiscuity determinants were dissected, revealing that the KS is remarkably tolerant to a diverse array of extender units, while the AT likely discriminates between extender units that are native to the producing organism. Our data provides a clear blueprint for future enzyme engineering efforts, and sets the stage for harnessing extender unit promiscuity by employing various in vivo polyketide diversification strategies.

Palaeoceanography: methane release in the Early Jurassic period.

Dramatic global warming, triggered by release of methane from clathrates, has been postulated to have occurred during the early Toarcian age in the Early Jurassic period. Kemp et al. claim that this methane was released at three points, as recorded by three sharp excursions of delta13C(org) of up to 3 per thousand magnitude. But they discount another explanation for the excursions: namely that some, perhaps all, of the rapid excursions could be a local signature of a euxinic basin caused by recycling of isotopically light carbon from the lower water column. This idea has been proposed previously (see ref. 3, for example) and is supported by the lack evidence for negative delta13C excursions in coeval belemnite rostra. Kemp et al. dismiss this alternative, claiming that each abrupt shift would have required the recycling of about double the amount of organic carbon that is currently present in the modern ocean; however, their measurements are not from an ocean but from a restricted, epicontinental seaway and so would not require whole-ocean mixing to achieve the excursions.

Tibial component overhang in total knee replacement: incidence and functional outcomes.

The aim of this study was to quantify the incidence and severity of tibial component overhang in total knee replacement and find any relationship between overhang and functional outcome. 532 anteroposterior radiographs of total knee replacements were reviewed retrospectively to identify medial or lateral overhang of the tibial component. Patients completed an Oxford knee score (OKS) questionnaire at mean follow-up of 36 months. Overhang of the tibial component was noted in 13.4% of patients with a mean OKS of 353. Severity of overhang did not correlate with OKS. Overhang of the tibial component is common. However, in this study it was not associated with a worse than expected outcome and increasing severity did not correlate with worse functional scores.