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.

A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides.

Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1-4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.

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.

Overlapping redox zones control arsenic pollution in Pleistocene multi-layer aquifers, the Po Plain (Italy).

Understanding the factors that control As concentrations in groundwater is vital for supplying safe groundwater in regions with As-polluted aquifers. Despite much research, mainly addressing Holocene aquifers hosting young (<100 yrs) groundwater, the source, transport, and fate of As in Pleistocene aquifers with fossil (>12,000 yrs) groundwaters are not yet fully understood and so are assessed here through an evaluation of the redox properties of the system in a type locality, the Po Plain (Italy). Analyses of redox-sensitive species and major ions on 22 groundwater samples from the Pleistocene arsenic-affected aquifer in the Po Plain shows that groundwater concentrations of As are controlled by the simultaneous operation of several terminal electron accepters. Organic matter, present as peat, is abundant in the aquifer, allowing groundwater to reach a quasi-steady-state of highly reducing conditions close to thermodynamic equilibrium. In this system, simultaneous reduction of Fe-oxide and sulfate results in low concentrations of As (median 7 µg/L) whereas As reaches higher concentrations (median of 82 µg/L) during simultaneous methanogenesis and Fe-reduction. The position of well-screens is an additional controlling factor on groundwater As: short screens that overlap confining aquitards generate higher As concentrations than long screens placed away from them. A conceptual model for groundwater As, applicable worldwide in other Pleistocene aquifers with reducible Fe-oxides and abundant organic matter is proposed: As may have two concentration peaks, the first after prolonged Fe-oxide reduction and until sulfate reduction takes place, the second during simultaneous Fe-reduction and methanogenesis.

TULIP: a randomised controlled trial of surgical versus non-surgical treatment of lateral compression injuries of the pelvis with complete sacral fractures (LC1) in the non-fragility fracture patient-a feasibility study protocol.

INTRODUCTION: Lateral compression type 1 (LC1) pelvic fractures are the most common type of pelvic fracture. The majority of LC1 fractures are considered stable. Fractures where a complete sacral fracture is present increases the degree of potential instability and have the potential to displace over time. Non-operative management of these unstable fractures may involve restricted weight bearing and significant rehabilitation. Frequent monitoring with X-rays is also necessary for displacement of the fracture. Operative stabilisation of these fractures may be appropriate to prevent displacement of the fracture. This may allow patients to mobilise pain-free, quicker. METHODS AND ANALYSIS: The study is a feasibility study to inform the design of a full definitive randomised controlled trial to guide the most appropriate management of these injuries. Participants will be recruited from major trauma centres and randomly allocated to either operative or non-operative management of their injuries. A variety of outcome instruments, measuring health-related quality of life, functional outcome and pain, will be completed at several time points up to 12 months post injury. Qualitative interviews will be undertaken with participants to explore their views of the treatments under investigation and trial processes.Eligibility and recruitment to the study will be analysed to inform the feasibility of a definitive trial. Completion rates of the measurement instruments will be assessed, as well as their sensitivity to change and the presence of floor or ceiling effects in this population, to inform the choice of the primary outcome for a definitive trial. ETHICS AND DISSEMINATION: Ethical approval for the study was given by the South West-Central Bristol NHS Research Ethics Committee on 2nd July 2018 (Ref; 18/SW/0135). The study will be reported in relevant specialist journals and through presentation at specialist conferences. TRIAL REGISTRATION NUMBER: ISRCTN10649958.

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).

Engineer P. multocida Heparosan Synthase 2 (PmHS2) for Size-Controlled Synthesis of Longer Heparosan Oligosaccharides.

Pasteurella multocida heparosan synthase 2 (PmHS2) is a dual-function polysaccharide synthase having both α1-4-N-acetylglucosaminyltransferase (α1-4-GlcNAcT) and ß1-4-glucuronyltransferase (ß1-4-GlcAT) activities located in two separate catalytic domains. We found that removing PmHS2 N-terminal 80-amino acid residues improved enzyme stability and expression level while retaining its substrate promiscuity. We also identified the reverse glycosylation activities of PmHS2 which complicated its application in size-controlled synthesis of oligosaccharides longer than hexasaccharide. Engineered Δ80PmHS2 single-function-glycosyltransferase mutants Δ80PmHS2_D291N (α1-4-GlcNAcT lacking both forward and reverse ß1-4-GlcAT activities) and Δ80PmHS2_D569N (ß1-4-GlcAT lacking both forward and reverse α1-4-GlcNAcT activities) were designed and showed to minimize side product formation. They were successfully used in a sequential one-pot multienzyme (OPME) platform for size-controlled high-yield production of oligosaccharides up to decasaccharide. The study draws attention to the consideration of reverse glycosylation activities of glycosyltransferases, including polysaccharide synthases, when applying them in the synthesis of oligosaccharides and polysaccharides. The mutagenesis strategy has the potential to be extended to other multifunctional polysaccharide synthases with reverse glycosylation activities to generate catalysts with improved synthetic efficiency.

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.

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.

The effects of irrigation on groundwater quality and quantity in a human-modified hydro-system: The Oglio River basin, Po Plain, northern Italy.

For several hundred years, farming in the Po Plain of Italy (46,000 km2, 20 million inhabitants) has been supported by intensive surface irrigation with lake and river water. Despite the longevity of irrigation, its effects on the quality and quantity of groundwater is poorly known and so is investigated here through seasonal measurements of hydraulic heads and water quality in groundwaters, rivers, lake, springs and rainwaters. In the north of the study region, an unconfined coarse-grained alluvial aquifer, infiltration of surface irrigation water, sourced from the Oglio River and low in NO3, contributes much to aquifer recharge (up to 88%, as evidenced by a δ2H-Cl/Br mixing model) and has positive effects on groundwater quality by diluting high concentrations of NO3 (decrease by 17% between June and September). This recharge also helps to maintain numerous local springs that form important local micro-environments. Any increase in water-use efficiency in irrigation will reduce this recharge, imperil the spring environments, and lessen the dilution of NO3 leading to increasing NO3 concentrations in groundwater. These findings can be extended by analogy to the entire Po Plain region and other surface-water-irrigated systems worldwide where inefficient irrigation methods are used and similar hydrogeological features occur.

Strategies for chemoenzymatic synthesis of carbohydrates.

Carbohydrates are structurally complex but functionally important biomolecules. Therefore, they have been challenging but attractive synthetic targets. While substantial progress has been made on advancing chemical glycosylation methods, incorporating enzymes into carbohydrate synthetic schemes has become increasingly practical as more carbohydrate biosynthetic and metabolic enzymes as well as their mutants with synthetic application are identified and expressed for preparative and large-scale synthesis. Chemoenzymatic strategies that integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions have been extremely powerful. Briefly summarized here are our experiences on developing one-pot multienzyme (OPME) systems and representative chemoenzymatic strategies from others using glycosyltransferase-catalyzed reactions for synthesizing diverse structures of oligosaccharides, polysaccharides, and glycoconjugates. These strategies allow the synthesis of complex carbohydrates including those containing naturally occurring carbohydrate postglycosylational modifications (PGMs) and non-natural functional groups. By combining these srategies with facile purification schemes, synthetic access to the diverse space of carbohydrate structures can be automated and will not be limited to specialists.

A Bacterial ß1-3-Galactosyltransferase Enables Multigram-Scale Synthesis of Human Milk Lacto-N-tetraose (LNT) and Its Fucosides.

ß1-3-Linked galactosides such as Galß1‒3GlcNAcßOR are common carbohydrate motifs found in human milk oligosaccharides (HMOSs), glycolipids, and glycoproteins. Efficient and scalable enzymatic syntheses of these structures have proven challenging due to the lack of access to a highly active ß1‒3-galactosyltransferase (ß3GalT) in large amounts. Previously reported E. coli ß3GalT (EcWbgO) has been identified as a limiting factor for producing a ß1-3-galactose-terminated human milk oligosaccharide lacto-N-tetraose (LNT) by fermentation. Here we report the identification of an EcWbgO homolog from C. violaceum (Cvß3GalT) which showed a high efficiency in catalyzing the formation of LNT from lacto-N-triose (LNT II). With the highly active Cvß3GalT, multigram-scale (>10 gram) synthesis of LNT from lactose was achieved using a sequential one-pot multienzyme (OPME) glycosylation process. The access to Cvß3GalT enabled enzymatic synthesis of several fucosylated HMOSs with or without further sialylation including LNFP II, S-LNF II, LNDFH I, LNFP V, and DiFuc-LNT. Among these, LNFP V and DiFuc-LNT would not be accessible by enzymatic synthesis if an active ß3GalT were not available.

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.

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.

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.

α2-6-Neosialidase: A Sialyltransferase Mutant as a Sialyl Linkage-Specific Sialidase.

The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here, we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 100-fold improved activity, high α2-6-sialyl linkage selectivity, and ability to cleave two common sialic acid forms, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). It is a valuable tool for sialoglycan structural analysis and functional characterization. The sequential saturation mutagenesis and screening strategy developed here can be explored to evolve other linkage-specific neoglycosidases from the corresponding glycosyltransferases.

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.

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.