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Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O -mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure—a ribitol in a phosphodiester linkage—for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains.

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), At FUT4, and At FUT6, are members of the plant-specific GT family 37 (GT37). At FUT4 and At FUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. At FUT4 and At FUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of At FUT4 and At FUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAc-Man₃GlcNAc₂-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cationdependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate “recognition arm.” Recognition arm interactions are similar to the enzyme–substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1) transfers galactose and arabinose to either extend or cap the β-1,4-galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBM95, and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a ‘handshake’ orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.The authors present a multidisciplinary approach investigating the mechanistic underpinnings of galactan synthase 1 and use their data to propose a new model for complex pectin biosynthesis.

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The fucosyltransferases (FUTs), FUT4, and FUT6, are members of the plant-specific GT family 37 (GT37). FUT4 and FUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. FUT4 and FUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of FUT4 and FUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that and gene expression is sub-localized in different parts of developing roots.

Xylan, the most abundant non-cellulosic polymer in plant cell walls, is structurally diverse, especially in grasses where it is heavily substituted with arabinofuranose and further modified by various residues. Common substitutions across species include glucuronic and 4- -methyl-glucuronic acid. Arabinose and xylose sidechains are synthesized by glycosyltransferase family 61 (GT61) proteins, many of which remain uncharacterized in plants, with limited structural and mechanistic understanding. In this study, we identified two novel GT61 enzymes in , functioning as xylan arabinosyltransferase (SbXAT) and xylan xylosyltransferase (SbXXT). We resolved the crystal structure of SbXAT, which exhibits a GT-B fold with two Rossmann-like domains linked by a cleft that accommodates the catalytic site. Structural comparison with a predicted SbXXT model revealed a substrate-binding residue critical for sugar donor specificity, validated through site-directed mutagenesis and enzymatic assays. These findings enhance understanding of xylan biosynthesis and provide a foundation for engineering glycosyltransferases and predicting their functions.

The excessive use of digital platforms with rapidly increasing users in the wireless domain enforces communication systems to provide information with high data rates, high reliability and strong transmission connection quality. Wireless systems with single antenna elements are not able to accomplish the desired needs. Therefore, multiple-input multiple-output (MIMO) antennas are getting more attention in modern high-speed communication systems and play an essential part in the current generation of wireless technology. However, along with their ability to significantly increase channel capacity, it is a challenge to achieve an optimal isolation in a compact size for fifth-generation (5G) terminals. Portable devices, automobiles, handheld gadgets, smart phones, wireless sensors, radio frequency identification and other applications use MIMO antenna systems. In this review paper, the fundamentals of MIMO antennas, the performance parameters of MIMO antennas, and different design approaches and methodologies are discussed to realize the three most commonly used MIMO antennas, i.e., ultra-wideband (UWB), dual-band and circularly polarized antennas. The recent MIMO antenna design approaches with UWB, dual band and circularly polarized characteristics are compared in terms of their isolation techniques, gain, efficiency, envelope correlation coefficient (ECC) and channel capacity loss (CCL). This paper is very helpful to design suitable MIMO antennas applicable in UWB systems, satellite communication systems, GSM, Bluetooth, WiMAX, WLAN and many more. The issues with MIMO antenna systems in the indoor environment along with possible solutions to improve their performance are discussed. The paper also focuses on the applications of MIMO characteristics for future sixth-generation (6G) technology.

Neonatal sepsis is a major cause of death in India, which needs hospital management but many families cannot access hospitals. The World Health Organization and the Government of India developed a guideline to manage possible serious bacterial infection (PSBI) when a referral is not feasible. We implemented this guideline to achieve high coverage of treatment of PSBI with low mortality. The implementation research study was conducted in over 50 villages of Palwal district, Haryana during August 2017-March 2019 and covered a population of 199143. Policy dialogue with central, state and district health authorities was held before initiation of the study. A baseline assessment of the barriers in the implementation of the PSBI intervention was conducted. The intervention was implemented in the program setting. The research team collected data throughout and also co-participated in the implementation of the intervention for the first six months to identify bottlenecks in the health system and at the community level. RE-AIM framework was utilized to document implementation strategies of PSBI management guideline. Implementation strategies by the district technical support unit (TSU) included: (i) empower mothers and families through social mobilization to improve care-seeking of sick young infants 0-59 days of age, (ii) build capacity through training and build confidence through technical support of health staff at primary health centers (PHC), community health centers (CHC) and sub-centers to manage young infants with PSBI signs and (iii) improve performance of accredited social health activists (ASHAs). A total of 370 young infants with signs of PSBI were identified and managed in 5270 live births. Treatment coverage was 70% assuming that 10% of live births would have PSBI within the first two months of life. Mothers identified 87.6% (324/370) of PSBI cases. PHCs and CHCs became functional and managed 150 (40%) sick young infants with PSBI. Twenty four young infants (7-59days) who had only fast breathing were treated with oral amoxicillin without a referral. Referral to a hospital was refused by 126 (84%); 119 had clinical severe infection (CSI), one 0-6 days old had fast breathing and six had critical illness (CI). Of 119 CSI cases managed on outpatient injection gentamicin and oral amoxicillin, 116 (96.7%) recovered, 55 (45.8%) received all seven gentamicin injections and only one died. All 7-59 day old infants with fast breathing recovered, 23 on outpatient oral amoxicillin treatment; and 19 (79%) received all doses. Of 65 infants managed at either district or tertiary hospital, two (3.1%) died, rest recovered. Private providers managed 155 (41.9%) PSBI cases, all except one recovered, but sub-classification and treatment were unknown. Sub-centers could not be activated to manage PSBI. The study demonstrated resolution of implementation bottlenecks with existing resources, activated PHCs and CHCs to manage CSI and fast breathers (7-59 day old) on an outpatient basis with low mortality when a referral was not feasible. TSU was instrumental in these achievements. We established the effectiveness of oral amoxicillin alone in 7-59 days old fast breathers and recommend a review of the current national policy.

This paper presents a novel, dual-band, four-port multi-input–multi-output (MIMO) antenna for 28/38 GHz millimeter wave 5G wearable applications. In the proposed work, we have used a novel design approach to get the dual-band behavior from a MIMO design with a small footprint of 18 × 8.5 × 0.25 mm 3 . For this purpose, each MIMO element is designed as a composite form of a circular and elliptical structure connected with a narrow strip and fed by a tapered feedline. The peak realized gains and total efficiencies of the antenna, evaluated in free space, are 4.15 dBi, 7.73 dBi and 80.13%, 85.44% at 28 GHz and 38 GHz frequencies, respectively. To appraise the thorough behavior of the MIMO antenna, we have evaluated all the parameters of the antenna: Envelope Correlation Coefficient (ECC), Diversity Gain (DG), Mean Effective Gain (MEG), Channel Capacity Loss (CCL), and Total Active Reflection Coefficient (TARC), and found them satisfactory. Channel capacity of the antenna at SNR = 20 dB is found to be 21.61 bps/Hz. For wearable applications, the proposed 4-port MIMO antenna is designed on a flexible Rogers 3003 substrate, and the performance is checked by evaluating bending analysis. The safety of the antenna is verified by analyzing the 1 g/10 g SAR at 28/38 GHz and the corresponding average SAR values are 0.11/0.08 W/kg and 0.05/0.04 W/kg, respectively. All the average SAR values for the proposed MIMO antenna are within the acceptable limits according to FCC/ICNIRP standards.