Sulfate Synthesis Research Articles

A novel mutation in ext2 caused hereditary multiple exostoses through reducing the synthesis of heparan sulfate.

Hereditary multiple exostoses (HME) is a rare skeletal disorder characterized by the formation of multiple benign cartilage-capped tumors, usually in the metaphyseal region of the long bones. Over 70% of HME cases arise from monoallelic mutations in either of the two genes encoding the heparan sulfate (HS) synthesis enzymes, ext1 and ext2. To identify more HME-associated mutations, genomic DNA from members of five independent consanguineous families with HME was sequenced with whole exome sequencing (WES). A novel heterozygous splice site mutation (c.1173+2T>A) in ext2 was detected in all three affected members of family V. Further study showed that the novel mutation caused exon 7 of ext2 mRNA to be skipped during splicing and caused a frameshift after the codon for Arg360, which results in the appearance of new 43 codons, followed by a termination codon. Although the resulting truncated protein was still localized to the Golgi, similar to the full-length EXT2, its HS synthesis activity decreased by 40%. In this study, a novel splice site mutation in ext2 was identified and suggested to be a pathogenic mutation of HME, which may expand the genetic etiology spectrum of HME and may be helpful for clinical genetic counseling and prenatal diagnosis.

A new nonlinear optical sulfate of layered structure: Cs2Zn2(SO4)3

Acentric structure is the indispensable condition for a compund to exhibit nonlinear optical (NLO) response. However, it’s difficult for inorganic sulfates to crystallize in acentric structures. In this communication, a new acentric sulfate, Cs2Zn2(SO4)3, has been synthesized by spontaneous crystallization method. The crystal structure of Cs2Zn2(SO4)3 is layered and is composed of Zn2(SO4)3 layers and Cs+ cations, which is different from A2B2(SO4)3 (A = K, Rb, B = Mg, Ca, Cd, Zn) type that owns three-dimensional framework. Powder NLO response suggest that Cs2Zn2(SO4)3 exhibits a moderate second-order NLO response of about 0.15 × KH2PO4. Theoretical calculations were performed and elucidated that the origination of NLO response is from SO4 groups and ZnOn (n = 4, 5) polyhedra. This work will enrich the diversity of sulfate structure and provide new desgin strategy on the synthesis of novel NLO sulfates.

Synthesis of Sodium Lauryl Sulfate (SLS) and Hexadecyltrimethylammonium Bromide (HDTMA-Br) Surfactant-Modified Activated Carbon as Adsorbent for Pb2+ and NO3-

The adsorption efficiency and selectivity of activated carbon as an adsorbent for ions can be improved. One way is to convert activated carbon into surfactant modified activated carbon (SMAC). The surfactants used in this study were the anionic surfactant Sodium Lauryl Sulfate (SLS) and the cationic surfactant hexadecyltrimethylammonium bromide (HDTMA-Br). This research aims to synthesize SMAC to obtain a material with a surface charge and absorb ions better than activated carbon. This research consisted of four stages. The first step was the carbonization of rice husks using a pyrolysis reactor at 400°C for 1 hour. The second stage was carbon activation using 30% ZnCl2 and microwave radiation for 5 minutes and 400 W. The third stage was the modification of activated carbon and characterization by FTIR, SEM, SAA. The fourth stage was the adsorption of Pb cations and nitrate anions by carbon, activated carbon, and SMAC. Several variables were applied, such as the type of surfactant, time, and method of modification. There are three ways of modification: (1) method A, in which activated carbon is brought into contact with SLS then HDTMA-Br. (2) Method B in which activated carbon was contacted with HDTMA-Br then SLS. (3) Method C in which activated carbon was brought into contact with SLS together with HDTMA-Br. All variables were investigated. The results showed that the optimum time for making SMAC for both surfactants was 4 hours, the optimum concentrations of SLS and HDTMA-Br were 60 and 300 ppm, respectively. SMAC made by the C method was the most effective at adsorbing Pb2+ and NO3- with adsorption capacities of 1.376 and 0.896 mg/g, respectively. The success of SMAC synthesis was evidenced by the S=O and (CH3)3N+ groups in the FTIR spectra. The SMAC surface area is smaller than activated carbon, 14.472 m2/g, but the surface morphology is smoother and more homogeneous.

Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.

Bi-allelic Pathogenic Variants in HS2ST1 Cause a Syndrome Characterized by Developmental Delay and Corpus Callosum, Skeletal, and Renal Abnormalities

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulfated linear polysaccharides. Heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report bi-allelic pathogenic variants in HS2ST1 in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2-O-sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2-O-sulfated domains but had an increase in N- and 6-O-sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a recognizable syndrome and emphasize a role for 2-O-sulfated heparan sulfate in human neuronal, skeletal, and renal development.

Genetic Manipulation Resulting in Decreased Donor Chondroitin sulfate Synthesis Mitigates Gvhd Following Allogeneic Hematopoietic Cell Transplantation in a Murine Model.

Introduction: Severe acute graft versus host disease (GVHD) represents a major risk associated with allogeneic hematopoietic cell transplantation (HSCT). Both acute and progressive GVHD following HSCT are in large part attributable to responses of donor T cells to host allo-antigens. Alloreactive T cell activation can be modulated by signals from the tissue microenvironment (TME). Glycosaminoglycans (GAGs) in the TME or modifying T cell surface proteoglycans are known to regulate T cell function. However, the roles of GAGs in acute GVHD following allogeneic HSCT remain to be elucidated, since a suitable murine model has not been available. We previously established a unique mouse model (T1KO), in which knockout of the chondroitin sulfate (CS) N-acetylgalactosaminyltransferase-1 (T1) gene - encoding the rate-limiting CS-synthesizing enzyme - decreases CS production. We also reported a role for CS in murine hematopoietic stem cells. In the present study, we focus on donor T cell CS levels to assess the role of T cell CS in acute GVHD following allogeneic HSCT. We were able to mitigate the clinical features of GVHD in a murine model using T1KO donors. Methods: Eight- to 12-week-old T1KO mice were generated from a C57BL/6N (WT) strain as donors (H-2b) for allogeneic bone marrow transplantation (BMT). Donor BM cells were then transplanted into eight- to 12-week-old BALB/c recipients (H-2d). Prior to transplantation, recipients were irradiated at a dose of 7 Gy. They then received 5 x 106 BM cells and splenocytes of identical genotype, as well as low (5 x 105), intermediate (1 x 106), or high (4 x 106) doses of CD90.2+ cells from either WT or T1KO donors. For five weeks following BMT, survival was monitored daily and clinical GVHD scores (incorporating body weight, activity, fur condition, alopecia, and tortoiseshell-like mucosae) were calculated three times per week. Results: The peripheral blood CD4+/CD8+ T cell ratio of T1KO mice was equivalent to that of WT mice. Following allogeneic BMT, histopathologic analysis confirmed onset of acute recipient GVHD on day 49 when donors were WT mice. As a result, significant lymphocyte infiltration was observed in target organs (liver, colon, and skin). High-dose transplantation of splenocytes from T1KO rather than WT mice significantly prolonged recipient median survival (27.0 versus 20.5 days, p = 0.02). A similar trend was observed for low- and intermediate-dose transplantation (low: 53.0 versus 29.0 days, p = 0.09; intermediate: 50.5 versus 30.5 days, p = 0.13. Similarly, a significant improvement in day 11 GVHD score was observed following high-dose splenocyte transplantation when donors were T1KO mice (mean scores: 1.00 versus 2.75, p < 0.01). However, no significant differences in GVHD score were observed following low- and intermediate-dose transplantation. Taken together, our results suggest that donor T cells producing lower CS levels alleviate acute GVHD following allogeneic HSCT in a murine model. Conclusion: Alloreactive T cell activation following allogeneic HSCT may be down-regulated by lower CS levels, thereby mitigating GVHD severity. Figure 1 Disclosures No relevant conflicts of interest to declare.

Glycogene expression profiles from a HaCaT cell line stably transfected with HPV16 E5 oncogene.

The altered expression of glycan antigens has been reported during cervix transformation, demonstrating increased mRNA levels of certain glycogenes. Human papillomavirus (HPV) is the aetiological agent of cervical cancer. High risk HPV E5 is considered an oncogene and has been implicated in cell transformation. E6 and E7 HPV oncoproteins modify the expression of certain glycogenes. The role of the E5 HPV protein in glycogene expression changes has not yet been reported. The aim of the present study was to determine the effects of HPV16 E5 oncoprotein on glycogene expression. For these, a microarray assay was performed using the HaCaT cell line and altered glycogenes were identified. The mRNA levels of certain glycogenes were determined via reverse transcription-quantitative PCR (RT-qPCR). Using in silico analysis, the present study identified that glycosylation pathways were altered by E5. Microarray analysis revealed alterations in certain glycogenes, including the upregulation of ST6GAL1, ST3GAL3, CHST2 and MANBA, and the downregulation of UGT2B15, GALNT11, NDST2 and UGT1A10. Increased mRNA levels were confirmed via RT-qPCR for sialyltransferases genes. Additionally, in silico analysis was performed to identify glycosylation networks altered in the presence of the E5 oncoprotein. The analysis revealed that E5 could modify glycan sialylation, the N-glycosylation pathway, keratan sulfate and glycosaminoglycan synthesis. To the best of our knowledge, the current study was the first to determine the role of the HPV16 E5 oncoprotein in glycogene expression changes. The results indicated that increased sialyltransferase mRNA levels reported in pre-malignant and malignant cervical tissues could be the result of E5 oncoprotein expression. The results provide a possible role of HPV infection on glycosylation changes reported during cervix transformation.

Synthesis, characterization, and thermal decomposition kinetics of copper hydroxide sulfate (Cu4(SO4)(OH)6) synthesized by chemical precipitation method

AbstractIn this study, copper hydroxide sulfate (Cu4(OH)6SO4) was synthesized by chemical precipitation using CuSO4 as the precursor material and the first time borax as basic precipitating agent. The synthesized samples were characterized using XRD, FTIR, and SEM. The decomposition kinetics of Cu4(OH)6SO4 were examined by the thermoanalytical procedures. It was obtained from TG‐DTG data that Cu4(OH)6SO4 has two‐region decomposition at elevated temperatures. Kinetic parameters of these regions were calculated by using both the model‐fitting and model‐free methods. In the decomposition of copper hydroxy sulfate, the mean activation energy for first region was calculated as 90.88 kJ mol−1 from KAS method and 91.47 kJ mol−1 from FWO method. The mean activation energy for the second region was 207.20 kJ mol−1 from KAS and 213.34 kJ mol−1 from FWO. The activation energies for first and second regions were obtained from Ortega method as 56.3 and 198.3 kJ mol−1, respectively.

Effects of syndecan-4 gene silencing by micro RNA interference in anoikis resistant endothelial cells: Syndecan-4 silencing and anoikis resistance.

The cell's resistance to cell death by adhesion loss to extracellular matrix (anoikis), contributes to tumor progression and metastasis. Various adhesion molecules are involved in the anoikis resistance, including the syndecan-4 (SDC4), a heparan sulfate proteoglycan (HSPG) present on the cell surface. Changes in the expression of SDC4 have been observed in tumor and transformed cells, indicating its involvement in cancer. In previous works, we demonstrated that acquisition of anoikis resistance resistance by blocking adhesion to the substrate up-regulates syndecan-4 expression in endothelial cells. This study investigates the role of SDC4 in the transformed phenotype of anoikis resistant endothelial cells. Anoikis-resistant endothelial cells (Adh1-EC) were transfected with micro RNA interference (miR RNAi) targeted against syndecan-4. The effect of SDC4 silencing was analyzed by real-time PCR, western blotting and immunofluorescence. Transfection with miRNA-SDC4 resulted in a sequence-specific decrease in syndecan-4 mRNA and protein levels. Furthermore, we observed a reduction in the number of heparan and chondroitin sulfate chains in the cell extract and culture medium. The SDC4 silencing led to downregulation of proliferative and invasive capacity and angiogenic abilities of anoikis-resistant endothelial cells. Compared with the parental cells (Adh1-EC), SDC4 silenced cells (SDC4 miR-Syn-4-1-Adh1-EC e miR-Syn-4-2-Adh1-EC) exhibited an increase in adhesion to collagen and laminin and also in the apoptosis rate. Moreover, transfection with miRNA-SDC4 caused a decrease in the number of cells in the S phase of the cell cycle. This is accompanied by an increase in the heparan sulfate synthesis after 12 h of simulation with fetal calf serum (FCS). SDC4 silencing cells are more dependent of growth factors present in the FCS to synthesize heparan sulfate than parental cells. Similar data were obtained for the wild-type cell line (EC). Our results indicated that downregulation of SDC4 expression reverses the transformed phenotype of anoikis resistant endothelial cells. These and other findings suggest that syndecan-4 is suitable for pharmacological intervention, making it an attractive target for cancer therapy.

N-glycosylation of the human \u03b21,4-galactosyltransferase 4 is crucial for its activity and Golgi localization

β1,4-galactosyltransferase 4 (B4GalT4) is one of seven B4GalTs that belong to CAZy glycosyltransferase family 7 and transfer galactose to growing sugar moieties of proteins, glycolipids, glycosaminoglycans as well as single sugar for lactose synthesis. Herein, we identify two asparagine-linked glycosylation sites in B4GalT4. We found that mutation of one site (Asn220) had greater impact on enzymatic activity while another (Asn335) on Golgi localization and presence of N-glycans at both sites is required for production of stable and enzymatically active protein and its secretion. Additionally, we confirm B4GalT4 involvement in synthesis of keratan sulfate (KS) by generating A375 B4GalT4 knock-out cell lines that show drastic decrease in the amount of KS proteoglycans and no significant structural changes in N- and O-glycans. We show that KS decrease in A375 cells deficient in B4GalT4 activity can be rescued by overproduction of either partially or fully glycosylated B4GalT4 but not with N-glycan-depleted B4GalT4 version.

A Genome-Wide CRISPR-Cas9 Screen Reveals the Requirement of Host Cell Sulfation for Schmallenberg Virus Infection.

Schmallenberg virus (SBV) is an insect-transmitted orthobunyavirus that can cause abortions and congenital malformations in the offspring of ruminants. Even though the two viral surface glycoproteins Gn and Gc are involved in host cell entry, the specific cellular receptors of SBV are currently unknown. Using genome-wide CRISPR-Cas9 forward screening, we identified 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 (PAPST1) as an essential factor for SBV infection. PAPST1 is a sulfotransferase involved in heparan sulfate proteoglycan synthesis encoded by the solute carrier family 35 member B2 gene (SLC35B2). SBV cell surface attachment and entry were largely reduced upon the knockout of SLC35B2, whereas the reconstitution of SLC35B2 in these cells fully restored their susceptibility to SBV infection. Furthermore, treatment of cells with heparinase diminished infection with SBV, confirming that heparan sulfate plays an important role in cell attachment and entry, although to various degrees, heparan sulfate was also found to be important to initiate infection by two other bunyaviruses, La Crosse virus and Rift Valley fever virus. Thus, PAPST1-triggered synthesis of cell surface heparan sulfate is required for the efficient replication of SBV and other bunyaviruses.IMPORTANCE SBV is a newly emerging orthobunyavirus (family Peribunyaviridae) that has spread rapidly across Europe since 2011, resulting in substantial economic losses in livestock farming. In this study, we performed unbiased genome-wide CRISPR-Cas9 screening and identified PAPST1, a sulfotransferase encoded by SLC35B2, as a host entry factor for SBV. Consistent with its role in the synthesis of heparan sulfate, we show that this activity is required for efficient infection by SBV. A comparable dependency on heparan sulfate was also observed for La Crosse virus and Rift Valley fever virus, highlighting the importance of heparan sulfate for host cell infection by bunyaviruses. Thus, the present work provides crucial insights into virus-host interactions of important animal and human pathogens.

Chemical O-sulfation of N-sulfoheparosan: a route to rare N-sulfo-3-O-sulfoglucosamine and 2-O-sulfoglucuronic acid.

Heparosan, the capsular polysaccharide of E. coli K5 is currently used as the starting material in the chemoenzymatic synthesis of heparan sulfate and the structurally related anticoagulant drug heparin. Base hydrolysis of N-acetyl groups and their subsequent N-sulfonation, are used to prepare N-sulfoheparosan an intermediate of biosynthesis. In the present study, when excess sulfonation reagent was used during N-sulfonation, some O-sulfation also took place in the N-sulfoheparosan product. After a nearly full digestion, a hexasaccharide fraction exhibited resistance to heparin lyase II. Excessive digestion by heparin lyase II and structural identification by NMR and mass spectroscopy indicated that the resistant hexasaccharide fraction has two structures, ΔUA-GlcNS-GlcA2S-GlcNS-GlcA-GlcNS and ΔUA-GlcNS-GlcA- GlcNS3S-GlcA-GlcNS in similar amounts. The 2-sulfated structure exhibited partial resistance to heparin lyase II; however the structure of ΔUA-GlcNS-GlcA-GlcNS3S was completely resistant to heparin lyase II.

RETRACTED: The synthesis of sulfate doped hydroxyapatite for enhanced drug loading and chemotherapeutic drug delivery towards lung cancer treatment

Abstract Tumor-targeting chemotherapeutic drug delivery platforms based on pH stimulus and “smart” drug nanocarriers have received increasing interest in precise control of spatial and temporal drug release to target lung cancer cells and reduce serious side effects compared to conventional cancer chemotherapy. Incorporation of bioactive targeting agent into drug nanocarriers offers new opportunities to improve the targeting specificity and maximize the anti-cancer therapeutic effects. Up to date, there has been limited study on the construction of effective pH-responsive tumor-targeting drug delivery systems based on bioactive targeting agents. Herein, we report the preparation of highly porous sulfate doped HAp (S-HAp) nanospheres via a facile hydrothermal approach, and apply surface-modified S-HAp nanospheres with PEG and folic acid (FA) as novel drug nanocarriers for effective pH-responsive chemotherapeutic drug delivery towards lung cancer treatment. The drug release profile shows anti-cancer doxorubicin (DOX) loaded S-HAp/PEG/FA/DOX display effective pH-triggered drug release under small pH variations. The extremely high surface area and FA surface functionalization enable a high drug loading capacity and strong anti-cancer killing effects. This is the first demonstration of highly porous sulfate doped HAp nanospheres with FA modification as biodegradable carriers for drug delivery. We believe this work can be extended to other drug carrier designs as well as pH-responsive nanoplatform construction for effective tumor-targeting cancer therapy.

Expression optimization and molecular modification of heparin C5 epimerase

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.

«Green» synthesis and characterization of galactomannan sulfates obtained using sulfamic acid

The “green” synthesis of galactomannan sulfates using a sulfamic acid–urea mixture has been studied for the first time. The effect of the time and temperature of the galactomannan sulfation process on the degree of substitution of galactomannan sulfates has been investigated. It is shown that, at a temperature of 70 °C with an increase in the process time up to 120 min, the degree of substitution increases up to 0.70. An increase in the process temperature up to 80 °C leads to the production of galactomannan sulfates with a degree of substitution of 1.67. With a further increase in the process temperature to 90 °C, the galactomannan structure is partially destructed, and the degree of substitution decreases. Embedding of the sulfate groups into the galactomannan structure has been confirmed by elemental analysis and Fourier-transform infrared spectroscopy. In addition, the initial and sulfated galactomannans have been characterized by X-ray diffraction, scanning electron microscopy, atomic force microscopy, and gel permeation chromatography. The thermal analysis shows that the initial galactomannan exhibits endothermic peaks at 254 and 294 °C and an exothermic peak at 315 °C, while sulfated galactomannan exhibits endothermic peaks at 209 and 275 °C and an exothermic peak at 281 °C. Using atomic force microscopy, it has been shown that the sulfated galactomannan film consists of spherical particles with an average diameter of 200–300 nm; according to the phase contrast data, it has the uniform composition without extraneous impurities.

Enzymatic Synthesis of Chondroitin Sulfate E to Attenuate Bacteria Lipopolysaccharide-Induced Organ Damage.

Chondroitin sulfate E (CS-E) is a sulfated polysaccharide that contains repeating disaccharides of 4,6-disulfated N-acetylgalactosamine and glucuronic acid residues. Here, we report the enzymatic synthesis of three homogeneous CS-E oligosaccharides, including CS-E heptasaccharide (CS-E 7-mer), CS-E tridecasaccharide (CS-E13-mer), and CS-E nonadecasaccharide (CS-E 19-mer). The anti-inflammatory effect of CS-E 19-mer was investigated in this study. CS-E 19-mer neutralizes the cytotoxic effect of histones in a cell-based assay and in mice. We also demonstrate that CS-E 19-mer treatment improves survival and protects against organ damage in a mouse model of endotoxemia induced by bacterial lipopolysaccharide (LPS). CS-E19-mer directly interacts with circulating histones in the plasma from LPS-challenged mice. CS-E 19-mer does not display anticoagulant activity nor react with heparin-induced thrombocytopenia antibodies isolated from patients. The successful synthesis of CS-E oligosaccharides provides structurally defined carbohydrates for advancing CS-E research and offers a potential therapeutic agent to treat life-threatening systemic inflammation.

Integration of transcriptomic profile of SARS-CoV-2 infected normal human bronchial epithelial cells with metabolic and protein-protein interaction networks.

A novel coronavirus (SARS-CoV-2, formerly known as nCoV-2019) that causes an acute respiratory disease has emerged in Wuhan, China and spread globally in early 2020. On January the 30th, the World Health Organization (WHO) declared spread of this virus as an epidemic and a public health emergency. With its highly contagious characteristic and long incubation time, confinement of SARS-CoV-2 requires drastic lock-down measures to be taken and therefore early diagnosis is crucial. We analysed transcriptome of SARS-CoV-2 infected human lung epithelial cells, compared it with mock-infected cells, used network-based reporter metabolite approach and integrated the transcriptome data with protein-protein interaction network to elucidate the early cellular response. Significantly affected metabolites have the potential to be used in diagnostics while pathways of protein clusters have the potential to be used as targets for supportive or novel therapeutic approaches. Our results are in accordance with the literature on response of IL6 family of cytokines and their importance, in addition, we find that matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) with keratan sulfate synthesis pathway may play a key role in the infection. We hypothesize that MMP9 inhibitors have potential to prevent "cytokine storm" in severely affected patients.

Altered Expression of Aggrecan, FAM20B, B3GALT6, and EXTL2 in Patients with Osteoarthritis and Kashin-Beck Disease

The objective of this study was to investigate the expression of enzymes involved in synthesis and modification of chondroitin sulfate (CS) in knee cartilage tissue of patients with osteoarthritis (OA) and Kashin-Beck disease (KBD). The knee articular cartilage samples were obtained from 18 age- and gender-matched donors with 6 each in KBD, OA, and control groups. Hematoxylin and eosin (HE) staining, toluidine blue (TB) staining, and immunohistochemical (IHC) staining were performed to estimate the expression level and localization of aggrecan, along with FAM20B, GalT-II, and EXTL2, which are associated with CS synthesis and modification. Rank-based analyses of variance test was used for the multiple comparisons of discrepancy in the positive staining rate among the 3 groups. In HE and TB staining results, damaged morphology, decreased chondrocyte numbers and proteoglycans were observed in OA and KBD groups compared with the control group. In line with these trends, the positive staining rates of aggrecan were lower in KBD and OA groups than in the control group. Meanwhile, the positive staining rates of CS chain modifying enzymes FAM20B, GalT-II, and EXTL2 decreased in OA and KBD groups. In conclusion, it was demonstrated that altered expression of CS chain modifying enzymes in OA and KBD groups influenced the synthesis procession of CS and could contribute to the damage of cartilage. Further investigation of these enzymes can provide new theoretical and experimental targets for OA and KBD pathogenesis studies.

One-pot synthesis of the organomodified layered double hydroxides - glibenclamide biocompatible nanoparticles

In this work, synthesis of sodium dodecyl sulfate (SDS) organomodified LDH Zn2Al carrying glibenclamide (GLIB) was performed in one step and in one-pot to obtain nanoparticles (NP). XRD data showed GLIB adsolubilization (d = 14.03 Å) and NP coating with Eudragit L100®. In addition, thermal and XRD data showed exfoliated/intercalated nanocomposite for NP S5 (LDH associated with SDS and Eudragit L100®). LDH organophilization and GLIB intercalation reduced surface area (SBET 23.58 m2/g) and NP size (469 nm). In addition, the change in zeta potential (−35.5 ζ) relative to pristine LDH (SBET 41.34 m2/g, 688.8 nm and +14 ζ) indicated that LDH functionalization seems an appropriate approach to produce NP with greater colloidal stability and enhanced functionality. The zinc release data from the LDH matrix (2.96 % ±0.002 ppm) showed the effectiveness of the coating in acid medium (pH 1.2) and the release data from GLIB showed the kinetics of release of zero order with release in simulated intestinal medium (pH 7.4) of 88 % and 73 % (24 h) for uncoated and coated NP, respectively. All NP were considered biocompatible in the WST-1 assay on BALB 3T3 fibroblast strains making these NP promising therapeutically.

O-GlcNAcylation Dampens Dpp/BMP Signaling to Ensure Proper Drosophila Embryonic Development.

BMP (bone morphogenetic protein) signaling activity is precisely controlled by both pathway agonists andantagonists. Here, we identify a previously unrecognized BMP signaling antagonist. We demonstrate that the Drosophila BMP type I receptor Sax(Saxophone) functions as a Dpp (Decapentaplegic) receptor in Drosophila embryos, but that its activity is normally inhibited by the O-linked glycosyltransferase Sxc (Super sex combs). In wild-type embryos, Sax activity is inhibited, and the BMP type I receptor Tkv (Thickveins) is the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal istransduced by both Tkv and Sax, and elevated Dpp signaling results in embryonic lethality. We also demonstrate that Sxc O-glycosylates Sax andobserve elevated Dpp signaling in response to maternal restriction of dietary sugar. These findings link fertility to nutritive environment and point to Sax signaling as the nutrient-sensitive branch of BMP signaling.