TREM2 on microglia cell surface binds to and forms functional binary complexes with heparan sulfate modified with 6-O-sulfation and iduronic acid.

The Triggering Receptor Expressed on Myeloid Cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including Surface Plasmon Resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease.

Identification of the grass carp interleukin-23 receptor and its proinflammatory role in intestinal inflammation.

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

Increased 3-O-sulfated heparan sulfate in Alzheimer's disease brain is associated with genetic risk gene HS3ST1.

HS3ST1 is a genetic risk gene associated with Alzheimer's disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3-O-sulfated HS displayed sevenfold increase in the AD group (n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3-O-sulfated HS is made by 3-O-sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3-O-sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3-O-sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.

Apolipoprotein†E Recognizes Alzheimer's Disease Associated 3-O Sulfation of Heparan Sulfate.

Apolipoprotein E (ApoE)'s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk.

Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis.

Roundabout 4 (Robo4) is a transmembrane receptor that expresses specifically in endothelial cells. Soluble Robo4 was reported in the human plasma and mouse serum and is inhibitory towards FGF- and VEGF-induced angiogenesis. It remains unknown how soluble Robo4 is generated and if soluble Robo4 regulates additional angiogenic signaling. Here, we report soluble Robo4 is the product of constitutive ectodomain shedding of endothelial cell surface Robo4 by disintegrin metalloproteinases ADAM10 and ADAM17 and acts to inhibit angiogenic Slit3 signaling. Meanwhile, the ligand Slit3 induces cell surface receptor Robo4 endocytosis to shield Robo4 from shedding, showing Slit3 inhibits Robo4 shedding to enhance Robo4 signaling. Our study delineated ADAM10 and ADAM17 are Robo4 sheddases, and ectodomain shedding, including negative regulation by its ligand Slit3, represents a novel control mechanism of Robo4 signaling in angiogenesis.

Application of a Mutant Cell Library to Determine the Structure-Function Relationship of Heparan Sulfate in Facilitating FGF2-FGFR1 Signaling.

Heparan sulfate (HS) is a linear polysaccharide with complex structures and modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Recently, we generated a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell individually or in their combination. Here, we describe the experimental procedure using the mutant cell library to determine the structure-function relationship of HS in the regulation of FGF2-FGFR1 signaling at the levels of cell surface FGF2 binding and the downstream intracellular signaling activation. Our results demonstrated that strictly defined fine structure is required for HS to act as a co-receptor for FGF2-FGFR1 signaling.

Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer.

The severe acute respiratory syndrome (SARS)-like coronavirus disease (COVID-19) is caused by SARS-CoV-2 and has been a serious threat to global public health with limited treatment. Cellular heparan sulfate (HS) has been found to bind SARS-CoV-2 spike protein (SV2-S) and co-operate with cell surface receptor angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 infection of host cells. In this study, we determined that host cell surface SV2-S binding depends on and correlates with host cell surface HS expression. This binding is required for SARS-Cov-2 virus to infect host cells and can be blocked by heparin lyase, HS antagonist surfen, heparin, and heparin derivatives. The binding of heparin/HS to SV2-S is mainly determined by its overall sulfation with potential, minor contribution of specific SV2-S binding motifs. The higher binding affinity of SV2-S G614 mutant to heparin and upregulated HS expression may be one of the mechanisms underlying the higher infectivity of the SARS-CoV-2 G614 variant and the high vulnerability of lung cancer patients to SARS-CoV-2 infection, respectively. The higher host cell infection by SARS-CoV-2 G614 variant pseudovirus and the increased infection caused by upregulated HS expression both can be effectively blocked by heparin lyase and heparin, and possibly surfen and heparin derivatives too. Our findings support blocking HS-SV2-S interaction may provide one addition to achieve effective prevention and/treatment of COVID-19.

STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes.

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Interleukin-17 suppresses grass carp reovirus infection in Ctenopharyngodon idellus kidney cells by activating NF-κB signaling.

The grass carp accounts for a large proportion of aquacultural production in China, but the hemorrhagic disease caused by grass carp reovirus (GCRV) infection often causes huge economic losses to the industry. Interleukin 17 (IL-17) is an important cytokine that plays a critical role in the inflammatory and immune responses. Although IL-17 family members have been extensively studied in mammals, our knowledge of the activity of IL-17 proteins in teleosts in response to viral infection is still limited. In this study, the role of IL-17 in GCRV infection and its mechanism were investigated. The expression levels of IL-17AF1, IL-17AF2, and IL-17AF3 in Ctenopharyngodon idella kidney (CIK) cells gradually increased from 6 h after infection with GCRV. The nuclear translocation of p65, which acts in the NF-κB signaling pathway, was also increased by GCRV infection. The overexpression of IL-17AF1, IL-17AF2, or IL-17AF3 also promoted the nuclear translocation of p65 and the levels of phospho-IκBα in CIK cells, and reduced the expression of the viral structural protein VP7. An NF-κB signal inhibitor abolished the inhibition of GCRV infection by IL-17 proteins. These results suggested that the NF-κB signaling pathway was activated by the overexpression of IL-17 proteins, resulting in the inhibition of viral infection. In conclusion, in this study, we demonstrated that IL-17AF1, IL-17AF2, and IL-17AF3 acted as immune cytokines, exerting an antiviral effect by activating the NF-κB signaling pathway.

3-O-Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake.

Prion-like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1-/- (HS 3-O-sulfotransferase-1 knockout) cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau-HS binding and likely the transcellular spread of tau, providing a novel target for disease-modifying treatment of AD and other tauopathies.

Dietary nano cerium oxide promotes growth, relieves ammonia nitrogen stress, and improves immunity in crab (Eriocheir sinensis).

Oxidative stress plays a crucial role in ammonia nitrogen toxicity. In this study, the beneficial effects of dietary nano cerium oxide (nano CeO2) as a potent antioxidant were examined in the Chinese mitten crab (Eriocheir sinensis). Crabs were fed a diet supplemented with 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, or 12.8 mg/kg nano CeO2 for 60 d. The optimum supplementation level of nano CeO2 that significantly increased weight gain rate and decreased feed coefficient was 0.8 mg/kg. This level also offered immune protection when crabs were kept under ammonia nitrogen stress and/or exposed to pathogen infection (Aeromonas hydrophila). Supplementation with 0.8 mg/kg of CeO2 (i) relieved pathological damage to the hepatopancreas; (ii) increased hemocyte counts, including total number of hemocytes, granulocytes, and hyalinocytes; (iii) decreased malondialdehyde content and increased antioxidant enzyme activities of superoxide dismutase and catalase in the hemolymph; (iv) increased the activities of lysozyme, acid phosphatase, and alkaline phosphatase in the hemolymph; and (v) increased gene and protein expression of cathepsin L in the hepatopancreas. Mortality increased when crabs were injected with bacteria under ammonia nitrogen stress, but dietary supplementation with 0.8 mg/kg nano CeO2 decreased the mortality rate. Thus, the results of this study suggested that dietary supplementation with nano CeO2 in crabs promoted growth and up-regulated immunity to bacterial infection under ammonia nitrogen stress.

Interleukin-12 receptor ß2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation.

Interleukin-12 receptor ß2 (IL-12Rß2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rß2 in teleost fish. In this work, we have cloned and characterized IL-12Rß2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rß2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rß2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rß2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rß2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rß2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rß2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rß2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rß2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rß2 is involved in the regulation of intestinal inflammation.

A transcriptome analysis focusing on inflammation-related genes of grass carp intestines following infection with Aeromonas hydrophila.

Inflammation is a protective response that is implicated in bacterial enteritis and other fish diseases. The inflammatory mechanisms behind Aeromonas hydrophila infections in fish remain poorly understood. In this study, we performed a de novo grass carp transcriptome assembly using Illumina's Solexa sequencing technique. On this basis we carried out a comparative analysis of intestinal transcriptomes from A. hydrophila-challenged and physiological saline solution (PSS/mock) -challenged fish, and 315 genes were up-regulated and 234 were down-regulated in the intestines infected with A. hydrophila. The GO enrichment analysis indicated that the differentially expressed genes were enriched to 12, 4, and 8 GO terms in biological process, molecular function, and cellular component, respectively. A KEGG analysis showed that 549 DEGs were involved in 165 pathways. Moreover, 15 DEGs were selected for quantitative real-time PCR analysis to validate the RNA-seq data. The results confirmed the consistency of the expression levels between RNA-seq and qPCR data. In addition, a time-course analysis of the mRNA expression of 12 inflammatory genes further demonstrated that the intestinal inflammatory responses to A. hydrophila infection simultaneously modulated gene expression variations. The present study provides intestine-specific transcriptome data, allowing us to unravel the mechanisms of intestinal inflammation triggered by bacterial pathogens.

Dietary nano-selenium relieves hypoxia stress and, improves immunity and disease resistance in the Chinese mitten crab (Eriocheir sinensis).

Hypoxia is a relevant physiological challenge for crab culture, and the hemolymph plays a crucial role in response to the hypoxia. In a 60 d feeding trial, Chinese mitten crabs (Eriocheir sinensis) fed a diet containing 0.2 mg/kg nano-selenium (nanoSe) showed a significantly increased weight gain rate (WGR) and a reduced feed coefficient (FC) compared to those fed diets with 0, 0.1, 0.4, 0.8, and 1.6 mg/kg nanoSe. Another 90 d feeding trial was conducted to determine the influence of dietary nanoSe on the immune response in juvenile Chinese mitten crabs kept under the condition of hypoxia. The results showed that hypoxia stress resulted in significantly increased hemocyte counts (THC, LGC, SGC, and HC), expression levels of the hemocyanin gene and protein, lactic acid level, and antioxidant capacity (T-AOC activities, SOD activities, GSH-Px and GSH content) in hemolymph supernatant. When these crabs were infected with Aeromonas hydrophila bacteria, hypoxia exposure increased mortality, but it was alleviated by a diet supplemented with 0.2 mg/kg nanoSe. The up-regulative effects of nanoSe (0.2 mg/kg) on antioxidant capacity, hemocyte counts, and hemocyanin expression under hypoxia exposure were further strengthened throughout, whereas lactic acid levels induced by hypoxia stress were restored. Thus, the observations in this study indicate that the level of dietary nanoSe is important in regulating immunity and disease resistance in crabs kept under hypoxia stress.

Characterization of interleukin-1ß as a proinflammatory cytokine in grass carp (Ctenopharyngodon idella).

Interleukin-1ß (IL-1ß) is a well-characterized cytokine that plays key roles in cellular responses to infection, inflammation, and immunological challenges in mammals. In this study, we identified and analyzed a grass carp (Ctenopharyngodon idella) ortholog of IL-1ß (gcIL-1ß), examined its expression patterns in various tissues in both healthy and lipopolysaccharide (LPS)-stimulated specimens, and evaluated its proinflammatory activities. The gcIL-1ß gene consists of seven exons and six introns. The full-length cDNA sequence contains an open reading frame of 813 nucleotides. The deduced amino acid sequence exhibits a characteristic IL-1 signature but lacks the typical IL-1ß converting enzyme cleavage site that is conserved in mammals. In the phylogenetic tree, IL-1ßs from grass carp and other members of the Cyprinidae family clustered into a single group. Expression pattern analysis revealed that gcIL-1ß is constitutively expressed in all 11 tissues examined, and LPS stimulation leads to significant up-regulation in muscle, liver, intestine, skin, trunk kidney, head kidney, and gill. Recombinant grass carp IL-1ß (rgcIL-1ß) was generated prokaryotically as a fusion protein of Trx-rgcIL-1ß. An anti-rgcIL-1ß polyclonal antibody (rgcIL-1ß pAb) was raised in mice against the purified Trx-rgcIL-1ß. Western blot analysis confirmed that rgcIL-1ß pAb reacted specifically with gcIL-1ß in C. idella kidney (CIK) cells. Quantitative real-time PCR data indicated that intestinal mRNA expression levels of endogenous IL-1ß, IL-1R2, and TNF-α were significantly up-regulated following Trx-rgcIL-1ß exposure. The inhibitory activities of rgcIL-1ß pAb against the inflammatory response were confirmed in a model of Aeromonas hydrophila-induced intestinal inflammation. Our immunohistochemical study revealed that the degree and intensity of inflammatory cell infiltration are fully consistent with the observed mRNA expression patterns of these key inflammatory genes. Taken together, these data suggest that gcIL-1ß plays a critical role in the proinflammatory response in the grass carp intestine.

Molecular characterization, expression analysis, and biological effects of interleukin-8 in grass carp Ctenopharyngodon idellus.

Interleukin-8 (IL-8) is a CXC chemokine that plays key regulatory roles in the immune and inflammatory responses implicated in many human diseases. In this study, we identified and characterized an IL-8 homologue from the grass carp, Ctenopharyngodon idellus. A sequence alignment of the full-length cDNA and genomic DNA showed that the exon/intron organization of grass carp IL-8 (gcIL-8) is identical to those of other known CXC chemokine genes. A multiple alignment analysis showed that gcIL-8 is an ELR(-)CXC chemokine, and its deduced amino acid sequence shares 81% and 36% identity with common carp IL-8s L1 (GenBank ID: ABE47600) and L2 (GenBank ID: AB470924), respectively, suggesting that it belongs to the lineage 1 group of fish IL-8 proteins. On a phylogenetic tree, gcIL-8 clustered with other teleost IL-8 proteins to form a fish-specific clade, clearly distinct from those of bird, mammal, and amphibian proteins. Real-time quantitative PCR analysis indicated that gcIL-8 is differentially expressed in various tissues under normal conditions and that the expression of gcIL-8 mRNA in immune-related tissues is clearly upregulated by Aeromonas hydrophila infection. To explore the biological effects of gcIL-8, we produced a recombinant protein, rgcIL-8, in a prokaryotic expression system. Purified rgcIL-8 was confirmed to be chemoattractive for head kidney neutrophils and mononuclear leukocytes in vitro. Our histopathological study also revealed that rgcIL-8 exerts proinflammatory effects by inducing neutrophil infiltration and erythrocyte extravasation. Overall, these results suggest that IL-8 is crucially involved in the inflammatory responses of fish.

Evaluation of the efficacy and safety of hyaluronic acid vaginal gel to ease vaginal dryness: a multicenter, randomized, controlled, open-label, parallel-group, clinical trial.

INTRODUCTION: Atrophic vaginitis is a common occurrence, particularly among postmenopausal women; however, few seek or receive treatment. One therapeutic solution is topically applied products. Estrogen-based treatments have been shown to be effective; however, many patients are reluctant to use such formulations due to health concerns, hence the need to assess the efficacy of acceptable alternatives. AIM: This multicenter, randomized, controlled, open-label, parallel-group clinical trial set out to evaluate the efficacy and safety of hyaluronic acid vaginal gel to treat vaginal dryness compared with estriol cream in postmenopausal women. METHODS: One hundred forty-four subjects were randomized, 72 to the test group treated with hyaluronic acid vaginal gel (Hyalofemme) and 72 to the control group treated with estriol cream (Ovestin). Treatment in both groups was applied by means of a device once every 3 days for a total of 10 applications over 30 days. MAIN OUTCOME MEASURES: Efficacy was measured by grading vaginal dryness and three other vaginal symptoms on a visual analog scale. Safety assessments included vital signs, laboratory examinations of the vaginal microecosystem, vaginal pH value, vaginal B ultrasound, and incidence of adverse events. Assessments were performed at baseline, by telephone after the third application, and at the final visit. RESULTS: Both hyaluronic acid vaginal gel and estriol cream can significantly improve the clinical symptoms of vaginal dryness in postmenopausal women, with improvement rate of 84.44% and 89.42%, respectively, after 10 applications, without statistically significant difference between them. CONCLUSION: Both hyaluronic acid vaginal gel and estriol cream are effective in the treatment of vaginal dryness. Hyaluronic acid vaginal gel may be considered as a valid alternative to estrogen-based treatments in relieving the symptoms of vaginal dryness.

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

The intermediate products in the degradation of 4-chlorophenol by pulsed high voltage discharge in water.

Degradation of 4-chlorophenol by pulsed high voltage discharge is an intricate process involving a series of complex chemical reactions. Hydroxylation of 4-chlorophenol to form hydroquinone, 4-chlororesorcinol and 4-chlorocatechol is the first step, though a very small amount of direct cleavage products of the C(1)-C(2) or C(5)-C(6) bond are observed. The yield of 4-chlorocatechol is about twice as much as that of hydroquinone. Less 4-chloresorcinol is produced. The free chloride ions dropped from the 4-chlorophenol degradation can obtain reactivity again from the discharge, and react with undegraded 4-chlorophenol to form 2,4-dichlorophenol. Some ring-opened products have also been identified and their possible reaction routes are proposed. Several compounds are verified by use of authentic samples. The more stable ring-opened products are low molecular weight (LMW) acids such as formic, acetic, oxalic, malonate, maleic and malic acid. By discharging 4-chlorophenol aqueous solution for 36 min, the amount of carbons obtained from organic acids is more than 50% while that of carbons from aromatic products less than 20% in the carbons of degraded 4-chlorophenol, which is about 94% of initial carbons. After 60 min of discharge, all the 4-chlorophenol and its aromatic intermediates have been removed completely and the organic carbons are mainly presented as organic acid such as acetic and oxalate acid. At the end of the 120 min discharge, the amount of the remaining organic carbons is not more than 14% of the initial carbons.

[Multicenter randomized, double-blind, placebo-controlled trial of prostaglandin E1 cream for female sexual arousal disorder].

OBJECTIVE: To investigate the efficacy and safety of alprostadil cream in management of female sexual arouse disorder (FSAD), and its appropriate dose for clinical prescription. METHODS: The volunteers were assigned randomly to four groups which received alprostadil cream in different dosage (500 µg, 700 µg and 900 µg) or placebo cream, respectively. The cream was applied to the clitoris and G-spot before coitus. The efficacy was assessed by comparing the satisfactory rate of sexual arousal, the score of female sexual function index (FSFI) and female sex disorder scale (FSDS) and the general appraised question (GAQ) before and after the treatment. The safety was evaluated by the adverse effects that appeared including symptoms, physical and biochemical examination. RESULTS: Totally, 400 women enrolled in this study with 374 assigned to the group for efficacy evaluation and 387 cases to the group for safety analysis. No significant difference was found among the four groups in the demographic characters and sexual baseline. The increase of satisfactory percentage of sexual arousal in the four groups (placebo, 500 µg, 700 µg and 900 µg) was 22.63%, 36.67%, 34.01%, and 44.29%, respectively (P<0.05), and the increase was statistically higher in the 900 µg group than in the placebo group (P<0.0167). The elevated FSFI score above the baseline in the treatment groups (900 µg 22.89, 700 µg 21.69, and 500 µg 20.71) were higher than that in the placebo group (14.68, P<0.05), while the reduced FSDS score below the baseline (900 µg 25.97, 700 µg 21.98, and 500 µg 20.27) were higher than that of the placebo (17.60, P<0.05). No significant difference was found in the four groups in GAQ (P=0.054). The main common adverse effect was topical stimulation. No adverse effect was reported in physical and biochemical examination, electrocardiogram (ECG) or Thinprep cytologic test (TCT). CONCLUSION: Alprostadil cream can treat female sexual arousal disorder effectively with the maximum effect at the dose of 900 µg and without significant adverse effect except for mild topical stimulation.