90-kDa Protein Research Articles

Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Like Agonist, Exendin-4-Fc

Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. However, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase IV. We engineered Exendin-4-Fc, a 66-kDa fusion protein by linking an IgG2 Fc to Exendin-4. A stably transfected Chinese hamster ovary cell line was obtained using electroporation. Exendin-4-Fc stimulated insulin secretion in INS-1 cells in a dose- and glucose-dependent manner and increased insulin mRNA expression. The plasma half-life of Exendin-4-Fc in cynomolgus monkeys was approximately 133.92 ± 25.1 h. In the KKAy mouse model of diabetes, one intraperitoneal injection of Exendin-4-Fc (1 mg/kg) reduced blood glucose levels for 5 days. A 4-week repeat-administration study identified sustained effects on blood glucose levels. Oral glucose tolerance tests conducted at the beginning and end of this 4-week period showed that Exendin-4-Fc produced a stable glucose lowering effect. In addition, KKAy mice treated with Exendin-4-Fc showed statistically significant weight loss from day 23. In conclusion, these properties of Exendin-4-Fc demonstrated that it could be a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes.

Clinical usefulness of 16S ribosomal RNA real-time PCR for the diagnosis of scrub typhus

Scrub typhus is a major acute febrile disease in the Asia–Pacific region. The purpose of the present study is to investigate the clinical usefulness of real-time PCR (Q-PCR) of 16S rRNA for the diagnosis of scrub typhus. We examined blood specimens from 148 adult patients who were confirmed to have scrub typhus from September 2008 to December 2009. Among the 148 scrub typhus patients, 36 patients were treated with antibiotics before admission. To evaluate the clinical usefulness of 16S rRNA Q-PCR, we compared its diagnostic accuracy to the accuracy of the following methods: nested PCR (N-PCR) targeting the gene encoding the 56-kDa protein, Q-PCR targeting the gene encoding the 47-kDa protein, and conventional PCR (C-PCR), targeting the 16S rRNA gene. According to 16S rRNA Q-PCR and 47-kDa Q-PCR, the mild group had copy numbers of 234.4 ± 261.9 and 130.5 ± 128.3, whereas the severe group had copy numbers of 584.4 ± 911.4 and 244.7 ± 210.9, respectively. In both tests, the mean copy numbers were significantly greater in the severe group (P = 0.037 and P = 0.035). 16S rRNA Q-PCR detected Orientia tsutsugamushi infections with a sensitivity of 91.9% (95% CI 86.3–95.7), and 56-kDa N-PCR, 47-kDa Q-PCR, and 16S rRNA C-PCR exhibited lower sensitivities of 81.1% (95% CI 73.8–87.0), 74.3% (95% CI 66.5–81.1), and 87.8% (95% CI 81.5–92.6), respectively, for all 148 patients. In addition, 16S rRNA Q-PCR exhibited a sensitivity of 99.1% (95% CI 95.1–100.0) in the 112 patients who were not treated with antibiotics before admission. 16S rRNA Q-PCR is clinically useful for the rapid diagnosis of scrub typhus and is more accurate than the 56-kDa N-PCR, 47-kDa Q-PCR, and 16S C-PCR methods.

Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

The flexible conformations of a multidomain protein are responsible for its biological functions. Although MurD, a 47-kDa protein that consists of three domains, sequentially changes its domain conformation from an open form to a closed form through a semiclosed form in its enzymatic reaction, the domain dynamics in each conformation remains unclear. In this study, we verify the conformational dynamics of MurD in the corresponding three states (apo and ATP- and inhibitor-bound states) with a combination of small-angle x-ray and neutron scattering (SAXS and SANS), dynamic light scattering (DLS), neutron backscattering (NBS), neutron spin echo (NSE) spectroscopy, and molecular dynamics (MD) simulations. Applying principal component analysis of the MD trajectories, twisting and open-closed domain modes are identified as the major collective coordinates. The deviations of the experimental SAXS profiles from the theoretical calculations based on the known crystal structures become smaller in the ATP-bound state than in the apo state, and a further decrease is evident upon inhibitor binding. These results suggest that domain motions of the protein are suppressed step by step of each ligand binding. The DLS and NBS data yield collective and self-translational diffusion constants, respectively, and we used them to extract collective domain motions in nanometer and nanosecond scales from the NSE data. In the apo state, MurD shows both twisting and open-closed domain modes, whereas an ATP binding suppresses twisting domain motions, and a further reduction of open-closed mode is seen in the inhibitor-binding state. These observations are consistent with the structure modifications measured by the small-angle scattering as well as the MD simulations. Such changes in the domain dynamics associated with the sequential enzymatic reactions should be related to the affinity and reaction efficiency with a ligand that binds specifically to each reaction state.

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.

Comparison of the Functional Characteristics and Post-Translational Modifications of Commercial Factor VIII Products

Introduction: The functional and biochemical characteristics of recombinant proteins are dependent on the amino acid sequence of the protein and the host cell line used to express the protein. We have previously reported the characterization of human cell line-derived recombinant factor VIII (rFVIII) simoctocog alfa (Nuwiq®, Octapharma AG, Switzerland) compared with three other recombinant FVIII products (Advate®, Kogenate FS® and Refacto®) derived from hamster cells and a human plasma-derived (pd) FVIII concentrate (octanate®, Octapharma AG, Switzerland) (Kannicht C et al. Thrombosis Res 2013; 131:78-88; Sandberg H et al. Thrombosis Res 2012; 130:808-17). Here we extend these analyses and compare the characteristics of simoctocog alfa (Nuwiq®) with 2 commercial moroctocog alfa (rFVIII-BDD) products, Octofactor® (Generium, Russian Federation) and ReFacto AF® (Pfizer, USA), to assess potential differences between expression systems and compared with a pdFVIII concentrate (Octanate®).Materials and methods: Commercially available batches of Octofactor®, ReFacto AF® and Nuwiq® were analyzed. The labeled potencies of all vials were 1000 IU. The reconstitution volumes were 5 mL (Octofactor®), 4 mL (ReFacto AF®), and 2.5 mL (Nuwiq®). FVIII activity was measured using chromogenic, one-stage and FVIII antigen assays. The protein content of the products was analyzed using SDS-PAGE followed by silver staining or Western blotting. Thrombin generation activity (TGA) was measured using Thrombogram at 1 IU. Glycosylation patterns and the extent of sulfation of Tyr1680 were analyzed by the liquid chromatography/mass spectrometry (LC/MS) method. Binding to von Willebrand Factor (VWF) was measured using surface plasmon resonance (SPR) analyses.Results: MS analysis confirmed that ReFacto AF® and Octofactor® share the same primary amino acid sequence, including that of the linker sequence between the light and heavy chains. The FVIII activities of each product per vial (labeled potency 1000 IU) are shown in Table 1. The higher FVIII antigen level and the lower specific activity for Octofactor® suggest that these samples contain higher levels of inactive FVIII protein. This is supported by results of the SDS-PAGE analyses, which revealed additional protein bands in the Octofactor® samples that reacted with anti-FVIII antibodies. These bands appear to be partially degraded 170-kDa and 80-kDa protein fragments, suggesting proteolytic cleavage of FVIII during the production of Octofactor®. No significant differences in TGA between the products were detected. The glycosylation patterns of Octofactor® and Refacto AF® were comparable, consistent with both products being expressed in hamster cell lines. Notably, the Octofactor® samples displayed a variable but significantly higher amount of the potentially antigenic non-human Neu5Gc glycan epitope than ReFacto AF®. Consistent with previous findings, no Neu5Gc was found in the samples of Nuwiq®. Nuwiq® is expressed in a human cell line, and it has been shown previously that the oligosaccharide and sialic acid content of Nuwiq® is comparable to pdFVIII.Conclusions: The Octofactor® samples showed lower specific activity and higher amounts of partially degraded and inactive FVIII protein. Analysis of the glycosylation patterns showed that Octofactor® and Refacto AF® both contain potentially antigenic non-human Neu5Gc glycan epitopes due to their expression in hamster cell lines. These epitopes are not present in Nuwiq®, which is produced in a human cell line, or in octanate®, which is derived from human plasma. [Display omitted] DisclosuresKohla:Octapharma Biopharmaceuticals GmbH: Employment. Belyanskaya:Octapharma AG: Employment. Tiemeyer:Octapharma Biopharmaceuticals GmbH: Employment. Züchner:Octapharma Biopharmaceuticals GmbH: Employment. Knappik:Octapharma AB: Employment. Rosenlöcher:Octapharma Biopharmaceuticals GmbH: Employment.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Immunohistochemical examination of p97/VCP expression in developing mouse pancreas and liver

Abstract
 Aim: The 97-kDa Valosin-containing protein has important functions in proteolysis. Although the expression of p97 / VCP has been studied in many types of cells and tissues, the localization of p97 / VCP at cellular level in developing mouse pancreas and liver has not been determined. Therefore, the aim of our study was investigate the immuolocalization of p97/VCP in the rat fetal and postnatal liver and pancreas. 
 Material Methods: The liver and pancreas from fetal, postnatal (1, 5, 15, 35 days old ) and adult (50 days old) mice were examined by using immunohistochemistry in order to determine the expression of p97/VCP. Furthermore the development of mouse pancreas and liver were histomorphologically analyzed under light microscope. 
 Results: The histological structures of the mouse pancreas and liver were maintained in postnatal period. The histological differences between endocrine and exocrine pancreas were significantly observed from the postnatal 5th day. The expression of p97/VCP in Langerhans islets was determined from day 5. p97/VCP expression was also seen in the exocrine pancreas in all postnatal days. p97/VCP was expressed in developing pancreatic and liver tissues and its expression was increased with the development.
 Conclusion: This study is the first to examine the distribution and the localization of p97/VCP in the fetal and postnatal rat liver and pancreas. This study forms the basis for clinical studies in order to determine the function of p97 / VCP in liver and pancreatic cells.

Protective effects of recombinant 53-kDa protein of Trichinella spiralis on acute lung injury in mice via alleviating lung pyroptosis by promoting M2 macrophage polarization.

Trichinella spiralis represents an effective treatment for autoimmune and inflammatory diseases. The effects of recombinant T. spiralis (TS) 53-kDa protein (rTsP53) on acute lung injury (ALI) remain unclear. Here, mice were divided randomly into a control group, LPS group, and rTsP53 + LPS group. ALI was induced in BALB/c mice by LPS (10 mg/kg) injected via the tail vein. rTsP53 (200 µl; 0.4 μg/μl) was injected subcutaneously three times at an interval of 5 d before inducing ALI in the rTsP53+LPS group. Lung pathological score, the ratio and markers of classic activated macrophages (M1) and alternatively activated macrophages (M2), cytokine profiles in alveolar lavage fluid, and pyroptosis protein expression in lung tissue were investigated. RTsP53 decreased lung pathological score. Furthermore, rTsP53 suppressed inflammation by increasing IL-4, IL-10, and IL-13. There was an increase in alveolar M2 macrophage numbers, with an increase in CD206 and arginase-1-positive cells and a decrease in alveolar M1 markers such as CD197 and iNOS. In addition, the polarization of M2 macrophages induced by rTsP53 treatment could alleviate ALI by suppressing lung pyroptosis. RTsP53 was identified as a potential agent for treating LPS-induced ALI via alleviating lung pyroptosis by promoting M2 macrophage polarization.

Expression and Characterization of P. aeruginosa Cytochrome P450 CYP168A1

BACKGROUND Pseudomonas aeruginosa (P. aeruginosa) is an encapsulated gram-negative bacterium regularly associated with the persistent opportunistic infections of the lungs of cystic fibrosis patients. Further, it is one of six pathogens that are the major causes of nosocomial infections in the United States and important worldwide due to increasing levels of multidrug resistance. Cytochrome P450 (CYP) from pathogenic bacteria such as M. tuberculosis have been defined as virulence factors and investigated for the ability to metabolize antibacterial drugs as well their roles in both cholesterol catabolism and fatty acid metabolism. However, the four putative P. aeruginosa CYPs have yet to be characterized. HYPOTHESIS P. aeruginosa CYPs regulate the bacterial metabolism of cholesterol, fatty acids and antibiotics resulting in selective advantages in both growth and the perpetuation of opportunistic bacterial colonization. METHODS The DNA sequence for the putative CYP168A1 was cloned and expressed in E. coli resulting in the expression of an approximately 49kDa protein. Total CYP168A1 was then assayed by the ferrous CO versus ferric difference spectrum resulting in 315 nmole/L of protein at a final concentration of 30µM. UV-visible spectroscopy was then utilized to characterize Type II binding to antifungal azoles, Type I binding to various fatty acids and reverse Type I binding to cholesterol. Further, CYP activity was investigated using LC-MS/MS to identify hydroxylation of fatty acid ligands. RESULTS UV-visible spectroscopy using revealed differential Type II binding affinities for ketoconazole, miconazole, and econazole defined by a Bmax of 0.045, 0.036 and 0.026 and a Kd of 0.697, 0.882 and 2.457µM, respectively. Interestingly, CYP168A1 showed no affinity for fluconazole. The Type I binding affinities of 1µM CYP168A1 have increasing Bmax and affinity corresponding to increasing carbon chain length for saturated fatty acids: lauric acid (12 carbons), myristic acid (14 carbons), and stearic acid (18 carbons). The binding of the monounsaturated oleic acid (18 carbons) results in Bmax = 0.0992 and Kd = 0.374µM and binding the polyunsaturated arachidonic acid (20 carbons) results in a Bmax = 0.058 and Kd = 0.9595µM. Further, CYP168A1 has been shown by LC-MS/MS to successfully hydroxylate lauric acid at the Ω and Ω-1 position utilizing either oxygen surrogates tert-butyl or cumene hydroperoxide. Initial work using spinach ferredoxin and ferredoxin reductase surrogates have also shown successful CYP168A1 metabolism of lauric acid. CONCLUSIONS As an initial step in defining the possible role that CYPs may play in P. aeruginosa growth and pathogenesis, a functional CYP168A1 was expressed in E. coli and characterized. The ability to differentially bind several fatty acids leads us to conclude that this CYP may be involved in energy metabolism. However, the ability of CYP168A1 to bind arachidonic acid leads us to postulate that CYP168A1 may also actively inhibit host lipid peroxidation shown to kill other opportunistic pathogens.

Molecular evolution of kalliklectin in teleost and identification of the novel type with eight apple domains in channel catfish, Ictalurus punctatus

Kalliklectin is a unique fish-specific lectin that demonstrates sequence similarity to mammalian plasma kallikrein and coagulation factor XI, which are not lectins but proteases. Reported fish kalliklectins and these mammalian proteases comprise four characteristic "apple domains" (APDs). Bioinformatics analysis revealed that Siluriformes species possess anomalous kalliklectins comprising 6 to 16 APDs. Complementary DNA cloning showed that the full-length nucleotide sequence of Ictalurus punctatus consists of 2240bp that encode 720 amino acid residues to produce a mature protein with a putative 18 amino acid N-terminus peptide sequence. This protein has a predicted molecular mass of 83,417.23Da. Reverse transcription-polymerase chain reaction (RT-PCR) showed that this lectin gene expresses in the liver but not in any other tissues, including the mucosal tissues. This differential expression pattern makes this lectin unique compared to other lectins described in previous studies. We successfully detected an 85-kDa protein in the serum using western blotting analysis, suggesting that this lectin protein is produced by the liver and secreted into the bloodstream. We characterized a novel cDNA sequence encoding a new type of kalliklectin with eight APDs isolated from channel catfish, I. punctatus. Based on phylogenetic analysis, we speculated that there was a duplication of the third and fourth APD set in a common Siluriformes ancestor at some point after its separation from the common teleost ancestor and that these duplications then underwent independent repeats in different lineages resulting in the generation of the [APD1]-[APD2]-{[APD3]-[APD-4]} × n structure in modern catfishes.

Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1–1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1–1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.

Exogenous toluene gas removal improvement in recombinant rabbit cytochrome P450 2E1 (CYP2E1)-transgenic Ardisia pusilla DC

The cytochrome P450 2E1 (CYP2E1) enzyme encoded by CYP2E1 plays an important role in the metabolism of organic compounds in mammalian liver cells. In this study, the plasmid pCAMBIA2300 harboring the rabbit CYP2E1 was transferred into Agrobacterium tumefaciens; this was then transferred into internodes of Ardisia pusilla. Shoot primordia, whose appearance resembles that of the in vitro protocorm-like body of orchids, regenerated from 121 of 332 internodes that were cultured in selection medium supplemented with 5 mg·L−1 kanamycin. PCR analysis confirmed that 25 of 89 putative transgenic plants (28.0%) stably harbored the CYP2E1 and NPTII genes, and Southern blot analysis revealed the presence of one to three copies of CYP2E1 within the genome of the 25 transgenic plants. The mRNA expression of the transgene was confirmed in 12 of 25 CYP2E1-transgenic plants by Northern blot analysis. Western blot analysis indicated the presence of a 53-kDa recombinant rabbit CYP2E1 protein in the 12 CYP2E1-transgenic A. pusilla plants. The aniline 4-hydroxylase activity of rabbit CYP2E1 in nine CYP2E1-transgenic A. pusilla plants was higher than that in non-transgenic A. pusilla plants. There was a statistically significant increase (5.1–6.1-fold) in the toluene removal ability of the transgenic line CYP2E1-2–2 compared to that observed in wild-type Ardisia plants at different times after exogenous toluene exposure.

Identification of allergens for food-dependent exercise-induced anaphylaxis to shrimp

Shrimp is a causative food that elicits food-dependent exercise-induced anaphylaxis (FDEIA). In this study, we sought to identify IgE-binding allergens in patients with shrimp-FDEIA. Sera were obtained from eight patients with shrimp-FDEIA and two healthy control subjects. Proteins were extracted from four shrimp species by homogenization in Tris buffer. Immunoblot analysis revealed that IgE from patient sera bound strongly to a 70-kDa and a 43-kDa protein in a preparation of Tris-soluble extracts from Litopenaeus vannamei. Mass spectrometry identified the 70-kDa and 43-kDa proteins as a P75 homologue and fructose 1,6-bisphosphate aldolase (FBPA), respectively. To confirm that the putative shrimp allergens were specifically recognized by serum IgE from shrimp-FDEIA patients, the two proteins were purified by ammonium sulfate precipitation followed by reversed-phase HPLC and/or anion-exchange hydrophobic interaction chromatography and then subjected to immunoblot analysis. Purified P75 homologue and FBPA were positively bound by serum IgE from one and three, respectively, of the eight patients with shrimp-FDEIA, but not by sera from control subjects. Thus, P75 homologue and FBPA are identified as IgE-binding allergens for shrimp-FDEIA. These findings could be useful for the development of diagnostic tools and desensitization therapy for shrimp-FDEIA patients.

Abstract P755: Perlecan Lg3 is Neuroprotective and Functionally Restorative in Experimental Ischemic Stroke

Despite promising advancements, ischemic stroke remains a leading cause of death and disability. We previously determined that human recombinant ~85-kDa C-terminal protein fragment of the vascular basement membrane proteoglycan perlecan termed domain V (DV) is neuroprotective, pro-angiogenic, neurogenic and functionally restorative when administered after experimental stroke (transient and permanent middle cerebral artery occlusion -MCAO) in mice and rats. As previous studies suggest, the 23-kDa subdomain of DV called laminin globular domain 3 (LG3) may confer most of DV’s biological activity and is more amenable to production, we tested whether human recombinant perlecan DV LG3 (also termed “DV LG3 ”) might be neuroprotective and functionally restorative in the transient MCAO (intraluminal filament model). Methods: Fifty male mice (C57BL/6J, 10-12 weeks old, 24–30 g) underwent MCAO for 60 min and were survived for seven days. At reperfusion mice were randomly allocated to one of three treatment groups and immediately received either human recombinant DV treatment (positive control, n=16 2 mg/kg, i.p.), LG3 (n=19 6 mg/kg, i.p.), or equivalent volume of PBS vehicle (n=15) ; and again on post-MCAO days 2 and 4. The DV:LG3 dose ratio was selected based on a preliminary in vivo dose evaluation study of therapeutic equivalence. Functional outcome measures (grip strength and grid-hang tests) were determined at baseline and on post-MCAO days 1, 3 and 7. Body weight was measured daily as per welfare. To determine infarct size on post-MCAO day 7, whole brain samples were harvested, sectioned and stained with 2,3,5- Triphenyltetrazolium chloride. The induction of MCAO, DV, DV LG3 or PBS treatments, functional tests and analyses were performed, blindly, by different individuals. While DV treatment significantly reduced mean infarct volume as expected (p<0.022, compared to PBS control group), DV LG3 further reduced infarct volumes (p<0.0001), weight loss (p<0.0002) and improved functional outcome measures (p<0.01) compared to PBS group. Our findings show that human recombinant DV LG3 is neuroprotective and functionally restorative, may be even more effective than DV, and is worthy of more study as a readily producible therapy for ischemic stroke.

Prevalence Determination of m. Hominis and m. Genitalium in the Semen Samples in the Northeast of Iran Using Culture and Multiplex Polymerase Chain Reaction.

Infertility has recently become a growing social and economic world problem. Genital mycoplasmas, such as Mycoplasma hominis and M. genitalium, are most frequently associated with several adverse effects on men’s fertility. The present study aimed to determine the prevalence of M. hominis and M. genitalium in the semen samples in thenortheast of Iran. During thiscross-sectional study from February to May, 2018, 100 semen samples were collected from 100 infertile men in Mashhad, Khorasan Razavi province, northeast of Iran. The presence of M. hominis and M. genitalium was detected by cultivation, polymerase chain reaction (PCR), and Multiplex PCR assays. The colony of mycoplasma was confirmed by Diene’s stain; moreover, arginine hydrolysis, glucose, and urea utilization were evaluated. The following semen indices were analyzed according to World Health Organization guidelines for semen analysis: color, volume, appearance, liquefaction, viscosity, concentration, pH, leukocyte concentration, progressive motility, morphological normality, motile sperm concentration, functional sperm concentration, sperm motility index, and functional sperm. The gene of 16SrRNA (GPO1& MGSO primers) was used as the target gene of the Mycoplasma genus in PCR assay. Multiplex-PCR was performed with a specific primer for conserved regions in the 16SrRNA gene for M. hominis (RNAH1& RNAH2 primers) and the 140-kDa Adhesion Protein Gene for M. genitalium (MG1 & MG2 primers).According to the results,9 (9%) samples were PCR-positive for Mycoplasma spp , while there were 7 (7%) cases isolated by cultivation. M. hominis was detected in 8 (8%) samples by Multiplex PCR, while there was no evidence for M. genitalium. The mean age scores of all infertile and infected men were obtained at 31 and 30 years, respectively. The study could not show any statistical correlation between mycoplasma infection and abnormal semen parameters. The heterogeneity of mycoplasma prevalence in the reports can be ascribed to differences in geographic areas, the sensitivity of the identification method, condition of the group (fertile/infertile), sample size, and operator proficiency. Various results have been reported in numerous studies conducted on the relationship between mycoplasma infection and abnormal semen parameters.

Evidence for existence of insulin-like factor 3 (INSL3) hormone-receptor system in the ovarian corpus luteum and extra-ovarian reproductive organs during pregnancy in goats.

Insulin-like factor 3 (INSL3), initially described as a male hormone, is expressed in female reproductive organs during the estrous cycle and pregnancy but its function has not yet been established. This study explores the function of INSL3 in pregnant Saanen goats by characterizing the expression dynamics of INSL3 and its receptor, relaxin family peptide receptor 2 (RXFP2) and by demonstrating specific INSL3 binding in reproductive organs, using molecular and immunological approaches and ligand-receptor interaction assays. We demonstrate that the corpus luteum (CL) acts as both a source and target of INSL3 in pregnant goats, while extra-ovarian reproductive organs serve as additional INSL3 targets. The expression of INSL3 and RXFP2 in the CL reached maximum levels in middle pregnancy, followed by a decrease in late pregnancy; in contrast, RXFP2 expression levels in extra-ovarian reproductive organs were higher in the mammary glands but lower in the uterus, cervix and placenta and did not significantly change during pregnancy. The functional RXFP2 enabling INSL3 to bind was identified as an ~85kDa protein in both the CL and mammary glands and localized in large and small luteal cells in the CL and in tubuloalveolar and ductal epithelial cells in the mammary glands. Additionally, INSL3 also bound to multiple cell types expressing RXFP2 in the uterus, cervix and placenta in a hormone-specific and saturable manner. These results provide evidence that an active intra- and extra-ovarian INSL3 hormone-receptor system operates during pregnancy in goats.

Characterization of Beta-2-Glycoprotein I Domain V interaction with Plasma Membrane

Beta-2-Glycoprotein I (β2GPI), also known as apolipoprotein H, is a 48kDa protein which circulates in blood at a high concentration [0.2 mg/ml]. β2GPI is the main target of autoantibodies in antiphospholipid syndrome (APS) which results in thrombosis and/or recurrent miscarriage. Therefore, investigating membrane binding mechanism of β2GPI is important to elucidate how this protein become immunogenic upon binding to plasma membrane and to help target specific drug development for APS. It is known that β2GPI binds to anionic phospholipids in the plasma membrane via its positively charged domain V (DV) but the mechanism of binding is still elusive. in this study, we performed multiple 500-ns molecular dynamics simulations of β2GPI-DV in the presence of membranes containing PC and PS in varying compositions employing the HMMM (highly mobile membrane mimetic) model. We observed stable membrane binding in eight out of ten replicas. Our results show that in seven out of eight replicas, β2GPI-DV interacts with the membrane via its flexible loop (311-317) which insert into the membrane below the PO4 level. interestingly, in one replica we observe insertion of the loop carrying three lysine residues, a mode which closely corresponds to an alternative binding mode reported previously. The orientation of membrane-bound states was characterized by measuring the tilt angle between the protein and the membrane. After insertion of the flexible loop, β2GPI-DV held an average tilt angle of 92 with respect to the membrane normal. This study establishes the significance of the flexible loop for membrane binding of β2GPI which could lead to a more rational approach in drug design for treatment of APS.

Isolation and Characterization of an Acidic, Salt-Tolerant Endoglucanase Cel5A from a Bacterial Strain Martelella endophytica YC6887 Genome

A Martelella endophytica (M. endophytica) strain YC6887 was previously isolated from the roots of a halophyte, Rosa rugosa, which was sequenced and characterized. The genomic and proteomic analysis showed a carbohydrate-degrading enzyme, endoglucanase Cel5A which was further characterized. The protein analysis revealed that this endoglucanase belongs to glycosidic hydrolase family 5 (GH5) with catalytic domain. This gene encodes 349-residue polypeptide and shows closest similarity with cellulases of other Martelella species. The protein was purified to homogeneity and shown that it was a 39kDa protein. The purified recombinant Cel5A endoglucanase exhibited maximum activity at 50°C and pH 4.5. The enzyme was salt tolerant and retained more than 50% residual activity up to 15% NaCl. The homology model structure of Cel5A displayed that it is stable and compact protein structure consisting of eleven α-helical structures and eight β-sheets. According to the predicted ligand binding site after superimposition with Pseudomonas stutzeri endoglucanase Cel5A (PDB ID: 4LX4), it consisted of five amino acid Asn157, Tyr116, Glu158, Glu270 and Trp303 that may be the expected active site of Cel5A from YC6887. This presented that our strain M. endophytica YC6887 that produces cellulase partially degrade the insoluble polysaccharides into reducing sugars.

Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT.

The intra-erythrocyte stage of P.falciparum relies primarily on glycolysis to generate adenosine triphosphate (ATP) and the energy required to support growth and reproduction. Lactic acid, a metabolic byproduct of glycolysis, is potentially toxic as it lowers the pH inside the parasite. Plasmodium falciparum formate-nitrite transporter (PfFNT), a 34-kDa transmembrane protein, has been identified as a novel drug target as it exports lactate from inside the parasite to the surrounding parasitophorous vacuole within the erythrocyte cytosol. The structure and detailed molecular mechanism of this membrane protein are not yet available. Here we present structures of PfFNT in the absence and presence of the functional inhibitor MMV007839 at resolutions of 2.56Å and 2.78Å using single-particle cryo-electron microscopy. Genetic analysis and transport assay indicate that PfFNT is able to transfer lactate across the membrane. Combined, our data suggest a stepwise displacement mechanism for substrate transport. The PfFNT membrane protein is capable of picking up lactate ions from the parasite's cytosol, converting them to lactic acids and then exporting these acids into the extracellular space.

The Choroid Plexus Is an Alternative Source of Prolactin to the Rat Brain.

Among the more than 300 functions attributed to prolactin (PRL), this hormone has been associated with the induction of neurogenesis and differentiation of olfactory neurons especially during pregnancy, which are essential for maternal behavior. Despite the original hypothesis that PRL enters the central nervous system through a process mediated by PRL receptors (PRLR) at the choroid plexus (CP), recent data suggested that PRL transport into the brain is independent of its receptors. Based on transcriptomic data suggesting that PRL could be expressed in the CP, this work aimed to confirm PRL synthesis and secretion by CP epithelial cells (CPEC). The secretion of PRL and the distribution of PRLR in CPEC were further characterized using an in vitro model of the rat blood-cerebrospinal fluid barrier. RT-PCR analysis of PRL transcripts showed its presence in pregnant rat CP, in CPEC, and in the rat immortalized CP cell line, Z310. These observations were reinforced by immunocytochemistry staining of PRL in CPEC and Z310 cell cytoplasm. A 63-kDa immunoreactive PRL protein was detected by Western blot in CP protein extracts as well as in culture medium incubated with rat pituitary and samples of rat cerebrospinal fluid and serum. Positive immunocytochemistry staining of PRLR was present throughout the CPEC cytoplasm and in the apical and basal membrane of these cells. Altogether, our evidences suggest that CP is an alternative source of PRL to the brain, which might impact neurogenesis of olfactory neurons at the subventricular zone, given its proximity to the CP.