Polymer-based antibody mimetics (iBodies) target human PD-L1 and function as a potent immune checkpoint blocker.

Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.

Photocatalytic activity of visible-light-driven L-Proline-TiO2/BiOBr nanostructured materials for dyes degradation: The role of generated reactive species.

L-Proline (2%)-TiO2/BiOBr (30%) nanocomposite was synthesized to obtain high photocatalytic performance in the visible light region and infrared radiation(IR) for methylene blue (MB) and congo red (CR) removal from the contaminated wastewater. L-Proline (2%)-TiO2/BiOBr (30%) photocatalyst with strong absorption near IR wavelength and high charge separation ability was fabricated for the first time. X-ray diffraction (XRD), Fourier transform infrared (FTIR), field-emission scanning electron microscope (FESEM)/Energy Dispersive X-ray (EDX), UV-Vis diffuse reflectance spectrum (DRS), photoluminescence (PL) and Brunauer-Emmett-Teller (BET) characterization techniques show that the visible driven nanocomposite was successfully synthesized. According to the UV-DRS analysis, the estimated band gaps for the L-proline (2%)-TiO2 and L-Proline (2%)-TiO2/BiOBr (30%) nanostructures were respectively 2.3 eV and 2.1 eV.The nanoparticles exhibited enhanced photocatalytic activity (93-100%) and high mineralization efficiency (71-89% TOC removal) for both the dyes. The best photocatalytic activity was achieved by adding 2 wt% of L-Proline and 30 wt% of BiOBr into TiO2 sol. Response surface methodology (RSM) was employed to find significant parameters and their optimum values for maximum degradation, which show pH, dye concentration, irradiation time, and catalyst dosage for both the dyes are significant. The best photocatalytic degradation efficiency was achieved at the optimum conditions of pH = 7.7, catalyst dosage = 0.71 g/L, irradiation time = 142 and dye concentration = 11 mg/L for MB. Scavenger study showed that •OH radicals are responsible for the degradation process.

Behçet's disease: An immunogenetic perspective.

Behçet's disease (BD) is a chronic and rare multisystemic disorder defined by autoimmunity and inflammatory characteristics, manifested by ocular lesions, recurrent genital and oral ulcers, skin symptoms and arthritis as well as neurological, intestinal, and vascular involvement. Despite the unknown cause of BD, there is some strong documentation for immunological, genetic, environmental, and infectious factors playing a role in the pathogenesis of BD. While the nature of the genetic variants remains unidentified, many genetic risk factors are considered to contribute to BD susceptibility. Along with human leukocyte antigen gene encoding B*51 (HLA-B*51) and areas including the major histocompatibility complex class I, genome-wide association studies have recognized numerous other BD susceptibility genes including those encoding interleukin (IL)-10, IL-12 receptor ß 2 (IL-12RB2), IL-23 receptor (IL-23R), C-C chemokine receptor 1 gene, signal transducer and activator of transcription 4 (STAT4), endoplasmic reticulum aminopeptidase (ERAP1), and genes encoding killer cell lectin-like receptor family members (KLRC4-KLRK1). It is believed that BD could be considered as a disorder lying in between autoimmune and autoinflammatory syndromes. The positive responses to classical immunosuppressive agents like azathioprine and cyclosporine and involvement of autoantigens in the initiation of the disorder are the main BD features that reflect the autoimmune nature of the disorder. In this review, we address recent findings on the role of common cytokines, antibodies and immunogenetic factors in BD.

Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach.

EndoglucanaseII (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 1.3 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant EndoglucanaseII may be increased the flexibility of protein, to form a suitable E-S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 °C were increased 2.4 and 2.34 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications.

An Interleukin-6 Single Nucleotide Polymorphism and Susceptibility to Prostate Adenocarcinoma and Bone Metastasis in an Iranian Population

Objective: Interleukin-6 (IL-6) is an inflammatory cytokine shown to be a strong factor for growth, proliferation and metastasis with many malignancies. The promoter single nucleotide polymorphism (SNPs) -174G>C (rs1800795) can alter the transcriptional pattern of this gene. The present study was aimed at assessing effects of the IL-6 (rs1800795) SNP on risk of benign prostate hyperplasia (BPH) and prostatic adenocarcinoma (PCa). Methods: The project was performed on 112 men with PCa, 118 with BPH and 250 healthy controls. After DNA extraction, genotyping of IL-6 (rs1800795) was performed using PCR TaqMan Allelic Discrimination (ABI MGB). Results: The G allele frequency for rs1800795 of the IL-6 gene was 74.1%, 68.6% and 67% in PCa patients, BPH patients and healthy men, respectively. PCa and control groups showed significant differences (P =0.030, OR = 1.73, 95% CI: 1.05-2.21). The GG genotype was more frequent in the PCa group, whereas the GC genotype was more common in the BPH in comparison to other groups. Conclusion: The current study identified IL-6 -174G>C (rs1800795) as a significant predictor of susceptibility for prostate cancer and bone metastasis in a northwest Iranian population.

PD-1 and cancer: molecular mechanisms and polymorphisms.

The programmed cell death protein 1 (PD-1) is expressed by activated T cells that act as an immunoregulatory molecule, and are responsible for the negative regulation of T cell activation and peripheral tolerance. The PD-1 gene also encodes an inhibitory cell surface receptor involved in the regulation of T cell functions during immune responses/tolerance. Beyond potent inhibitory effects on T cells, PD-1 also has a role in regulating B cell and monocyte responses. An overexpression of PD-1 has been reported to contribute to immune system avoidance in different cancers. In particular, PD-1 over-expression influences tumor-specific T cell immunity in a cancer microenvironment. Blocking the PD-1/PD-1 ligand (PD-L1) pathway could potentially augment endogenous antitumor responses. Along these lines, the use of PD-1/PD-L1 inhibitors has been applied in clinical trials against diverse forms of cancer. It was believed that antibodies targeting PD-1/PD-L1 might synergize with other treatments that enhance endogenous antitumor immunity by blocking inhibitory receptor-ligand interactions. However, in all cases, the host genetic status (as well as that of the tumor) is likely to have an impact on the expected outcomes. Various investigations have evaluated the association between PD-1 polymorphisms and the risk of various types of cancer. Frequently studied PD-1 polymorphisms, PD-1.1 (rs36084323), PD-1.3 (rs11568821), PD-1.5 (rs2227981), PD-1.9 (rs2227982), and PD-1 rs7421861, and their associations in the risk of susceptibility to different types of cancer are mentioned in this review, as are studies highlighting the significance of conducting genetic association studies in different ethnic populations.

An interleukin 12 B single nucleotide polymorphism increases IL-12p40 production and is associated with increased disease susceptibility in patients with relapsing-remitting multiple sclerosis.

BACKGROUND: Through mounting genetic investigations, it has been established that IL12B and IL23R gene single nucleotide polymorphisms have significant associations with autoimmune diseases including inflammatory bowel disease, psoriasis, and ankylosing spondylitis. IL-12/IL-23 pathway plays a pivotal role in etiopathogenesis of multiple sclerosis (MS), suggested by studies both in patients and animal models. METHODS: In a case-control study, 145 MS patients and 200 healthy subjects were genotyped for polymorphisms in IL12B and IL23R genes using Real-Time PCR allelic discrimination approach. Additionally, quantitative analysis of mRNA expression of IL12B in Peripheral Blood Mononuclear Cells from patients and controls was conducted through Real-Time PCR using the TaqMan Gene Expression Assay. RESULTS: The rs6887695 single-nucleotide polymorphism (SNP) in IL12B gene showed an association with susceptibility to MS. GG genotype of this variation was more frequent in patients. mRNA expression of IL12B was upregulated in patients. Expression of IL12B mRNA in both MS patients collectively and those with GG genotype for rs6887695 SNP correlated negatively with onset age of MS patients. CONCLUSIONS: The GG genotype of rs6887695 SNP in IL12B gene plays a role in etiopathogenesis of MS.

Downregulation of Immunosuppressive Molecules, PD-1 and PD-L1 but not PD-L2, in the Patients with Multiple Sclerosis.

Programmed cell death-1 (PD-1) and its ligands, PD-L1 and PD-L2, have been regarded as important immune system regulatory molecules. The aberrant expression of the molecules has been related to several autoimmune disorders. This study is aimed to assess the mRNA expression level of PD-1, PD-L1, and PD-L2 molecules in the peripheral blood mononuclear mells (PBMCs) from multiple sclerosis (MS) patients. PBMCs were isolated from the whole blood of 50 MS and 50 healthy individuals. Total RNA content of the leukocytes was extracted. Then, cDNA was synthesized from the extracted RNA. Afterwards, quantitative analysis of PD-1, PD-L1 and PD-L2 was carried out through Real Time PCR using the TaqMan gene expression assays. Relative expression of PD-1 and PD-L1 in PBMCs from MS patients was significantly lower compared with the healthy control group (p=0.003 and 0.012, respectively). However, no significant difference was observed in the expression level of PD-L2 between patients and healthy individuals. Relative expression of PD-1 correlated with expanded disability status scale score (EDSS) of the patients (r=-0.763, p=0.008). Downregulation of the immunosuppressive molecules, PD-1 and PD-L1, may imply that over-activation of immune cells in multiple sclerosis occurs through signaling dysfunction of these molecules and PD-L2 plays no important role in this context.

PD-1/PD-L and autoimmunity: A growing relationship.

Programmed death 1 (PD-1) and its ligands, namely PD-L1 and PD-L2, are one of the key factors responsible for inhibitory T cell signaling, mediating the mechanisms of tolerance and providing immune homeostasis. Mounting evidence demonstrates that impaired PD-1:PD-L function plays an important role in a variety of autoimmune diseases such as Type 1 diabetes (T1D), encephalomyelitis, inflammatory bowel diseases (IBD), Rheumatoid Arthritis (RA), autoimmune hepatitis (AIH), Behcet's disease (BD), myasthenia gravis (MG), autoimmune uveitis (AU), Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), myocarditis, and ankylosing spondylitis (AS). By investigating the candidate genes, genome-wide association studies, and identification of single nucleotide polymorphisms (SNPs) in PD-1 gene in humans, it has been shown that there is a higher risk in relevant genetic associations with developing autoimmune diseases in certain ethnic groups. In this review we have tried to present a comprehensive role of PD-1:PD-L in all recently studied autoimmune diseases.

Cancer/Testis Antigens: Expression, Regulation, Tumor Invasion, and Use in Immunotherapy of Cancers.

UNLABELLED: Cancer/testis antigens (CTAs) are named based on their expression pattern that is restricted in a number of normal and abnormal tissues. Tumor cells frequently express antigens whose expression is typically restricted to germ cells. Their unique expression pattern is guaranteed by precise epigenetic regulatory mechanisms. Because of their tumor-limited, high immunogenicity, and biased expression, discovery of these molecules provides unprecedented opportunities for further research and clinical development in the field of cancer diagnosis and immunotherapy. Evolving evidence reveals that a number of CTAs stimulate epithelial mesenchymal transition (EMT) and generation of cancer stem-like cells, intensifying metastasis, invasion, and tumorigenesis. Based on these features, CTAs attract attention to be considered as ideal targets for developing several clinical trials, many of them concentrating on CTA vaccine therapy. According to recent practical clinical interest, more characterizations of CTA regulation are identified. CTA expression has been demonstrated in a variety of human cancer tissues, and some of them have been found to elicit humoral and/or cellular immune responses in cancer patients. CTAs are brilliant targets for anticancer drug discovery, targeted tumor therapy, and diagnostic biomarkers, furthermore, valued genes in the study of immunotherapy, promoting tumorigenesis, and malignant progression. This review outlines and categorizes our current understanding of the complex and biased process of CTAs mRNA and protein expression in cancer, and supplies the most recent information on their regulation and function. Besides, a concise synopsis of the major clinical trials involving CTAs, as therapeutic avenues, is discussed. ABBREVIATIONS: AIRE: autoimmune regulator; cAMP: cyclic adenosine 3',5'-cyclic monophosphate; CEA: carcinoembryonic antigen; CML: chronic myeloid leukemia; CREB: cyclicamp response element binding; CSCs: cancer stem cells; CTAs: cancer/testis antigens; CTL: cytotoxic T lymphocyte; DCs: dendritic cells; EMT: epithelial-mesenchymal transition; ERK: extracellular signal-regulated kinase; ESCC: esophageal squamous cell carcinoma; ETS: E26 transformation-specific; His: histidine; HLA: human leukocyte antigen; HNSCC: head and neck squamous cell carcinoma; IFN-γ: interferon-γ; IHC: Immunohistochemistry; IL-7: Interleukin7; MHC: major histocompatibility complex; MMP2: matrix metalloproteinase 2; mTECs: medullary thymus epithelial cells; MUC1: mucin 1; NSCLC: non-small cell lung cancer; PRAME: preferentially expressed antigen in melanoma; RDA: representational difference analysis; SEREX: serological analysis of cDNA expression; SSX: synovial sarcoma X chromosome; TAAs: tumor-associated antigens; TCR: T-cell receptor; TCGA: The Cancer Genome Atlas; TGF-ß: transforming growth factor-ß.

STAT4 single nucleotide gene polymorphisms and susceptibility to endometriosis-related infertility.

INTRODUCTION: Endometriosis is a multifactorial benign gynecologic disorder, characterized by the ectopic growth of misplaced endometrial cells with complex genetic inheritance and changing of some immune based factors and also shares some autoimmune characteristics. However, it is not clear yet that how and when these immunological factors affect the initiation or progression of the disease. It has been shown that STAT4 is a predisposing gene in the development of some autoimmune diseases. METHOD: The study group comprised 114 patients with endometriosis and 92 sex-, age-, and ethnicity-matched healthy controls of Iranian ancestry. Four SNPs (rs7574865, rs7601754, rs7582694 and rs11889341) were genotyped using the MGB TaqMan. RESULTS: A significant association in rs7582694 between C allele (P=0.002, OR=1.986, 95% CI: 1.262-3.126) and endometriosis was found in our study, while the G allele (P=0.002, OR=0.0503, 95% CI: 0.319-0.792) was significantly decreased in the patients population. The GC genotype (P=0.004, OR=2.234, 95% CI: 1.301-4.150) was also significantly overrepresented in the patients with endometriosis, while the frequency of GG genotype was significantly lower in the patient group, compared to the controls (P=0.007, OR=0.457, 95% CI: 0.256-0.813). CONCLUSIONS: Our results for the first time showed a significant association between rs7582694 alleles and genotypes and susceptibility to endometriosis in a population.

Enhanced resistance to Sclerotinia sclerotiorum in Brassica napus by co-expression of defensin and chimeric chitinase genes.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the major fungal diseases of Brassica napus L. To develop resistance against this fungal disease, the defensin gene from Raphanus sativus and chimeric chit42 from Trichoderma atroviride with a C-terminal fused chitin-binding domain from Serratia marcescens were co-expressed in canola via Agrobacterium-mediated transformation. Twenty transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR), with 4.8 % transformation efficiency. The chitinase activity of PCR-positive transgenic plants were measured in the presence of colloidal chitin, and five transgenic lines showing the highest chitinase activity were selected for checking the copy number of the transgenes through Southern blot hybridisation. Two plants carried a single copy of the transgenes, while the remainder carried either two or three copies of the transgenes. The antifungal activity of two transgenic lines that carried a single copy of the transgenes (T4 and T10) was studied by a radial diffusion assay. It was observed that the constitutive expression of these transgenes in the T4 and T10 transgenic lines suppressed the growth of S. sclerotiorum by 49 % and 47 %, respectively. The two transgenic lines were then let to self-pollinate to produce the T2 generation. Greenhouse bioassays were performed on the transgenic T2 young leaves by challenging with S. sclerotiorum and the results revealed that the expression of defensin and chimeric chitinase from a heterologous source in canola demonstrated enhanced resistance against sclerotinia stem rot disease.

Co-expression of chimeric chitinase and a polygalacturonase-inhibiting protein in transgenic canola (Brassica napus) confers enhanced resistance to Sclerotinia sclerotiorum.

OBJECTIVES: Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is one of the major fungal diseases of canola. To develop resistance against this fungal disease, the chit42 from Trichoderma atroviride with chitin-binding domain and polygalacturonase-inhibiting protein 2 (PG1P2) of Phaseolus vulgaris were co-expressed in canola via Agrobacterium-mediated transformation. RESULTS: Stable integration and expression of transgenes in T0 and T2 plants was confirmed by PCR, Southern blot and RT-PCR analyses. Chitinase activity and PGIP2 inhibition were detected by colorimetric and agarose diffusion assay in transgenic lines but not in untransformed plants. The crude proteins from single copy transformant leaves having high chitinase and PGIP2 activity (T16, T8 and T3), showed up to 44 % inhibition of S. sclerotiorum hyphal growth. The homozygous T2 plants, showing inheritance in Mendelian fashion (3:1), were further evaluated under greenhouse conditions for resistance to S. sclerotiorum. Intact plants contaminated with mycelia showed resistance through delayed onset of the disease and restricted size and expansion of lesions as compared to wild type plants. CONCLUSIONS: Combined expression of chimeric chit42 and pgip2 in Brassica napus L. provide subsequent protection against SSR disease and can be helpful in increasing the canola production in Iran.

Molecular Characterization of KRAS, BRAF, and EGFR Genes in Cases with Prostatic Adenocarcinoma; Reporting Bioinformatics Description and Recurrent Mutations.

BACKGROUND: Prostate cancer is one of the most common cancers which develops by mutations and/or other genetic alterations in specific genes. Regarding the previous studies in literature predominant mutations take place in KRAS, BRAF, and EGFR genes in special types of cancers. In this research, we attempt to identify the prevalence and significant role of the possible mutations in EGFR exons 18-21, KRAS codon 12, 13, and 61, and BRAF codon 600 mutations in tumoral tissue specimens from patients with prostatic adenocarcinoma. Furthermore, in this research, it has been attempted to investigate the molecular characteristics of these genes in terms of bioinformatic aspects. METHODS: A total of 35 prostatic adenocarcinoma fresh tissue samples, enriched in neoplastic cells, were obtained from the Cancer Institute of Iran. The presence of mutations at codons 12, 13 and 61 of KRAS, codon 600 of BRAF and EGFR exons 18-21 were determined by direct Sanger sequencing. To evaluate the molecular features, structure, and post-translation modification of those genes, a bioinformatics survey was performed using the SWISS-MODEL (http://swissmodel.expasy.org) and NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) Server. Also, using bioinformatics software, the phylogeny tree of the mutations was drawn. RESULTS: Mutations of codons 12 and 13 of KRAS were found in 2 of the 35 prostatic adenocarcinomas. Two cases carried homozygous mutations on exon 2 in codon 12 (G12V) and codon 13 (G13D). Also, no mutation was detected at BRAF codon 600 and EGFR exons 18-21 in any of the samples. CONCLUSIONS: Based on the group of patients with prostate adenocarcinoma, our research shows that the mutations in codons 12 and 13 of KRAS are the most common in prostate carcinomas. Noting these results and the molecular pathway of this gene, there is a possible more perceptible role for this gene in the pathogenesis of prostatic carcinoma. However, according to our finding, as in previous studies, the role of BRAF and EGFR gene mutations in prostate adenocarcinoma are less than in the KRAS gene and, therefore, we assume that these common mutations of the KRAS gene can be used as an early determining marker for early diagnosis of prostate adenocarcinoma. In the future, due to the complexity of etiological parameters in prostate cancer development, the case specific tumor molecular identification and treatment for each affected subject are urgently needed.

Selectable Marker Gene Removal and Expression of Transgene by Inducible Promoter Containing FFDD Cis-Acting elements in Transgenic Plants.

BACKGROUND: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression. OBJECTIVES: This study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter. MATERIALS AND METHODS: In pG-IPFFDD-creint-gusint construct, cre recombinase and selectable marker gene (nptII) cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and ß-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation. RESULTS: The results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay. CONCLUSIONS: The established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene.

Association between a PD-1 gene polymorphism and antisperm antibody-related infertility in Iranian men.

OBJECTIVE: Programmed cell death-1 (PD-1, Pdcd1), an immunoreceptor belonging to the CD28/CTLA-4 family negatively regulates antigen receptor signalling by recruiting protein tyrosine phosphatase, SHP-2 upon interacting with either of two ligands, PD-L1 or PD-L2. This study investigates PD-1 gene polymorphism in patients with antisperm antibody-related infertility METHODS: Genotyping was performed by polymerase chain reaction and restriction enzyme digestion (PCR-RFLP), this polymorphism was genotyped in 145 Iranian subjects (61 patients with antisperm antibody-related infertility and 84 healthy controls). RESULTS: Patients frequencies of the G/A genotype in comparison with healthy controls (38.2 % vs. 32.7 %, OR =1.21, P = 0.35) were not significantly different. However, G/G and A/A genotype frequencies between patients and healthy controls were significantly different (P = 0.042, P = 0.00001, respectively). Also, allele frequencies of this polymorphism were significantly different (P = 0.0012) in patients compared to healthy controls. CONCLUSION: According to these results, there is a correlation between PD-1 gene polymorphism and susceptibility to antisperm antibody-related infertility in our study group.

Characterization and high level expression of acidic endoglucanase in Pichia pastoris.

Bioconversion of cellulosic material into glucose needs cellulase enzymes. One of the most important organisms that produces cellulases is Trichoderma reesei, whose cellulose enzymes are probably the most widely used in the industry. However, these enzymes are not stable enough at high pH and temperatures. The optimized synthetic endoglucanase II gene with Pichia pastoris codon preferences was secretary expressed in P. pastoris. Recombinant enzyme characterization showed maximum activity at pH 4.8 and temperature 75 °C, and it demonstrated increasing thermal stability in high temperature. The enzyme maintained its activity in a wide pH range from 3.5 to 6.5. The optimization of fermentation medium was carried out in shaking flasks. Recombinant protein expression at optimum conditions (pH 7, temperature 25 °C, and 1 % methanol induction) for 72 h demonstrated 2,358.8 U/ml endoglucanase activity units. To our knowledge, this is the highest acidic thermophilic endoglucanase activity that is reported in crude intracellular medium in P. pastoris. We conclude that P. pastoris is an appropriate host for high-level expression of optimized endoglucanase gene with improved thermal stability.