Identification of candida species in patients with vulvovaginitis presenting different clinical symptoms

Abstract: Background and Objective: Candidiasis is one of the most important and common agents of vulvovaginitis in women. Various clinical manifestations of candidiasis in patients may be associated with different species of Candida. The present study was designed to accurately determine Candida species isolates from patients using culture methods and molecular analysis. Materials and Methods: In this cross-sectional analytical study, 350 patients with suspected vulvovaginal diseases who manifested various clinical symptoms were entered. Vaginal specimens from the patients were collected. Direct microscopy, primary and specific cultures using SDA, CRA and CMA media, germ tube test, as well as DNA based techniques targeting ITS1-5S-ITS2 fragment, were used for diagnosis and identification of Candida species. Results: 165 out of 350 patients (47.14%) were positive with Candida species including 71.5% recurrent candidiasis and 28.5% acute candidiasis. Characterization of the isolates in the specific sub-cultures and by PCR-RFLP resulted in identification of six different species consisted of Candida albicans (60.6%), C. dubliniensis (3.6%), C. glabrata (23%), C. krusei (10.9%), C. parapsilosis (0.6%) and C. tropicalis (1.2%). In the group consisting of 100 patients with C. albicans, 44% and 56% presented severe and mild to moderate clinical vulvovaginitis, respectively. In patients with non albicans candidiasis, 61.5% showed severe and 38.5% showed mild to moderate vulvovaginitis, significantly different from those of the former group (P =0.028). The results indicated significant involvement of some risk factors (i.e. diabetes and antibiotic consumption) in clinically different vaginal infections (P<0.0001) Conclusion: The study showed a high prevalence of candida infection in patients with vulvovaginitis in Southeast of Iran, involving several species, mostly C. albicans and C. glabrata. Considering the increasing prevalence of non-albicans species among these patients, precise determination of causative Candida species with more reliable methods such as molecular techniques are recommended

Transient Expression of a Recombinant Monoclonal Antibody in HEK293T Cells

Background: Monoclonal antibodies (mAbs) are considered the most important and financially successful category of the biopharmaceuticals. Extensive optimization of the expression vector, host system and culture parameters are required for the successful production of active monoclonal antibodies in mammalian cells. In this regards, transient expression enables rapid and cost-effective production of recombinant proteins for initial characterization. Methods: In the present study, an internal ribosome entry site (IRES) based bicistronic expression system has been evaluated for the transient expression of an anti-CD52 monoclonal antibody in mammalian cells. The IRES based bicistronic vector was generated through sequential cloning of the Light chain (LC), IRES, and Heavy chain (HC) in an intermediate vector and transfer of the resulting fragment to the expression vector. Transfection of the HEK293T cells was performed and antibody expression was analyzed in cell culture supernatant. Results: Restriction enzyme analysis indicated successful cloning of the antibody coding unit in the expression vector. Analysis of EGFP expression indicated successful transfection of the HEK293T cells. Production levels of 220 μg/L of antibody were achieved in HEK293T cells during three days of culture. Conclusion: Our results show the convenience and efficiency of the bicistronic expression system for transient expression of the whole monoclonal antibodies in mammalian cells

Evaluation of dot immunogold filtration assay (DIGFA) by recombinant protein EPC1 for anti- echinococcus granulosus IGG antibody

Background: Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method. Methods: Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA. Results: Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA. Conclusion: DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis

The CRISPR growth spurt: from bench to clinic on versatile small RNAs

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) in association with CRISPR associated protein (Cas) is an adaptive immune system, playing a pivotal role in the defense of bacteria and archaea. Ease of handling and cost effectiveness make CRISPR-Cas system an ideal programmable nuclease tool. Recent advances in understanding the CRISPR-Cas system have tremendously improved its efficiency. For instance, it is possible to recapitulate the chronicle CRISPR-Cas from its infancy and inaugurate a developed version by generating novel variants of Cas proteins, subduing off-target effects and optimization of innovative strategies. In summary, CRISPR-Cas system could be employed in a number of applications including providing model systems, rectification of detrimental mutations, and antiviral therapies

Humanizing glycosylation pathways in eukaryotic expression systems

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system

Humanizing glycosylation pathways in eukaryotic expression systems

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system

Expression of the Mouse HSP27 Chaperone in CHO-K1 Cells for the Enhancement of Viable Cell Density in Batch Culture: Mouse HSP27-expressing CHO cells

Chinese hamster ovary (CHO) cells are extremely vulnerable to cell viability loss in culture despite the availability of different nutrients supplementation strategies. As a result, extending the culture lifetime can profoundly increase recombinant protein expression. Overexpression of HSP27 and its anti-apoptotic effects have been shown in human cell lines in previous studies. In the current study, mouse HSP27 (mHSP27) was cloned in pcDNA 3.1 hygro expression vector and was expressed in CHO-K1 cells to assess its impacts on cell viability and growth. Expression of mHSP27 in CHO-K1 cells was confirmed using RT-PCR. A 3-fold enhancement in peak viable cell density of mHSP27 transfected clones was observed, and culture viability loss was delayed by 2 days compared to un-transfected cells. In future studies, the resulting mHSP27 CHO-K1 cells could be employed as a novel host system for the transient and stable expression of therapeutic recombinant proteins. HIGHLIGHTS Cell engineering is an effective strategy to cope with apoptosis in CHO cells. HSP27 is involved in mammalian cell apoptosis. Expression of the mouse HSP27 increased the viability and cell density of CHO-K1 cells

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial-Dependent Apoptosis in GBM Cells, Which Is Regulated by Autophagy

Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have had more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol-lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple-combination therapy of TMZ, Simvastatin (Simva), and acetylshikonin, and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species, mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that a TMZ/Simva/ASH combination therapy induced significantly more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to the partial inhibition of autophagy flux (accumulation of LC3β-II and a decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux, as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for a more effective treatment of GBM, but further investigations are required for a better identification of the mechanisms