Engineered ClearColi™-derived outer membrane vesicles as functional carriers for development of HIV-1 therapeutic vaccine candidate.

Bacteria-derived outer membrane vesicles (OMVs) can be engineered to incorporate foreign antigens. This study explored the potential of ClearColi™-derived OMVs as a natural adjuvant and a carrier (recombinant OMVs or rOMVs) for development of an innovative therapeutic vaccine candidate harboring HIV-1 Nef and Nef-Tat antigens. Herein, the rOMVs containing CytolysinA (ClyA)-Nef and ClyA-Nef-Tat fusion proteins were isolated from ClearColi™ strain. The presence of Nef and Nef-Tat proteins on their surface (rOMVNef and rOMVNef-Tat) was confirmed by western blotting after proteinase K treatment. Immune responses induced by Nef and Nef-Tat proteins emulsified with Montanide® ISA720 or mixed with OMVs, and also rOMVNef and rOMVNef-Tat were investigated in BALB/c mice. Additionally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was assessed for the secretion of cytokines in vitro. Our findings showed that the rOMVs as an antigen carrier (rOMVNef and rOMVNef-Tat) induced higher levels of IgG2a, IFN-γ and granzyme B compared to OMVs as an adjuvant (Nef + OMV and Nef-Tat + OMV), and also Montanide® ISA720 (Nef + Montanide and Nef-Tat + Montanide). Moreover, IFN-γ level in splenocytes isolated from mice immunized with rOMVNef-Tat was higher than other regimens after exposure to SCR virions. Generally, ClearColi™-derived rOMVs can serve as potent carriers for developing effective vaccines against HIV-1 infection.

Recent advances in various bio-applications of bacteria-derived outer membrane vesicles.

Outer membrane vesicles (OMVs) are spherical nanoparticles released from gram-negative bacteria. OMVs were originally classified into native 'nOMVs' (produced naturally from budding of bacteria) and non-native (produced by mechanical means). nOMVs and detergent (dOMVs) are isolated from cell supernatant without any detergent cell disruption techniques and through detergent extraction, respectively. Growth stages and conditions e.g. different stress factors, including temperature, nutrition deficiency, and exposure to hazardous chemical agents can affect the yield of OMVs production and OMVs content. Because of the presence of bacterial antigens, pathogen-associated molecular patterns (PAMPs), various proteins and the vesicle structure, OMVs have been developed in many biomedical applications. OMVs due to their size can be phagocytized by APCs, enter lymph vessels, transport antigens efficiently, and induce both T and B cells immune responses. Non-engineered OMVs have been frequently used as vaccines against different bacterial and viral infections, and various cancers. OMVs can also be used in combination with different antigens as an attractive vaccine adjuvant. Indeed, foreign antigens from target microorganisms can be trapped in the lumen of nonpathogenic vesicles or can be displayed on the surface through bacterial membrane protein to increase the immunogenicity of the antigens. In this review, different factors affecting OMV production including time of cultivation, growth media, stress conditions and genetic manipulations to enhance vesiculation will be described. Furthermore, recent advances in various biological applications of OMVs such as vaccine, drug delivery, cancer therapy, and enzyme carrier are discussed. Generally, the application of OMVs as vaccine carrier in three categories (i.e., non-engineered OMVs, OMVs as an adjuvant, recombinant OMVs (rOMVs)), as delivery system for small interfering RNA and therapeutic agents, and as enzymes carrier will be discussed.

A novel loss of function mutation in the PHD domain of the RAG2 gene, affecting zinc-binding affinity.

BACKGROUND: Severe combined immune deficiencies (SCIDs) are genetically heterogeneous disorders that lead to the absence or malfunction of adaptive immune cells, including T- and B-cells. Pathogenic variants in the RAG2 gene are associated with this disease. METHODS: A couple with consanguineous marriage from the Iranian-Azeri-Turkish ethnic group was referred to the genetic lab. Two children of this family died due to SCID disease with symptoms of skin granulomas, lack of developed T- and B-cells, and intact NK cells. To infer their genotypes, DNA samples obtained from the parents were subjected to whole-exome sequencing (WES). RESULTS: WES data analysis revealed that both parents were carriers of a pathogenic variant, NC_000011.10 (NM_000536.4):c.1268G > C, in the RAG2 gene. This variant was absent in our cohort of 400 healthy individuals from the same ethnic group. To gain insight into the consequence of the variant on the protein function, further analysis was performed by applying bioinformatics tools. This study revealed that the replacement of cysteine with serine at the zinc-binding domain diminished the domain's affinity to zinc ion, resulting in the loss of the mutant protein's ability to bind to the recombination signal sequence (RSS). The formation of the RAG2-RSS complex is vital for T- and B-cell development. CONCLUSION: The identification of a novel pathogenic variant, c.1268G > C, revealed that this variant in the zinc-binding domain diminished the affinity of the zinc ion to the mutant protein and consequently led to the loss of its ability to bind to the RSS.

Frequency of Cytomegalovirus Viral Load in Iranian Human Immunodeficiency Virus-1-Infected Patients with CD4+ Counts <100 Cells/mm3.

OBJECTIVES: The aim of present work was to assess cytomegalovirus (CMV) viremia in Iranian human immunodeficiency virus (HIV)-1-infected patients with a CD4+ count <100 cells/mm3 and to explore whether CMV DNA loads correlate with CD4+ cell counts or associated retinitis. METHODS: This study was conducted at the AIDS research center in Iran on HIV-1-infected patients with CD4+ count <100 cells/mm3, antiretroviral therapy-naive, aged ≥18 years with no previous history of CMV end-organ disease (CMV-EOD). RESULTS: Thirty-nine of 82 patients (47.56%) had detectable CMV viral load ranging from 66 to 485,500 IU/mL. CMV viral load in patients with retinitis ranges from 352 to 2,720 IU/mL, and it was undetectable in 2 patients. No significant associations between CMV viremia and CD4+ cell count was found (p value = 0.31), whereas significant association of CMV viremia in HIV-infected patients with retinitis was found (p < 0.02). CONCLUSIONS: We estimated the frequency of CMV viral load infection in Iranian HIV-1-infected patients with a CD4+ cell count <100 mm3/mL in the largest national referral center for HIV-1 infection in Iran. Further research is required on the relevance of CMV viral load in diagnostic and prognostic value of CMV-EOD.

Effects of Delphinidin on Pathophysiological Signs of Nucleus Basalis of Meynert Lesioned Rats as Animal Model of Alzheimer Disease.

Alzheimer's disease (AD) is an advanced neurodegenerative disorder greatly accompanied by cognitive deficits, oxidative stress, inflammation, amyloid plaques deposition, and acetylcholinesterase (AChE) hyper-activation. Growing evidence suggests natural compounds with antioxidant and anti-inflammatory features improve pathophysiological signs of AD. The present study was designed to investigate the effects of Delphinidin (25, 50 mg/kg) as an anthocyanidin on spatial memory impairment and AD hallmarks such as hippocampal AChE activity, amyloid plaques deposition, oxidative stress and expression of amyloid precursor protein (APP), AChE, and amyloid beta (Aß) proteins in nucleus basalis of Meynert (NBM) lesioned rats as the most prevalent animal model of AD. Interestingly, Delphinidin-treated animals showed a significant decrease in escape latency and distance moved. Furthermore, in probe test, NBM lesioned rats treated with both doses of Delphinidin spent more time in the target quadrant zone in Morris water maze task. It could also interact with catalytic site of AChE enzyme and inhibits acetylcholine hydrolysis in in vitro and in vivo conditions. In addition, Delphinidin could scavenge additional produced reactive oxygen molecules dose dependently. Our immunoblotting analysis confirmed high dose of Delphinidin reduced AChE, APP and Aß contents in AD model. Staining of hippocampus tissue revealed that Delphinidin treatment decreased amyloid plaques formation in NBM lesion rats. It seems that Delphinidin is a plate-like molecule intercalated between ß-plated sheets related to Aß molecules and inhibited amyloid fibril formation. Altogether, Delphinidin and Delphinidin-rich fruits could be suggested as a therapeutic adjuvant in AD and other related cognitive disorders.

The impact of water molecules on binding affinity of the anti-diabetic thiazolidinediones for catalase: Kinetic and mechanistic approaches.

Water molecules play a vital role in efficient drug binding to its target. Thiazolidinediones (TZDs), a class of anti-diabetic drugs, are widely used for treatment of type 2 diabetes mellitus. In the present study, the possible contribution of water molecules to the binding of TZDs to catalase, a potential target in the liver, was investigated by different experimental and theoretical methods. These studies indicated that TZDs could significantly improve the catalase catalytic function with a significant contribution from water molecules. As a probe for the differential number of released water molecules during the catalase transition from E to E* states, the activity of TZDs-catalase complexes was demonstrated to be mainly dependent on water activity. However, free catalase decomposed the substrate more independently. In addition, the spectrofluorimetry studies showed that the binding of TZDs to catalase needed the release of water molecules from the enzyme's binding pocket. The thermodynamic studies indicated that the binding enthalpy and entropy of TZDs for catalase were decreased with lower water activity. The favorable process contributes to release of water molecules from the binding pocket through the formation of hydrophobic interactions between catalase and TZDs in an enthalpic manner. Molecular docking simulations confirmed that the depletion of water molecules from the binding cavity is essential for effective interactions between TZDs and catalase.

Evaluation of Acquired HIV Drug Resistance among People Living with HIV Who Have Taken Antiretroviral Therapy for 9-15 Months in 14 Triangular Clinics in Iran, 2015-2016.

AIMS: The aim of this study was to evaluate drug resistance patterns among Iranian people living with HIV who have taken antiretroviral therapy for 9-15 months. METHODS: A cross-sectional study was conducted between December 2015 and May 2016. Two hundred fifty-two blood samples were collected from all eligible HIV-infected patients at fourteen healthcare settings, located in major provinces in Iran. The samples were examined for presence of drug resistance strains and viral load level. Moreover, a phylogenetic tree, using neighbor joining, was constructed and HIV subtypes were determined. RESULTS: The most common subtypes were CRF35-AD (47.6%) and A1 (42.8%), followed by 45_CPX (4.8%) and C (4.8%). The resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), and protease inhibitors was reported as 19.2, 19.2, and 10.3%, respectively. M184I/V mutation was the most frequent (31.6%) mutation among NRTI-based regimens. Moreover, K103E/N was the most frequent (34.2%) NNRTI mutation. CONCLUSIONS: This is the first study to illuminate the emergence of the CPX genotype among Iranian patients. The drug resistance rate of NNRTIs was similar to that of NRTIs. By assessing drug resistance, it is possible to evaluate the efficacy of treatment and patient adherence to treatment.

Improving effects of Echium amoenum aqueous extract on rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder greatly accompanied by oxidative stress and acetylcholine reduction in synaptic cleft that leads to dementia. Previously approved there is correlation between nucleus basalis of Meynert (NBM) degeneration and loss of memory, learning ability and thought. The aim of this study was to investigate improving effects of Echium amoenum aqueous extract on memory deficient, pathophysiological and oxidative damages imposed by NBM lesion in rats as documented AD model. Results showed NBM destruction causes hash oxidative stress that possibly leads to neurodegeneration in hippocampus tissue. Orally administration of plant extract significantly reduced oxidative stress by reactive molecules scavenging that resulted to decrease lipid peroxidation also. Plant extract treatment inhibited acetylcholine esterase enzyme (more than 5 folds) in hippocampus tissue related to NBM lesioned rats. Histological studies approved NBM lesion causes harsh neurodegeneration in hippocampus tissue possibly by acetylcholine reduction that was compensated by plant extract protective effects. Interestingly improving effects of plant in molecular level causes improved spatial learning ability in Morris water maze test. By considering pathophysiological and molecular similarities between AD and NBM lesion model, E. amoenum could be used as a therapeutic adjuvant in patients suffering from Alzheimer or similar cognitive disorders.

Chrysin as an Anti-Cancer Agent Exerts Selective Toxicity by Directly Inhibiting Mitochondrial Complex II and V in CLL B-lymphocytes.

We investigated the effect of chrysin on isolated normal and chronic lymphocytic leukemia (CLL) B-lymphocytes and their isolated mitochondria. We report that a selective and significant increase in cytotoxicity, intracellular reactive oxygen species, mitochondrial membrane potential collapse, ADP/ATP ratio, caspase 3 activation and finally apoptosis in chrysin-treated CLL B- lymphocytes. Also we determined that chrysin selectively inhibits complex II and ATPases in cancerous mitochondria. In this study we proved that the ability of chrysin to promote apoptosis in CLL B-lymphocytes performed by selectively targeting of mitochondria. Our findings may provide a potential therapeutic approach for using chrysin to target mitochondria in CLL B-lymphocytes.

Selective Anticancer Activity of Acacetin Against Chronic Lymphocytic Leukemia Using Both In Vivo and In Vitro Methods: Key Role of Oxidative Stress and Cancerous Mitochondria.

The present study investigates the in vitro and in vivo effect of acacetin (4'-methoxy-5,7-dihydroxyflavone) on chronic lymphocytic leukemia (CLL) B-lymphocytes and mitochondria. CLL B-lymphocytes and healthy B-lymphocytes were obtained from CLL patients and healthy donors, respectively. Mitochondria were isolated from B-lymphocytes of both groups. Xenografts in severe combined immune deficient mice were used to examine the toxicity and anti CLL activity of acacetin. We evaluated and compared the mechanism of action of acacetin on CLL and healthy B-lymphocytes and their mitochondria. We have found that acacetin (10 µM) can selectively induce apoptosis on CLL B-lymphocyte (25% at 24 h) by directly targeting mitochondria, through increased reactive oxygen species (ROS) formation, MMP collapse, MPT, release of cytochrome c, caspase 3 activation, and finally apoptosis, while sparing normal healthy B-lymphocytes unaffected at similar concentrations. Besides, oral administration of acacetin showed a potent in vivo anticancer activity in CLL xenograft mouse models. Our in vivo findings indicate that acacetin accumulates and kills CLL B-lymphocyte in a rather selective way through targeting cancerous mitochondria and ROS formation, which ends in CLL therapy. Finally, we can recommend acacetin as a promising compound for further drug development assays for the CLL treatment.

HIV Drug Resistance and Phylogeny Profile in Naïve and Antiretroviral-Experienced Patients in Tehran, Iran.

BACKGROUND: Increasing the accessibility of antiretroviral therapy (ART) has caused the emergence of drug resistance in patients receiving ART and in naïve patients. The aim of this study was to evaluate HIV subtype and drug resistance between naïve patients and ART-experienced patients. METHODS: Blood samples were collected from 78 antiretroviral and naïve HIV-1 patients; antiretroviral-resistant mutations and subtyping were then determined by sequencing pol regions. RESULTS: Phylogenetic analysis revealed that 96.1% of sequences belong to the CRF35-AD subtype. Transmitted drug resistance was determined in 14% of drug-naïve patients and 40% of ART-experienced patients. CONCLUSION: The findings of this study illustrated the importance of resistance testing before and during ART treatment. This study can be used to set up a best medicine strategy in Iranian guidelines.

Antioxidant effects of alfalfa can improve iron oxide nanoparticle damage: Invivo and invitro studies.

Medicago sativa Linn. or alfalfa (Leguminosae) has been used traditionally as an effective cure for CNS, heart and metabolic disorders and digestive aids. Alfalfa is a resistant plant against stress due to small antioxidant molecules and enzymes. Our previous work demonstrated that 100 µg/ml of 50 nm size Fe2O3-NP causes harsh oxidative stress in HepG2 cells and 100 mg/kg of same nanoparticle causes extreme damage in rat's liver. Therefore it can be used as a useful model for invivo and invitro studies of oxidative stress. This study assessed the effects of two concentration of alfalfa on the mentioned invivo and invitro damage. Our results showed alfalfa reduced reactive oxygen species (ROS) production and enhanced reduced glutathione (GSH) that cause reduction of DNA fragmentation and prevent apoptosis pathway so improve viability of the cells. Results also showed alfalfa decreased hepatic enzymes penetrating and lipid peroxidation in rat's liver. Note that Fe2O3-NP potentially has widespread biological application but its usage is limited due to bio incompatibility. A suitable antioxidant compound that reduce nanoparticle side effects can be used as an effective adjuvant with iron oxide nanoparticle (and may be other nanomaterials) in biological applications.

Production of alkaline protease by Aspergillus niger in a new combinational paper waste culture medium.

Enzymes derived from microbial sources have gained increasing popularity in industrial applications over the past decades. Despite the high production cost, alkaline proteases have wide applications in industries such as tanneries, food production, and detergents. In recent years, there has been a shift towards utilizing natural carbon sources for cultivating microorganisms and extracting proteases in order to reduce production costs. This study aimed to investigate the biochemical and kinetic properties of protease enzymes obtained from Aspergillus niger cultivated in a paper waste medium and compare with the enzyme produced in a basal medium. Glucose is a more favorable carbon source compared to cellulose, so paper waste was pretreated with cellulose-degrading bacteria to convert cellulose into smaller carbohydrates. After the growth of A. niger in basal and combinational media, the enzymatic properties were compared between the extracted enzymes by using casein as substrate. The results demonstrated that A. niger could produce protease enzymes in the paper waste medium similar to the basal medium with more than 5-fold cost saving. The specific activity of the enzymes isolated from the basal and paper waste media was calculated to be 184.95 ± 10.56 U ml-1 and 169.88 ± 11.05 U ml-1, respectively. Carbon sources did not affect the optimum pH and temperature of the protease enzyme, which were found to be 8 and 37 °C, respectively. This study provides valuable insights into the production of alkaline protease from A. niger using a combinational medium (paper waste pretreated by cellulose-degrading bacteria), offering a cost-effective approach for industrial applications.

Physio-biochemical and DNA methylation analysis of the defense response network of wheat to drought stress.

In the present work, physio-biochemical and DNA methylation analysis were conducted in wheat (Triticum aestivum L.) cultivars "Bolani" (drought-tolerant) and "Sistan" (drought-sensitive) during drought treatments: well-watered (at 90% field capacity (FC)), mild stress (at 50% FC, and severe stress (at 25% FC). During severe stress, O2•- and H2O2 content in cultivar Sistan showed significant increase (by 1.3 and 2.5-fold, respectively) relative to cultivar Bolani. In Bolani, the increased levels of radical scavenging activity (by 32%), glycine betaine (GB) (by 11.44%), proline (4-fold), abscisic acid (by 63.76%), and more stability of relative water content (RWC) (2-fold) were observed against drought-induced oxidative stress. Methylation level significantly decreased from 70.26% to 60.64% in Bolani and from 69.06% to 59.85% in Sistan during stress, and higher decreased tendency was related to CG and CHG in Bolani but CG in Sistan under severe stress. Methylation patterns showed that the highest polymorphism in Bolani was mainly as CG. As the intensity of stress increased, the enhanced physio-biochemical responses of Bolani cultivar were accompanied by a more decrease in the number of unchanged bands. According to heat map analysis, the highest difference (84.38%) in methylation patterns was observed between control and severe stress. Multivariate analysis using principal component analysis (PCA) showed a cultivar-specific methylation during stress and that methylation changes between cultivars are much higher than that of within a cultivar. Higher methylation to demethylation in Bolani (30.06 vs. 22.12%) compared to that of cultivar Sistan (23.21 vs. 30.15%) indicated more demethylation did not induce tolerance responses in Sistan. Sequencing differentially methylated fragments along with qRT-PCR analysis showed the efficient role of various DNA fragments, including demethylated fragments such as phosphoenol pyruvate carboxylase (PEPC), beta-glucosidase (BGlu), glycosyltransferase (GT), glutathione S-transferase (GST) and lysine demethylase (LSD) genes and methylated fragments like ubiquitin E2 enzyme genes in the development of drought tolerance. These results suggested the specific roles of DNA methylation in development of drought tolerance in wheat landrace.

The effects of Fe2O3 nanoparticles on catalytic function of human acetylcholinesterase: size and concentration role.

Introduction: Fe2O3 NPs can enter cells quickly, pass through the blood-brain barrier and interact with macromolecules. These materials are widely used in different fields, so their risk assessment is among the most critical issues. Acetylcholinesterase (AChE) is a cholinergic enzyme in central and peripheral nervous systems. Methods: In this work, the possible effects of Fe2O3 NPs on the structure and catalytic activity of AChE were investigated using circular dichroism (CD), surface plasmon resonance (SPR), and fluorescence spectroscopies. Results: The outcomes demonstrated that 5 nm Fe2O3 NPs inhibit AChE activity through mixed mechanism. While 50 nm Fe2O3 NPs caused an enhancement in the catalytic activity up to 60 nM. However, higher concentrations of Fe2O3 NPs (above 60 nM) hindered the enzyme activity via mixed mechanism. Fluorescence analysis showed that NPs can quench the fluorescence intensity of AChE that refer to conformational changes. Furthermore, CD results showed that Fe2O3 NPs can reduce the α-helix and ß-sheet contents of the enzyme and decrease the stability of AChE. Also, the SPR data analysis showed that the affinity between AChE and Fe2O3 NPs decreased with rising temperature. After treatment with Fe2O3 NPs, the catalytic activity of AChE was assessed in HepG2 cell lines, and the results confirmed the inhibitory effects of Fe2O3 NPs on AChE activity in vivo. Conclusion: These findings provide helpful information about the impact of Fe2O3 NPs on the structure and function of AChE and could offer new insights into the risk assessment of the medical application of nanoparticles.

The link between serum neuregulin-1 and atherogenic index in type 2 diabetes mellitus.

BACKGROUND: Neuregulin-1(NRG-1) is a protein that belongs to the group of epidermal growth factors. It plays vital roles in anti-fibrotic effects on the myocardium. The current paper explores the role of NRG-1 in type 2 diabetes mellitus (T2DM) and its relation to atherogenic index as a factor for increasing cardiovascular disease(CVD) risk. MATERIAL AND METHODS: In this study, 79 diabetes mellitus patients are independent insulin. These patients consisted of 53 females and 26 males their age were ranged 40-67 years. They were divided into two groups depending on the atherogenic index of plasma (AIP). Group I including48 diabetic patient with high risk of CVD and group II including 31 diabetic patients without risk CVD. Forty healthy individuals were included as control. RESULT: When compared to the control group, the serum levels of NRG-1 were significantly lower (p = 0.01). Additionally, group I had a much lower NRG-1level than group II. The results of multiple stepwise regression showed that the only independent predictor for NRG-1 level prediction was AIP (ß = - 0.600, P = 0.040). When comparing the diabetic patients with high risk factors for CVD to the healthy subject group, the AUC was outstanding (AUC = 0.889, P = 0.001) and had a high diagnosis. CONCLUSIONS: We proved low NRG-1 levels in diabetic patients and the association of highest NRG-1 amounts to a better AIP. Moreover, the measurement of NRG-1 levels could be beneficial as laboratory markers to monitor for increasing CVD risk in type 2 diabetes mellitus.

Gastrointestinal cancer resistance to treatment: the role of microbiota.

The most common illnesses that adversely influence human health globally are gastrointestinal malignancies. The prevalence of gastrointestinal cancers (GICs) is relatively high, and the majority of patients receive ineffective care since they are discovered at an advanced stage of the disease. A major component of the human body is thought to be the microbiota of the gastrointestinal tract and the genes that make up the microbiome. The gut microbiota includes more than 3000 diverse species and billions of microbes. Each of them has benefits and drawbacks and been demonstrated to alter anticancer medication efficacy. Treatment of GIC with the help of the gut bacteria is effective while changes in the gut microbiome which is linked to resistance immunotherapy or chemotherapy. Despite significant studies and findings in this field, more research on the interactions between microbiota and response to treatment in GIC are needed to help researchers provide more effective therapeutic strategies with fewer treatment complication. In this review, we examine the effect of the human microbiota on anti-cancer management, including chemotherapy, immunotherapy, and radiotherapy.

Investigation of amyloid­ß peptide production and clearance pathways in different stages of Alzheimer's disease.

Alzheimer's disease (AD) is an age­related, progressive decline in cognitive ability. Accumulation and deposition of amyloid­ß (Aß) is still the best­known cause of AD that worsens over time. It is unclear whether the increase in Aß production or the inefficiency of the degradation system causes the accumulation of ß­fibrils during AD development. This research investigated Aß­producing and clearance pathways in different stages of AD. For this purpose, patients were categorized into four experimental groups: patients with mild cognitive impairment, patients with moderate cognitive decline, patients with very severe cognitive decline, and healthy patients as control. Levels of Aß­40, soluble amyloid precursor protein beta (sAPPß), matrix metalloproteinase­9 (MMP­9), matrix metalloproteinase­3 (MMP­3), neprilysin (NEP), angiotensin­converting enzyme (ACE), and insulin­degrading enzyme (IDE) were determined by ELISA kits and immunoblotting in serum samples. According to the results, the levels of Aß­40 and sAPPß increased in AD patients from an early stage, and levels were maintained in progressive AD stages. MMP­9 also increased in the early stage, but its content decreased with disease development. MMP­3 was significantly higher in the three stages of AD compared to the control patients. However, IDE, NEP, and ACE enzymes as clearing systems decreased in all studied AD samples, with their reductions more remarkable in the middle and late stages. The results showed that multiple Aß­degrading enzymes such as NEP and IDE in AD patients decline as AD progresses, while Aß­40 and sAPPß increased from the early stage of the disease. Therefore, it could be concluded that detection of the dementia phase is a critical step for therapeutic strategies.

Heterologous DNA Prime/Protein Boost Immunization Targeting Nef-Tat Fusion Antigen Induces Potent T-cell Activity and in vitro Anti-SCR HIV-1 Effects.

BACKGROUND: Heterologous combinations in vaccine design are an effective approach to promote T cell activity and antiviral effects. The goal of this study was to compare the homologous and heterologous regimens targeting the Nef-Tat fusion antigen to develop a human immunodeficiency virus-1 (HIV-1) therapeutic vaccine candidate. METHODS: At first, the DNA and protein constructs harboring HIV-1 Nef and the first exon of Tat as linked form (pcDNA-nef-tat and Nef-Tat protein) were prepared in large scale and high purity. The generation of the Nef-Tat protein was performed in the E. coli expression system using an IPTG inducer. Then, we evaluated and compared immune responses of homologous DNA prime/ DNA boost, homologous protein prime/ protein boost, and heterologous DNA prime/protein boost regimens in BALB/c mice. Finally, the ability of mice splenocytes to secret cytokines after exposure to single-cycle replicable (SCR) HIV-1 was compared between immunized and control groups in vitro. RESULTS: The nef-tat gene was successfully subcloned in eukaryotic pcDNA3.1 (-) and prokaryotic pET-24a (+) expression vectors. The recombinant Nef-Tat protein was generated in the E. coli Rosetta strain under optimized conditions as a clear band of ~ 35 kDa detected on SDS-PAGE. Moreover, transfection of pcDNA-nef-tat into HEK-293T cells was successfully performed using Lipofectamine 2000, as confirmed by western blotting. The immunization studies showed that heterologous DNA prime/protein boost regimen could significantly elicit the highest levels of Ig- G2a, IFN-γ, and Granzyme B in mice as compared to homologous DNA/DNA and protein/protein regimens. Moreover, the secretion of IFN-γ was higher in DNA/protein regimens than in DNA/DNA and protein/protein regimens after exposure of mice splenocytes to SCR HIV-1 in vitro. CONCLUSION: The chimeric HIV-1 Nef-Tat antigen was highly immunogenic, especially when applied in a heterologous prime/ boost regimen. This regimen could direct immune response toward cellular immunity (Th1 and CTL activity) and increase IFN-γ secretion after virus exposure.

Investigation of Coronavirus disease 2019 virus in vaginal fluid and menses blood and the effect on menstrual cycle duration and sexual desire: A cross-sectional study.

Background: Coronavirus disease 2019 (COVID-19) was detected in the throat, urine, and feces but has little evidence documented of sexual transmission. Objective: Here, we aimed to diagnose the presence of COVID-19 in vaginal fluids and menses blood. Menstrual cycle duration and sexual desire were the other aims. Materials and Methods: In this cross-sectional study, 300 individuals with clinical approval of COVID-19 infection who were referred to the Alzahra hospital of Tabriz University of Medical Sciences, Tabriz, Iran were divided into mild (n = 178, partial pressure of oxygen ≥ 91) and severe (n = 122, partial pressure of oxygen ≤ 91) groups, also based on clinical signs and hospitalization, from January to May 2021. Demographic characteristics, menstruation, and sexual desire of individuals were recorded in the questionnaire blood sampling was done on days 2-4 for menses, and vaginal fluid after menses for polymerase chain reaction by using a Dacron tip swab. Results: Participants were studied in the mild (mean age: 43.32 ± 7.41) and severe (mean age: 47.15 ± 6.9) groups. COVID-19 infection resulted in shortening the menstrual cycle duration in the severe group (30.15 ± 2.9 vs. 25.12 ± 2.1 days, p = 0.01). Polymerase chain reaction test for vaginal fluid and menses blood was negative for all cases. Sexual desire declined in both groups, significantly. Conclusion: This virus was not present in the menses blood and vaginal fluid of women with COVID-19 infection, which proposed a low risk of virus transmission via vaginal tracts. Severe COVID-19 infection may affect the menstrual duration.