Mosaicism in CRISPR/Cas9-mediated genome editing

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.     

A Chronic Autochthonous Fifth Clade Case of Candida auris Otomycosis in Iran

Candida auris, a multidrug-resistant nosocomial pathogen, has emerged globally with high morbidity and mortality among immunocompromised individuals and COVID19 hospitalized patients. Five major clades of C. auris have been previously described. The fifth clade is exclusively found in Iran where C. auris isolates are genetically distinct from other clades by?>?200,000 single-nucleotide polymorphisms. The origin of C. auris remains unclear, and limited clinical data are available at present regarding clade V infection or colonization. Herein, another case of otomycosis in Iran caused by an isolate of C. auris belonging to the fifth clade is reported. Genotyping revealed that the obtained C. auris isolate from Isfahan clustered with earlier clade V isolates from Babol, cities around 600 km separated, which indicates that C. auris clade V is established in Iran. C. auris is thought to exist more commonly in Iran, given that limited diagnostic capacity in the country has probably curbed the identification of more C. auris cases. Therefore, surveillance of the environment, patients and healthcare facilities in different geographical regions in Iran is urgently required.

     

A computational model of hemodynamic parameters in cortical capillary networks

The analysis of hemodynamic parameters and functional reactivity of cerebral capillaries is still controversial. To assess the hemodynamic parameters in the cortical capillary network, a generic model was created using 2D voronoi tessellation in which each edge represents a capillary segment. This method is capable of creating an appropriate generic model of cerebral capillary network relating to each part of the brain cortex because the geometric model is able to vary the capillary density. The modeling presented here is based on morphometric parameters extracted from physiological data of the human cortex. The pertinent hemodynamic parameters were obtained by numerical simulation based on effective blood viscosity as a function of hematocrit and microvessel diameter, phase separation and plasma skimming effects. The hemodynamic parameters of capillary networks with two different densities (consistent with the variation of the morphometric data in the human cortical capillary network) were analyzed. The results show pertinent hemodynamic parameters for each model. The heterogeneity (coefficient variation) and the mean value of hematocrits, flow rates and velocities of the both network models were specified. The distributions of blood flow throughout the both models seem to confirm the hypothesis in which all capillaries in a cortical network are recruited at rest (normal condition). The results also demonstrate a discrepancy of the network resistance between two models, which are derived from the difference in the number density of capillary segments between the models.     

Deep analysis of N-cadherin/ADH-1 interaction: a computational survey

Due to the considerable role of N-cadherin in cancer metastasis, tumor growth, and progression, inhibition of this protein has been highly regarded in recent years. Although ADH-1 has been known as an appropriate inhibitor of N-cadherin in clinical trials, its chemical nature and binding mode with N-cadherin have not been precisely specified yet. Accordingly, in this study, quantum mechanics calculations were used to investigate the chemical nature of ADH-1. These calculations clarify the molecular properties of ADH-1 and determine its reactive sites. Based on the results, the oxygen atoms are suitable for electrophilic reactivity, while the hydrogen atoms that are connected to nitrogen atoms are the favorite sites for nucleophilic reactivity. The higher electronegativity of the oxygen atoms makes them the most reactive portions in this molecule. Molecular docking and molecular dynamics (MD) simulation have also been applied to specify the binding mode of ADH-1 with N-cadherin and determine the important residues of N-cadherin involving in the interaction with ADH-1. Moreover, the verified model by MD simulation has been studied to extract the free energy value and find driving forces. These calculations and molecular electrostatic potential map of ADH-1 indicated that hydrophobic and electrostatic interactions are almost equally involved in the implantation of ADH-1 in the N-cadherin binding site. The presented results not only enable a closer examination of N-cadherin in complex with ADH-1 molecule, but also are very beneficial in designing new inhibitors for N-cadherin and can help to save time and cost in this field.     

Synthesis of a unique high-performance poly-horseradish peroxidase complex to enhance sensitivity of immunodetection systems

Because early detection is the first step in successful therapy, increasing the sensitivity of detection systems has always been considered as one of the major trends in development of these technologies. Therefore, we have fabricated a high-performance poly-horseradish peroxidase (HRP) complex and analyzed it in different formats of immunodetection systems. To construct this complex, dextran-aldehyde was prepared by oxidation of dextran in the presence of sodium periodate. Activated polymer was then coupled to lysine amino acids and accomplishment of the process was evaluated with trinitrobenzenesulfonic acid. Following conjugation of HRP to free amino groups of lysine, the stage's accuracy and the rate of conjugation were demonstrated by SDS-PAGE. Then, conjugation of poly-HRP complex to streptavidin by biotin was performed. The results of a series of experiments confirmed the complete synthesis of streptavidin-poly-HRP complex by this procedure. Finally, we compared our harvested complex with the golden standard complex available for ELISA and immunohistochemistry (IHC). The results showed the high efficiency of the synthesized complex. Consequently, this complex can be applicable in highly sensitive detection technologies. Conjugating this complex to any antibody by using biotin-streptavidin bridging and preparing poly-HRP-labeled antibodies will be a valuable multifold approach to increase the sensitivity of detection systems, which can be applicable in ELISA, immunocytochemistry, and IHC methods.     

Molecular perception of interactions between bis(7)tacrine and cystamine-tacrine dimer with cholinesterases as the promising proposed agents for the treatment of Alzheimer’s disease

The infamous chronic neurodegenerative disease, Alzheimer’s, that starts with short-term memory loss and eventually leads to gradual bodily function decline which has been attributed to the deficiency in brain neurotransmitters, acetylcholine, and butylcholine. As a matter of fact, design of compounds that can inhibit cholinesterases activities (acetylcholinesterase and butylcholinesterase) has been introduced as an efficient method to treat Alzheimer’s. Among proposed compounds, bis(7)tacrine (B7T) is recognized as a noteworthy suppressor for Alzheimer’s disease. Recently a new analog of B7T, cystamine-tacrine dimer is offered as an agent to detain Alzheimer’s complications, even better than the parent compound. In this study, classical molecular dynamic simulations have been employed to take a closer look into the modes of interactions between the mentioned ligands and both cholinesterase enzymes. According to our obtained results, the structural differences in the target enzymes active sites result in different modes of interactions and inhibition potencies of the ligands against both enzymes. The obtained information can help to investigate those favorable fragments in the studied ligands skeletons that have raised the potency of the analog in comparison with the parent compound to design more potent multi target ligands to heal Alzheimer’s disease.     

Adipose derived stem cells (ASCs) isolated from breast cancer tissue express IL-4, IL-10 and TGF-β1 and upregulate expression of regulatory molecules on T cells: do they protect breast cancer cells from the immune response?

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25^highFoxp3⁺ T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells.     

Effects of indoleamine 2, 3-dioxygenase (IDO) silencing on immunomodulatory function and cancer-promoting characteristic of adipose-derived mesenchymal stem cells (ASCs)

Indoleamine 2, 3-dioxygenase (IDO) catabolizes tryptophan, mediates immunomodulatory functions, and is released by stromal cells such as mesenchymal stem cells. The aims of this study were to investigate the effects of IDO silencing on immunosuppressive function of adipose-derived mesenchymal stem cells (ASCs), T cells phenotype, and the proliferation/migration of tumor cells. ASCs isolated from adipose tissues of healthy women were transfected with IDO-siRNA. Galectin-3, transforming growth factor-β1, hepatocyte growth factor, and interleukin-10 as immunomodulators were measured in ASCs using qRT-PCR. T cells phenotype, interferon-γ, and interleukin-17 expression were evaluated in peripheral blood lymphocytes (PBLs) cocultured with IDO silenced-ASCs by flow cytometry and qRT-PCR, respectively. Scratch assay was applied to assess the proliferation/migration of MDA-MB-231 cell line. Galectin-3 was upregulated (p ˂ 0.05) while hepatocyte growth factor was downregulated (p ˂ 0.05) in IDO-silenced ASCs compared to control groups. Regulatory T cells were inhibited in PBLs cocultured with IDO-silenced ASCs; also T helper2 was decreased in PBLs cocultured with IDO-silenced ASCs relative to the scramble group. IDO-silenced ASCs caused interferon-γ overexpression but interleukin-17 downregulation in PBLs. The proliferation/migration of MDA-MB-231 was suppressed after exposing to condition media of IDO-silenced ASCs compared with condition media of untransfected (p < 0.01) and scramble-transfected ASCs (p < 0.05). The results exhibited the weakened capacity of IDO-silenced ASCs for suppressing the immune cells and promoting the tumor cells' proliferation/migration. IDO suppression may be utilized as a strategy for cancer treatment. Simultaneous blocking of immunomodulators along with IDO inhibitors may show more effects on boosting the efficiency of immune-based cancer therapies.     

Computational approach to suggest a new multi-target-directed ligand as a potential medication for Alzheimer’s disease

Abstract

Acetylcholinesterase (AChE) enzyme and myeloid differentiation 2 protein (MD2) are two critical proteins involved in Alzheimer’s disease (AD). Since the nature of the active site of AChE and the binding pocket of MD2 are similar, some ligands can inhibit both of them appropriately. Oxidative stress has also been known as an important cause of AD. Designing an effective common inhibitor with antioxidant activity to inhibit AChE and MD2 proteins is the main goal of this work. In this regard, we used tacrine molecule with a high ligand efficiency (LE) and dehydrozingerone (DHZ) with anti-inflammatory, antioxidant and anti-Alzheimer activities. Some modifications on DHZ structure can increase its antioxidant activity. So, tacrine molecule was combined with modified DHZ to present a new multi-target-directed ligand (MTDL). The ability of the designed ligand to inhibit AChE and MD2 proteins was confirmed by molecular docking, molecular dynamics (MD) simulation, and binding-free energy calculations. Therefore, the designed ligand can target two proteins involved in AD. It can also act as a potent antioxidant. In general, three important causative agents of AD are targeted by the designed ligand. Moreover, the inhibition of MD2, as the main source of oxidative stress, significantly reduces the production of free radicals.     

HPV16 Seropositivity and Subsequent HPV16 Infection Risk in a Naturally Infected Population: Comparison of Serological Assays

Background

Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays.

Methodology/Principal Findings

Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27–0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28–0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection.

Conclusions/Significance

Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity.