CRISPR, CAR-T, and NK: Current applications and future perspectives.

Chimeric antigen receptor T (CAR-T) cell therapy represents a breakthrough in personalized cancer treatments. In this regard, synthetic receptors comprised of antigen recognition domains, signaling, and stimulatory domains are used to reprogram T-cells to target tum or cells and destroy them. Despite the success of this approach in refractory B-cell malignancies, the optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been validated. Natural killer cells are powerful cytotoxic lymphocytes specialized in recognizing and dispensing the tumor cells in coordination with other anti-tumor immunity cells. Based on these studies, many investigations are focused on the accurate designing of CAR T-cells with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system or other novel gene editing tools that can induce hereditary changes with or without the presence of a double-stranded break into the genome. These methodologies can be specifically focused on negative controllers of T-cells, induce modifications to a particular gene, and produce reproducible, safe, and powerful allogeneic CAR T-cells for on-demand cancer immunotherapy. The improvement of the CRISPR/Cas9 innovation offers an adaptable and proficient gene-editing capability in activating different pathways to help natural killer cells interact with novel CARs to particularly target tumor cells. Novel achievements and future challenges of combining next-generation CRISPR-Cas9 gene editing tools to optimize CAR T-cell and natural killer cell treatment for future clinical trials toward the foundation of modern cancer treatments have been assessed in this review.

Effect of Pioglitazone and Cetuximab on Colon Cancer Stem-like Cell (CCSLCs) Properties.

BACKGROUND: One of the main reasons for cancer resistance to chemotherapy is the presence of cancer stem cells (CSCs) in cancer tissues. It is also believed that CSCs are the unique originators of all tumor cells. On the other hand, the Epithelial-Mesenchymal Transition pathway (EMT) can act as the main agent of metastasis. Therefore, it is possible that targeting CSCs as well as the EMT pathway could help in cancer therapy. Considering that CSCs constitute only a small percentage of the total tumor mass, enrichment before study is necessary. In our previous study, CSCs were enriched in the human colon cancer cell line HT29 by induction of EMT. These CSC-enriched HT29 cells with mesenchymal morphology were named "HT29-shE". In the present study, these cells were used to investigate the effect of pioglitazone (Pio) and Cetuximab (Cet) in order to find CSC and EMT targeting agents. METHOD: The viability and IC50 rate of cells treated with different concentrations of Pio and Cet were evaluated using the MTT test. EMT and CSC markers and cell morphology were assessed in Pio and Cet treated and untreated HT29-shE cells using flow cytometry, realtime PCR, immunocytochemistry, and microscopic monitoring. RESULTS: The findings showed that Pio and Cet at concentrations of 250 µM and 40 µg/ml, respectively, decrease cell viability by 50%. Also, they were able to reduce the expression of CSC markers (CD133 and CD44) in the CSC enriched HT29 cell line. Furthermore, Pio and Cet could efficiently reduce the expression of vimentin as a mesenchymal marker and significantly upregulate the expression of E-cadherin as an epidermal marker of EMT and its reverse mesenchymal-- to-epithelial transition (MET). In addition, the mesenchymal morphology of HT29-shE changed into epithelial morphology after Cet treatment. CONCLUSION: Pio and Cet could inhibit EMT and reduce CSC markers in the EMT induced/CSC enriched cell line. We expect that focus on finding EMT/CSC-targeting agents like these drugs can be helpful for cancer treatment.

In vivo study of anticancer activity of ginsenoside Rh2-containing arginine-reduced graphene in a mouse model of breast cancer.

Objectives: This study aims to evaluate the in vivo anticancer activity of arginine-reduced graphene (Gr-Arg) and ginsenoside Rh2-containing arginine-reduced graphene (Gr-Arg-Rh2). Materials and Methods: Thirty-two mice with breast cancer were divided into four groups and treated every three days for 32 days: Group 1, PBS, Group 2, Rh2, Group 3, Gr-Arg, and Group 4, Gr-Arg-Rh2. The tumor size and weight, gene expression (IL10, INF-γ, TGFß, and FOXP3), and pathological properties of the tumor and normal tissues were assessed. Results: Results showed a significant decrease in TGFß expression for all drug treatment groups compared with the controls (P=0.04). There was no significant difference among the groups regarding IL10 and FOXP3 gene expression profiles (P>0.05). Gr-Arg-Rh2 significantly inhibited tumor growth (size and weight) compared with Rh2 and control groups. The highest survival rate and the highest percentage of tumor necrosis (87.5%) belonged to the Gr-Arg-Rh2 group. Lungs showed metastasis in the control group. No metastasis was observed in the Gr-Arg-Rh2 group. Gr-Arg-Rh2 showed partial degeneration of hepatocytes and acute cell infiltration in the portal spaces and around the central vein. The Gr-Arg group experienced a moderate infiltration of acute cells into the port spaces and around the central vein. The Rh2 group also showed a mild infiltration of acute and chronic cells in portal spaces. Conclusion: Based on the results, Gr-Arg-Rh2 can reduce tumor size, weight, and growth, TGF-ß gene expression, and increase tumor necrosis and survival time in mice with cancer.

Investigating the Antimicrobial Activity of Vancomycin-Loaded Soy Protein Nanoparticles.

Developing targeted and slow-release antibiotic delivery systems can effectively reduce drug overdose and side effects. This study aimed to investigate the antimicrobial activity of vancomycin-loaded soy protein nanoparticles (vancomycin-SPNs). For the preparation of SPNs, the desolvation method was applied in different concentrations of vancomycin and soy protein (15:5, 10:15, 6:20, 8:25, and 10:30 of vancomycin:soy protein). Scanning electron microscope (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and FTIR were used for nanoparticle characterization. Antibacterial activity was evaluated by the radial diffusion assay (RDA) and absorbance methods. Proper synthesis was demonstrated by characterization. The best drug loading (% entrapment efficiency = 90.2%), the fastest release rate (% release = 88.2%), and the best antibacterial activity were observed in ratio 10:30 of vancomycin:SPNs. Results showed that SPNs are a potent delivery system for antibiotic loading and slow release to control antibiotic use.

Slow release curcumin-containing soy protein nanoparticles as anticancer agents for osteosarcoma: synthesis and characterization.

Curcumin-containing soy protein nanoparticles (curcumin-SPNs) were synthesized by desolvation (coacervation) method and characterized by SEM, DLS, FTIR, and XRD. For anticancer evaluation, osteogenic sarcoma (SAOS2) cell lines were incubated with different concentrations of nanostructures. The dialysis method was used for assessment of drug release. Intracellular reactive oxygen species (ROS) were evaluated in IC50 dose after 24 h of exposure to free curcumin and curcumin-SPNs. Characterization data showed that the size of drug-free SPNs and curcumin-SPNs were 278.2 and 294.7 nm, respectively. The zeta potential of drug-free SPNs and curcumin-SPNs were - 37.1 and - 36.51 mv, respectively. There was no significant difference between the control and drug-free SPNs groups in terms of cell viability (p > 0.05). The viability of cells in different concentrations of the designed curcumin-SPNs in Saos2 cell line was significantly higher than free drug (p < 0.05). The release of curcumin showed that more than 50% of the drug was released in the first 2 h of incubation. After this time, the slow release of drug was continued to 62-83% of drug. IC50 values of free curcumin and curcumin-SPNs (1/10) were 156.8 and 65.9 µg/mL, respectively (a free curcumin IC50 was 2.4 times more than curcumin-SPNs). Slow-release of the curcumin causes the cell to be exposed to the anticancer drug for a longer period of time. The intracellular ROS levels significantly increased in an IC50 dose after 24 h of exposure to both free curcumin and curcumin-SPNs compared with controls (p < 0.05).

Advantages and disadvantages of using Carbon Nanostructures in Reproductive Medicine: two sides of the same coin.

Carbon nanostructures are important nanomaterial with interesting physical and chemical properties. These nanostructures have been assessed for application in different fields of medicine, such as cancer detection and treatment, Parkinson disease, reproductive medicine, etc. This nanomaterial can be used in reproductive medicine as a drug delivery system, antifungal, antiviral, and antibacterial agent, condom-coating agent, enhancer of sperm fertilizing ability, ectopic pregnancy treatment, trophoblastic diseases, endometriosis, uterine fibroids, and Assisted Reproduction Techniques (ART) improvement. The other side of this coin involves various side effects of carbon nanostructures, especially negative effects on reproductive systems. All carbon nanostructures showed toxicity on the reproductive system by producing reactive oxygen species and oxidative stress. Less attention has been given to the unique properties of carbon nanostructures, except for their practical attractiveness, the other side of this coin, namely the risks and side effects of these compounds - especially in the case of a reproductive system that supports the survival and health of future generations. Therefore, we suggest paying particular attention to the negative aspects of the increasing use of carbon nanostructures.

Effect of Nanoalumina on Sex Hormones and Fetuses in Pregnant Rats.

OBJECTIVE: This study aimed at investigating the effect of nanoalumina on sex hormones, and fetuses in pregnant rats. METHODS: In this study, sixty-four pregnant rats were divided into eight groups. The control and the injection-control group received normal food and water, and 0.5 ml of distilled water, respectively. Treatment groups were treated with 25, 50, 100, 250, 500, and 1000µg/ml concentrations of Nanoalumina from the 7th day until the 18th day of pregnancy. On the 18th day, the rats were investigated in terms of their hormone levels. We evaluated the number of healthy and aborted offspring, as well as fetus size. RESULTS: Nanoalumina caused an increase in progesterone hormones at the concentrations of 250, and 500µg/ml, and a significant reduction in estrogen hormone and aborted fetuses at the concentrations of 250 and 500µg/ml (p<0.05). The largest and smallest size of fetuses were observed in 500µg/ml and 1000µg/ml, respectively. The highest number of aborted fetuses was observed in the group treated with the 500µg/ml concentration. There was no aborted fetuses with 25, 50,100, control, and injection-control groups. CONCLUSIONS: Due to nanoalumina toxicity, it must be used with caution.

Antimicrobial Peptides from Amphibian Innate Immune System as Potent Antidiabetic Agents: A Literature Review and Bioinformatics Analysis.

Antimicrobial peptides, as an important member of the innate immune system, have various biological activities in addition to antimicrobial activity. There are some AMPs with antidiabetic activity, especially those isolated from amphibians. These peptides can induce insulin release via different mechanisms based on peptide type. In this review study, we collected all reported AMPs with antidiabetic activity. We also analyze the sequence and structure of these peptides for evaluation of sequence and structure effect on their antidiabetic activity. Based on this review, the biggest peptide family with antidiabetic activity is temporins with nine antidiabetic peptides. Frogs are the most abundant source of antidiabetic peptides. Bioinformatics analysis showed that an increase of positive net charge and a decrease of hydrophobicity can improve the insulinotropic effect of peptides. Peptides with higher positive net charge and Boman index showed higher activity. Based on this review article, AMPs with antidiabetic activity, especially those isolated from amphibians, can be used as novel antidiabetic drug for type 2 diabetes disease. So, amphibians are potential sources for active peptides which merit further evaluation as novel insulin secretagogues. However, strategy for the increase of stability and positive activity as well as the decrease of negative side effects must be considered.

The Development and Validation of a Scale for the Activities of Daily Living in Iranian Children.

Introduction: Activities of Daily Living (ADL), as an ultimate goal of rehabilitation, rely on cultural and environmental factors. This study aimed to develop a questionnaire based on the occupational therapy practice frame to accurately evaluate Iranian children's occupational performance. Methods: This scale was developed in two phases of planning and construction. The planning phase involved a literature review and a collection of the available evaluation tools in the area. The advice of two expert panels was used to develop a preliminary 87-item questionnaire. In the construction phase, 40 parents were surveyed to assess the popularity of the activities in Iran. After a face to content validation, the final version of the questionnaire was prepared with 93 items. Results: The final 93-item questionnaire was used to assess the ADL of 3-6-year-old children. The 93 items, selected according to criteria found in the literature and the panel of experts, were categorized into six ranges of occupational therapy practice framework (bathing/washing/personal hygiene, toileting, dressing, eating/feeding, functional mobility, and others). Conclusion: The ADL in Iranian children is a practical and culturally relevant tool for measuring the occupational performance of Iranian children. It can be used in clinical and population-based research. Highlights: ADLs are essential and routine tasks required to independent and healthy living.The active participation of the child in ADL during development has a positive effect on the development of sensory and cognitive functions of the brain.ADLs are highly dependent on contextual and ecological factors, such as parenting style, the environment and given context.The ADLIC tool is a standard, culturally adapted tool that can be used by occupational therapists to assess ADLS in Iranian children. Plain Language Summary: The ADLs referred to Self Care Skills, which play a major role in a child overall functional growth, confidence and independence. The healthy individuals can perform these life skills without assistance. The inability to accomplish essential ADLs may lead to unsafe conditions and poor quality of life. Due to the importance of these activities during the development of the child, it is necessary for Iranian children to be evaluated in terms of independence in these activities so that in case of disorder, the necessary counseling and education can be provided by occupational therapists.

Fluorimetric detection of methylated DNA of Sept9 promoter by silver nanoclusters at intrastrand 6C-loop.

Methylation of DNA at carbon 5 of cytosines is the most common epigenetic modification of human genome. Due to its critical role in many normal cell processes such as growth and development, any aberrant methylation pattern in a particular locus may lead to abnormal functions and diseases such as cancer. Development of methods to detect methylation state of DNA which may eliminate labor-intensive chemical or enzymatic treatments has received considerable attention in recent years. Herein, we report a DNA methylation detection procedure based on fluorescence turn-on strategy. Target sequence was selected from Sept9 promoter region that has been reported as one of the most frequently methylated sites in colorectal cancer. Probe DNA was designed to be complementary to this sequence with an additional six cytosines in the middle to form an internal loop to host silver nanoclusters. The fluorescence intensity of the synthesized silver nanoclusters with the duplexes of probe-non-methylated target was significantly different from that of probe-methylated target. The fluorescence enhanced with increasing the methylated DNA concentration with a linear relation in the range of 1.0 × 10-8 M to 5.0 × 10-7 M with the detection limit of 8.2 × 10-9 M, and quenched with non-methylated ones. The method was very specific in the presence of non-complementary sequences with maximum similarity of 40%. Circular dichroism spectra indicated that silver ions significantly affected the structure of methylated and non-methylated DNA into different extents which could further influence the nanocluster fluorescence. Finally, a method was introduced to meet the concerns in the applicability of the proposed method in real situation.

Evaluating the Toxicity and Histological Effects of Al2O3 Nanoparticles on Bone Tissue in Animal Model: A Case-Control Study.

The applications of nanostructures have been limited by their different toxicities. So, the investigation of these toxicities is necessary before nanostructure application. This study aimed to evaluate the effect of aluminum oxide (Al2O3) nanoparticles on bone density in Wistar rat. Al2O3 nanoparticle was prepared by the sol-gel method. Characterization was done by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Sixty-four male adult Wistar rats were divided into eight groups including six groups intravenously treated with Al2O3 nanoparticle at concentrations of 25, 50, 100, 250, 500, and 1000 µg/ml: one group received food and water as the control group, and one group received food and water as well as intravenously distilled water as an injection control group. After 41 days, bone density was analyzed by dual-energy X-ray absorptiometry (DEXA). According to X-ray diffraction, the average particle size for Al2O3 nanoparticles was 20.85 nm. The data of densitometry showed that the bone density of right and left foot was reduced in concentrations of 250, 500, and 1000 µg/ml that were statistically significant in comparison with the control group. The reduction of bone density was increased with the enhancement of nanostructures concentration. The effect of Al2O3 nanoparticles on bone density was similar in the left and right legs. Histopatholological assessment also showed that Al2O3 nanoparticles (250, 500, and 1000 µg/ml) lead to significant reduction of trabeculae. Empty lacunae are observed in these three groups. Considering that high concentrations of Al2O3 nanoparticles had toxicity on bone tissue, it must be used by more caution, especially its use as a coating in different devices such as implants, surgical instruments, and bone prostheses.

Assessment of a New Ginsenoside Rh2 Nanoniosomal Formulation for Enhanced Antitumor Efficacy on Prostate Cancer: An in vitro Study.

INTRODUCTION: Ginsenoside Rh2, purified from the Panax ginseng root, has been demonstrated to possess anticancer properties against various cancerous cells including colorectal, breast, skin, ovarian, prostate, and liver cancerous cells. However, the poor bioavailability, low stability on gastrointestinal systems, and fast plasma elimination limit further clinical applications of Ginsenoside Rh2 for cancer treatments. In this study, a novel formulation of niosomal Ginsenoside Rh2 was prepared using the thin film hydration technique. METHODS: The niosomal formulation contained Span 60 and cholesterol, and cationic lipid DOTAP was evaluated by determining particle size distribution, encapsulation efficiency, the polydispersity index (PDI), and surface morphology. The cytotoxic effects of free Ginsenoside Rh2 and Ginsenoside Rh2-loaded niosomes were determined using the MTT method in the PC3 prostate cancer cell line. For the investigation of the in vitro cellular uptake of Ginsenoside Rh2-loaded niosome, two formulations were prepared: the Ginsenoside Rh2-loaded niosomal formula containing 5% DOTAP and the Ginsenoside Rh2-loaded niosomal formula without DOTAP. RESULTS: The mean size, DPI, zeta potential, and encapsulation efficiency of the Ginsenoside Rh2-loaded nanoniosomal formulation containing DOTAP were 93.5±2.1 nm, 0.203±0.01, +4.65±0.65, and 98.32% ±2.4, respectively. The niosomal vesicles were found to be round and have a smooth surface. The release profile of Ginsenoside Rh2 from niosome was biphasic. Furthermore, a two-fold reduction in the Ginsenoside Rh2 concentration was measured when Ginsenoside Rh2 was administered in a nanoniosomal form compared to free Ginsenoside Rh2 solutions in the PC3 prostate cancer cell line. After storage for 90 days, the encapsulation efficiency, vesicle size, PDI, and zeta potential of the optimized formulation did not significantly change compared to the freshly prepared samples. The cellular uptake experiments of the niosomal formulation demonstrated that by adding DOTAP to the niosomal formulation, the cellular uptake was enhanced. DISCUSSION: The enhanced cellular uptake and cytotoxic activity of the Ginsenoside Rh2 nanoniosomal formulation on the PC3 cell make it an attractive candidate for application as a nano-sized delivery vehicle to transfer Ginsenoside Rh2 to cancer cells.

Coronavirus Disease 2019 (COVID-19) in Children: Prevalence, Diagnosis, Clinical Symptoms, and Treatment.

In this article, we have reviewed the prevalence, diagnosis, symptoms, and treatment of COVID-19 in children. The incidence of COVID-19 among children under 18 years was 2.1% based on the reported studies, where the mortality rate in the same age group was 0.2%. No death has been reported in children under 9-years old. There are some articles that report children with COVID-19 having symptoms similar to Kawasaki's disease. In these cases, heart complications were observed. The best markers for diagnosing the severity of the disease in children are the levels of bilirubin and hepatic enzymes. Large number of angiotensin converting enzyme 2 (ACE2) receptors on cell surfaces, effective innate immune system, and high level of blood lymphocyte have been reported to be the potent reasons for lower incidence of severe symptoms of COVID-19 among children. Children can very well be the carriers of this virus. Children with severe COVID-19 clinical symptoms, especially those suffering from pneumonia, must be hospitalized similar to adults, while quarantine is required for those having mild symptoms. Antiviral medication (lopinavir, darunavir, favipiravir, remdesivir, ribavirin, oseltamivir, tocilizumab, and umifenovir), ACE inhibitors, interferon-α2b, co-therapy with azithromycin, inhaling iNO, and oxygen therapy can be used for treatment. For the treatment of children without any clinical and infection symptoms, home isolation protocol has been recommended.

Application of intercalating molecules in detection of methylated DNA in the presence of silver ions.

The extent of DNA structural perturbation by silver ions is different in methylated and non-methylated DNA. Here, we explored the interaction of eight convenient DNA interacting molecules with methylated and non-methylated short GC rich oligonucleotides in the presence and absence of silver ions. Acridine orange, DAPI, Doxorubicin, Ethidium bromide, Hoechst 33342, Methylene blue, PicoGreen, and Propidium iodide are tested for their ability to distinguish methylated and non-methylated DNA. Among them, Ethidium bromide, Methylene blue, and PicoGreen were able to discriminate between methylated and non-methylated DNA, while DAPI and Hoechst 33342 were only able to discriminate with the aid of silver ions. A detection method is proposed using Ethidium bromide in which the silver-treated sample of DNA exposed different fluorescence intensity from the untreated one on the base of its methylation state. This phenomenon was sequence-dependent and could provide a sensing platform with a detection limit of about 4fi0 nM.

Fluorescence enhancement of silver nanocluster at intrastrand of a 12C-loop in presence of methylated region of sept 9 promoter.

Determining methylation state of a particular DNA sequence is an essential task in many epigenetic investigations. Here a facile method based on silver nanocluster (AgNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region that its hypermethylation is demonstrated as a reliable biomarker of colorectal cancer. Probe DNA was complementary to a 25 nucleotide of the target region and possessed twelve additional cytosines in the middle to grant the formation of AgNCs. After probe strands were hybridized with methylated and non-methylated targets separately, AgNCs were synthesized, and their fluorescence intensities were recorded. Fluorescence intensity enhanced when the target strands were methylated and quenched when they were non-methylated. The Linear range of fluorescence enhancement was from 1.0 × 10-7 M to 5.0 × 10-7 M with the detection limit of 7.6 × 10-8 M. Sensor specificity was checked with non-complementary strands with the maximum similarity of 40%. Further experiments explored various characteristics of methylated and non-methylated DNAs carrying AgNC and indicated that structure of methylated and non-methylated DNAs was affected differently by silver ions that could then influence AgNC fluorescence. This effect was strongly sequence-dependent, and either fluorescence enhancement or quenching was observed with two different sequences.

Discrimination of methylated and nonmethylated region of a colorectal cancer related promoter using fluorescence enhancement of gold nanocluster at intrastrand of a 9C-loop.

Among epigenetic modifications of DNA, methylation of cytosine at its carbon 5 is the most common mark that is usually associated with gene silencing in human. Determining whether a particular DNA molecule is methylated or not, is an indispensable task in many epigenetic investigations. Presenting detection methods with less labor-intensive and time-consuming procedures has substantial value. Here a facile method based on gold nanocluster (AuNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region as its hypermethylation is demonstrated as a reliable biomarker of colorectal cancer. DNA probe was complementary to a 25 nucleotide of the target region and possessed 9 additional cytosines in the middle to allow the formation of AuNCs. Probe-AuNCs strands were first hybridized with methylated and non-methylated targets separately, and then their fluorescence intensities were recorded. Fluorescence intensity was higher with methylated targets than non-methylated one. Applying silver ions reversed the situation and fluorescence intensities of non-methylated DNA got higher than methylated one.