Prospective Prediction of Treatment Response in High-Grade Glioma Patients using Pre-Treatment Tumor ADC Value and miR-222 and miR-205 Expression Levels in Plasma.

Background: Treatment response in High-grade Glioma (HGG) patients changes based on their genetic and biological characteristics. MiRNAs, as important regulators of drug and radiation resistance, and the Apparent Diffusion Coefficients (ADC) value of tumor can be used as a prognostic predictor for glioma. Objective: This study aimed to identify some of the pre-treatment individual patient features for predicting the treatment response in HGG patients. Material and Methods: In this prospective study, 18 HGG patients, who were candidated for chemo-radiation treatment, participated after informed consent of the patients. The investigated features were the expression level of miR-222 and miR-205 in plasma, the ADC value of tumor, Body Mass Index (BMI), and age. Treatment response was assessed, and Least Absolute Shrinkage and Selection Operator (LASSO) regression was used to obtain a model to predict the treatment response. Mann-Whitney U test was also applied to select the variables with a significant relationship with patients' treatment response. Results: The LASSO coefficients for miR-205, miR-222, tumor's mean ADC value, BMI, and age were 3.611, -1.683, 2.468, -0.184, and -0.024, respectively. Mann-Whitney U test results showed miR-205 and tumor's mean ADC significantly related to treatment response (P-value<0.05). Conclusion: The miR-205 expression level of the patient in plasma and tumor's mean ADC value has the potential for prognostic predictors in HGG.

In-Utero exposure to potential sources of indoor air pollution and umbilical cord blood leukocyte telomere length.

Indoor air pollution (IAP) has been associated with various adverse health effects. However, the evidence regarding such an association with leukocyte telomere length (LTL) in cord blood samples is still scarce. Therefore, the present study aimed to assess the relationship between exposure to indicators of IAP and LTL in umbilical cord blood samples. This cross-sectional study was based on 188 mother-newborn pairs who participated in our study between 2020 and 2022 in Isfahan, Iran. Umbilical LTL was measured by quantitative real-time polymerase chain reaction (qRT-PCR) technique. Linear mixed-effect models were used to assess the relationship between IAP indicators and umbilical LTL, adjusted for relevant covariates. The median (interquartile range (IQR)) of umbilical LTL was 0.92 (0.47). In fully adjusted models, frequency of using degreasing spray during pregnancy (times per month) (ß = -0.047, 95% CI:0.09, -0.05, P-value = 0.02), using air freshener spray during pregnancy (ß = -0.26, 95% CI: -0.5, -0.02, P-value = 0.03) and frequency of using insecticides during pregnancy (times per month) (ß = -0.025, 95% CI: -0.047, -0.003, P-value = 0.02) were significantly associated with shorter umbilical LTL. There was a positive significant relationship between the frequency of using cleaning spray during pregnancy (times per month) with umbilical LTL (ß = 0.019, 95% CI: 0.005, 0.033, P-value = 0.01). Furthermore, the direct connection of the parking with home and the frequency of using barbecue (times per week) were marginally associated with shorter umbilical LTL. For other indicators of IAP, we did not observe any statistically significant associations. Overall, this study suggested a negative association between prenatal exposure to IAP during pregnancy and umbilical LTL.

Expression of Matrix Metalloproteinases in Human Cystic Echinococcosis.

BACKGROUND: Cystic echinococcosis (CE) is a zoonotic disease caused by the Echinococcus granulosus senso lato (E. granulosus s.l.) larval stages. Parasitederived products have been shown to regulate host matrix metalloproteinases (MMPs), contributing to CE pathogenesis and progressive liver fibrosis in intermediate hosts. The current study aimed to investigate the potential role of MMP1, 7, 8, and 13 in E. granulosus s.l-induced liver fibrosis. METHODS: Thirty CE patients with active, transitional, or inactive hydatid cysts were enrolled in this study to determine the inductive effects of E. granulosus on the expression of MMP-1, MMP-7, MMP-8, and MMP-13 in healthy liver tissue and fibrotic liver tissue using qRT-PCR. RESULTS: According to the WHO-IWGE classification, patients with functional cysts (CE1 and CE2) had the highest percentage (46.6%). MMP-1, MMP-7, MMP-8, and MMP-13 expression levels were significantly higher in fibrotic liver than in normal liver tissue. MMP-13 and MMP-1 had the highest and lowest expression levels among MMPs. Compared to the normal group, the fold change for MMP-13 in the fibrotic group was greater than 12 and had the highest AUC value (AUC= 0.8283). CONCLUSION: Our findings suggest that E. granulosus-derived products might be involved in regulating host MMPs. Thus, MMPs may be considered potential biomarkers for predicting CE prognosis. Because of the non-normal distribution of our patients' CE types, further research, particularly on circulation MMPs, is needed to confirm the potential role of MMPs in CE pathogenesis and to follow up on CE patients.

Whole exome sequencing revealed variants in four genes underlying X-linked intellectual disability in four Iranian families: novel deleterious variants and clinical features with the review of literature.

AIM AND OBJECTIVE: Intellectual disability (ID) is a heterogeneous condition affecting brain development, function, and/or structure. The X-linked mode of inheritance of ID (X-linked intellectual disability; XLID) has a prevalence of 1 out of 600 to 1000 males. In the last decades, exome sequencing technology has revolutionized the process of disease-causing gene discovery in XLIDs. Nevertheless, so many of them still remain with unknown etiology. This study investigated four families with severe XLID to identify deleterious variants for possible diagnostics and prevention aims. METHODS: Nine male patients belonging to four pedigrees were included in this study. The patients were studied genetically for Fragile X syndrome, followed by whole exome sequencing and analysis of intellectual disability-related genes variants. Sanger sequencing, co-segregation analysis, structural modeling, and in silico analysis were done to verify the causative variants. In addition, we collected data from previous studies to compare and situate our work with existing knowledge. RESULTS: In three of four families, novel deleterious variants have been identified in three different genes, including ZDHHC9 (p. Leu189Pro), ATP2B3 (p. Asp847Glu), and GLRA2 (p. Arg350Cys) and also with new clinical features and in another one family, a reported pathogenic variant in the L1CAM (p. Glu309Lys) gene has been identified related to new clinical findings. CONCLUSION: The current study's findings expand the existing knowledge of variants of the genes implicated in XLID and broaden the spectrum of phenotypes associated with the related conditions. The data have implications for genetic diagnosis and counseling.

Phage based vaccine: A novel strategy in prevention and treatment.

The vaccine was first developed in 1796 by a British physician, Edward Jenner, against the smallpox virus. This invention revolutionized medical science and saved lives around the world. The production of effective vaccines requires dominant immune epitopes to elicit a robust immune response. Thus, applying bacteriophages has attracted the attention of many researchers because of their advantages in vaccine design and development. Bacteriophages are not infectious to humans and are unlikely to bind to cellular receptors and activate signaling pathways. Phages could activate both cellular and humoral immunity, which is another goal of an effective vaccine design. Also, phages act as an effective adjuvant, along with the antigens, and induce a robust immune response. Phage-based vaccines can also be administered orally because of their stability in the gastrointestinal tract, in contrast to common vaccination routes, which are intradermal, subcutaneous, or intramuscular. This review presents the current improvements in phage-based vaccines and their applications as preventive or therapeutic vaccines.

Unraveling the potential of M13 phages in biomedicine: Advancing drug nanodelivery and gene therapy.

M13 phages possessing filamentous phage genomes offer the benefits of selective display of molecular moieties and delivery of therapeutic agent payloads with a tolerable safety profile. M13 phage-displayed technology for resembling antigen portions led to the discovery of mimetic epitopes that applied to antibody-based therapy and could be useful in the design of anticancer vaccines. To date, the excremental experiences have engaged the M13 phage in the development of innovative biosensors for detecting biospecies, biomolecules, and human cells with an acceptable limit of detection. Addressing the emergence of antibiotic-resistant bacteria, M13 phages are potent for packaging the programmed gene editing tools, such as CRISPR/Cas, to target multiple antimicrobial genes. Moreover, their display potential in combination with nanoparticles inspires new approaches for engineering targeted theragnostic platforms targeting multiple cellular biomarkers in vivo. In this review, we present the available data on optimizing the use of bacteriophages with a focus on the to date experiences with M13 phages, either as monoagent or as part of combination regimens in the practices of biosensors, vaccines, bactericidal, modeling of specific antigen epitopes, and phage-guided nanoparticles for drug delivery systems. Despite increasing research interest, a deep understanding of the underlying biological and genetic behaviors of M13 phages is needed to enable the full potential of these bioagents in biomedicine, as discussed here. We also discuss some of the challenges that have thus far limited the development and practical marketing of M13 phages.

Unmasking the complex roles of hypocalcemia in cancer, COVID-19, and sepsis: Engineered nanodelivery and diagnosis.

Calcium (Ca2+) homeostasis is essential for maintaining physiological processes in the body. Disruptions in Ca2+ signaling can lead to various pathological conditions including inflammation, fibrosis, impaired immune function, and accelerated senescence. Hypocalcemia, a common symptom in diseases such as acute respiratory distress syndrome (ARDS), cancer, septic shock, and COVID-19, can have both potential protective and detrimental effects. This article explores the multifaceted role of Ca2+ dysregulation in inflammation, fibrosis, impaired immune function, and accelerated senescence, contributing to disease severity. Targeting Ca2+ signaling pathways may provide opportunities to develop novel therapeutics for age-related diseases and combat viral infections. However, the role of Ca2+ in viral infections is complex, and evidence suggests that hypocalcemia may have a protective effect against certain viruses, while changes in Ca2+ homeostasis can influence susceptibility to viral infections. The effectiveness and safety of Ca2+ supplements in COVID-19 patients remain a subject of ongoing research and debate. Further investigations are needed to understand the intricate interplay between Ca2+ signaling and disease pathogenesis.

Toxocara Infection in Dogs and Cats in Isfahan Province of Iran in 2021.

Background: Toxocariasis is an acute or chronic disease caused by parasites of the Ascaridae family, especially Toxocara of dogs and cats. Eggs are excreted out by feces of these animals on soil. Infective eggs develop on soil which can be infective to human. In this study, infection rate of Toxocara spp. in dogs and cats of urban and rural areas of Isfahan province of Iran has been investigated. Materials and Methods: Three hundred and seventy-five stray dog feces and 230 stray cat feces were collected from the public environment (slaughterhouses, parks, children's playgrounds, student dormitories, university environment, streets and squares) in Isfahan province of Iran. At first, dogs' and cats' feces were examined for the presence of Toxocara spp. eggs using formalin ether method. In the second stage, by using molecular methods, Toxocara eggs spp. (Toxocara canis or Toxocara cati) were identified. Results: From 375 dog fecal samples, 39 (10.40%) and from 230 cat fecal samples, 38 (16.52%) were positive for presence of the Toxocara eggs. Conclusion: Dogs and cats in Isfahan province of Iran were infected with Toxocara parasite. These infections can be potential risk for human toxocariasis.

A novel METTL5 variant disrupting a donor splice site leads to primary microcephaly-related intellectual disability in an Iranian family: clinical features and literature review.

Intellectual disability (ID) is a highly heterogeneous disorder, affecting 1-3% of the world's population, which is associated with a significant disorder in cognitive development, adaptive functioning and behavioural problems in human life. In this study, due to the genetic heterogeneity of the disease, the whole-exome sequencing (WES) was performed on a 13-year-old boy suffering from microcephaly. In addition, Sanger sequencing, cosegregation analysis, and structural modelling were performed to identify and verify the causative variant in the proband and obligate carriers in the family. WES revealed a novel, homozygote 10-bp deletion in the donor splice site of 2nd exon of METTL5 gene (NM_014168:c.223_224?8del), which was found segregating with the phenotype in the pedigree. This variant meets the criteria of being pathogenic according to the American College of Medical Genetics (ACMG) variant interpretation guideline. Up to now, four pathogenic homozygous variants of the METTL5 gene have been reported that are associated with ID. A comparison of the clinical characteristics of our patient with previously reported cases revealed variability in the disease severity and some clinical presentations, including overall growth, dysmorphic facial features and behavioural psychiatric manifestations. The clinical findings of the case reported in this study extend the spectrum of genetic variations and phenotypes associated with ID and provide a better insight of the disease pathogenesis.

Human recombinant soluble PD1 can interference in T cells and Treg cells function in response to MDA-MB-231 cancer cell line.

OBJECTIVES: PD1/PDL1 pathway targeting using antibodies shows immune related adverse events in patients with tumors. The masking of PD1 ligand by soluble human PD-1 (shPD-1) probably inhibits the PD1/PDL1 interaction between T cells and tumor cells. Accordingly, the goal of this study was to produce human recombinant PD-1-secreting cells and find out how soluble human PD-1 affects T lymphocyte function. METHODS: An inducible construct of the human PD-1 secreting gene under hypoxia condition was synthesized. The construct was transfected into the MDA-MB-231 cell line. In six groups exhausted T lymphocytes were co-cultured with transfected or non-transfected MDA-MB-231 cell lines. The effect of shPD-1 on IFNγ production, Treg cell's function, CD107a expression, apoptosis, and proliferation was assessed by ELISA and flow cytometry, respectively. RESULTS: The results of this study showed that shPD-1 inhibits PD-1/PD-L1 interaction and enhances T lymphocyte responses through a significant increase in IFNγ production and CD107a expression. In addition, in the presence of shPD-1, the percentage of Treg cells decreased, while MDA-MB-231 cell apoptosis increased. CONCLUSIONS: We concluded that the human PD-1 secreting construct induced under hypoxia condition inhibits the interaction of PD-1/PD-L1 and enhances T lymphocyte responses in tumor environments and chronic infections.

Anti-leishmaniasis Effect of Staphylococcus Aureus Protein A on the Size of the Lesion and Parasitic Load.

Background: Many studies in the past have evaluated the role of immune system boosters in the treatment of leishmania major infection. Protein A (PA) is one of the structural components in peptidoglycan cell wall of gram-negative bacteria such as staphylococcus aurous which functions as a stimulator in the cellular immune system. The present study aims to evaluate the anti-inflammatory effect of PA on the recovery of leishmania major infection. Materials and Methods: This study was conducted on 24 female Balb/c-infected mice. The experimental group received PA at a dose of 60 mg/kg for four weeks. There was no intervention for the negative control group; the third group received the solvent of PA and sterile H2O; and the positive control group received Amphotericin B at a dose of 1 mg/kg body weight. At the end of the treatment period, a real-time polymerase chain reaction (PCR) assay was performed to determine parasitic burden, and the size of the lesions was measured by caliper with an accuracy of 0.01 mm. Results: Results showed that PA did slightly decrease the wound spread and growth but not to an extent that can be considered statistically significant. Also, differences in cycle threshold (Ct) values between the treated group and the untreated group was not impressive. Conclusions: Although findings showed that PA isn't such a good candidate for leishmania treatment, it may still be suitable for therapies that use multiple drugs in combination to speed up the healing of leishmaniosis, an issue that merits evaluation in future studies.

Pyrimido[4,5-b]indole derivatives bearing 1,2,4-oxadiazole moiety as MDM2 inhibitor candidates in cancer treatment.

Aim: In this study, novel hybrid structures of pyrimido-indole-oxadiazole were developed as MDM2 inhibitors for restoring the regular function of the p53. Materials & methods: A multistep chemical pathway was used to synthesize the derivatives. Nutlin-3a was used as a standard lead in molecular docking and molecular dynamics simulations. Finally, cytotoxicity was evaluated against MCF-7 cancer cells versus Doxorubicin. Results: The most promising candidate was 12c, which had an NO2 group in the para position of the oxadiazole ring (IC50: 1.1 µM). A satisfactory result was obtained with the combined application of 12c and Doxorubicin (IC50 decreased to 0.63 µM), which could be potentially attributed to MDM2 inhibition. Conclusion: These hybrid structures can be further investigated as potential MDM2 inhibitors.

The potential therapeutic impacts of trehalose on cardiovascular diseases as the environmental-influenced disorders: An overview of contemporary findings.

Cardiovascular diseases (CVDs) as environmental-influenced disorders, are a major concern and the leading cause of death worldwide. A range of therapeutic approaches has been proposed, including conventional and novel methods. Natural compounds offer a promising alternative for CVD treatment due to their ability to regulate molecular pathways with minimal adverse effects. Trehalose is natural compound and disaccharide with unique biological functions and cardio-protective properties. The cardio-protective effects of trehalose are generated through its ability to induce autophagy, which is mediated by the transcription factors TFEB and FOXO1. The stimulation of TFEB plays a significant role in regulating autophagy genes and autophagosome formation. Activation of FOXO1 through dephosphorylation of Foxo1 and blocking of p38 mitogen-activated protein kinase (p38 MAPK) also triggers autophagy dramatically. Trehalose has been shown to reduce CVD risk factors, including atherosclerosis, cardiac remodeling after a heart attack, cardiac dysfunction, high blood pressure, and stroke. It also reduces structural abnormalities of mitochondria, cytokine production, vascular inflammation, cardiomyocyte apoptosis, and pyroptosis. This review provides a molecular overview of trehalose's cardioprotective functions, including its mechanisms of autophagy and its potential to improve CVD symptoms based on clinical evidence.

Enhanced in vivo anti-tumor efficacy of whole tumor lysate in combination with whole tumor cell-specific polyclonal antibody.

Background and purpose: Despite the widespread utilization of cancer vaccines with specified antigens, the use of whole tumor cell lysates in tumor immunotherapy would be a very promising approach that can overcome several significant obstacles in vaccine production. Whole tumor cells provide a broad source of tumor-associated antigens and can activate cytotoxic T lymphocytes and CD4+ T helper cells concurrently. On the other hand, as an effective immunotherapy strategy, recent investigations have shown that the multi-targeting of tumor cells with polyclonal antibodies, which are also more effective than monoclonal antibodies at mediating effector functions for target elimination, might minimize the escape variants. Experimental approach: We prepared polyclonal antibodies by immunizing rabbits with the highly invasive 4T1 breast cancer cell line. Findings/Results: In vitro investigation indicated that the immunized rabbit serum inhibited cell proliferation and induced apoptosis in target tumor cells. Moreover, in vivo analysis showed enhanced anti-tumor efficacy of whole tumor cell lysate in combination with tumor cell-immunized serum. This combination therapy proved beneficial in significant inhibition of the tumor growth and the established tumor was entirely eradicated in treated mice. Conclusion and implications: Serial intravenous injections of tumor cell immunized rabbit serum significantly inhibited tumor cell proliferation and induced apoptosis in vitro and in vivo in combination with whole tumor lysate. This platform could be a promising method for developing clinical-grade vaccines and open up the possibility of addressing the effectiveness and safety of cancer vaccines.

Crosstalk of Transcriptional Regulators of Adaptive Immune System and microRNAs: An Insight into Differentiation and Development.

MicroRNAs (miRNAs), as small regulatory RNA molecules, are involved in gene expression at the post-transcriptional level. Hence, miRNAs contribute to gene regulation of various steps of different cell subsets' differentiation, maturation, and activation. The adaptive immune system arm, which exhibits the most specific immune responses, is also modulated by miRNAs. The generation and maturation of various T-cell subsets concomitant with B-cells is under precise regulation of miRNAs which function directly on the hallmark genes of each cell subset or indirectly through regulation of signaling pathway mediators and/or transcription factors involved in this maturation journey. In this review, we first discussed the origination process of common lymphocyte progenitors from hematopoietic stem cells, which further differentiate into various T-cell subsets under strict regulation of miRNAs and transcription factors. Subsequently, the differentiation of B-cells from common lymphocyte progenitors in bone marrow and periphery were discussed in association with a network of miRNAs and transcription factors.

Viewpoints on the Role of Transient Receptor Potential Melastatin Channels in Cardiovascular System and Disease: A Systematic Review.

Transient receptor potential (TRP) family play critical roles in cardiovascular system. TRPM family as largest TRP subfamily is non-voltage Ca2+-activated selective channels which has 8 members. This study aimed to discuss the role of TRPM family in cardiovascular system and diseases. Systematic search was performed covering PubMed, ISI Web of Science, and Google Scholar from inception until June 2021 using related keywords and Mesh terms for English studies with human, animal and in-vitro subjects. Finally 10 studies were selected for data extraction. Reviewing the articles showed that TRPM2, TRPM4, TRPM5, TRPM6 and TRPM7 play important roles in cardiovascular system and diseases. TRPM2 could be activated by reactive oxygen species (ROS) and effects on cardiac injury and cardiac fibrosis. TRPM7 and TRPM6 also have been reported to be associated with cardiac fibrosis and atrial fibrosis development respectively. TRPM4 channels contributed to resting membrane potential of cerebral artery smooth muscle cells and atrial contraction. TRPM5 channels are bitter taste sensors and prevent high salt intake and consequently high blood pressure due to the high salt intake. In conclusion based on the proof of the effectiveness of some members of TRPM family in the cardiovascular system, research on other members of this channel group seems to be useful and necessary to find their possible connection to the cardiovascular system.

Bio-Membrane SELEX as a New Approach for Selecting ss-DNA Aptamers that Bind to the Hydatid Cyst Laminated Layer.

BACKGROUND: Hydatid cyst (HC) is the larval stage of the canine intestinal tapeworm (cestode), Echinococcus granulosus. In addition to the high global economic cost of livestock farming, the infection can lead to dangerous problems for human health. Therefore, research into new diagnosis and treatment approaches is valuable. This study is set out to explore aptamers that bind to HC antigens. METHODS: The similarity between HC genotype in sheep and humans was that sheep HCs were collected and used as a biological membrane for aptamer selection. Four Bio- Membrane SELEX rounds were conducted, and ssDNA aptamers were selected. Selected aptamers' affinity and specificity to the laminated layer antigens were evaluated using membrane staining by fluorescein primer as a probe. Biotinylated primer was used as a probe for aptahistochemistry and dot blot techniques. Subsequently, cloning and plasmid extraction was conducted. The affinity and specificity of sequenced aptamers were examined with the dot blot method. RESULTS: Selected aptamers reacted with HC wall in aptahistochemistry, aptahistofluorescent, and dot blot experiments. Following cloning and sequencing, 20 sequences were achieved. A strong reaction between HC total antigens and sequenced aptamers has emerged in the dot blot method. CONCLUSION: We propose a novel method to determine specific aptamers in this investigation. Bio-Membrane SELEX could be assumed as a practical and sensitive method for aptamer selection. Selected aptamers in this study possibly may be used for specific HC antigens detection.

Evaluation of the antileishmanial effect of polyclonal antibodies and cationic antimicrobial peptides.

Leishmaniasis is one of the tropical and subtropical diseases which, according to WHO, has the priority of control. The list of anti-leishmanial drugs is limited and requires side effects, high costs, and long-term treatments. Various species, parasite resistance, and simultaneous diseases are among the factors that affect the effectiveness of treatment. Due to these problems and based on satisfactory records of previous studies using antimicrobial peptides (AMPs) against infectious diseases, this study aimed to evaluate the antileishmanial effect of Leishmania-infected macrophage polyclonal antibody (LIMPA) with or without different concentrations (2, 4, 6, 8, 10, 20, 40, 60, and 100 µg/ml) of CM11 and (40, 80, and 100 µg/ml) BufIIIb, two AMPs, in vitro and their therapeutic effects against CL of Balb/c mice. Results showed that LIMPA induced an anti-proliferative effect on Leishmania major growth in macrophages in vitro and intramacrophage-amastigotes in vivo. CM11 with IC50 of 8.73 and 10.10 µg/ml at 48 hours, and BufIIIb with IC50 of 66.83 and 80.26 µg/ml, at 24 hours showed the most significant inhibition of L. major promastigotes and amastigotes. In addition, the CM11 and BufIIIb, with a CC50 of 9.7 µg/ml and 40.34 µg/ml, showed the most significant inhibition effect on the J774.A1 cell line at 48 hours, respectively. In addition, in vivo experiments using LIMPA with a 0.01 mg/kg dosage showed a significant difference (p < 0.001) in the last week of the measurement compared to the control. The results of this study may be a promising prospect for further investigations.

Environmental Contamination of Different Areas of Isfahan Province of Iran with Toxocara spp. Eggs using Molecular Methods.

Background: Toxocariasis is a parasitic disease caused by the larval stage of Toxocara canis and Toxocara cati. Infective stage of this parasite for human develops on soil. So, in this work contamination of the soil of public environments in five geographical areas of Isfahan province of Iran has been investigated. Materials and Methods: In this descriptive study, 355 soil samples were collected from parks, children's playgrounds, student dormitories, and university environments, and examined by Flotation method. The samples were then inspected using microscopic and molecular methods. Results: From the 355 examined soil samples in 77 (21.69%), and 87 (24.50%) cases Toxocara eggs were detected by microscopic and molecular methods, respectively. In the molecular method, 31 (8.70%) cases of T. cati and 44 (12.39%) cases of T. canis were identified. Conclusion: Toxocara eggs were identified in all areas of Isfahan province, although contamination rate was higher in Fereydun Shahr and Semirum counties.