Cloning, expression and molecular analysis of recombinant Netrin-A protein of Lucilia sericata Meigen (Diptera: Calliphoridae) larvae.

Objectives: Lucilia sericata (Diptera: Calliphoridae) is used in larval therapy for wound healing. Netrin-A is an enzyme secreted from the salivary glands of these larvae, and has a central role in neural regeneration and angiogenesis. This study aimed to produce the recombinant Netrin-A protein from Lucilia sericata larvae by the baculovirus expression vector system in the Sf9 insect cell line. Methods: The coding sequence of Netrin-A was cloned, amplified in the pTG19 vector, and then cloned in the pFastBac HTA vector. It was then transformed into DH10Bac, and the recombinant Bacmid was subsequently transfected into Sf9 cells. The recombinant Netrin-A was purified by Ni-NTA agarose. The evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay. Results: The molecular weight of this protein was 52 kDa with 404 amino acids. The signal peptide was located between amino acids 24 and 25. The concentration of Netrin-A was calculated to be 48.8 µg/ml. It reaffirmed the characterized gene codes of Lucilia sericata Netrin-A in a previous study. Conclusions: The generation of recombinant Netrin-A could be used in larval therapy, and as a biomarker in certain diseases. The netrin-A of Lucilia sericata was unprecedentedly cloned and expressed in a eukaryotic cell line. Given that this larva is FDA-approved, and non-pathogenic, it conduces to research on the development of maggot therapy in future.

Nitrate concentrations and health risks in cow milk from Iran: Insights from deterministic, probabilistic, and AI modeling.

Excessive nitrate consumption has been linked to potential health risks in humans. Thus, understanding nitrate levels in staple foods such as cow milk can provide insights into their health implications. This study meticulously examined nitrate concentrations in 70 cow milk samples from traditional and industrialized cattle farming systems in Fars province, Iran. A combination of deterministic modeling, a probabilistic approach, and six artificial intelligence algorithms was employed to determine health risk assessments. The data disclosed average nitrate concentrations of 32.63 mg/L in traditional farming and 34.95 mg/L in industrialized systems, presenting no statistically significant difference (p > 0.05). The Hazard Quotient (HQ) was deployed to gauge potential health threats, underscoring heightened vulnerability in children, who exhibited HQ values ranging from 0.05 to 0.58 (mean = 0.19) in contrast to adults, whose values spanned 0.01 to 0.16 (mean = 0.05). Monte Carlo simulations enriched the risk assessment, demarcating the 5th and 95th percentile nitrate concentrations for children at 0.07 and 0.39, respectively. In children, pivotal interactions that influenced HQ encompassed those between nitrate concentration and consumption rate, as well as nitrate concentration and body weight. The interplay between nitrate concentration and consumption rate was most consequential for the adult cohort. Among the algorithms assessed for HQ prediction, Gaussian Naive Bayes (GNB) was optimal for children and eXtreme Gradient Boosting (XGB) for adults, with nitrate concentration being a key determinant. The results underscore the imperative for rigorous oversight of milk nitrate concentrations, highlighting the enhanced susceptibility of children and emphasizing the need for preventive strategies and enlightened consumption.

Modeling, quality assessment, and Sobol sensitivity of water resources and distribution system in Shiraz: A probabilistic human health risk assessment.

Given water's vital role in supporting life and ecosystems, global climate change and human activities have significantly diminished its availability and quality. This study explores the health risks of drinking water consumption in the shiraz county water resources and distribution system. The result showed that the water was slightly alkaline. However, the average pH values during the study were within the permissible range. The area's abundance of total hardness and calcium was due to the high concentration of minerals in rocks and soils. The nitrate and fluoride concentrations in drinking groundwater varied from 0.02 to 116.70 mg/L and 0.10-1.85 mg/L, respectively. Although the water quality index indicated that 52.63, 45.03, and 20.3 percent of samples were of excellent, good, and poor quality in 2020, those percentages obtained 46.05, 52.09, and 14.0 percent in 2021. The regression values of training, testing, validation, and the proposed artificial neural network model were 0.93, 0.92, 0.85, and 0.92. The maximum levels of hazard quotient of nitrate and fluoride (except for adults) were higher than 1 in all age groups, indicating a high non-carcinogenic risk by exposure to nitrate. Furthermore, according to the Monte Carlo simulation, the 95th percentile hazard index in all groups was more than 1. Children and infants were more inclined towards risk than teens and adults based on the intake of nitrate and fluoride from drinking water. The Sobol sensitivity reflected that the nitrate concentration and ingestion rate are vital parameters that influence the outcome of the oral exposure model for all age groups. The interaction of ingestion rate with a concentration of nitrate and fluoride is an important parameter affecting the health risk assessment. In conclusion, these findings suggest that precise measures can reduce health risks and guarantee safe drinking water for residents of Shiraz County.

Entomological survey of malaria vectors in Dashtestan County, South of Iran.

Anopheline mosquitoes are responsible for transmission of some diseases such as malaria. This study was carried out in five villages of Dashtestan county, Bushehr province, south of Iran with mountainous and plain areas. Anopheles larvae were sampled once a month from May to July 2021 by dipping method using standard dippers. Adults were captured by the total catch technique. In this study, 1062 Anopheles mosquitoes were collected including 850 adults and 212 larvae. Samples were A. superpictus, A. stephensi, A. dthali and A. fluviatilis. The dominant species at all sites (larvae and adults) were A. dthali (31.35%), A. superpictus (28.93%), A. stephensi (27.77%), and A. fluviatilis (11.95%), respectively. Among adults, A. stephensi was the most frequent species, but among larvae, A. dthali was the dominant species. The highest number of A. dthali was captured from Dalaki village with 35%, while A. superpictus and A. fluviatilis were not caught at this station. The minimum collected adults of all species occurred in Bashirabad. This result showed that A. stephensi plays an important role in south Iran, which is under the elimination phase. The fauna and niches of Anopheles has different patterns depending on ecological, climatic, and topographic features. These items affect host preferences, feeding behaviors, and distribution of these species. A monthly or annual entomological survey is necessary in regions with mobile populations since imported malaria is a problematic issue in the elimination programs of Iran.

First molecular detection of SARS-CoV-2 virus in cockroaches.

Coronavirus is one of the main pathogens that primarily targets the human respiratory system. There are several ways to transmit this virus, such as direct contact or droplets spread by coughing or sneezing, and direct contact with fomites and surfaces is another way. This cross-sectional study was conducted in Shiraz, southern Iran, in 2021. 5 locations, including 3 hospitals and 2 dormitories, were selected for the survey. The cockroaches were collected from selected locations and transferred to the Laboratory of Medical Entomology at Shiraz University of Medical Sciences. All specimens were identified morphologically. The external and gastrointestinal washouts of collected samples with sterile phosphate-buffered saline separately were used for molecular analysis. An RT-qPCR assay, which suggests the possible insect­borne transmission, was used. External and gastrointestinal washout of B. germanica from Dastgheyb Dormitory and P. americana from Ali-Asghar Hospital were positive for contamination with the SARS-CoV-2. Cockroaches spread the virus in the environment and contaminate human food and various surfaces of buildings. Their role will be more important in crowded places such as hotels, lodging houses, restaurants, and hospitals; vector control programs should be carried out with more accuracy in such places.

Molecular Characterization and In Silico Analyses of Maurolipin Structure as a Secretory Phospholipase A 2 (sPLA2) from Venom Glands of Iranian Scorpio maurus (Arachnida: Scorpionida).

The venom is a mixture of various compounds with specific biological activities, such as the phospholipase A 2 (PLA2) enzyme present in scorpion venom. PLA2 plays a key role in inhibiting ryanodine receptor channels and has neurotoxic activity. This study is the first investigation of molecular characterization, cloning, and in silico analyses of PLA2 from Iranian Scorpio maurus, named Maurolipin. After RNA extraction from S. maurus venom glands, cDNA was synthesized and amplified through RT-PCR using specific primers. Amplified Maurolipin was cloned in TA cloning vector, pTG19. For in silico analyses, the characterized gene was analyzed utilizing different software. Maurolipin coding gene with 432 base pair nucleotide length encoded a protein of 144 amino acid residues and 16.34 kilodaltons. Comparing the coding sequence of Maurolipin with other characterized PLA2 from different species of scorpions showed that this protein was a member of the PLA2 superfamily. According to SWISS-MODEL prediction, Maurolipin had 38.83% identity with bee venom PLA2 with 100% confidence and 39% identity with insect phospholipase A 2 family, which Phyre2 predicted. According to the three-dimensional structure prediction, Maurolipin with five disulfide bonds has a very high similarity to the structure of PLA2 that belonged to the group III subfamily. The in silico analyses showed that phospholipase A 2 coding gene and protein structure is different based on scorpion species and geographical condition in which they live.

Effects of probiotic and magnesium co-supplementation on mood, cognition, intestinal barrier function and inflammation in individuals with obesity and depressed mood: A randomized, double-blind placebo-controlled clinical trial.

Background: The co-occurrence of obesity and mood impairments named as "metabolic mood syndrome" (MMS) is often neglected in the obesity management. This study aimed to evaluate effects of Probio-Tec ®BG-VCap-6.5 and magnesium co-supplementation on mood, cognition, intestinal barrier function and serum C reactive protein (CRP) levels in participants with obesity and depressed mood. Design: Seventy-four eligible participants were randomly allocated to either Probio-Tec®BG-VCap-6.5 [containing Lactobacillus rhamnosus (LGG®) and Bifidobacterium animalis subsp. Lactis (BB-12®)] + Magnesium chloride or placebo for 9 weeks. Sociodemographic data were collected in the beginning. Anthropometric, dietary and physical activity (PA) assessments were carried out. Beck Depression Inventory-II (BDI-II) and Montreal Cognitive Assessment (MoCA) scores were assessed through validated questionnaires. Fasting plasma zonulin, lipopolysaccharide (LPS) and (CRP) were measured by ELIZA kits. Results: Of seventy-four participants (mean age 37.51 ± 8.10), 52 completed the study. Changes in serum LPS and zonulin were not different significantly between groups (-3.04 ± 44.75 ng/dl, 0.11 ± 5.13, ng/dl, p > 0.05 for LPS and 1.40 ± 48.78 ng/dl, -0.17 ± 6.60, p > 0.05 for zonulin, respectively). CRP levels reduced significantly in intervention group compared to placebo [-474.75 (-1,300.00, -125.00) mg/l vs. 175.20 (-957.75, 1,683.25) mg/l, p = 0.016]. Changes in BDI-II and MoCA scores were not significantly different between intervention (-7.13 ± 5.67, 1.20 ± 2.16, respectively) and placebo (-5.42 ± 6.71, 1.94 ± 1.86, respectively) groups (p > 0.05). Conclusion: Nine weeks of probiotic and magnesium co-supplementation resulted in decreased CRP levels as an indicator of inflammatory state with no significant effects on mood, cognition and intestinal integrity in individuals with obesity and depressed mood.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Therapeutic Potential of Bee and Scorpion Venom Phospholipase A2 (PLA2): A Narrative Review.

Venomous arthropods such as scorpions and bees form one of the important groups with an essential role in medical entomology. Their venom possesses a mixture of diverse compounds, such as peptides, some of which have toxic effects, and enzymatic peptide Phospholipase A2 (PLA2) with a pharmacological potential in the treatment of a wide range of diseases. Bee and scorpion venom PLA2 group III has been used in immunotherapy, the treatment of neurodegenerative and inflammatory diseases. They were assessed for antinociceptive, wound healing, anti-cancer, anti-viral, anti-bacterial, anti-parasitic, and anti-angiogenesis effects. PLA2 has been identified in different species of scorpions and bees. The anti-leishmania, anti-bacterial, anti-viral, and anti-malarial activities of scorpion PLA2 still need further investigation. Many pieces of research have been stopped in the laboratory stage, and several studies need vast investigation in the clinical phase to show the pharmacological potential of PLA2. In this review, the medical significance of PLA2 from the venom of two arthropods, namely bees and scorpions, is discussed.

Mosquito surveillance and the first record of morphological and molecular-based identification of invasive species Aedes (Stegomyia) aegypti (Diptera: Culicidae), southern Iran.

Ae. aegypti is an important vector for transmission of some dangerous arboviral diseases, including Dengue Fever. The present study was conducted (from August 2017 to January 2020) to survey the fauna of Culicine mosquitoes, emphasizing the existence of this invasive species in oriental parts of the country located near the Persian Gulf. Different sampling methods were used to collect all life stages of the mosquito. After morphological identification, a molecular study based on Cytochrome Oxidase (COI) gene-specific primers was performed. Then, the COI gene was sequenced via the Sanger method. A total of 4843 adults and 11,873 larvae were collected (8 species of Culex, one species of Culiseta, and 5 species of Aedes). Fifty-five Ae. aegypti specimens (8 adults and 47 larvae) were identified. Based on the biology and ecological requirements of Ae. aegypti, the possibility of the permanent establishment of this species in the tropical climate of the region is very likely. Considering the detection of this invasive vector mosquito species in Iran and the high incidence of some arboviral diseases in the neighboring countries, and continuous movements of the settlers of these areas, potential outbreaks of arboviral diseases can be predicted. Planning and implementing an immediate surveillance and control program of the vector mosquito is vital to prevent the permanent establishment of this invasive vector mosquito species in southern Iran.

Monitoring of synthetic insecticides resistance and mechanisms among malaria vector mosquitoes in Iran: A systematic review.

BACKGROUND: In Iran, the prospect of malaria control relies mainly on insecticides used against the genus Anopheles (Diptera: Culicidae) as important vectors of malaria, arboviruses, and so on. Only eight out of 30 malaria mosquito vectors (Anopheles species) have been examined for insecticide resistance in Iran. This study aimed to review articles related to the incremental trend in insecticide resistance and their mechanisms among anopheline malaria vectors in Iran. METHODS: A literature review was conducted based on such search engines as Iran doc, Web of Science, SID, PubMed, Scopus, and Google Scholar websites using the following keywords: "Anopheles," "Malaria," "Resistance," "Vectors," "Insecticide Resistance," and "Iran" for data collection. Published papers in English or Persian covering 1980 to 2020 were reviewed. RESULTS: A total of 1125 articles were screened, only 16 of which were filtered to be pertinent in this review. While most of the mosquito vectors of malaria, such as Anopheles stephensi, were resistant to DDT, dieldrin, malathion, and becoming less susceptible to deltamethrin and other synthetic pyrethroid insecticides, few like Anopheles fluviatilis s. l. were susceptible to all insecticides. A disseminating trend in insecticide resistance among different anopheline mosquito vector species was evident. Metabolic and insecticide target-site resistance mechanisms were involved with organochlorines and pyrethroids, respectively. CONCLUSIONS: Insecticide resistance is becoming a severe scourge to the effectiveness of vector-borne disease management measures. This event is especially critical in developing and marginalized communities that applied chemical-based vector elimination programs for malaria; therefore, it is crucial to monitor insecticide resistance in malaria vectors in Iran using biochemical and molecular tools.

Phytochemical Composition and Bioassay on Iranian Teucrium Polium Extracts against Anopheles Stephensi (Diptera: Culicidae).

Background: Anopheles stephensi is an important malaria vector mosquito in Iran and other western Asian countries. In many human communities, plant products have been used traditionally instead of synthetic pesticides for mosquito control due to their minimal hazardous effects. Teucrium polium, known popularly as felty germander, has been introduced in Persian Medicine (PM) as an insect repellent from a long time ago. Methods: The present study was undertaken to evaluate repellent and larvicidal activity of dichloromethane (DCMETP) and ethanolic extracts (EE-TP) of T. polium against An. stephensi under laboratory conditions. The possible chemical components of the extracts were also investigated through gas chromatography/mass spectrometry (GC-MS) technique. Results: Based on the results, DCME-TP showed better repellent activity than EE-TP with 56.67 and 28.33 % protection, respectively. Larvicidal activity of DCME-TP with 49.41% mortality was also higher than EE-TP (20.24%). The main identified constituents of DCME-TP were long chain alkanes, phenol, aromatic ester, oxaspiro and triterpenoid. While phenolic and aliphatic acid were only the identified components in EE-TP. It is notable that lupeol was detected in DCME of T. polium for the first time. Conclusion: DCME-TP can be considered as a new herbal candidate to control An. stephensi mosquitoes. Further studies are required on this extract for the fractionation and identification of the active compounds, and the evaluation of their bioactivity in the laboratory and field.

Frequency of pyrethroid resistance in human head louse treatment: systematic review and meta-analysis.

Head lice (Pediculus humanus capitis) are one of the most common insects causing infestations in humans worldwide, and infestation is associated with adverse socio-economic and public health effects. The development of genetic insensitivity (e.g., target site insensitivity = knockdown resistance or kdr) to topical insecticides has impaired effective treatment. Therefore, this study was undertaken to review and meta-analyze the frequency of pyrethroid resistance in treated head louse populations from the beginning of 2000 to the end of June 2021 worldwide. In order to accomplish this, all English language articles published over this period were extracted and reviewed. Statistical analyses of data were performed using fixed and random effect model tests in meta-analysis, Cochrane, meta-regression and I2 index. A total of 24 articles from an initial sample size of 5033 were accepted into this systematic review. The mean frequency of pyrethroid resistance was estimated to be 76.9%. In collected resistant lice, 64.4% were homozygote and 30.3% were heterozygote resistant. Globally, four countries (Australia, England, Israel, and Turkey) have 100% kdr gene frequencies, likely resulting in the ineffectiveness of pyrethrin- and pyrethroid-based pediculicides. The highest resistance recorded in these studies was against permethrin. This study shows that pyrethroid resistance is found at relatively high frequencies in many countries. As a result, treatment with current insecticides may not be effective and is likely the cause of increased levels of infestations. It is recommended that resistance status be evaluated prior to insecticide treatment, to increase efficacy.

TITLE: Fréquence de la résistance aux pyréthroïdes dans le traitement du pou de tête chez l'homme : revue systématique et méta-analyse. ABSTRACT: Les poux de tête (Pediculus humanus capitis) sont l'un des insectes les plus courants à l'origine d'infestations chez l'homme dans le monde, et l'infestation est associée à des effets socio-économiques et de santé publique néfastes. Le développement d'une insensibilité génétique (par exemple, l'insensibilité au site cible = résistance knockdown ou kdr) aux insecticides topiques a altéré l'efficacité de leur traitement. Par conséquent, cette étude a été entreprise pour examiner et méta-analyser la fréquence de la résistance aux pyréthroïdes dans les populations de poux de tête étudiées du début 2000 à la fin juin 2021 dans le monde. Pour ce faire, tous les articles en anglais publiés au cours de cette période ont été extraits et examinés. Les analyses statistiques des données ont été effectuées à l'aide de tests de modèles à effets fixes et aléatoires dans la méta-analyse, Cochrane, méta-régression et indice I2. Un total de 24 articles provenant d'un échantillon initial de 5033 ont été acceptés dans cette revue systématique. La fréquence moyenne de la résistance aux pyréthroïdes a été estimée à 76,9 %. Chez les poux résistants collectés, 64,4 % étaient homozygotes résistants et 30,3 % étaient hétérozygotes résistants. À l'échelle mondiale, quatre pays (Australie, Angleterre, Israël et Turquie) ont des fréquences de gène kdr de 100 %, ce qui entraîne probablement une inefficacité des pédiculicides à base de pyréthrine et de pyréthrinoïde. La résistance la plus élevée enregistrée dans ces études était celle contre la perméthrine. Cette étude montre que la résistance aux pyréthroïdes est trouvée à des fréquences relativement élevées dans de nombreux pays. En conséquence, le traitement avec les insecticides actuels peut ne pas être efficace et est probablement la cause d'une augmentation des niveaux d'infestation. Il est recommandé d'évaluer le statut de résistance avant le traitement insecticide, pour augmenter son efficacité.

In vitro evaluation of CRISPR PX-LmGP63 vector effect on pathogenicity of Leishmania major as a primary step to control leishmaniasis.

Cutaneous leishmaniasis (CL) is caused by intracellular obligate parasites (Leishmania spp.) carried by the blood-sucking of female sandflies and transmitted between mammalian hosts. Despite the high incidence and prevalence of Leishmania cases in many countries, it has been a neglected tropical disease. The current treatment approaches are limited by the complications such as loss of fertility and drug resistance. It is, therefore, essential to find new medicines to treat leishmaniasis. CRISPR/Cas9 as a powerful genome-editing tool provides the opportunity to create precise genetic manipulation to investigate the molecular basis of different leishmaniasis cases. Therefore, our main goal was to evaluate the CRISPR PX-LmGP63 vector effect on pathogenicity of Leishmania majorin vitroto challenge for using CRISPR/Cas9 as a therapeutic CL through the reduction of L. major pathogenicity by manipulating the GP63 gene. In this study, L. major parasites were transfected with CRISPR/Cas9 vectors constructed by electroporation and then added to macrophage cells on RPMI. The effect of CRISPR/Cas9 constructs on GP63 mutation, viability, and status of L. major was investigated by counting phagocytic parasites into macrophages and DNA sequence analysis. Our data validate that the use of CRISPR/Cas9 in L. major creates a new stop codon and disrupts the frame sheet of the gene by creating a new insertion (thymine), which prevents its expression. In addition, the parasite count was significantly different in the case and control of infected macrophages (P < 0.05). This study shows the successfully targeted manipulation of the L. major GP63 gene via the adaptation of the CRISPR/Cas9 editing tool. The manipulation of GP63 revealed a reduction in the infection load compared to wild-type parasite infection. Therefore, more studies are necessary for this field to help achieve a new method for the prevention and treatment of CL disease.

Potential co-infection of Wolbachia with Leishmania among sand fly vectors caught from endemic leishmaniasis foci in Fars province, southern Iran.

Leishmaniasis is one of the Neglected Tropical Diseases in the tropical region of many countries in the world. The etiological agents (Leishmania parasites) of the disease are transmitted to human and other vertebrate hosts by infectious bites of female phlebotomine sand flies. On the other hand, some symbiotic microorganisms such as Wolbachia (Rickettsiales: Anaplasmataceae) may be transmitted vertically in many arthropods and may cause synergistic or antagonistic effects on epidemiology of the vector-borne diseases. Hence, in the present study, potential coinfection of Wolbachia with Leishmania in the sand fly vectors will be examined by PCR technique in the important leishmaniasis foci of Fars province in southern Iran, as a new feature for the disease long-term control. Sand flies were collected by sticky traps from indoor and outdoor locations of 5 different areas of Fars province during 2018 and 2019. DNAs of sand flies were extracted and PCR method was performed based on primers which were designed from surface proteins (WSP) genome region for Wolbachia and minicircle kDNA gene for Leishmania detections. At last, PCR products were sequenced and recorded in the GenBank. Out of 1002 sand flies caught from 5 different foci of Fars province, 909 male and female and 386 female sand flies' DNAs were extracted for detection of Wolbachia and Leishmania by PCR, respectively. Accordingly, out of the total 44 pools prepared from sand flies, 6 out of 28 pools form P. papatasi female were positive for Wolbachia in Shiraz, Marvdasht and Kharameh. However, none of female sand flies were positive for Leishmania. The study also focused on monitoring of sand flies co-infection of Wolbachia with Leishmania, which was not found in any of the studied samples. The negative results may be due to control strategies implemented which were done against Leishmaniasis in the studied areas during last years.

Association between genotoxic properties of inhalation anesthetics and oxidative stress biomarkers.

Exposure to inhalation anesthetics (IAs) has been associated with DNA damage as reflected in the increased frequency of micronuclei (MN) and chromosomal aberrations (CAs). The present study was undertaken to ascertain whether there was any correlation between increased MN and CA and the extent of oxidative stress as well as the antioxidant status of a group of operating room personnel exposed to a mixture of IAs, including nitrous oxide, isoflurane, and sevoflurane. In this cross-sectional study, 60 operating room personnel (exposed group) in whom the frequencies of MN and CA had already been shown to be significantly higher than those of a referent group, as well as 60 unexposed nurses, were studied. Venous blood samples were taken from all participants, and malondialdehyde (MDA) levels as an index of oxidative stress (OS) and the activity of superoxide dismutase (SOD) and levels of total antioxidant capacity (TAC) as indices of antioxidant status were measured. The level of TAC (1.76 ± 0.59 mM vs. 2.13 ± 0.64 mM, p = 0.001) and the activity of SOD (11.22 ± 5.11 U/ml vs. 13.36 ± 4.12 U/ml, p = 0.01) were significantly lower, while the mean value of MDA was significantly higher (2.46 ± 0.66 µM vs. 2.19 ± 0.68 µM, p = 0.03) in the exposed group than in the nonexposed group. After adjusting for potential confounders, there were statistically significant associations between exposure to IAs, gender, SOD, and TAC with MN frequency and between exposure to IAs and SOD with numbers of CA. The findings of the present study indicated that exposure to IAs was associated with OS, and this, in turn, may be causally linked with DNA damage.

Wound healing potential: evaluation of molecular profiling and amplification of Lucilia sericata angiopoietin-1 mRNA mid-part.

OBJECTIVE: High prevalence of chronic ulcers and the burden of disease necessitate the increasingly significant production of new recombinant proteins in the world. The angiopoietin-1 enzyme is a part of the growth factors group which is secreted by Lucilia sericata (Diptera: Calliphoridae) larvae when they meet lesions to ensure maggot therapy. It is one of the most potent proteins in wound healing. Given its essential role, the angiopoietin-1 gene of L. sericata was characterized, which provided some necessary information on its identity. RESULTS: The mid-part of the angiopoietin-1 mRNA sequence was thus characterized based on the design of different primers such as exon-exon junction, conserved regions, and specific region primers via conventional polymerase chain reaction (PCR). Its structural features were configured by in silico method. The sequence of mid-part (390 bp) of angiopoietin-1 was determined empirically, and BLAST analysis unraveled its high identity (85%) with the sequence of angiopoietin-1 mRNA of the larval housefly, Musca domestica. The homology of this enzyme also exhibited that its nucleic acid sequence was very similar to the domains of angiopoietin-1 in Lucilia cuprina. The current data are instructive and critical to evaluate the action of this enzyme in recombinant protein production in future molecular studies on wound healing.

Molecular characterization of the netrin-1 UNC-5 receptor in Lucilia sericata larvae.

Larval therapy with Lucilia sericata is a promising strategy in wound healing. Axon guidance molecules play vital roles during the development of the nervous system and also regulate the capacity of neuronal restoration in wound healing. Netrin-1, one of the proteins that larvae secrete, plays a useful role in cell migration and nerve tissue regeneration. The UNC-5 receptor combines with a netrin-1 signal and transmits the signal from one side of the membrane to the other side, initiating a change in cell activity. In the current study, we identified the full length of the UNC-5 receptor mRNA in L. sericata using different sets of primers, including exon junction and specific region primers. The coding sequence (CDS) of the UNC-5 receptor was sequenced and identified to include 633 base-pair nucleic acids, and BLAST analysis on its nucleotide sequence revealed 96% identity with the Lucilia cuprina netrin-1 UNC-5 receptor. The protein residue included 210 amino acids (aa) and coded for a protein with 24 kD weight. This gene lacked the signal peptide. Furthermore, the UPA domain is conserved in UNC-5. It lied at the interval of 26-131 aa. We identified the CDS of netrin-1 UNC-5 receptor in L. sericata. It could be applied to research activities implementing a new essential component design in wound healing.

Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing.

BACKGROUND: Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia Sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy. OBJECTIVES: In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac). MATERIALS AND METHODS: cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose. RESULTS: The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL-1 based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme. CONCLUSION: Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future.

Genotoxicity of inhalational anesthetics and its relationship with the polymorphisms of GSTT1, GSTM1, and GSTP1 genes.

Due to their wide applications, concern exists regarding possible genotoxic effects of inhalational anesthetics (IAs) among operating room personnel. This study was undertaken to examine genotoxic properties of co-exposure to nitrous oxide, sevoflurane, and isoflurane on induction of micronucleus (MN) and chromosomal aberrations (CAs) and to determine whether any associations exist between polymorphisms of GST genes and the level of genomic damage measured by MN and CAs assays. Sixty operating room personnel and 60 unexposed referent nurses were studied. The workers' exposure to the IAs was determined. DNA damage was evaluated by MN and CAs assays. Additionally, the GSTM1, GSTT1, and GSTP1 polymorphisms were detected. The mean concentrations of nitrous oxide, isoflurane, and sevoflurane were found to be 850.92 ± 919.78, 2.40 ± 0.86, and 0.18 ± 0.14 ppm, respectively. The frequency of MN and CAs in the exposed group was significantly higher than that of the non-exposed group. The frequency of MN was significantly higher in referent nurses with null GSTT1, compared to referent nurses with positive GSTT1. The frequency of MN was significantly higher in exposed individuals carrying the combined genotype of GSTT1 (-), GSTM1 (-), and GSTP1 AG as compared with subjects carrying a combination of GSTT1 (+), GSTM1 (+), and GSTP1 AA. Statistically significant associations were noted between exposure to the IAs, gender, and the combination of the three GSTs genotypes with MN frequency. These findings indicate that inhalation exposure to IAs induces genotoxic response and the polymorphisms of GSTs genes might modulate the effect of exposure to IAs on MN.