Search results for: genetic circuit

Authors: Banafsheh Nikmehr, Mohsen Bahrami, Yueqiang Song, Anuradha Koduru, Ayse K. Vuruskan, Hongkun Lu, Mallory Pitts, Tolga B. Mesen, Tamer M. Yalcinkaya

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The number of people who receive in vitro fertilization (IVF) treatment has increased on a startling trajectory over the past two decades. Despite advances in this field, particularly the introduction of intracytoplasmic sperm injection (ICSI) and the preimplantation genetic screening (PGS), the IVF success remains low. A major factor contributing to IVF failure is embryonic aneuploidy (abnormal chromosome content), which often results in miscarriage and birth defects. Although PGS is often used as the standard diagnostic tool to identify aneuploid embryos, it is an invasive approach that could affect the embryo development, and yet inaccessible to many patients due its high costs. As such, there is a clear need for a non-invasive cost-effective approach to identify euploid embryos for single embryo transfer (SET). The reported differences between morphokinetic behaviors of aneuploid and euploid embryos has shown promise to address this need. However, current literature is inconclusive and further research is urgently needed to translate current findings into clinical diagnostics. In this ongoing study, we found significant differences between morphokinetic behaviors of euploid and aneuploid embryos that provides important insights and reaffirms the promise of such behaviors for developing non-invasive methodologies. Methodology—A total of 242 embryos (euploid: 149, aneuploid: 93) from 74 patients who underwent IVF treatment in Carolinas Fertility Clinics in Winston-Salem, NC, were analyzed. All embryos were incubated in an EmbryoScope incubator. The patients were randomly selected from January 2019 to June 2021 with most patients having both euploid and aneuploid embryos. All embryos reached the blastocyst stage and had known PGS outcomes. The ploidy assessment was done by a third-party testing laboratory on day 5-7 embryo biopsies. The morphokinetic variables of each embryo were measured by the EmbryoViewer software (Uniesense FertiliTech) on time-lapse images using 7 focal depths. We compared the time to: pronuclei fading (tPNf), division to 2,3,…,9 cells (t2, t3,…,t9), start of embryo compaction (tSC), Morula formation (tM), start of blastocyst formation (tSC), blastocyst formation (tB), and blastocyst expansion (tEB), as well as intervals between them (e.g., c23 = t3 – t2). We used a mixed regression method for our statistical analyses to account for the correlation between multiple embryos per patient. Major Findings— The average age of the patients was 35.04 yrs. The average patient age associated with euploid and aneuploid embryos was not different (P = 0.6454). We found a significant difference in c45 = t5-t4 (P = 0.0298). Our results indicated this interval on average lasts significantly longer for aneuploid embryos - c45(aneuploid) = 11.93hr vs c45(euploid) = 7.97hr. In a separate analysis limited to embryos from the same patients (patients = 47, total embryos=200, euploid=112, aneuploid=88), we obtained the same results (P = 0.0316). The statistical power for this analysis exceeded 87%. No other variable was different between the two groups. Conclusion— Our results demonstrate the importance of morphokinetic variables as potential biomarkers that could aid in non-invasively characterizing euploid and aneuploid embryos. We seek to study a larger population of embryos and incorporate the embryo quality in future studies.

Keywords: IVF, embryo, euploidy, aneuploidy, morphokinteic

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Authors: Julia Kapr, Andrea Rossi, Haribaskar Ramachandran, Marius Pollet, Ilka Egger, Selina Dangeleit, Katharina Koch, Jean Krutmann, Ellen Fritsche

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It is widely acknowledged that animal models do not always represent human disease. Especially human brain development is difficult to model in animals due to a variety of structural and functional species-specificities. This causes significant discrepancies between predicted and apparent drug efficacies in clinical trials and their subsequent failure. Emerging alternatives based on 3D in vitro approaches, such as human brain spheres or organoids, may in the future reduce and ultimately replace animal models. Here, we present a human induced pluripotent stem cell (hiPSC)-based 3D neural in a vitro disease model for the Cockayne Syndrome B (CSB). CSB is a rare hereditary disease and is accompanied by severe neurologic defects, such as microcephaly, ataxia and intellectual disability, with currently no treatment options. Therefore, the aim of this study is to investigate the molecular and cellular defects found in neural hiPSC-derived CSB models. Understanding the underlying pathology of CSB enables the development of treatment options. The two CSB models used in this study comprise a patient-derived hiPSC line and its isogenic control as well as a CSB-deficient cell line based on a healthy hiPSC line (IMR90-4) background thereby excluding genetic background-related effects. Neurally induced and differentiated brain sphere cultures were characterized via RNA Sequencing, western blot (WB), immunocytochemistry (ICC) and multielectrode arrays (MEAs). CSB-deficiency leads to an altered gene expression of markers for autophagy, focal adhesion and neural network formation. Cell migration was significantly reduced and electrical activity was significantly increased in the disease cell lines. These data hint that the cellular pathologies is possibly underlying CSB. By induction of autophagy, the migration phenotype could be partially rescued, suggesting a crucial role of disturbed autophagy in defective neural migration of the disease lines. Altered autophagy may also lead to inefficient mitophagy. Accordingly, disease cell lines were shown to have a lower mitochondrial base activity and a higher susceptibility to mitochondrial stress induced by rotenone. Since mitochondria play an important role in neurotransmitter cycling, we suggest that defective mitochondria may lead to altered electrical activity in the disease cell lines. Failure to clear the defective mitochondria by mitophagy and thus missing initiation cues for new mitochondrial production could potentiate this problem. With our data, we aim at establishing a disease adverse outcome pathway (AOP), thereby adding to the in-depth understanding of this multi-faced disorder and subsequently contributing to alternative drug development.

Keywords: autophagy, disease modeling, in vitro, pluripotent stem cells

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Authors: Simone Raimondi De Souza, Glaucia Maria Moraes De Oliveira, Ronir Raggio Luiz, Glorimar Rosa

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Introduction: Obesity is among the main risk factors for cardiovascular disease (CVD). Genesis is multifactorial, including genetic, hormonal and environmental factors disorders, among which inadequate feeding pattern, for which nutritional counseling strategies have proven effective. The consumption of beta-glucans (soluble fibers that reportedly promote satiety) present in oat bran can be an effective strategy for preventing and treating obesity. Other benefits have been observed with oat bran consumption, such as reduction of hypercholesterolemia and hyperglycemia, two other risk factors for CVD. Objectives: To analyze the effect of oat bran consumption associated with nutritional counseling in reducing body mass index (BMI), blood cholesterol, glucose profile, waist and neck circumference in obese individuals, and to evaluate the change in eating pattern. Methods: clinical trial, randomized, double-blind, placebo-controlled, lasting 90 days with adults of both genders, with BMI ≥30kg/m2. The study was approved by the Ethics in Research involving human beings in a public institute of cardiology, in Rio de Janeiro, Brazil. Individuals were invited to participate and accepted formally by signing the Terms of Consent. Participants were randomized into oat bran group (gOB) or placebo group (gPCB) and received, respectively: morning prepared consisting of 40g oat bran, 30g of skimmed milk powder and 1g sweetener sucralose; refined flour 40g rice, 30g of milk powder and 1g sweetener sucralose. The Ten Steps to Healthy Eating, of Brazilian Ministry of Health were used to support the nutritional counseling. Variables analyzed: gender; age; BMI, waist circumference (WC) neck circumference (NC); systolic blood pressure (SBP); diastolic blood pressure (DBP); food consumption, total cholesterol (TC), LDL-cholesterol (LDL-c), HDL-cholesterol (HDL-c), non-HDL cholesterol (nHDLc), triglycerides (TG), fasting glucose (FG), fasting insulin (FI) and HOMA-IR. Dietary intake was assessed by 24-hour dietary recall. The Diet Quality Index revised for the Brazilian population (IQD-R) assessed quality of feeding pattern. Statistical analyzes were performed using SPSS version 21, considering statistically significant p-value less than 0.05. Results: A total of 38 participants were included, age = 50 ± 7,6years, 63% women. 19 subjects were placed in gOB and 19 in gPCB. After intervention, statistically significant reductions were observed in the following parameters: in gOB: IQD-R, TC, LDL-c, nHDL-c, FI, SBP, DBP, BMI, WC, NC; in gPCB: IQD-R, LDL-c, SBP, DBP, BMI, WC, NC. No statistically significant differences were observed in the results between groups. Conclusion: Our results reinforce nutritional counseling as important strategy for prevention and treatment of obesity and suggest that inclusion of oat bran in daily diet can bring additional benefits controlling risk factors for CVD. More studies are needed to establish all benefits of oat bran to human health as well as the ideal daily dose for consumption.

Keywords: oat bran, cardiovascular disease, nutritional counseling, obesity

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Authors: Nisha Sharma, Mili Kaur, Ashutosh Halder, Seema Kaushal, Manoj Kumar, Manish Jain

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Purpose: To investigate microRNAs (miRNA) as an epigenomic etiological factor in idiopathic non-obstructive azoospermia (NOA). In order to achieve the same, an association was seen between occupational exposure to radiation, thermal, and chemical factors and idiopathic cases of non-obstructive azoospermia, and later, testicular differential miRNA expression profiling was done in exposure group NOA cases. Method: It is a prospective study in which 200 apparent idiopathic male factor infertility cases, who have been advised to undergo testicular fine needle aspiration (FNA) evaluation, are recruited. A detailed occupational history was taken to understand the possible type of exposure due to the nature and duration of work. A total of 26 patients were excluded upon XY-FISH and Yq microdeletion tests due to the presence of genetic causes of infertility, 6 hypospermatogeneis (HS), six Sertoli cell-only syndrome (SCOS), and six normospermatogeneis patients testicular FNA samples were used for RNA isolation followed by small RNA sequencing and nCounter miRNA expression analysis. Differential miRNA expression profile of HS and SCOS patients was done. A web-based tool, miRNet, was used to predict the interacting compounds or chemicals using the shortlisted miRNAs with high fold change. The major limitation encountered in this study was the insufficient quantity of testicular FNA sample used for total RNA isolation, which resulted in a low yield and RNA integrity number (RIN) value. Therefore, the number of RNA samples admissible for differential miRNA expression analysis was very small in comparison to the total number of patients recruited. Results: Differential expression analysis revealed 69 down-regulated and 40 up-regulated miRNAs in HS and 66 down-regulated and 33 up-regulated miRNAs in SCOS in comparison to normospermatogenesis controls. The miRNA interaction analysis using the miRNet tool showed that the differential expression profiles of HS and SCOS patients were associated with arsenic trioxide, bisphenol-A, calcium sulphate, lithium, and cadmium. These compounds are reproductive toxins and might be responsible for miRNA-mediated epigenetic deregulation leading to NOA. The association between occupational risk factor exposure and the non-exposure group of NOA patients was not statistically significant, with ꭓ2 (3, N= 178) = 6.70, p= 0.082. The association between individual exposure groups (radiation, thermal, and chemical) and various sub-types of NOA is also not significant, with ꭓ2 (9, N= 178) = 15.06, p= 0.089. Functional analysis of HS and SCOS patients' miRNA profiles revealed some important miR-family members in terms of male fertility. The miR-181 family plays a role in the differentiation of spermatogonia and spermatocytes, as well as the transcriptional regulation of haploid germ cells. The miR-34 family is expressed in spermatocytes and round spermatids and is involved in the regulation of SSCs differentiation. Conclusion: The reproductive toxins might adopt the miRNA-mediated mechanism of disease development in idiopathic cases of NOA. Chemical compound induced; miRNA-mediated epigenetic deregulation can give a future perspective on the etiopathogenesis of the disease.

Keywords: microRNA, non-obstructive azoospermia (NOA), occupational exposure, hypospermatogenesis (HS), Sertoli cell only syndrome (SCOS)

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Authors: Kamyar Tolouei, Ehsan Moosavi

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In open-pit mines, the long-term production scheduling optimization problem (LTPSOP) is a complicated problem that contains constraints, large datasets, and uncertainties. Uncertainty in the output is caused by several geological, economic, or technical factors. Due to its dimensions and NP-hard nature, it is usually difficult to find an ideal solution to the LTPSOP. The optimal schedule generally restricts the ore, metal, and waste tonnages, average grades, and cash flows of each period. Past decades have witnessed important measurements of long-term production scheduling and optimal algorithms since researchers have become highly cognizant of the issue. In fact, it is not possible to consider LTPSOP as a well-solved problem. Traditional production scheduling methods in open-pit mines apply an estimated orebody model to produce optimal schedules. The smoothing result of some geostatistical estimation procedures causes most of the mine schedules and production predictions to be unrealistic and imperfect. With the expansion of simulation procedures, the risks from grade uncertainty in ore reserves can be evaluated and organized through a set of equally probable orebody realizations. In this paper, to synthesize grade uncertainty into the strategic mine schedule, a stochastic integer programming framework is presented to LTPSOP. The objective function of the model is to maximize the net present value and minimize the risk of deviation from the production targets considering grade uncertainty simultaneously while satisfying all technical constraints and operational requirements. Instead of applying one estimated orebody model as input to optimize the production schedule, a set of equally probable orebody realizations are applied to synthesize grade uncertainty in the strategic mine schedule and to produce a more profitable and risk-based production schedule. A mixture of metaheuristic procedures and mathematical methods paves the way to achieve an appropriate solution. This paper introduced a hybrid model between the augmented Lagrangian relaxation (ALR) method and the metaheuristic algorithm, the Harris Hawks optimization (HHO), to solve the LTPSOP under grade uncertainty conditions. In this study, the HHO is experienced to update Lagrange coefficients. Besides, a machine learning method called Random Forest is applied to estimate gold grade in a mineral deposit. The Monte Carlo method is used as the simulation method with 20 realizations. The results specify that the progressive versions have been considerably developed in comparison with the traditional methods. The outcomes were also compared with the ALR-genetic algorithm and ALR-sub-gradient. To indicate the applicability of the model, a case study on an open-pit gold mining operation is implemented. The framework displays the capability to minimize risk and improvement in the expected net present value and financial profitability for LTPSOP. The framework could control geological risk more effectively than the traditional procedure considering grade uncertainty in the hybrid model framework.

Keywords: grade uncertainty, metaheuristic algorithms, open-pit mine, production scheduling optimization

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Authors: Sharma Aseem, Dhurat Rachita, Pawar Varsha, Khalse Manisha

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Introduction: Androgenetic alopecia (AGA) is a prevalent condition characterized by progressive hair thinning driven by genetic and androgen-related factors. The current FDA-approved treatments include oral finasteride and topical minoxidil, though many patients seek combination therapies to enhance results. This study aims to evaluate the effectiveness of a combination therapy involving Minoxidil, Finasteride, and Capixyl™ based on feedback from dermatologists. Methodology: A survey, validated by experts, was distributed to 29 leading dermatologists across India (in Tier 1 and 2 cities). The survey examined real-world clinical experiences, focusing on patient outcomes and the overall effectiveness of the mentioned formulation. Results: Among the surveyed dermatologists, 41.4% identified women aged 35-40 as the most frequently diagnosed with female pattern hair loss. The combination therapy with Minoxidil, Finasteride, and Capixyl™ was utilized by 34.5% of dermatologists for over 60 patients per month. The majority highlighted the benefits of this combination therapy, which acts via multiple mechanisms, such as vasodilation and dihydrotestosterone (DHT) receptor blockade, resulting in improved hair regrowth. Additionally, patients demonstrated better clinical outcomes, enhanced compliance, and fewer side effects. Demographically, younger patients, particularly those with AGA for less than 10 years, responded more positively to the treatment. Early intervention led to quicker and more significant results. Overall satisfaction among dermatologists was high, with 86.2% expressing positive feedback on the therapy. In terms of treatment outcomes, 51.7% of dermatologists observed visible results within 4-6 months, while 34.5% noticed a significant reduction in hair fall within 8-12 weeks. Improvements in scalp health were reported by 48.3%, and 51.7% saw an increased hair density within 3-4 months. Despite mild side effects such as scalp irritation, dryness, flaking, and occasional issues like folliculitis, headaches, itching, and redness, patient satisfaction remained high. Dermatologists reported that 93.1% of patients experienced faster and better hair regrowth with Capixyl™ compared to Minoxidil alone. Suggestions for improving the formulation included incorporating peptides like Saw Palmetto and enhancing product packaging to better meet patient needs. Discussion: The combination of Minoxidil, Finasteride, and Capixyl™ yielded positive clinical outcomes, especially in improving hair density, scalp health, and overall patient satisfaction. Dermatologists found that Capixyl™ peptides enhanced the therapeutic effect, promoting hair regrowth and improving compliance. While side effects were generally mild, there were suggestions to further improve the formulation by adding additional peptides like Saw Palmetto. Conclusion: The combination of Minoxidil and Finasteride fortified with Capixyl™ presents a promising therapeutic option for managing AGA. Dermatologists reported significant improvements in hair density, scalp health, and patient satisfaction. With its favorable efficacy and manageable side effects, this formulation proves to be a valuable addition to the treatment landscape for AGA.

Keywords: androgenetic alopecia, combination therapy, minoxidil, finasteride, capixyl

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Authors: Arup Dutta, Badrul Alam, Sayed M. Wazed, Taslima Newaz, Srobonti Dutta

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Background: In today's world, with modern science and contemporary technology, a lot of diseases may be quickly identified and ruled out, but children's fever of unknown origin (FUO) still presents diagnostic difficulties in clinical settings. Any fever that reaches 38 °C and lasts for more than seven days without a known cause is now classified as a fever of unknown origin (FUO). Despite tremendous progress in the medical sector, fever of unknown origin, or FOU, persists as a major health issue and a major contributor to morbidity and mortality, particularly in children, and its spectrum is sometimes unpredictable. The etiology is influenced by geographic location, age, socioeconomic level, frequency of antibiotic resistance, and genetic vulnerability. Since there are currently no known diagnostic algorithms, doctors are forced to evaluate each patient one at a time with extreme caution. A persistent fever poses difficulties for both the patient and the doctor. This prospective observational study was carried out in a Bangladeshi tertiary care hospital from June 2018 to May 2019 with the goal of identifying the epidemiological patterns of fever of unknown origin in pediatric patients. Methods: It was a hospital-based prospective observational study carried out on 106 children (between 2 months and 12 years) with prolonged fever of >38.0 °C lasting for more than 7 days without a clear source. Children with additional chronic diseases or known immunodeficiency problems were not allowed. Clinical practices that helped determine the definitive etiology were assessed. Initial testing included a complete blood count, a routine urine examination, PBF, a chest X-ray, CRP measurement, blood cultures, serology, and additional pertinent investigations. The analysis focused mostly on the etiological results. The standard program SPSS 21 was used to analyze all of the study data. Findings: A total of 106 patients identified as having FUO were assessed, with over half (57.5%) being female and the majority (40.6%) falling within the 1 to 3-year age range. The study categorized the etiological outcomes into five groups: infections, malignancies, connective tissue conditions, miscellaneous, and undiagnosed. In the group that was being studied, infections were found to be the main cause in 44.3% of cases. Undiagnosed cases came in at 31.1%, cancers at 10.4%, other causes at 8.5%, and connective tissue disorders at 4.7%. Hepato-splenomegaly was seen in people with enteric fever, malaria, acute lymphoid leukemia, lymphoma, and hepatic abscesses, either by itself or in combination with other conditions. About 53% of people who were not diagnosed also had hepato-splenomegaly at the same time. Conclusion: Infections are the primary cause of PUO (pyrexia of unknown origin) in children, with undiagnosed cases being the second most common cause. An incremental approach is beneficial in the process of diagnosing a condition. Non-invasive examinations are used to diagnose infections and connective tissue disorders, while invasive investigations are used to diagnose cancer and other ailments. According to this study, the prevalence of undiagnosed diseases is still remarkable, so extensive historical analysis and physical examinations are necessary in order to provide a precise diagnosis.

Keywords: children, diagnostic challenges, fever of unknown origin, pediatric fever, undiagnosed diseases

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Authors: Mariam Khaled, Noha Farag, Ghada Mohamed Abdel Salam, Khaled Abu-Aisha, Mohamed El-Azizi

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Gastric and colorectal cancers are among the most frequent causes of cancer-associated mortalities in Africa. They have been considered as a global public health concern, as nearly one million new cases are reported per year. IL-1β is a pro-inflammatory cytokine-produced by activated macrophages and monocytes- and a member of the IL-1 family. The inactive IL-1β precursor is cleaved and activated by caspase-1 enzyme, which itself is activated by the assembly of intracellular structures defined as NLRP3 (Nod Like receptor P3) inflammasomes. Activated IL-1β stimulates the Interleukin-1 receptor type-1 (IL-1R1), which is responsible for the initiation of a signal transduction pathway leading to cell proliferation. It has been proven that the IL-1β gene is a highly polymorphic gene in which single nucleotide polymorphisms (SNPs) may affect its expression. It has been previously reported that SNPs including base transitions between C and T at positions, -511 (C-T; dbSNP: rs16944) and -31 (C-T; dbSNP: rs1143627), from the transcriptional start site, contribute to the pathogenesis of gastric and colorectal cancers by affecting IL-1β levels. Altered production of IL-1β due to such polymorphisms is suspected to stimulate an amplified inflammatory response and promote Epithelial Mesenchymal Transition leading to malignancy. Allele frequency distribution of the IL-1β-31 and -511 SNPs, in different populations, and their correlation to the incidence of gastric and colorectal cancers, has been intriguing to researchers worldwide. The current study aims to investigate allele distributions of the IL-1β SNPs among gastric and colorectal cancers Egyptian patients. In order to achieve to that, 89 Biopsy and surgical specimens from the antrum and corpus mucosa of chronic gastritis subjects and gastric and colorectal carcinoma patients was collected for DNA extraction followed by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). The amplified PCR products of IL-1β-31C > T and IL-1β-511T > C were digested by incubation with the restriction endonuclease enzymes ALu1 and Ava1. Statistical analysis was carried out to determine the allele frequency distribution in the three studied groups. Also, the effect of the IL-1β -31 and -511 SNPs on nuclear factor binding was analyzed using Fluorescence Electrophoretic Mobility Shift Assay (EMSA), preceded by nuclear factor extraction from gastric and colorectal tissue samples and LPS stimulated monocytes. The results of this study showed that a significantly higher percentage of Egyptian gastric cancer patients have a homozygous CC genotype at the IL-1β-31 position and a heterozygous TC genotype at the IL-1β-511 position. Moreover, a significantly higher percentage of the colorectal cancer patients have a homozygous CC genotype at the IL-1β-31 and -511 positions as compared to the control group. In addition, the EMSA results showed that IL-1β-31C/T and IL-1β-511T/C SNPs do not affect nuclear factor binding. Results of this study suggest that the IL-1β-31 C/T and IL-1β-511 T/C may be correlated to the incidence of gastric cancer in Egyptian patients; however, similar findings couldn’t be proven in the colorectal cancer patients group for the IL-1β-511 T/C SNP. This is the first study to investigate IL-1β -31 and -511 SNPs in the Egyptian population.

Keywords: colorectal cancer, Egyptian patients, gastric cancer, interleukin-1β, single nucleotide polymorphisms

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Authors: Sachin Mulmi Shrestha, Xin Fang, Hui Ye, Lihua Ren, Qinghua Ji, Ruihua Shi

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Background: Esophageal squamous cell carcinoma (ESCC) is a tumor arising from esophageal epithelial cells and is one of the major disease subtype in Asian countries, including China. Esophageal cancer is the 7th highest incidence based on the 2020 data of GLOBOCAN. The pathogenesis of cancer is still not well understood as many molecular and genetic basis of esophageal carcinogenesis has yet to be clearly elucidated. Circular RNAs are RNA molecules that are formed by back-splicing covalently joined 3′- and 5′-endsrather than canonical splicing, and recent data suggest circular RNAs could sponge miRNAs and are enriched with functional miRNA binding sites. Hence, we studied the mechanism of circular RNA, its biological function, and the relationship between microRNA in the carcinogenesis of ESCC. Methods: 4 pairs of normal and esophageal cancer tissues were collected in Zhongda hospital, affiliated to Southeast University, and high-throughput RNA sequencing was done. The result revealed that circ_0032746 was upregulated, and thus we selected circ_0032746 for further study. The backsplice junction of circRNA was validated by sanger sequence, and stability was determined by RNASE R assay. The binding site of circRNA and microRNA was predicted by circinteractome,mirandaand RNAhybrid database. Furthermore, circRNA was silenced by siRNA and then by lentivirus. The regulatory axis of circ0032746/miR4270 was validated by shRNA, mimic, and inhibitor transfection. Then, in vitro experiments were performed to assess the role of circ0032746 on proliferation (CCK-8 assay and colon formation assay), migration and invasion (Transewell assay), and apoptosis of ESCC. Results: The upregulated circ0032746 was validated in 9 pairs of tissues and 5 types of cell lines by qPCR, which showed high expression and was statistically significant (P<0.005) ). Upregulated circ0032746 was silenced by shRNA, which showed significant knockdown in KYSE 30 and TE-1 cell lines expression compared to control. Nuclear and cytoplasmic mRNA fraction experiment displayed the cytoplasmic location of circ0032746. The sponging of miR4270 was validated by co-transfection of sh-circ0032746 and mimic or inhibitor. Transfection with mimic showed the decreased expression of circ_0032746, whereas inhibitor inhibited the result. In vitro experiments showed that silencing of circ_0032746 inhibited the proliferation, migration, and invasion compared to the negative control group. The apoptosis was seen higher in a knockdown group than in the control group. Furthermore, 11 common mircoRNA target mRNAs were predicted by Targetscan, MirTarbase, and miRanda database, which may further play role in the pathogenesis. Conclusion: Our results showed that novel circ_0032746 is upregulated in ESCC and plays role in itsoncogenicity. Silencing of circ_0032746 inhibits the proliferation and migration of ESCC whereas increases the apoptosis of cancer cells. Hence, circ0032746 acts as an oncogene in ESCC by sponging with miR4270 and could be a potential biomarker in the diagnosis of ESCC in the future.

Keywords: circRNA, esophageal squamous cell carcinoma, microRNA, upregulated

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Authors: Meiling Yu, Nadia Rouatbi, Khuloud T. Al-Jamal

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Background: Treatment of neurological disorders with modern medical and surgical approaches remains difficult. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. The treatment of brain diseases with gene therapy requires the gene-editing tool to be delivered efficiently to the central nervous system. In this study, we explored the efficiency of different delivery routes, namely intravenous (i.v.), intra-cranial (i.c.), and intra-nasal (i.n.), to deliver stable nucleic acid-lipid particles (SNALPs) containing gene-editing tools namely Cas9 mRNA and sgRNA encoding for GFP as a reporter protein. We hypothesise that SNALPs can reach the brain and perform gene-editing to different extents depending on the administration route. Intranasal administration (i.n.) offers an attractive and non-invasive way to access the brain circumventing the blood–brain barrier. Successful delivery of gene-editing tools to the brain offers a great opportunity for therapeutic target validation and nucleic acids therapeutics delivery to improve treatment options for a range of neurodegenerative diseases. In this study, we utilised Rosa26-Cas9 knock-in mice, expressing GFP, to study brain distribution and gene-editing efficiency of SNALPs after i.v.; i.c. and i.n. routes of administration. Methods: Single guide RNA (sgRNA) against GFP has been designed and validated by in vitro nuclease assay. SNALPs were formulated and characterised using dynamic light scattering. The encapsulation efficiency of nucleic acids (NA) was measured by RiboGreen™ assay. SNALPs were incubated in serum to assess their ability to protect NA from degradation. Rosa26-Cas9 knock-in mice were i.v., i.n., or i.c. administered with SNALPs to test in vivo gene-editing (GFP knockout) efficiency. SNALPs were given as three doses of 0.64 mg/kg sgGFP following i.v. and i.n. or a single dose of 0.25 mg/kg sgGFP following i.c.. knockout efficiency was assessed after seven days using Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis. In vivo, the biodistribution of DiR labelled SNALPs (SNALPs-DiR) was assessed at 24h post-administration using IVIS Lumina Series III. Results: Serum-stable SNALPs produced were 130-140 nm in diameter with ~90% nucleic acid loading efficiency. SNALPs could reach and stay in the brain for up to 24h following i.v.; i.n. and i.c. administration. Decreasing GFP expression (around 50% after i.v. and i.c. and 20% following i.n.) was confirmed by optical imaging. Despite the small number of mice used, ICE analysis confirmed GFP knockout in mice brains. Additional studies are currently taking place to increase mice numbers. Conclusion: Results confirmed efficient gene knockout achieved by SNALPs in Rosa26-Cas9 knock-in mice expressing GFP following different routes of administrations in the following order i.v.= i.c.> i.n. Each of the administration routes has its pros and cons. The next stages of the project involve assessing gene-editing efficiency in wild-type mice and replacing GFP as a model target with therapeutic target genes implicated in Motor Neuron Disease pathology.

Keywords: CRISPR, nanoparticles, brain diseases, administration routes

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Authors: Faheema Khan

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To investigate the ability of sensitive and tolerant genotype of Glycine max to adapt to a saline environment in a field, we examined the growth performance, water relation and activities of antioxidant enzymes in relation to photosynthetic rate, chlorophyll a fluorescence, photosynthetic pigment concentration, protein and proline in plants exposed to salt stress. Ten soybean genotypes (Pusa-20, Pusa-40, Pusa-37, Pusa-16, Pusa-24, Pusa-22, BRAGG, PK-416, PK-1042, and DS-9712) were selected and grown hydroponically. After 3 days of proper germination, the seedlings were transferred to Hoagland’s solution (Hoagland and Arnon 1950). The growth chamber was maintained at a photosynthetic photon flux density of 430 μmol m−2 s−1, 14 h of light, 10 h of dark and a relative humidity of 60%. The nutrient solution was bubbled with sterile air and changed on alternate days. Ten-day-old seedlings were given seven levels of salt in the form of NaCl viz., T1 = 0 mM NaCl, T2=25 mM NaCl, T3=50 mM NaCl, T4=75 mM NaCl, T5=100 mM NaCl, T6=125 mM NaCl, T7=150 mM NaCl. The investigation showed that genotype Pusa-24, PK-416 and Pusa-20 appeared to be the most salt-sensitive. genotypes as inferred from their significantly reduced length, fresh weight and dry weight in response to the NaCl exposure. Pusa-37 appeared to be the most tolerant genotype since no significant effect of NaCl treatment on growth was found. We observed a greater decline in the photosynthetic variables like photosynthetic rate, chlorophyll fluorescence and chlorophyll content, in salt-sensitive (Pusa-24) genotype than in salt-tolerant Pusa-37 under high salinity. Numerous primers were verified on ten soybean genotypes obtained from Operon technologies among which 30 RAPD primers shown high polymorphism and genetic variation. The Jaccard’s similarity coefficient values for each pairwise comparison between cultivars were calculated and similarity coefficient matrix was constructed. The closer varieties in the cluster behaved similar in their response to salinity tolerance. Intra-clustering within the two clusters precisely grouped the 10 genotypes in sub-cluster as expected from their physiological findings.Salt tolerant genotype Pusa-37, was further analysed by 2-Dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the Present study, 173 protein spots were identified. Of these, 40 proteins responsive to salinity were either up- or down-regulated in Pusa-37. Proteomic analysis in salt-tolerant genotype (Pusa-37) led to the detection of proteins involved in a variety of biological processes, such as protein synthesis (12 %), redox regulation (19 %), primary and secondary metabolism (25 %), or disease- and defence-related processes (32 %). In conclusion, the soybean plants in our study responded to salt stress by changing their protein expression pattern. The photosynthetic, biochemical and molecular study showed that there is variability in salt tolerance behaviour in soybean genotypes. Pusa-24 is the salt-sensitive and Pusa-37 is the salt-tolerant genotype. Moreover this study gives new insights into the salt-stress response in soybean and demonstrates the power of genomic and proteomic approach in plant biology studies which finally could help us in identifying the possible regulatory switches (gene/s) controlling the salt tolerant genotype of the crop plants and their possible role in defence mechanism.

Keywords: glycine max, salt stress, RAPD, genomic and proteomic variability

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Authors: Massimiliano Biagini, Marco Spinsanti, Gabriella De Angelis, Sara Tomei, Ilaria Ferlenghi, Maria Scarselli, Alessia Biolchi, Alessandro Muzzi, Brunella Brunelli, Silvana Savino, Marzia M. Giuliani, Isabel Delany, Paolo Costantino, Rino Rappuoli, Vega Masignani, Nathalie Norais

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The gram-negative bacterium Neisseria meningitidis serogroup B (MenB) is an exclusively human pathogen representing the major cause of meningitides and severe sepsis in infants and children but also in young adults. This pathogen is usually present in the 30% of healthy population that act as a reservoir, spreading it through saliva and respiratory fluids during coughing, sneezing, kissing. Among surface-exposed protein components of this diplococcus, factor H binding protein is a lipoprotein proved to be a protective antigen used as a component of the recently licensed Bexsero vaccine. fHbp is a highly variable meningococcal protein: to reflect its remarkable sequence variability, it has been classified in three variants (or two subfamilies), and with poor cross-protection among the different variants. Furthermore, the level of fHbp expression varies significantly among strains, and this has also been considered an important factor for predicting MenB strain susceptibility to anti-fHbp antisera. Different methods have been used to assess fHbp expression on meningococcal strains, however, all these methods use anti-fHbp antibodies, and for this reason, the results are affected by the different affinity that antibodies can have to different antigenic variants. To overcome the limitations of an antibody-based quantification, we developed a quantitative Mass Spectrometry (MS) approach. Selected Reaction Monitoring (SRM) recently emerged as a powerful MS tool for detecting and quantifying proteins in complex mixtures. SRM is based on the targeted detection of ProteoTypicPeptides (PTPs), which are unique signatures of a protein that can be easily detected and quantified by MS. This approach, proven to be highly sensitive, quantitatively accurate and highly reproducible, was used to quantify the absolute amount of fHbp antigen in total extracts derived from 105 clinical isolates, evenly distributed among the three main variant groups and selected to be representative of the fHbp circulating subvariants around the world. We extended the study at the genetic level investigating the correlation between the differential level of expression and polymorphisms present within the genes and their promoter sequences. The implications of fHbp expression on the susceptibility of the strain to killing by anti-fHbp antisera are also presented. To date this is the first comprehensive fHbp expression profiling in a large panel of Neisseria meningitidis clinical isolates driven by an antibody-independent MS-based methodology, opening the door to new applications in vaccine coverage prediction and reinforcing the molecular understanding of released vaccines.

Keywords: quantitative mass spectrometry, Neisseria meningitidis, vaccines, bexsero, molecular epidemiology

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Authors: Changhan Ouyang, Zhonglin Xie

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In response to proatherosclerotic factors such as oxidized lipids, or to therapeutic interventions such as angioplasty, stents, or bypass surgery, vascular smooth muscle cells (VSMCs) migrate from the media to the intima, resulting in intimal hyperplasia, restenosis, graft failure, or atherosclerosis. These proatherosclerotic factors also activate autophagy in VSMCs. However, the functional role of autophagy in vascular health and disease remains poorly understood. In the present study, we determined the role of autophagy in the regulation of VSMC migration. Autophagy activity in cultured human aortic smooth muscle cells (HASMCs) and mouse carotid arteries was measured by Western blot analysis of microtubule-associated protein 1 light chain 3 B (LC3B) and P62. The VSMC migration was determined by scratch wound assay and transwell migration assay. Ex vivo smooth muscle cell migration was determined using aortic ring assay. The in vivo SMC migration was examined by staining the carotid artery sections with smooth muscle alpha actin (alpha SMA) after carotid artery ligation. To examine the relationship between autophagy and neointimal hyperplasia, C57BL/6J mice were subjected to carotid artery ligation. Seven days after injury, protein levels of Atg5, Atg7, Beclin1, and LC3B drastically increased and remained higher in the injured arteries three weeks after the injury. In parallel with the activation of autophagy, vascular injury-induced neointimal hyperplasia as estimated by increased intima/media ratio. The en face staining of carotid artery showed that vascular injury enhanced alpha SMA staining in the intimal cells as compared with the sham operation. Treatment of HASMCs with platelet-derived growth factor (PDGF), one of the major factors for vascular remodeling in response to vascular injury, increased Atg7 and LC3 II protein levels and enhanced autophagosome formation. In addition, aortic ring assay demonstrated that PDGF treated aortic rings displayed an increase in neovessel formation compared with control rings. Whole mount staining for CD31 and alpha SMA in PDGF treated neovessels revealed that the neovessel structures were stained by alpha SMA but not CD31. In contrast, pharmacological and genetic suppression of autophagy inhibits VSMC migration. Especially, gene silencing of Atg7 inhibited VSMC migration induced by PDGF. Furthermore, three weeks after ligation, markedly decreased neointimal formation was found in mice treated with chloroquine, an inhibitor of autophagy. Quantitative morphometric analysis of the injured vessels revealed a marked reduction in the intima/media ratio in the mice treated with chloroquine. Conclusion: Autophagy activation increases VSMC migration while autophagy suppression inhibits VSMC migration. These findings suggest that autophagy suppression may be an important therapeutic strategy for atherosclerosis and intimal hyperplasia.

Keywords: autophagy, vascular smooth muscle cell, migration, neointimal formation

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Authors: Abiodun Amusan, Olugbenga Akinola, Kazeem Akano, María Hernández-Castañeda, Jenna Dick, Akintunde Sowunmi, Geoffrey Hart, Grace Gbotosho

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Introduction: Artemisinin-based Combination Therapy (ACTs) is the cornerstone malaria treatment option in most malaria-endemic countries. Unfortunately, the malaria control effort is constantly being threatened by resistance of Plasmodium falciparum to ACTs. The recent evidence of artemisinin resistance in East Africa and its possibility of spreading to other African regions portends an imminent health catastrophe. This study aimed at evaluating the occurrence, prevalence, and influence of artemisinin-resistance markers on treatment outcomes in Ibadan before and after post-adoption of artemisinin combination therapy (ACTs) in Nigeria in 2005. Method: The study involved day zero dry blood spot (DBS) obtained from malaria patients during retrospective (2000-2005) and prospective (2021) studies. A cohort in the prospective study received oral dihydroartemisinin-piperaquine and underwent a 42-day follow-up to observe treatment outcomes. Genomic DNA was extracted from the DBS samples using a QIAamp blood extraction kit. Fragments of P. falciparum kelch13 (Pfkelch13), P. falciparum coronin (Pfcoronin), P. falciparum multidrug resistance 2 (PfMDR2), and P. falciparum chloroquine resistance transporter (PfCRT) genes were amplified and sequenced on a sanger sequencing platform to identify artemisinin resistance-associated mutations. Mutations were identified by aligning sequenced data with reference sequences obtained from the National Center for Biotechnology Information. Data were analyzed using descriptive statistics and student t-tests. Results: Mean parasite clearance time (PCT) and fever clearance time (FCT) were 2.1 ± 0.6 days (95% CI: 1.97-2.24) and 1.3 ± 0.7 days (95% CI: 1.1-1.6) respectively. Four mutations, K189T [34/53(64.2%)], R255K [2/53(3.8%)], K189N [1/53(1.9%)] and N217H [1/53(1.9%)] were identified within the N-terminal (Coiled-coil containing) domain of Pfkelch13. No artemisinin resistance-associated mutation usually found within the β-propeller domain of the Pfkelch13 gene was found in these analyzed samples. However, K189T and R255K mutations showed a significant correlation with longer parasite clearance time in the patients (P<0.002). The observed Pfkelch13 gene changes did not influence the baseline mean parasitemia (P = 0.44). P76S [17/100 (17%)] and V62M [1/100 (1%)] changes were identified in the Pfcoronin gene fragment without any influence on the parasitological parameters. No change was observed in the PfMDR2 gene, while no artemisinin resistance-associated mutation was found in the PfCRT gene. Furthermore, a sample each in the retrospective study contained the Pfkelch13 K189T and Pfcoronin P76S mutations. Conclusion: The study revealed absence of genetic-based evidence of artemisinin resistance in the study population at the time of study. The high frequency of K189T Pfkelch13 mutation and its correlation with increased parasite clearance time in this study may depict geographical variation of resistance mediators and imminent artemisinin resistance, respectively. The study also revealed an inherent potential of parasites to harbour drug-resistant genotypes before the introduction of ACTs in Nigeria.

Keywords: artemisinin resistance, plasmodium falciparum, Pfkelch13 mutations, Pfcoronin

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Authors: Wen-Yang Hu, Ranli Lu, Mark Maienschein-Cline, Danping Hu, Larisa Nonn, Toshi Shioda, Gail S. Prins

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Background: Genetic mutations are highly associated with increased prostate cancer risk. In addition to whole genome sequencing, somatic mutations can be identified by aligning transcriptome sequences to the human genome. Here we analyzed bulk RNAseq and single cell RNAseq data of human prostate cancer cells and their matched non-cancer cells in benign regions from 4 individual patients. Methods: Sequencing raw reads were aligned to the reference genome hg38 using STAR. Variants were annotated using Annovar with respect to overlap gene annotation information, effect on gene and protein sequence, and SIFT annotation of nonsynonymous variant effect. We determined cancer-specific novel alleles by comparing variant calls in cancer cells to matched benign cells from the same individual by selecting unique alleles that were only detected in the cancer samples. Results: In bulk RNAseq data from 3 patients, the most common variants were the noncoding mutations at UTR3/UTR5, and the major variant types were single-nucleotide polymorphisms (SNP) including frameshift mutations. C>T transversion is the most frequently presented substitution of SNP. A total of 222 genes carrying unique exonic or UTR variants were revealed in cancer cells across 3 patients but not in benign cells. Among them, transcriptome levels of 7 genes (CITED2, YOD1, MCM4, HNRNPA2B1, KIF20B, DPYSL2, NR4A1) were significantly up or down regulated in cancer stem cells. Out of the 222 commonly mutated genes in cancer, 19 have nonsynonymous variants and 11 are damaged genes with variants including SIFT, frameshifts, stop gain/loss, and insertions/deletions (indels). Two damaged genes, activating transcription factor 6 (ATF6) and histone demethylase KDM3A are of particular interest; the former is a survival factor for certain cancer cells while the later positively activates androgen receptor target genes in prostate cancer. Further, single cell RNAseq data of cancer cells and their matched non-cancer benign cells from both primary 2D and 3D tumoroid cultures were analyzed. Similar to the bulk RNAseq data, single cell RNAseq in cancer demonstrated that the exonic mutations are less common than noncoding variants, with SNPs including frameshift mutations the most frequently presented types in cancer. Compared to cancer stem cell enriched-3D tumoroids, 2D cancer cells carried 3-times higher variants, 8-times more coding mutations and 10-times more nonsynonymous SNP. Finally, in both 2D primary and 3D tumoroid cultures, cancer stem cells exhibited fewer coding mutations and noncoding SNP or insertions/deletions than non-stem cancer cells. Summary: Our study demonstrates the usefulness of bulk and single cell RNAseaq data in identifying somatic mutations in prostate cancer, providing an alternative method in screening candidate genes for prostate cancer diagnosis and potential therapeutic targets. Cancer stem cells carry fewer somatic mutations than non-stem cancer cells due to their inherited immortal stand DNA from parental stem cells that explains their long-lived characteristics.

Keywords: prostate cancer, stem cell, genomic mutation, RNAseq

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Authors: Mei Chen, Yingping Hou, Michelle Hao, Soheil Aghamohammadzadeh, Esteban Terzo, Roger Clark, Vijay Modur

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Background: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a genetic skin condition characterized by skin tearing and unremitting blistering upon minimal trauma. Repeated blistering, fibrosis, and scarring lead to aggressive squamous cell carcinoma later in life. RDEB is caused by mutations in the COL7A1 gene encoding collagen type VII (C7), the major component of anchoring fibrils mediating epidermis-dermis adherence. Nonsense mutations in the COL7A1 gene of a subset of RDEB patients leads to premature termination codons (PTC). Similarly, most Junctional Epidermolysis Bullosa (JEB) cases are caused by nonsense mutations in the LAMB3 gene encoding the β3 subunit of laminin 332. Currently, there is an unmet need for the treatment of RDEB and JEB. Zikani Therapeutics has discovered an array of macrocyclic compounds with ring structures similar to macrolide antibiotics that can facilitate readthrough activity of nonsense mutations in the COL7A1 and LAMB3 genes by acting as Ribosome Modulating Agents (RMAs). The medicinal chemistry synthetic advancements of these macrocyclic compounds have allowed targeting the human ribosome while preserving the structural elements responsible for the safety and pharmacokinetic profile of clinically used macrolide antibiotics. Methods: C7 expression was used as a measure of readthrough activity by immunoblot assays in two primary human fibroblasts from RDEB patients (R578X/R578X and R163X/R1683X-COL7A1). Similarly, immunoblot assays in C325X/c.629-12T > A-LAMB3 keratinocytes were used to measure readthrough activity for JEB. The relative readthrough activity of each compound was measured relative to Gentamicin. An imaging-based fibroblast migration assay was used as an assessment of C7 functionality in RDEB-fibroblasts over 16-20 hrs. The incubation period for the above experiments was 48 hrs for RDEB fibroblasts and 72 hours for JEB keratinocytes. Results: 9 RMAs demonstrated increased protein expression in both patient RDEB fibroblasts. The highest readthrough activity at tested concentrations without cytotoxicities increased protein expression up to 179% of Gentamicin (400 µg/ml), with favored readthrough activity in R163X/R1683X-COL7A1 fibroblasts. Concurrent with protein expression, fibroblast hypermotility phenotype observed in RDEB was rescued by reducing motility by ~35% to WT levels (the same level as 690 µM Gentamicin treated cells). Laminin β3 expression was also shown to be increased by 6 RMAs in keratinocytes to 33-83% of (400 µg/ml) Gentamicin. Conclusions: To date, 9 RMAs have been identified that enhance the expression of functional C7 in a mutation-dependent manner in two different RDEB patient fibroblast backgrounds (R578X/R578X and R163X/R1683X-COL7A1). A further 6 RMAs have been identified that enhance the readthrough of C325X-LAMB3 in JEB patient keratinocytes. Based on the clinical trial conducted by us with topical gentamycin in 2017, Zikani’s RMAs achieve clinically significant levels of read-through for the treatment of recessive dystrophic and Junctional Epidermolysis Bullosa.

Keywords: epidermolysis bullosa, nonsense mutation, readthrough, ribosome modulation

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Authors: Joanna Marie A. Paulino-Morente, Vaneza Valentina L. Penolio, Grace Sabado

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Ovarian cancer is extremely hard to diagnose in its early stages, and those afflicted at the time of diagnosis are typically asymptomatic and in the late stages of the disease, with metastasis to other organs. Ovarian cancers often occur sporadically, with only 5% associated with hereditary mutations. Mutations in the BRCA1 and BRCA2 tumor suppressor genes have been found to be responsible for the majority of hereditary ovarian cancers. One type of ovarian tumor is Malignant Mixed Mullerian Tumor (MMMT), which is a very rare and aggressive type, accounting for only 1% of all ovarian cancers. Reported is a case of a 43-year-old G3P3 (3003), who came into our institution due to a 2-month history of difficulty of breathing. Family history reveals that her eldest and younger sisters both died of ovarian malignancy, with her younger sister having a histopathology report of endometrioid ovarian carcinoma, left ovary stage IIIb. She still has 2 asymptomatic sisters. Physical examination pointed to pleural effusion of right lung, and presence of bilateral ovarian new growth, which had a Sassone score of 13. Admitting Diagnosis was G3P3 (3003), Ovarian New Growth, bilateral, Malignant; Pleural effusion secondary to malignancy. BRCA was requested to establish a hereditary mutation; however, the patient had no funds. Once the patient was stabilized, TAHBSO with surgical staging was performed. Intraoperatively, the pelvic cavity was occupied by firm, irregularly shaped ovaries, with a colorectal metastasis. Microscopic sections from both ovaries and the colorectal metastasis had pleomorphic tumor cells lined by cuboidal to columnar epithelium exhibiting glandular complexity, displaying nuclear atypia and increased nuclear-cytoplasmic ratio, which are infiltrating the stroma, consistent with the features of Malignant Mixed Mullerian Tumor, since MMMT is composed histologically of malignant epithelial and sarcomatous elements. In conclusion, discussed is the clinic-pathological feature of a patient with primary ovarian Malignant Mixed Mullerian Tumor, a rare malignancy comprising only 1% of all ovarian neoplasms. Also, by understanding the hereditary ovarian cancer syndromes and its relation to this patient, it cannot be overemphasized that a comprehensive family history is really fundamental for early diagnosis. The familial association of the disease, given that the patient has two sisters who were diagnosed with an advanced stage of ovarian cancer and succumbed to the disease at a much earlier age than what is reported in the general population, points to a possible hereditary syndrome which occurs in only 5% of ovarian neoplasms. In a low-resource setting, being in a third world country, the following will be recommended for monitoring and/or screening women who are at high risk for developing ovarian cancer, such as the remaining sisters of the patient: 1) Physical examination focusing on the breast, abdomen, and rectal area every 6 months. 2) Transvaginal sonography every 6 months. 3) Mammography annually. 4) CA125 for postmenopausal women. 5) Genetic testing for BRCA1 and BRCA2 will be reserved for those who are financially capable.

Keywords: BRCA, hereditary breast-ovarian cancer syndrome, malignant mixed mullerian tumor, ovarian cancer

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Authors: Sarwan Dhir, Suma Basak, Dipika Parajulee

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Alfalfa is renowned for its nutritional and biopharmaceutical value as a perennial forage legume. However, establishing a rapid plant regeneration protocol using somatic embryogenesis and efficient transformation frequency are the crucial prerequisites for gene editing in alfalfa. This study was undertaken to establish and improve the protocol for somatic embryogenesis and subsequent plant regeneration. The experiments were conducted in response to natural sensitivity using various antibiotics such as cefotaxime, carbenicillin, gentamycin, hygromycin, and kanamycin. Using 3-week-old leaf tissue, somatic embryogenesis was initiated on Gamborg’s B5 basal (B5H) medium supplemented with 3% maltose, 0.9µM Kinetin, and 4.5µM 2,4-D. Embryogenic callus (EC) obtained from the B5H medium exhibited a high rate of somatic embryo formation (97.9%) after 3 weeks when the cultures were placed in the dark. Different developmental stages of somatic embryos and cotyledonary stages were then transferred to Murashige and Skoog’s (MS) basal medium under light, resulting in a 94% regeneration rate of plantlets. Our results indicate that leaf segments can grow (tolerate) up to 450 mg/L of cefotaxime and 400 mg/L of carbenicillin in the culture medium. However, the survival threshold for hygromycin at 12.5 mg/L, kanamycin at 250 mg/L, gentamycin at 50 mg/L, and timentin (300 mg/L). The experiment to improve the protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector was also conducted. The vector contains two reporter genes such as β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene (HPT), all driven under the CaMV 35s promoter. Various transformation parameters were optimized using 3-week-old in vitro-grown plantlets. The different parameters such as types of explant, leaf ages, preculture days, segment sizes, wounding types, bacterial concentrations, infection periods, co-cultivation periods, different concentrations of acetosyringone, silver nitrate, and calcium chloride were optimized for transient gene expression. The transient gene expression was confirmed via histochemical GUS and GFP visualization under fluorescent microscopy. The data were analyzed based on the semi-quantitative observation of the percentage and number of blue GUS spots on different days of agro-infection. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old leaf segments wounded using a scalpel blade of 11 size- after 3 days of post-incubation at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of bacterial-leaf segment co-cultivation, with the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. Our results suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The stable gene expression in the putative transgenic tissue was confirmed using PCR amplification of both marker genes, indicating that gene expression in explants was not solely due to Agrobacterium, but also from transformed cells. The improved protocol could be used for generating transgenic alfalfa plants using genome editing techniques such as CRISPR/Cas9.

Keywords: Medicago sativa l. (Alfalfa), agrobacterium tumefaciens, β-glucuronidase, green fluorescent protein, transient gene

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Authors: Majed Al Otaibi, Melissa Lessard-Beaudoin, Amel Loudghi, Raphael Chouinard-Watkins, Melanie Plourde, Frederic Calon, C. Alexandre Castellano, Stephen Cunnane, Helene Payette, Pierrette Gaudreau, Denis Gris, Rona K. Graham

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Introduction: Alzheimer disease (AD) is a chronic disorder that affects millions of individuals worldwide. Symptoms include memory dysfunction, and also alterations in attention, planning, language and overall cognitive function. Olfactory dysfunction is a common symptom of several neurological disorders including AD. Studying the mechanisms underlying the olfactory dysfunction may therefore lead to the discovery of potential biomarkers and/or treatments for neurodegenerative diseases. Objectives: To determine if olfactory dysfunction predicts future cognitive impairment in the aging population and to characterize the olfactory system in a murine model expressing a genetic factor of AD. Method: For the human study, quantitative olfactory tests (UPSIT and OMT) have been done on 93 subjects (aged 80 to 94 years) from the Quebec Longitudinal Study on Nutrition and Successful Aging (NuAge) cohort accepting to participate in the ORCA secondary study. The telephone Modified Mini Mental State examination (t-MMSE) was used to assess cognition levels, and an olfactory self-report was also collected. In a separate cohort, olfactory cortical volume was calculated using MRI results from healthy old adults (n=25) and patients with AD (n=18) using the AAL single-subject atlas and performed with the PNEURO tool (PMOD 3.7). For the murine study, we are using Western blotting, RT-PCR and immunohistochemistry. Result: Human Study: Based on the self-report, 81% of the participants claimed to not suffer from any problem with olfaction. However, based on the UPSIT, 94% of those subjects showed a poor olfactory performance and different forms of microsmia. Moreover, the results confirm that olfactory function declines with age. We also detected a significant decrease in olfactory cortical volume in AD individuals compared to controls. Murine study: Preliminary data demonstrate there is a significant decrease in expression levels of the proform of caspase-3 and the caspase substrate STK3, in the olfactory bulb of mice expressing human APOE4 compared with controls. In addition, there is a significant decrease in the expression level of the caspase-9 proform and caspase-8 active fragment. Analysis of the mature neuron marker, NeuN, shows decreased expression levels of both isoforms. The data also suggest that Iba-1 immunostaining is increased in the olfactory bulb of APOE4 mice compared to wild type mice. Conclusions: The activation of caspase-3 may be the cause of the decreased levels of STK3 through caspase cleavage and may play role in the inflammation observed. In the clinical study, our results suggest that seniors are unaware of their olfactory function status and therefore it is not sufficient to measure olfaction using the self-report in the elderly. Studying olfactory function and cognitive performance in the aging population will help to discover biomarkers in the early stage of the AD.

Keywords: Alzheimer's disease, APOE4, cognition, caspase, brain atrophy, neurodegenerative, olfactory dysfunction

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Authors: Tatiana Butkova, Nikolai Kibrik, Kristina Malsagova, Alexander Izotov, Alexander Stepanov, Anna Kaysheva

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Relevance. The genetic risk of developing schizophrenia is determined by two factors: single nucleotide polymorphisms and gene copy number variations. The search for serological markers for early diagnosis of schizophrenia is driven by the fact that the first five years of the disease are accompanied by significant biological, psychological, and social changes. It is during this period that pathological processes are most amenable to correction. The aim of this study was to analyze single nucleotide polymorphisms (SNPs) that are hypothesized to potentially influence the onset and development of the endogenous process. Materials and Methods It was analyzed 73 single nucleotide polymorphism variants. The study included 48 patients undergoing inpatient treatment at "Psychiatric Clinical Hospital No. 1" in Moscow, comprising 23 females and 25 males. Inclusion criteria: - Patients aged 18 and above. - Diagnosis according to ICD-10: F20.0, F20.2, F20.8, F21.8, F25.1, F25.2. - Voluntary informed consent from patients. Exclusion criteria included: - The presence of concurrent somatic or neurological pathology, neuroinfections, epilepsy, organic central nervous system damage of any etiology, and regular use of medication. - Substance abuse and alcohol dependence. - Women who were pregnant or breastfeeding. Clinical and psychopathological assessment was complemented by psychometric evaluation using the PANSS scale at the beginning and end of treatment. The duration of observation during therapy was 4-6 weeks. Total DNA extraction was performed using QIAamp DNA. Blood samples were processed on Illumina HiScan and genotyped for 652,297 markers on the Infinium Global Chips Screening Array-24v2.0 using the IMPUTE2 program with parameters Ne=20,000 and k=90. Additional filtration was performed based on INFO>0.5 and genotype probability>0.5. Quality control of the obtained DNA was conducted using agarose gel electrophoresis, with each tested sample having a volume of 100 µL. Results. It was observed that several SNPs exhibited gender dependence. We identified groups of single nucleotide polymorphisms with a membership of 80% or more in either the female or male gender. These SNPs included rs2661319, rs2842030, rs4606, rs11868035, rs518147, rs5993883, and rs6269.Another noteworthy finding was the limited combination of SNPs sufficient to manifest clinical symptoms leading to hospitalization. Among all 48 patients, each of whom was analyzed for deviations in 73 SNPs, it was discovered that the combination of involved SNPs in the manifestation of pronounced clinical symptoms of schizophrenia was 19±3 out of 73 possible. In study, the frequency of occurrence of single nucleotide polymorphisms also varied. The most frequently observed SNPs were rs4849127 (in 90% of cases), rs1150226 (86%), rs1414334 (75%), rs10170310 (73%), rs2857657, and rs4436578 (71%). Conclusion. Thus, the results of this study provide additional evidence that these genes may be associated with the development of schizophrenia spectrum disorders. However, it's impossible cannot rule out the hypothesis that these polymorphisms may be in linkage disequilibrium with other functionally significant polymorphisms that may actually be involved in schizophrenia spectrum disorders. It has been shown that missense SNPs by themselves are likely not causative of the disease but are in strong linkage disequilibrium with non-functional SNPs that may indeed contribute to disease predisposition.

Keywords: gene polymorphisms, genotyping, single nucleotide polymorphisms, schizophrenia.

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Authors: Peggy M. Randon, Lisa Randon

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Introduction: The post-COVID-19 era has presented unique challenges for addressing complex mental health issues, particularly due to exacerbated stress, increased social isolation, and disrupted continuity of care. This article outlines relevant health disparities and policy implications within the context of the United States while maintaining international relevance. Methods: A comprehensive literature review (including studies, reports, and policy documents) was conducted to examine concerns related to childhood-onset schizophrenia and the impact on patients and their families. Qualitative and quantitative data were synthesized to provide insights into the complex etiology of schizophrenia, the effects of the pandemic, and the challenges faced by socioeconomically disadvantaged populations. Case studies were employed to illustrate real-world examples and areas requiring policy reform. Results: Early intervention in childhood is crucial for preventing or mitigating the long-term impact of complex psychotic disorders, particularly schizophrenia. A comprehensive understanding of the genetic, environmental, and physiological factors contributing to the development of schizophrenia is essential. The COVID-19 pandemic worsened symptoms and disrupted treatment for many adolescent patients with schizophrenia, emphasizing the need for adaptive interventions and the utilization of virtual platforms. Health disparities, including stigma, financial constraints, and language or cultural barriers, further limit access to care, especially for socioeconomically disadvantaged populations. Policy implications: Current US health policies inadequately support patients with schizophrenia. The limited availability of longitudinal care, insufficient resources for families, and stigmatization represent ongoing policy challenges. Addressing these issues necessitates increased research funding, improved access to affordable treatment plans, and cultural competency training for healthcare providers. Public awareness campaigns are crucial to promote knowledge, awareness, and acceptance of mental health disorders. Conclusion: The unique challenges faced by children and families in the US affected by schizophrenia and other psychotic disorders have yet to be adequately addressed on institutional and systemic levels. The relevance of findings to an international audience is emphasized by examining the complex factors contributing to the onset of psychotic disorders and their global policy implications. The broad impact of the COVID-19 pandemic on mental health underscores the need for adaptive interventions and global responses. Addressing policy challenges, improving access to care, and reducing the stigma associated with mental health disorders are crucial steps toward enhancing the lives of adolescents and young adults with schizophrenia and their family members. The implementation of virtual platforms can help overcome barriers and ensure equitable access to support and resources for all patients, enabling them to lead healthy and fulfilling lives.

Keywords: childhood, schizophrenia, policy, United, States, health, disparities

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Authors: Natalia Hordynska, Magdalena Szechynska-Hebda, Miroslaw Sobczak, Elzbieta Rozanska, Joanna Troczynska, Zofia Banaszak, Maria Wedzony

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Albinism is a common problem encountered in wheat and triticale breeding programs, which require in vitro culture steps e.g. generation of doubled haploids via androgenesis process. Genetic factor is a major determinant of albinism, however, environmental conditions such as temperature and media composition influence the frequency of albino plant formation. Cold incubation of wheat and triticale spikes induced a switch from gametophytic to sporophytic development. Further, androgenic structures formed from anthers of the genotypes susceptible to androgenesis or treated with cold stress, had a pool of structurally primitive plastids, with small starch granules or swollen thylakoids. High temperature was a factor inducing andro-genesis of wheat and triticale, but at the same time, it was a factor favoring the formation of albino plants. In genotypes susceptible to albinism or after heat stress conditions, cells formed from anthers were vacuolated, and plastids were eliminated. Partial or complete loss of chlorophyll pigments and incomplete differentiation of chloroplast membranes result in formation of tissues or whole plant unable to perform photosynthesis. Indeed, susceptibility to the andro-genesis process was associated with an increase of total concentration of photosynthetic pigments in anthers, spikes and regenerated plants. The proper balance of the synthesis of various pigments, was the starting point for their proper incorporation into photosynthetic membranes. In contrast, genotypes resistant to the androgenesis process and those treated with heat, contained 100 times lower content of photosynthetic pigments. In particular, the synthesis of violaxanthin, zeaxanthin, lutein and chlorophyll b was limited. Furthermore, deregulation of starch and lipids synthesis, which led to the formation of very complex starch granules and an increased number of oleosomes, respectively, correlated with the reduction of the efficiency of androgenesis. The content of other sugars varied depending on the genotype and the type of stress. The highest content of various sugars was found for genotypes susceptible to andro-genesis, and highly reduced for genotypes resistant to androgenesis. The most important sugars seem to be glucose and fructose. They are involved in sugar sensing and signaling pathways, which affect the expression of various genes and regulate plant development. Sucrose, on the other hand, seems to have minor effect at each stage of the androgenesis. The sugar metabolism was related to metabolic activity of microspores. The genotypes susceptible to androgenesis process had much faster mitochondrium- and chloroplast-dependent energy conversion and higher heat production by tissues. Thus, the effectiveness of metabolic processes, their balance and the flexibility under the stress was a factor determining the direction of microspore development, and in the later stages of the androgenesis process, a factor supporting the induction of androgenic structures, chloroplast formation and the regeneration of green plants. The work was financed by Ministry of Agriculture and Rural Development within Program: ‘Biological Progress in Plant Production’, project no HOR.hn.802.15.2018.

Keywords: androgenesis, chloroplast, metabolism, temperature stress

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Authors: Adriana Haulica

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Powered by Machine Learning, Precise medicine is tailored by now to use genetic and molecular profiling, with the aim of optimizing the therapeutic benefits for cohorts of patients. As the majority of Machine Language algorithms come from heuristics, the outputs have contextual validity. This is not very restrictive in the sense that medicine itself is not an exact science. Meanwhile, the progress made in Molecular Biology, Bioinformatics, Computational Biology, and Precise Medicine, correlated with the huge amount of human biology data and the increase in computational power, opens new healthcare challenges. A more accurate diagnosis is needed along with real-time treatments by processing as much as possible from the available information. The purpose of this paper is to present a deeper vision for the future of Artificial Intelligence in Precise medicine. In fact, actual Machine Learning algorithms use standard mathematical knowledge, mostly Euclidian metrics and standard computation rules. The loss of information arising from the classical methods prevents obtaining 100% evidence on the diagnosis process. To overcome these problems, we introduce MEDICOMPILLS, a new architectural concept tool of information processing in Precise medicine that delivers diagnosis and therapy advice. This tool processes poly-field digital resources: global knowledge related to biomedicine in a direct or indirect manner but also technical databases, Natural Language Processing algorithms, and strong class optimization functions. As the name suggests, the heart of this tool is a compiler. The approach is completely new, tailored for omics and clinical data. Firstly, the intrinsic biological intuition is different from the well-known “a needle in a haystack” approach usually used when Machine Learning algorithms have to process differential genomic or molecular data to find biomarkers. Also, even if the input is seized from various types of data, the working engine inside the MEDICOMPILLS does not search for patterns as an integrative tool. This approach deciphers the biological meaning of input data up to the metabolic and physiologic mechanisms, based on a compiler with grammars issued from bio-algebra-inspired mathematics. It translates input data into bio-semantic units with the help of contextual information iteratively until Bio-Logical operations can be performed on the base of the “common denominator “rule. The rigorousness of MEDICOMPILLS comes from the structure of the contextual information on functions, built to be analogous to mathematical “proofs”. The major impact of this architecture is expressed by the high accuracy of the diagnosis. Detected as a multiple conditions diagnostic, constituted by some main diseases along with unhealthy biological states, this format is highly suitable for therapy proposal and disease prevention. The use of MEDICOMPILLS architecture is highly beneficial for the healthcare industry. The expectation is to generate a strategic trend in Precise medicine, making medicine more like an exact science and reducing the considerable risk of errors in diagnostics and therapies. The tool can be used by pharmaceutical laboratories for the discovery of new cures. It will also contribute to better design of clinical trials and speed them up.

Keywords: bio-semantic units, multiple conditions diagnosis, NLP, omics

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Authors: L. Abubakar, B. M. Sokoto, I. U. Mohammed, M. S. Na’allah, A. Mohammad, A. N. Garba, T. S. Bubuche

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Onion (Allium cepa var. cepa L.), is the most important species of the Allium group belonging to family Alliaceae and genus Allium. It can be regarded as the single important vegetable species in the world after tomatoes. Despite the similarities, which bring the species together, the genus is a strikingly diverse one, with more than five hundred species, which are perennial and mostly bulbous plants. Out of these, only seven species are in cultivation, and five are the most important species of the cultivated Allium. However, Allium cepa (onion) and Allium sativum (Garlic) are the two major cultivated species grown all over the world of which the onion crop is the most important. Heritability defined as the proportion of the observed total variability that is genetic, and its estimates from variance components give more useful information of genotypic variation from the total phenotypic differences and environmental effects on the individuals or families. It therefore guide the breeder with respect to the ease with which selection of traits can be carried out. Heritability estimates guide the breeder with respect to ease of selection of traits while correlations suggest how selection among characters can be practiced. Correlations explain relationship between characters and suggest how selection among characters can be practiced in breeding programmes. Highly significant correlations have been reported, between yield, maturity, rings/bulb and storage loss in onions. Similarly significant positive correlation exists between total bulb yield and plant height, leaf number/plant, bulb diameter and bulb yield/plant. Moderate positive correlations have been observed between maturity date and yield, dry matter content was highly correlated with soluble solids, and higher correlations were also observed between storage loss and soluble solids. The objective of the study is to determine heritability estimates and correlations for characters among onion cultivars of North Western Nigeria. This is envisaged will assist in the breeding of superior onion cultivars within the zone. Thirteen onion cultivars were collected during an expedition covering north western Nigeria and Southern part of Niger Republic during 2013, which are areas noted for onion production. The cultivars were evaluated at two locations; Sokoto, in Sokoto State and Jega in Kebbi State all in Nigeria during the 2013/14 onion season (dry season) under irrigation. Combined analysis of the results revealed fresh bulb yield is highly significantly positively correlated with bulb height and cured bulb yield, and significant positive correlation with plant height and bulb diameter. It also recorded significant negative correlation with mean No. of leaves/plant and non significant negative correlation with bolting %. Cured bulb yield (marketable yield) had highly significant positive correlation with mean bulb weight and fresh bulb yield/ha, with significant positive correlation with bulb height. It also recorded highly significant negative correlation with No. of leaves/plant and significant negative correlation with bolting % and non significant positive correlation with plant height and non significant negative correlation with bulb diameter. High broad sense heritability estimates were recorded for plant height, fresh bulb yield, number of leaves/plant, bolting % and cured bulb yield. Medium to low broad sense heritabilities were also observed for mean bulb weight, plant height and bulb diameter.

Keywords: correlation, heritability, onions, North Western Nigeria

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Authors: Zakia Hbellaq

Abstract:

The Mediterranean region is considered a hotbed for climate change. Morocco is a semi-arid Mediterranean country facing water shortages and poor water quality. Its limited water resources limit the activities of various economic sectors. Most of Morocco's territory is in arid and desert areas. The potential water resources are estimated at 22 billion m3, which is equivalent to about 700 m3/inhabitant/year, and Morocco is in a state of structural water stress. Strictly speaking, the Kingdom of Morocco is one of the “very riskiest” countries, according to the World Resources Institute (WRI), which oversees the calculation of water stress risk in 167 countries. The surprising results of the Institute (WRI) rank Morocco as one of the riskiest countries in terms of water scarcity, ranking 3.89 out of 5, thus occupying the 23rd place out of a total of 167 countries, which indicates that the demand for water exceeds the available resources. Agriculture with a score of 3.89 is most affected by water stress from irrigation and places a heavy burden on the water table. Irrigation is an unavoidable technical need and has undeniable economic and social benefits given the available resources and climatic conditions. Irrigation, and therefore the agricultural sector, currently uses 86% of its water resources, while industry uses 5.5%. Although its development has undeniable economic and social benefits, it also contributes to the overfishing of most groundwater resources and the surprising decline in levels and deterioration of water quality in some aquifers. In this context, REUSE is one of the proposed solutions to reduce the water footprint of the agricultural sector and alleviate the shortage of water resources. Indeed, wastewater reuse, also known as REUSE (reuse of treated wastewater), is a step forward not only for the circular economy but also for the future, especially in the context of climate change. In particular, water reuse provides an alternative to existing water supplies and can be used to improve water security, sustainability, and resilience. However, given the introduction of organic trace pollutants or, organic micro-pollutants, the absorption of emerging contaminants, and decreasing salinity, it is possible to tackle innovative capabilities to overcome these problems and ensure food and health safety. To this end, attention will be paid to the adoption of an integrated and attractive approach, based on the reinforcement and optimization of the treatments proposed for the elimination of the organic load with particular attention to the elimination of emerging pollutants, to achieve this goal. , membrane bioreactors (MBR) as stand-alone technologies are not able to meet the requirements of WHO guidelines. They will be combined with heterogeneous Fenton processes using persulfate or hydrogen peroxide oxidants. Similarly, adsorption and filtration are applied as tertiary treatment In addition, the evaluation of crop performance in terms of yield, productivity, quality, and safety, through the optimization of Trichoderma sp strains that will be used to increase crop resistance to abiotic stresses, as well as the use of modern omics tools such as transcriptomic analysis using RNA sequencing and methylation to identify adaptive traits and associated genetic diversity that is tolerant/resistant/resilient to biotic and abiotic stresses. Hence, ensuring this approach will undoubtedly alleviate water scarcity and, likewise, increase the negative and harmful impact of wastewater irrigation on the condition of crops and the health of their consumers.

Keywords: water scarcity, food security, irrigation, agricultural water footprint, reuse, emerging contaminants

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Authors: Alexander A. Stakheev, Larisa V. Samokhvalova, Sergey K. Zavriev

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Fusarium fungi are among the most devastating plant pathogens distributed all over the world. Significant reduction of grain yield and quality caused by Fusarium leads to multi-billion dollar annual losses to the world agricultural production. These organisms can also cause infections in immunocompromised persons and produce the wide range of mycotoxins, such as trichothecenes, fumonisins, and zearalenone, which are hazardous to human and animal health. Identification of Fusarium fungi based on the morphology of spores and spore-forming structures, colony color and appearance on specific culture media is often very complicated due to the high similarity of these features for closely related species. Modern Fusarium taxonomy increasingly uses data of crossing experiments (biological species concept) and genetic polymorphism analysis (phylogenetic species concept). A number of novel Fusarium sibling species has been established using DNA barcoding techniques. Species recognition is best made with the combined phylogeny of intron-rich protein coding genes and ribosomal DNA sequences. However, the internal transcribed spacer of (ITS), which is considered to be universal DNA barcode for Fungi, is not suitable for genus Fusarium, because of its insufficient variability between closely related species and the presence of non-orthologous copies in the genome. Nowadays, the translation elongation factor 1 alpha (TEF1α) gene is the “gold standard” of Fusarium taxonomy, but the search for novel informative markers is still needed. In this study, we used two novel DNA markers, frataxin (FXN) and heat shock protein 90 (HSP90) to discover phylogenetic relationships between Fusarium species. Multilocus phylogenetic analysis based on partial sequences of TEF1α, FXN, HSP90, as well as intergenic spacer of ribosomal DNA (IGS), beta-tubulin (β-TUB) and phosphate permease (PHO) genes has been conducted for 120 isolates of 19 Fusarium species from different climatic zones of Russia and neighboring countries using maximum likelihood (ML) and maximum parsimony (MP) algorithms. Our analyses revealed that FXN and HSP90 genes could be considered as informative phylogenetic markers, suitable for evolutionary and taxonomic studies of Fusarium genus. It has been shown that PHO gene possesses more variable (22 %) and parsimony informative (19 %) characters than other markers, including TEF1α (12 % and 9 %, correspondingly) when used for elucidating phylogenetic relationships between F. avenaceum and its closest relatives – F. tricinctum, F. acuminatum, F. torulosum. Application of novel DNA barcodes confirmed the fact that F. arthrosporioides do not represent a separate species but only a subspecies of F. avenaceum. Phylogeny based on partial PHO and FXN sequences revealed the presence of separate cluster of four F. avenaceum strains which were closer to F. torulosum than to major F. avenaceum clade. The strain F-846 from Moldova, morphologically identified as F. poae, formed a separate lineage in all the constructed dendrograms, and could potentially be considered as a separate species, but more information is needed to confirm this conclusion. Variable sites in PHO sequences were used for the first-time development of specific qPCR-based diagnostic assays for F. acuminatum and F. torulosum. This work was supported by Russian Foundation for Basic Research (grant № 15-29-02527).

Keywords: DNA barcode, fusarium, identification, phylogenetics, taxonomy

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Authors: Layla Haidar, Shari Esquenazi-Karonika

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Introduction: Mental health includes one’s emotional, psychological, and interpersonal well-being; it ranges from “good” to “poor” on a continuum. At the individual-level, it affects how a person thinks, feels, and acts. Moreover, it determines how they cope with stress, relate to others, and interface with their surroundings. Research has yielded that mental health is directly related with short- and long-term physical health (including chronic disease), health risk behaviors, education-level, employment, and social relationships. As is the case with physical conditions like diabetes, heart disease, and cancer, mitigating the behavioral and genetic risks of debilitating mental health conditions like anxiety and depression can nurture a healthier quality of mental health throughout one’s life. In order to maximize the benefits of prevention, it is important to identify modifiable risks and develop protective habits earlier in life. Methods: The Adolescent Behaviors and Experiences Survey (ABES) dataset was used for this study. The ABES survey was administered to high school students (9th-12th grade) during January 2021- June 2021 by the Centers for Disease Control and Prevention (CDC). The data was analyzed to identify any associations between feelings of sadness, hopelessness, or increased suicidality among high school students with relation to their participation on one or more sports teams and their average daily consumed screen time. Data was analyzed using descriptive and multivariable analytic techniques. A multinomial logistic regression of each variable was conducted to examine if there was an association, while controlling for grade-level, sex, and race. Results: The findings from this study are insightful for administrators and policymakers who wish to address mounting concerns related to student mental health. The study revealed that compared to a student who participated on zero sports teams, students who participated in 1 or more sports teams showed a significantly increased risk of depression (p<0.05). Conversely, the rate of depression in students was significantly less in those who consumed 5 or more hours of screen time per day, compared to those who consumed less than 1 hour per day of screen time (p<0.05). Conclusion: These findings are informative and highlight the importance of understanding the nuances of student participation on sports teams (e.g., physical exertion, social dynamics of team, and the level of competitiveness within the sport). Likewise, the context of an individual’s screen time (e.g., social media, engaging in team-based video games, or watching television) can inform parental or school-based policies about screen time activity. Although physical activity has been proven to be important for emotional and physical well-being of youth, playing on multiple teams could have negative consequences on the emotional state of high school students potentially due to fatigue, overtraining, and injuries. Existing literature has highlighted the negative effects of screen time; however, further research needs to consider the type of screen-based consumption to better understand its effects on mental health.

Keywords: behavioral science, mental health, adolescents, prevention

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Authors: Olga V. Chervyakova, Elmira T. Tailakova, Vitaliy M. Strochkov, Kulyaisan T. Sultankulova, Nurlan T. Sandybayev, Lev G. Nemchinov, Rosemarie W. Hammond

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Sheep pox is a highly contagious infection that OIE regards to be one of the most dangerous animal diseases. It causes enormous economic losses because of death and slaughter of infected animals, lower productivity, cost of veterinary and sanitary as well as quarantine measures. To control spread of sheep pox infection the attenuated vaccines are widely used in the Republic of Kazakhstan and other Former Soviet Union countries. In spite of high efficiency of live vaccines, the possible presence of the residual virulence, potential genetic instability restricts their use in disease-free areas that leads to necessity to exploit new approaches in vaccine development involving recombinant DNA technology. Vaccines on the basis of recombinant proteins are the newest generation of prophylactic preparations. The main advantage of these vaccines is their low reactogenicity and this fact makes them widely used in medical and veterinary practice for vaccination of humans and farm animals. The objective of the study is to produce recombinant immunogenic proteins for development of the high-performance means for sheep pox prophylaxis. The SPV proteins were chosen for their homology with the known immunogenic vaccinia virus proteins. Assay of nucleotide and amino acid sequences of the target SPV protein genes. It has been shown that four proteins SPPV060 (ortholog L1), SPPV074 (ortholog H3), SPPV122 (ortholog A33) and SPPV141 (ortholog B5) possess transmembrane domains at N- or C-terminus while in amino acid sequences of SPPV095 (ortholog А 4) and SPPV117 (ortholog А 27) proteins these domains were absent. On the basis of these findings the primers were constructed. Target genes were amplified and subsequently cloned into the expression vector рЕТ26b(+) or рЕТ28b(+). Six constructions (pSPPV060ΔТМ, pSPPV074ΔТМ, pSPPV095, pSPPV117, pSPPV122ΔТМ and pSPPV141ΔТМ) were obtained for expression of the SPV genes under control of T7 promoter in Escherichia coli. To purify and detect recombinant proteins the amino acid sequences were modified by adding six histidine molecules at C-terminus. Induction of gene expression by IPTG was resulted in production of the proteins with molecular weights corresponding to the estimated values for SPPV060, SPPV074, SPPV095, SPPV117, SPPV122 and SPPV141, i.e. 22, 30, 20, 19, 17 and 22 kDa respectively. Optimal protocol of expression for each gene that ensures high yield of the recombinant protein was identified. Assay of cellular lysates by western blotting confirmed expression of the target proteins. Recombinant proteins bind specifically with antibodies to polyhistidine. Moreover all produced proteins are specifically recognized by the serum from experimentally SPV-infected sheep. The recombinant proteins SPPV060, SPPV074, SPPV117, SPPV122 and SPPV141 were also shown to induce formation of antibodies with virus-neutralizing activity. The results of the research will help to develop a new-generation high-performance means for specific sheep pox prophylaxis that is one of key moments in animal health protection. The research was conducted under the International project ISTC # K-1704 “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants” and under the Grant Project RK MES G.2015/0115RK01983 "Recombinant vaccine for sheep pox prophylaxis".

Keywords: prophylactic preparation, recombinant protein, sheep pox virus, subunit vaccine

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Authors: Kaaryn Cater

Abstract:

People vary in how they perceive, process, and react to internal, external, social, and emotional environmental factors; some are more sensitive than others. Compassionate people have a highly reactive nervous system and are more impacted by positive and negative environmental conditions (Differential Susceptibility). Further, some sensitive individuals are disproportionately able to benefit from positive and supportive environments without necessarily suffering negative impacts in less supportive environments (Vantage Sensitivity). Environmental sensitivity is underpinned by physiological, genetic, and personality/temperamental factors, and the phenotypic expression of high sensitivity is Sensory Processing Sensitivity. The hallmarks of Sensory Processing Sensitivity are deep cognitive processing, emotional reactivity, high levels of empathy, noticing environmental subtleties, a tendency to observe new and novel situations, and a propensity to become overwhelmed when over-stimulated. Several educational advantages associated with high sensitivity include creativity, enhanced memory, divergent thinking, giftedness, and metacognitive monitoring. High sensitivity can also lead to some educational challenges, particularly managing multiple conflicting demands and negotiating low sensory thresholds. A mixed methods study was undertaken. In the first quantitative study, participants completed the Perceived Success in Study Survey (PSISS) and the Highly Sensitive Person Scale (HSPS-12). Inclusion criteria were current or previous postsecondary education experience. The survey was presented on social media, and snowball recruitment was employed (n=365). The Excel spreadsheets were uploaded to the statistical package for the social sciences (SPSS)26, and descriptive statistics found normal distribution. T-tests and analysis of variance (ANOVA) calculations found no difference in the responses of demographic groups, and Principal Components Analysis and the posthoc Tukey calculations identified positive associations between high sensitivity and three of the five PSISS factors. Further ANOVA calculations found positive associations between the PSISS and two of the three sensitivity subscales. This study included a response field to register interest in further research. Respondents who scored in the 70th percentile on the HSPS-12 were invited to participate in a semi-structured interview. Thirteen interviews were conducted remotely (12 female). Reflexive inductive thematic analysis was employed to analyse data, and a descriptive approach was employed to present data reflective of participant experience. The results of this study found that compassionate students prioritize work-life balance; employ a range of practical metacognitive study and self-care strategies; value independent learning; connect with learning that is meaningful; and are bothered by aspects of the physical learning environment, including lighting, noise, and indoor environmental pollutants. There is a dearth of research investigating sensitivity in the educational context, and these studies highlight the need to promote widespread education sector awareness of environmental sensitivity, and the need to include sensitivity in sector and institutional diversity and inclusion initiatives.

Keywords: differential susceptibility, highly sensitive person, learning, neurosensitivity, sensory processing sensitivity, vantage sensitivity

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Authors: Shagufta Jabeen, Faez J. Firdaus Abdullah, Zunita Zakaria, Nurulfiza M. Isa, Yung C. Tan, Wai Y. Yee, Abdul R. Omar

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Pasteurella multocida (PM) is an important veterinary opportunistic pathogen particularly associated with septicemic pasteurellosis, pneumonic pasteurellosis and hemorrhagic septicemia in cattle and buffaloes. P. multocida serotype A has been reported to cause fatal pneumonia and septicemia. Pasteurella multocida subspecies multocida of serotype A Malaysian isolate PMTB2.1 was first isolated from buffaloes died of septicemia. In this study, the genome of P. multocida strain PMTB2.1 was sequenced using third-generation sequencing technology, PacBio RS2 system and analyzed bioinformatically via de novo analysis followed by in-depth analysis based on comparative genomics. Bioinformatics analysis based on de novo assembly of PacBio raw reads generated 3 contigs followed by gap filling of aligned contigs with PCR sequencing, generated a single contiguous circular chromosome with a genomic size of 2,315,138 bp and a GC content of approximately 40.32% (Accession number CP007205). The PMTB2.1 genome comprised of 2,176 protein-coding sequences, 6 rRNA operons and 56 tRNA and 4 ncRNAs sequences. The comparative genome sequence analysis of PMTB2.1 with nine complete genomes which include Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli and five P. multocida complete genome sequences including, PM70, PM36950, PMHN06, PM3480, PMHB01 and PMTB2.1 was carried out based on OrthoMCL analysis and Venn diagram. The analysis showed that 282 CDs (13%) are unique to PMTB2.1and 1,125 CDs with orthologs in all. This reflects overall close relationship of these bacteria and supports the classification in the Gamma subdivision of the Proteobacteria. In addition, genomic distance analysis among all nine genomes indicated that PMTB2.1 is closely related with other five Pasteurella species with genomic distance less than 0.13. Synteny analysis shows subtle differences in genetic structures among different P.multocida indicating the dynamics of frequent gene transfer events among different P. multocida strains. However, PM3480 and PM70 exhibited exceptionally large structural variation since they were swine and chicken isolates. Furthermore, genomic structure of PMTB2.1 is more resembling that of PM36950 with a genomic size difference of approximately 34,380 kb (smaller than PM36950) and strain-specific Integrative and Conjugative Elements (ICE) which was found only in PM36950 is absent in PMTB2.1. Meanwhile, two intact prophages sequences of approximately 62 kb were found to be present only in PMTB2.1. One of phage is similar to transposable phage SfMu. The phylogenomic tree was constructed and rooted with E. coli, A. pleuropneumoniae and H. parasuis based on OrthoMCL analysis. The genomes of P. multocida strain PMTB2.1 were clustered with bovine isolates of P. multocida strain PM36950 and PMHB01 and were separated from avian isolate PM70 and swine isolates PM3480 and PMHN06 and are distant from Actinobacillus and Haemophilus. Previous studies based on Single Nucleotide Polymorphism (SNPs) and Multilocus Sequence Typing (MLST) unable to show a clear phylogenetic relatedness between Pasteurella multocida and the different host. In conclusion, this study has provided insight on the genomic structure of PMTB2.1 in terms of potential genes that can function as virulence factors for future study in elucidating the mechanisms behind the ability of the bacteria in causing diseases in susceptible animals.

Keywords: comparative genomics, DNA sequencing, phage, phylogenomics

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