Abstracts of the 2nd International Electronic Conference on Genes ^†

Abstract

The 2nd International Electronic Conference on Genes (IECGE2024) was organized by the MDPI open access journal Genes and held online from the 11th to 13th December 2024. This conference provided leading scientists with an online platform to share their latest research and engage in exciting discussions. The main topics and sessions of the conference were as follows: Non-coding RNAs in Health and Diseases; Genetic Diagnosis and Targeted Therapy in Cancer; Microbial Genetics and Genomics; Technologies and Resources for Genetics Research; Human Genomics and Genetic Diseases. This was an excellent opportunity for researchers and scientists to interact with each other, communicate with their colleagues, learn from each other, share ideas and results, help solve problems, and suggest alternative options for a better future.

1. Session: Non-Coding RNAs in Health and Diseases

1.1. Association of the Expression Levels of lncRNAs GAS5, HAND2-AS1, LINC00152 and LINC00339 and Tumor Size with Lymphogenous Metastasis in Clear-Cell Renal-Cell Carcinoma

• Irina Valerievna Pronina ^1,2, Vitaly Loginov ^1,3

¹ 

Institute of General Pathology and Pathophysiology, Russia

² 

Department of Physiology, Human Ecology and Medical and Biological Sciences, State University of Education, 105005 Moscow, Russia

³ 

Research Center for Medical Genetics, Russia

The frequency of clear-cell renal-cell carcinoma (ccRCC) metastasis has reached 25–30%, which makes the search for prognostic biomarkers urgent. Long non-coding RNAs (lncRNAs) regulate biological processes and may serve as prognostic markers. The aim of our study was to compare the expression levels of lncRNAs GAS5, HAND2-AS1, LINC00152, and LINC00339 in tumors and normal kidney tissue and analyze the association of their changes with clinicopathological parameters of ccRCC. We used archival RNA samples (RIN > 7) from 70 primary tumors with a confirmed diagnosis of ccRCC and 70 adjacent normal kidney tissues. Expression analysis was performed by RT-qPCR. Statistical analysis was performed using a multivariate ANOVA test (p ≤ 0.05). A decrease in the expression of GAS5 by 5.25, HAND2-AS1 by 5.92, LINC00152 by 2.04, and LINC00339 by 2.34 times was revealed in the tumor tissues. The expression of LINC00152 did not change at T1, T2, and T3 (the TNM classification) and decreased by 33.43 times when the tumor grew beyond Gerota’s fascia (T4). LINC00152 expression was lower in T4 tumors compared to tumors of a smaller size: T1/T4—10.86 times; T2/T4—22.76 times; and T3/T4—27.11 times. LINC00152 expression decreased by 4.71 times in cases with multiple lesions of regional lymph nodes (normal/N2), including compared with tumors with no metastases in the lymph nodes (N0/N2) or one affected lymph node (N1/N2), by 4.36 and 7.26 times, respectively. HAND2-AS1 had a lower level of expression in tumors with multiple lesions of regional lymph nodes: normal/N2—15.02 times; N0/N2—5.84 times; N1/N2—4.04 times. The significant decrease in the expression of HAND2-AS1 and LINC00152 when more than one regional lymph node is affected (N2 according to the TNM classification) in ccRCC allows us to nominate them as prognostic markers of lymphogenous metastasis in the absence of the ability to evaluate regional lymph nodes (Nx).

1.2. Decoding miRNA Interactions and SNP Variability in the 3′UTR of NF-κB: Implications for Gene Regulation and Cancer

• Muhammad Safdar
• Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan

Nuclear factor-κB (NF-κB) is a pivotal transcription factor family involved in key biological processes such as inflammation, immune response, cell survival, apoptosis, cellular stress reactions, and tumorigenesis. The dysregulation of NF-κB is increasingly recognized as a critical factor in the initiation and progression of various cancers. Polymorphisms in miRNA genes or their target sites (miRSNPs) can significantly impact miRNA activity. While polymorphisms in miRNA genes are rare, studies have shown that SNPs at miRNA target sites can either enhance or reduce the strength of miRNA–target interactions. The objective of this study was to identify miRSNPs in the NF-κB gene and SNPs in miRNA genes targeting their 3′UTR and to evaluate their potential roles in apoptosis and cancer using computational tools. We identified 121 miRNA-binding sites corresponding to 101 distinct miRNAs, as well as 16 SNPs located within miRNA-binding regions of the NF-κB 3′UTR. Notably, a binding site for miR-6826-5p in the NF-κB 3′UTR harbors an SNP (rs960795970, A/G), and the same miRNA’s genomic sequence contains an SNP (rs6771809, C/T) at the same nucleotide position as rs960795970. Additionally, miR-6826-5p has three other SNPs (rs757908839, A/G; rs746350709, C/T; and rs115693266, A/C), with the first positioned directly at its binding site. This cross-matching between miRSNP (rs960795970) in the NF-κB 3′UTR and an SNP (rs6771809) in the miR-6826-5p genomic sequence in the same binding region suggests potential functional interplay. Moreover, miR-6826-5p was found to target several genes associated with cancer and apoptosis, including HIP1, TRIAP1, GSKIP, NIN, DAP, CAAP1, XIAP, TMBIM1, TMBIM4, TNFRSF10A, RAD21, AKT1, BAG1, and BAG4, despite having no previously established cancer-related interactions. These findings imply that miR-6826-5p may play a critical role in apoptosis through pathways beyond NF-κB, potentially influencing cancer progression via alternative mechanisms.

1.3. Identification of Biomarkers for Early Diagnosis and Prognosis in Sepsis: A Comprehensive Analysis

• Kumari Astha Rupali, Dhiraj Kishore
• Department of General Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh-221005, India

Introduction: Sepsis is a life-threatening disease caused by an excessive immune response to infection, causing inflammation, tissue damage, and organ failure. Globally, sepsis caused 19.7% of all fatalities in 2017, with India having one of the highest rates, with over 4 million cases annually. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression by binding to messenger RNAs (mRNAs) and blocking their transcription. Identifying miRNAs as predictive biomarkers could significantly reduce the worldwide burden of sepsis by enhancing patient care, management, and treatment. This study aims to discover potential diagnostic biomarkers implicated in the dysregulated immune response and provide insights for mechanistic illness progression research.

Methods: The Gene Expression Omnibus (GEO) datasets GSE134364, GSE134358, GSE94717, GSE236713, and GSE131761 were used to identify the differentially expressed genes involved in sepsis. Biological targets were identified that coincide with the identified differentially expressed miRNAs. The biological pathways were analyzed using KEGG and GO. Hub genes were identified for further functional and signaling pathway analysis.

Results: Several differentially expressed miRNA biomarkers were identified between the control and sepsis groups (p0.05 and fold change >2). MiR-146a regulates the immune response, miR-155 promotes pro-inflammatory cytokine expression, miR-150 regulates immune cell function and inflammation, and miR-21 is involved in apoptosis and inflammation. Moreover, we identified matrix metalloproteinase (MMP8 and MMP9) as being involved in neutrophil degranulation.

Conclusions: MiRNAs regulating MMP-8 and MMP-9 in sepsis regulate immune responses and inflammation, leading to tissue damage. MiR-21 downregulates MMP-9 expression, while miR-146a indirectly affects MMP expression by targeting upstream signaling molecules. Targeting specific miRNAs that regulate MMPs offers a potential therapeutic strategy for managing sepsis. By modulating the levels of these miRNAs, it may be possible to control the activity of MMPs and reduce the harmful effects of excessive inflammation and tissue damage.

1.4. Predicting Mimotopes of Amyloid Beta (Aβ₄₂) from Non-Coding DNA as Candidates for Synthetic Peptide Vaccine Design Against Alzheimer’s Disease

• Navya Raj ¹, Shidhi P R ², Deepthi Varughese ³, Achuthsankar S Nair ⁴, Pawan Dhar ⁵

¹ 

Department of Health Informatics, College of Health Sciences, Saudi Electronic University, Dammam, Saudi Arabia

² 

Department of Zoology, University of Kerala, Thiruvananthapuram, Kerala, India

³ 

Centre for Development and Aging Research, Inter University Centre for Biomedical Research & Super Specialty Hospital, Mahatma Gandhi University Campus at Thalappady, Kerala, India

⁴ 

Department of Computational Biology and Bioinformatics, University of Kerala, India

⁵ 

School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.

Introduction: What could be an ideal raw material for developing a drug or a vaccine? In line with the research on different biomolecules, like DNA, RNA, proteins, and peptides, we explore the potential use of non-coding DNA in designing lifesaving vaccines and drugs.

Methods: Using computational approaches, a multi-parametric virtual library of novel peptides from the intergenic regions of the Escherichia coli genome was generated, and their therapeutic potential was studied. The potential antigens from the peptides were analyzed for their B cell epitopes and MHC-binding T cell epitopes, which find application in epitope-based vaccine design. Alzheimer’s disease (AD) immunotherapy was selected as an example to demonstrate the possible application of the non-coding DNA-derived peptides as promising candidates for synthetic peptide vaccine design.

Results: Sequence and structure comparison studies between toxic amyloid beta (Aβ₄₂) peptides and the non-coding DNA-derived peptides revealed promising matches. The structural mimics of Aβ₄₂ with potential mimotopes were further studied through mimotope–antibody docking and molecular dynamic simulation for their affinity with the antibody’s antigen-binding fragment (Fab).

Conclusions: Our study put forward a novel method to identify therapeutic peptides from non-coding DNA, which offers a non-obvious source of potential mimotope candidates for designing novel synthetic vaccines against Alzheimer’s Disease.

1.5. The Role of Non-Coding RNAs (miRNA and lncRNA) in the Diagnosis and Prognosis of Rheumatoid Arthritis

• Mamatha Bhanu L S
• Yuvaraj’s College, University of Mysore, India

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting multiple joints and causing adverse effects on organs. Early diagnosis, aggressive treatment, and disease-modifying anti-rheumatic drugs (DMARDs) have improved the management and long-term prognosis of the disease. The global prevalence of RA is increasing, primarily in women, and is associated with disability and mortality. The prognosis depends on the disease stage at diagnosis; early diagnosis can prevent or delay disease progression in 90% of patients, preventing irreversible joint damage and disability. However, non-coding RNA (ncRNA), including microRNA and long ncRNA (lncRNA), which are small, non-coding segments of RNA that regulate gene expression, has been discovered to be deregulated in diseases like RA, potentially contributing to its pathogenesis, progression, and treatment. Pre-clinical RA, characterized by auto-antibodies and biomarkers before clinical RA, is a stage of RA research aimed at the early diagnosis and control of immunological abnormalities. For instance, SNP rs2850711 in lnc00305 links RA susceptibility to the response of miR-10a to methotrexate (MTX) treatment, and ncRNA plays a crucial role in refractory RA by regulating drug sensitivity. The reduced miR-20a expression in rheumatoid arthritis synovial fibroblasts (RASFs) activates Janus Kinase (JAK)-mediated inflammation, promoting cell proliferation and apoptosis resistance. This study highlights changes in ncRNAs, disease progression, and novel therapeutic targets and strategies.

2. Session: Genetic Diagnosis and Targeted Therapy in Cancer

2.1. Recurrent Clinically Significant Mutations in Acute Myeloid Leukemia: Analysis of the Cancer Genomics Database and Proprietary Data on the CBio Portal

• Elena Voropaeva ^1,2, Irina Chuhontseva ², Vladimir Maximov ^1,3, Tatyana Pospelova ²

¹ 

Research Institute of Internal and Preventive Medicine-Branch of the Federal State Budget Scientific Institution The Federal Research Center Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences

² 

Department of Hematology, Novosibirsk State Medical University

³ 

Novosibirsk State Medical University

According to the literature, the frequency of mutations in genes associated with the development of acute myeloid leukemia (AML) varies significantly.

The objective of this study was to obtain up-to-date data on the prevalence of mutations in the “hot spots” of the FLT3, NPM1, IDH1, IDH2, and DNMT3A genes in AML.

Methods: An analysis of NGS data from 1567 patients with AML presented in the cancer genomics database on C-Bioportal and 124 Russian patients with AML was performed.

Results: According to the database analysis, at the time of the diagnosis of the disease, 46.6% of patients had mutations in DNMT3A p.R882, NPM1 p.W288Cfs*12, FLT3-ITD, FLT3-TKD1, IDH1 p.R132, and IDH2 p.R140. Only in a third of cases (30.1%) did the DNMT3A mutation of R.R882 occur in patients in an isolated variant. In 47.4% of cases, this was combined with NPM1 p.W288Cfs*12, in 34.1% with mutations in the hot spots of the FLT3 gene, and in 23.0% with recurrent mutations in IDH1 and IDH2. At the same time, the combination revealed by our data remains highly significant (p0.001) even after adjusting for the multiplicity of comparisons (q0.001). In Russian AML patients, the mutation rates in the “hot spots” of genes generally corresponded to the data from the C-Bioportal cancer genomics database and amounted to the following: DNMT3A p.R882—7.3%; NPM1 p.W288Cfs*12—15.3%; FLT3-ITD—14.5%; FLT3-TKD1—4.0%; IDH1 p.R132—5.6%; IDH2 p.R140—10.5%.

Conclusions: The data obtained indicate that in 40–50% of AML cases, clinically significant recurrent mutations in one or more of the studied genes are detected at the onset of the disease. Mutations for which targeted drugs (FLT3, IDH1, and IDH2 inhibitors) have been developed occur in 35% of patients. In one-fifth of cases (18.1%) of AML, NPM1 p.W288Cfs*12 is detected, which can be used as an independent target for the molecular assessment of minimal residual disease.

2.2. RUNX1 Mutations in Stomach Adenocarcinoma: Unveiling a New Player in Solid Tumors and Its Immune Microenvironment Impact

• Swarna Beesetti
• St. Jude Children’s Research Hospital, USA

Introduction: RUNX1, a transcription factor crucial for hematopoiesis and frequently mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), has recently been implicated in solid tumors. This study investigates the role of RUNX1 mutations in stomach adenocarcinoma and their impact on the tumor immune microenvironment.

Methods: Bioinformatic approaches using GEPIA (Gene Expression Profiling Interactive Analysis) and TIMER (Tumor Immune Estimation Resource) were used to analyze gene expression data and immune cell infiltration in stomach adenocarcinoma samples with and without RUNX1 mutations.

Results: Stomach adenocarcinomas harboring RUNX1 mutations exhibited significantly higher levels of macrophage infiltration compared to wild-type tumors. Gene expression analysis revealed upregulation of key inflammatory mediators, including IFNG, TNF, IL1B, and NLRP3, in RUNX1-mutated samples. These findings suggest a strong association between RUNX1 mutations and an enhanced inflammatory tumor microenvironment.

Conclusions: This study establishes a potential link between RUNX1 mutations, increased immune cell recruitment, and elevated expression of pro-inflammatory genes in stomach adenocarcinoma. This altered immune landscape may have important implications for disease progression and the development of targeted therapeutic approaches. Furthermore, these findings extend our understanding of RUNX1’s role beyond hematological malignancies, highlighting its significance in solid tumors and opening new avenues for research and potential therapeutic interventions.

2.3. Study of SUM159 Cell Lines Untreated and Treated with the Drug Mebendazole and Its Effect on Transcriptome Level (Rna-Seq Analysis)

• Pramodkumar P Gupta ¹, Mala M Parab ¹, Janhavi Tripathi ¹, Mayur Sonkusare ¹, Hridhya Yogesh Nair ¹, Mrunal Gokhale ¹, Amit Kumar Shrivastava ², Prerona Boruah ¹, Debjani Dasgupta ¹

¹ 

School of Biotechnology and Bioinformatics, D Y Patil Deemed to be University, Navi Mumbai, Maharashtra, India

² 

Department of Pharmacology, Universal College of Medical Sciences, Bhairahawa, Rupandehi, Nepal.

Breast cancer is a leading cause of cancer-related deaths worldwide. While significant progress has been made in its diagnosis and treatment, it remains a major public health concern. This study aimed to investigate the transcriptomic effects of Mebendazole, an antiparasitic drug, on SUM159 cell lines, a model for triple-negative breast cancer (TNBC). RNA-Seq analysis was conducted to identify differentially expressed genes (DEGs) between untreated and Mebendazole-treated cells. Our analysis revealed significant transcriptional alterations in Mebendazole-treated SUM159 cells using data collected from the NCBI GEO database. The GALAXY server NGS data analysis framework was used, followed by FASTQC, FASTP, HISAT2, SAMTOOL_Sort, SAMTOOLs_Dataset, the Uploading GTF for Humans standard file from UCSC, Hiseq-Count, and analysis in R-reported DEGs. The list of 820 DEGs were analyzed for enrichment using EnrichR, David tools, to understand the involvement of DEGs in biological processes. DEGs were enriched in pathways related to cell cycle regulation, apoptosis, survival signaling, and metabolism, suggesting that Mebendazole exerts its effects through multiple mechanisms. Notably, the identified DEGs were associated with various diseases, including breast cancer and neurodegenerative disorders. The downregulated genes from the analysis reported to be involved in liver cirrhosis, atherosclerosis, neurological disorder, cellular adhesion, the extracellular matrix, the transcription factor, and the assembly of collagens in humans, whereas the upregulated genes were more involved in the pathways of breast cancer, ovarian cancer, the cell cycle and the cell division process. These findings highlight the potential for Mebendazole to be repurposed as a therapeutic agent beyond its traditional use in parasitic infections. Further research is needed to validate these in vitro findings in vivo and explore the clinical implications of Mebendazole for cellular mechanisms, the signal cascade, TNBC and neurodegenerative diseases.

2.4. The Role of Iroquois-Class Homeobox Genes in Cancer Stemness

• Amali Thannkoon ^1,2, Achala Vitharanage ^1,2, Jyotsna Batra ^1,2,3

¹ 

School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, QLD 4102, Australia

² 

The Centre for Genomics and Personalised Health, Queensland University of Technology, Brisbane, QLD 4059, Australia

³ 

Translational Research Institute, Queensland University of Technology, Brisbane, QLD 4102, Australia

Introduction: Cancer poses a global challenge due to its aggressive metastasis, treatment resistance, and relapse, often driven by cancer stem cells (CSCs). CSCs evade conventional therapies, emphasizing the need for CSC-targeted strategies. The Iroquois homeobox (IRX) gene family is linked to cancer stemness, but the exact roles and therapeutic potential of IRX genes in CSC promotion remain unclear.

Methods: Normalized gene expression data from all six IRX genes were downloaded from publicly available databases. A comparison of IRX expression with cancer stages and drug sensitivity was conducted. All data processing and analyses were performed using GraphPad Prism (10.0.2). A Student’s t-test was used to compare the differences between two groups and a one-way ANOVA test was applied to compare multiple groups. The functional role of these IRXs in CSC regulation will be assessed using in vitro and in vivo models.

Results: In silico analyses revealed an elevated expression of Iroquois homeobox 3 (IRX3) and Iroquois homeobox 5 (IRX5) in castration-resistant prostate cancer (PCa) patient samples and patient-derived xenografts (PDXs) compared to primary tumors, with further upregulation after enzalutamide treatment. Gene set enrichment analysis identified significant enrichment of stemness-associated gene signatures in PCa patients with high IRX3 expression. Additionally, expression data revealed IRX5 upregulation with therapy resistance in several breast cancer (BCa) cell lines. Future studies will explore the roles of IRX3 and IRX5 in CSC maintenance, tumor progression, metastasis, and therapeutic resistance in PCa and BCa models.

Conclusions: Given the link between CSCs, therapy resistance, and metastasis, we hypothesize that elevated IRX3 and IRX5 expression contributes to CSC maintenance in PCa and BCa. If validated, these IRXs could serve as novel therapeutic targets, inhibiting CSC-driven tumor growth and progression. This would represent a significant advancement, as no therapies currently target CSCs and ultimately preventing metastasis, drug resistance, and relapse in PCa and BCa patients.

3. Session: Human Genomics and Genetic Diseases

3.1. Relevance of Pharmacogenomics, CYP450 Genes and Their Genetic Variations in Drug Metabolism and Toxicity

• Surbhi Malhotra, Manik Kaushal, Gauri Awasthi
• Genomics and Personalized Medicine, Tata Consultancy Services

Background: Variations in drug responses are related to the inherited characteristics of the genome, causing wide variability in individual responses to drugs. Pharmacogenetics or pharmacogenomics analyses help us to understand the DNA variations that are related to drug action (pharmacodynamics) and drug disposition (pharmacokinetics). Using a pharmacogenomics (PGx) approach, we studied different CYP450 genes that are associated with drug metabolism, along with their genomic variants, thus strengthening our understanding of personalized medicines.

Methods: We downloaded the PGx database from the FDA website and collated the data utilizing various evolutionary tools and software. We also provided an overview of the current progress in computational approaches to the prediction of drug metabolism and toxicity using a combination of knowledge graph- and AI-based approaches. Utilizing ethnicity data from published sources, we also correlated the clinical implications of drug metabolism and toxicity variability in different population cohorts.

Results: We observed that 42 unique drugs catering to nine different therapeutic areas are associated with six CYP450 (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A5) genes/biomarkers and 32 unique variants/SNPs. The allelic frequencies (AFs) of the 32 variants were also studied in different population and ethnicity groups (from a published database). We observed unique combinations of drugs, biomarkers, and variant associations that highlight how the metabolism of a drug is controlled/regulated/metabolized by the genetic basis of an individual. We observed 32 unique combinations where the same CYP biomarker and its variant were shared by different drugs associated with different therapeutic areas.

Conclusions: PGx studies should be inclusive, and policy makers/drug regulators should include such approaches to better understand the genetic basis of drug-metabolizing genes to improve drug response. The PGx approach also highlights a significant association between Precision Medicine and Pharmacovigilance by understanding the drug metabolism and toxicity in accordance with an individual’s genetic makeup.

3.2. A Leaky Deep Intronic Splice Variant in CLRN1 Is Associated with Non-Syndromic Retinitis Pigmentosa

• Asodu Sandeep Sarma ¹, Maria Abu Elasal ¹, Samer Khateb ¹, Daan M Panneman ², Frans PM Cremers ², Eyal Banin ¹, Dror Sharon ¹

¹ 

Division of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120 Jerusalem, Israel

² 

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands

Introduction: Inherited retinal diseases (IRDs) are clinically complex and genetically heterogeneous visual impairment disorders with varying penetrance and severity. Disease-causing variants in at least 289 nuclear and mitochondrial genes have been implicated in their pathogenesis. Methods: Genomic DNA was isolated from peripheral blood lymphocytes. Exome sequencing was performed on an Illumina platform, and splicing analysis was performed using a pET01 minigene plasmid in Hela cells. Results: In the current study, we performed exome sequencing on a 51-year-old Ashkenazi Jewish patient with non-syndromic retinitis pigmentosa (RP) and identified compound heterozygous variants in the CLRN1 gene: a known pathogenic missense [p.(N48K)] and a novel deep intronic variant, c.254-643G>T. A minigene splicing assay was performed, aiming to study the effect of the c.254-643G>T variant on CLRN1 pre-mRNA splicing, and this revealed the inclusion of a pseudo-exon that was also reported to be included in the transcript due to an adjacent variant, c.254-649T>G. However, unlike the reported c.254-649T>G variant, c.254-643G>T showed aberrant splicing in a leaky manner, implying that the identified variant is not totally penetrant. Conclusions: The non-syndromic phenotype observed in this index case may be attributed to the leaky nature of this variant, which causes some normal transcripts to be produced. To conclude, we report on a novel deep intronic variant in CLRN1 that causes non-syndromic RP due to the leaky nature of the identified variant.

3.3. ACE Insertion/Deletion Polymorphism as a Potential Risk Factor for Congenital Heart Disease Among North Indians: Insights from a Tertiary Pediatric Cardiac Care Centre Study

• Shadab Ahamad ¹, Prachi Kukshal ¹, Ajay Kumar ¹, Subramanian Chellappan ², Yogesh Sathe ², Prabhatha Rashmi Murthy ³

¹ 

Sri Sathya Sai Sanjeevani Research Foundation, Palwal, Haryana, India-121102

² 

Sri Sathya Sai Sanjeevani International Centre for Child Heart Care & Research, Palwal, Haryana, India-121102

³ 

Sri Sathya Sai Sanjeevani Centre for Child Heart Care & Training in Paediatric Cardiac Skills, Navi Mumbai, Maharashtra, India-410210

Background: Congenital heart disease (CHD) is a global health concern, particularly in low- to middle-income countries like India. The renin–angiotensin–aldosterone system (RAAS) plays a crucial role in the development of cardiovascular disorders and hypertension, with the angiotensin-converting enzyme insertion/deletion (ACE I/D) polymorphism being a key genetic factor. Our study aims to elucidate the genetic influence of ACE I/D polymorphism on CHD in a north Indian cohort.

Methods: A total of 667 CHD cases, including 433 individuals with parental data and 104 controls, were enrolled and genotyped via polymerase chain reaction. Case–control association, parental transmission tests, and the association of patients’ and parents’ clinical parameters with ACE I/D were explored.

Results: The frequencies of the DD, ID, and II genotypes were 0.23, 0.47, and 0.30, respectively. Our findings highlight significant associations, notably the increased CHD risk conferred by the DD genotype in females (p = 0.036; OR = 1.68), its correlation with abnormal hemoglobin (p = 0.049; OR = 1.68), and its impact on primigravida (p = 0.05). Conversely, the II genotype was found to significantly elevate the risk of CHD in the offspring of tobacco-consuming fathers by 2.5-fold (p = 0.029). Notably, cyanotic cases exhibited a heightened prevalence of ACE I/D mutations (p = 0.059), with the Tetralogy of Fallot showing the strongest association (p = 0.024). Additionally, the DD genotype’s involvement in conditions such as stenosis (p = 0.026) and pulmonary artery hypertension (p = 0.05) underscores its clinical relevance. The parent-of-origin test showed maternal transmission of the D allele in combined (p = 0.037) and acyanotic cases (p = 0.039) and paternal transmission in ventricular septal defect (p = 0.021).

Conclusions: This is the first study from India and possibly the only study globally that reports a significant association between ACE I/D and CHD, highlighting the importance of genetic factors in CHD susceptibility.

3.4. Bioinformatics Analysis of Gymnema Sylvestre and Withania Somnifera on Insulin Resistance Pathway Targets

• Maulshree Shree, Dhiraj Kishore
• Dept. of Gen. Medicine, IMS, Banaras Hindu University

Ayurvedic medicine offers a holistic approach to managing complex diseases such as Type 2 diabetes mellitus, which is primarily driven by insulin resistance. Bioinformatics has emerged as a powerful tool for understanding the molecular mechanisms underlying the effects of Ayurvedic drugs on insulin resistance pathway targets. This study uses bioinformatics techniques to analyze the pharmacological potential of Ayurvedic herbs traditionally used to treat diabetes, such as Gymnema sylvestre and Withania somnifera. The key bioactive compound in G. sylvestre has been shown to suppress the taste of sugar and reduce glucose absorption in the intestines, promoting better blood sugar control. Also, recent studies highlight W. somnifera’s role in improving insulin sensitivity and reducing blood sugar levels. Using databases like DrugBank, STITCH, and PubChem, active compounds in these herbs were identified, and their interactions with key targets in the insulin signaling pathway, including IRS1, PI3K, and GLUT4, were analyzed. Molecular docking, network pharmacology, and gene expression analyses revealed that these phytochemicals potentially modulate glucose uptake, insulin sensitivity, and inflammation, key processes involved in insulin resistance. Our findings suggest that the synergistic effects of these herbs could offer a complementary approach to managing insulin resistance, warranting further experimental validation. The study paves the way for rational drug design based on traditional knowledge. It contributes to the growing field of systems biology in exploring the efficacy of multi-compound, multi-target therapeutic strategies.

3.5. Genetic Insights and Therapeutic Approaches for Epidermolysis Bullosa in the Saudi Arabian Population

• Nancy Shehata ¹, Noor A. Shaikh ², Husna Irfan Thalib ³, Bayan Al Zoabi ³

¹ 

Department of Dermatology, King Abdullah Medical Complex, Jeddah, Saudi Arabia

² 

Department of Genetic Medicine, Faculty of Medicine at King Abdul Aziz University, Jeddah, Saudi Arabia

³ 

General Medicine Practice Program, Batterjee Medical College, Jeddah, Saudi Arabia

Introduction: Epidermolysis bullosa (EB) is a genetic skin disorder characterized by extreme skin fragility and recurrent blister formation, which significantly impacts the quality of life of patients afflicted by this disease. In Saudi Arabia, the diagnosis of EB relies heavily on thorough clinical assessments, as well as genetic testing to confirm specific EB subtypes.

Methods: In this review, we conducted a systematic literature search focusing on genetic implications and management strategies for Epidermolysis bullosa within the Saudi Arabian population. We analyzed peer-reviewed studies and clinical guidelines to synthesize insights into their prevalence, genetic mutations, and therapeutic approaches specific to this demographic.

Results: Although prenatal diagnosis is an option for families with a history of EB, its application is limited in Middle Eastern countries due to a variety of factors, including access to genetic services and cultural considerations. Current management strategies primarily focus on symptomatic relief. However, recent years have seen a significant increase in the development of innovative therapeutics. For example, gene modification and stem cell therapy are currently under extensive investigation. These experimental approaches promise far more effective long-term solutions but face numerous challenges.

Conclusions: Further research is necessary to improve diagnostic accuracy and treatment options, as well as to enhance the overall standard of care for patients with EB. This review delves into the genetic basis of EB and its management within the Saudi population, highlighting the critical need for genetic insights to facilitate accurate diagnosis, tailor targeted treatments, and provide effective counseling for patients and their families.

3.6. Non-Coding Single-Nucleotide and Structural Variants Affecting the EYS Putative Promoter Cause Autosomal Recessive Retinitis Pigmentosa

• Dror Sharon ¹, Shai Ovadia ², Jaya Krishnan ³, Manon Bouckaert ⁴, Daan M. Panneman ⁵, Milton English ³, Johanna Valensi ¹, Frans P.M. Cremers ⁵, Tamar Ben-Yosef ⁶, L Ingeborgh van den Born ⁷, Suzanne E. de Bruijn ⁵, Susanne Roosing ⁵, Eyal Banin ¹, Samer Khateb ¹, Ruth Ashery-Padan ², Frauke Coppieters ^4,8, Anand Swaroop ³, Tamar Hayman ¹

¹ 

Division of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, Israel.

² 

Department of Human Molecular Genetics and Biochemistry, Faculty of Medical & Health Sciences and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

³ 

Neurobiology, Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD, USA

⁴ 

Center for Medical Genetics Ghent (CMGG), Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

⁵ 

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands

⁶ 

Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

⁷ 

The Rotterdam Eye Hospital, Rotterdam, The Netherlands

⁸ 

Department of Pharmaceutics, Ghent University, Ghent, Belgium

Purpose: To characterize the effect of variants identified in the 5′-untranslated region of the EYS gene in patients with autosomal recessive retinitis pigmentosa (ARRP).

Methods: The variant screening included gene panel, Sanger, exome, and genome sequencing. Functional validation included an electrophoretic mobility shift assay (EMSA) and various luciferase assays. Clinical examination included visual acuity testing, electroretinography (ERG) testing, and retinal imaging.

Results: Patients with RP from six EYS biallelic Arab-Muslim families harbored a 5′-non-coding EYS variant, c.-453G>T, and four harbored a structural variant affecting the 5′-non-coding exons. The EMSA analysis revealed an effect on the binding of transcription factors for c.-453G>T and a neighboring variant, c.-454G>T, which was reported previously in a patient with RP. Dual luciferase assays using the overexpression of various transcription factors showed distinct effects on expression. c.-453G>T was associated with higher luciferase expression with CRX overexpression, and c.-454G>C was associated with higher luciferase expression with OTX2 overexpression. In addition, these two variants were found to influence translation by affecting upstream initiation codons. Interestingly, the visual functions (including age of onset, visual acuity, and ERG responses) of EYS RP patients who harbor c.-453G>T is better than that of those with biallelic null EYS mutations.

Conclusions: Our analysis revealed both single-nucleotide and structural variants in the EYS promoter as the cause of ARRP. These variants may affect EYS expression via a dual mechanism by altering transcription factor binding affinity at the EYS promoter and by affecting upstream open reading frames.

3.7. The CXCR4/CXCL12 Axis Contributes to Cerebrolysin-Induced Neuroprotection Against Staurosporine-Treated Cortical Neurons at 7 Days In Vitro and Prevents Inflammation in a N2a Cell Line Exposed to LPS

• Jose Joaquin Merino
• UCM (Universidad Complutense de Madrid), Dpto. Farmacología, Farmacognosia y Botánica (UCM), IUIN, Spain

Introduction: Oxidative stress and inflammation are hallmarks of neurodegenerative diseases, including a reduced repair capacity. Neural progenitor cells (NPCs) from the subventricular zone (SVZ), the dentate gyrus, and the olfactory bulb can migrate and differentiate into neurons or glial cells. CXCL12 chemokine binds to CXCR4, and this axis contributes to neuroinflammation. Following CNS insult, chemokines recruit stem cells for repair while the aberrant CXCR4 activation promotes cell death. In fact, NPCs, endothelial cells, neurons (and glia) express CXCR4, which enhances the homing of stem cells for neuronal repair. CXCL12 attracts neuroblasts and it is also secreted at sites of injury.

Aim: This study evaluated whether cerebrolysin (brain porcine peptide) or recombinant chemokines may protect cortical neurons at 7 DIV against staurosporine-induced cell death or LPS-induced inflammation in a N2a cell line. For this purpose, extracts from cortical neurons or N2a cells were isolated and several inflammatory markers were quantified through pCR (IL-1 beta and CXCR4/SDF1 alpha). Staurosporine or LPS treatment were added over 6 h in the medium and cerebrolysin or recombinant proteins were o/n added in the presence of these treatments.

Results: The antiapoptotic role of chemokines and/or cerebrolysin was demonstrated in staurosporine-treated cortical neurons at 7 DIV using an XTT assay. Cerebrolysin prevented staurosporine-induced apoptosis in the N2A cell line and decreased Il-1 beta levels in an N2a cell line exposed to LPS during 6 h.

Conclusions: The observed correlation between cerebrolysin and the neuroprotective effect of chemokines against apoptosis suggests that CXCR4 chemokine receptor activation by cerebrolysin prevented staurosporine-induced cell death in cortical neurons from rats and also reduced IL-1 beta levels in a N2a murine cell line exposed to LPS (lipopolysaccharide)). Although the clinical efficacy of cerebrolysin against dementia requires more clinical evidence, some clinical trials have confirmed its efficacy against neurological diseases.

3.8. The Early Diagnostic of Niemann-Pick Disease Type a Caused by c.996del and c.1252C>T in SMPD1 Gene: A Case Study

• Natalya N Mazanova ¹, Dmitry S Demianov ², Goar B Movsisyan ³, Anastasya A Rusakova ², Daria A Chudakova ², Alexander A Pushkov ², Ilya S Zhanin ², Alexandr S Potapov ², Kirill V Savostyanov ², Andrey P Fisenko ⁴

¹ 

Laboratory of Medical Genomics, National Medical Research of Children’s Health Federal State Autonomous Institution of the Ministry of Health of the Russian Federation, Moscow, 119991, Russian Federation

² 

National Medical Research of Children’s Health Federal State Autonomous Institution of the Ministry of Health of the Russian Federation

³ 

Gastroentherology Department, National Medical Research of Children’s Health Federal State Autonomous Institution of the Ministry of Health of the Russian Federation, Moscow, 119991, Russian Federation

⁴ 

Director of National Medical Research of Children’s Health Federal State Autonomous Institution, National Medical Research of Children’s Health Federal State Autonomous Institution of the Ministry of Health of the Russian Federation, Moscow, 119991

Niemann-Pick disease Type A (MIM 257200, NPD type A) is a rare congenital autosomal recessive hereditary condition. It is caused by the presence of causal nucleotide variants in the gene SMPD1 (NM_000543.5), resulting in a deficiency of the enzyme Acid Sphingomyelinase (ASM) encoded by SMPD1. This, in turn, leads to an abnormal lysosomal accumulation of sphingomyelin, causing tissue and organ damage. If untreated, it results in severe complications and death. Therefore, the early diagnosis of NPD type A is crucial. The multi-methodological diagnostic of NPD type A includes measurements of the enzyme activity of ASM, a genetic analysis aimed at detecting causal nucleotide variants in SMPD1, and a genetic and genealogical study of the proband’s family. The activity of ASM is commonly measured in Dried Blood Spots (DBS) using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS), as this method outperforms others. The gold standard for the detection of nucleotide variants in SMPD1 is Sanger sequencing. Finally, a genetic and genealogical study of the proband’s family is recommended when possible. As an example of such an approach, here, we present the diagnostic journey of one family. The patient was referred to our center based on the overall clinical picture. The HPLC-MS/MS detected a significant decrease in ASM activity in the patient’s DBS. The sequencing of SMPD1 revealed that the proband had two heterozygous nucleotide variants: c.996del, (p.Phe333Serfs*52), and c.1252C>T, (p.Arg418*). Further segregation analysis showed that each variant was inherited from one of the genetically unrelated healthy parents. The family received genetic counseling regarding the findings. Another two siblings were not carriers of these variants in SMPD1 and their ASM activity was in the normal range. However, the same nucleotide variants were found in the third sibling, whose ASM activity levels were below the normal range. Without the multi-methodological approach, this patient would have missed a timely diagnosis of NPD type A.

3.9. Whole-Genome Profile of Greek Patients with Asthenozoospermia: Identification of Candidate Variants and Genes

• Maria-Anna Kyrgiafini ¹, Chrysi Kontse ¹, Alexia Chatziparasidou ^1,2, Zissis Mamuris ¹

¹ 

Laboratory of Genetics, Comparative and Evolutionary Biology, Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece

² 

Embryolab IVF Unit, Thessaloniki, Greece

Introduction: At present, infertility represents a multifaceted health issue that significantly impacts numerous couples, giving rise to notable psychological and social complexities. Notably, one in six couples experience infertility, with approximately 50% of cases attributed to male factors. Male infertility, a complex disorder influenced by both environmental factors and genetic predisposition, involves the interplay of various genes contributing to its manifestation. It can be categorized into specific subtypes, including asthenozoospermia. The primary aim of this study was to identify novel variants associated with asthenozoospermia within the Greek population and to elucidate the roles of the genes involved. Materials and Methods: Whole-genome sequencing (WGS) was conducted on both normozoospermic and asthenozoospermic individuals. Following the identification of variants exclusively present in asthenozoospermic men, an extensive range of tools, functional assessments, and predictive algorithms were employed to prioritize these variants. Results: The investigation unveiled numerous polymorphisms, comprising 155 classified as high-impact and 715 classified as moderate-impact. While several of these variants were found within genes previously linked to male infertility, a notable subset was associated with asthenozoospermia for the first time. Furthermore, pathway enrichment analysis and gene ontology (GO) analyses revealed polymorphisms on genes implicated in teratozoospermia through various mechanisms and pathways. Conclusions: This study reaffirms the involvement of previously studied genes in male infertility, while also shedding light on novel molecular mechanisms. By providing a comprehensive list of variants and candidate genes associated with asthenozoospermia within the Greek population, this research contributes significantly to our understanding of male infertility and paves the road for future studies.

4. Session: Technologies and Resources for Genetics Research

4.1. A Draft Transcriptome of the Climate Resilient California Native Plant Ribes Malvaceum

• Savanah Senn ¹, Xochitl Estrada ¹, Steven Carrell ², Karu Smith ¹, Meika Best ¹, Daila Melendez ^1,3, John Hsieh ¹, Gerald Presley ⁴

¹ 

Los Angeles Pierce College Department of Agriculture Sciences, Plant Science program, Woodland Hills, CA 91371

² 

Oregon State University, Center for Quantitative Life Sciences, Corvallis, OR 97331

³ 

Oregon State University, Department of Horticulture, Corvallis, OR 97331

⁴ 

Oregon State University, Department of Wood Science and Engineering, Corvallis, OR 97331

Ribes malvaceum is commonly known as Chaparral Currant. This pilot project aimed to create genomic resources for R. malvaceum for phylogenomic studies of secondary metabolite production. The Ribes sp. are of interest in this study due to the production of bioactive compounds for human health. R. malvaceum is of interest due to its desirable climate-resiliency traits. Based on the known characteristics of closely related cultivated species Ribes nigrum and the best BLASTX results, we hypothesize that we will find transcriptomic evidence of similar secondary metabolites produced by R. malvaceum in the transcripts.

Ribes malvaceum tissue was collected on the Red Trail at Gold Creek Preserve, Angeles National Forest. Leaf and fruit tissue was snap-frozen in the field and sent to BGI Americas for extraction, library preparation, and sequencing. The data pipeline for plant RNAseq consisted of QC, de novo assembly, BUSCO evaluation, quantification, and annotation. The shell script find_transcript_ids.sh was used to find lant transcripts with TPM > 10 (Transcripts Per Million) and length > 1000. The downstream analysis was performed in order to narrow down interesting secondary metabolite genes which had a long length and high TPM. The BUSCO results showed that the transcriptome was 90.63% complete.

Delta (8)-fatty-acid desaturase sequences were closely related to Helianthus (70.43% similar), TPM = 42.85, length = 2115. Transcripts closely matched Chalcone synthase from Humulus, with 90.69% similarity and a length of 1506, and were elevated (TPM = 644.85). A transcript related to Flavonoid 3′-monooxygenase in Petunia was identified (77.57% similar), length 2075, TPM = 132.70. Anthocyanidin reductase ((2S)-flavan-3-ol-forming) was highly similar to Vitis (84.57%), with TPM = 39.75; length = 1249. Monothiol glutaredoxin was related to the sequence Arabidopsis (70.67% similar; TPM = 49.77; effective length = 3616). Transcripts related to 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 2 were identified that were 87.69% similar to Vitis (TPM = 231.54; effective length = 1029).

These results are a starting point for further investigation into the species’ resiliency and natural products.

4.2. An Approach to Identifying Aggression Profiles via Machine Learning

• Sakeena Bairamova, Konstantin Pavlov
• V. Serbsky National Medical Research Centre of Psychiatry and Narcology, Kropotkinsky per. 23, 119034 Moscow, Russia

The complex nature of aggression and challenges in phenotype description often make it difficult to identify genetic variations associated with aggressive behavior. The use of machine learning algorithms and multivariate analysis makes it possible to construct more accurate diagnostic schemes to characterize the underlying causes of aggression. The aim of this study was to develop a method to differentiate aggression profiles among subjects in a heterogeneous sample and to facilitate the association of the obtained profiles with genetic variations in BDNF rs6265, BDNF rs10835210, HTR2A rs6313, and DRD4 1800955 later.

Participants’ aggressiveness was assessed using the Buss–Durkee Hostility Inventory (BDHI). On the basis of the 10 scales of the BDHI, 1013 unique subspaces were formed, in which data were clustered. “Cluster neighborhood” plots were constructed for each participant in the study, allowing us to identify the content of each category of subjects in all of the clusters into which the participant fell. This approach demonstrated two dramatically different phenotypes based on all the possible combinations of characteristics. However, many study participants exhibited mixed phenotypes, which were separated using the DBSCAN spatial clustering algorithm.

Individuals initially classified into a control group, addiction-prone group, or inmate group (convicted of felony) were often adjacent to each other in the newly formed clusters, suggesting the need to reevaluate the sample: 13 individuals from the addiction-prone group and 17 individuals from the inmate group were assigned to the control group, and 24 individuals from the inmate group and 6 individuals from the control group were assigned to the addiction-prone group. A total of 140 individuals belonged to the monotype group, while the remaining 261 individuals belonged to the mixed phenotype group.

Phenotypes defined by indirect traits often create an erroneous picture. It is necessary to identify similar groups of patients based on their psychometric data and then examine the genotypes within these groups.

4.3. Elucidation of the Molecular Basis of Phenotypes in Seri-Genetic Resources (Bombyx mori L. and Morus spp.) Using Repository RNA-Seq Datasets: A Self-Service Data-Mining Approach

• Raju Mondal ¹, Dr. Ritwika Sur Chaudhuri ¹, Dr. Lokesh Gangadharaiah ¹, Dr. Nishitha Naik V. ¹, Dr. Manthira Moorthy ²

¹ 

CSB-Central Sericultural Germplasm Resources Centre, Central Silk Board, Hosur, Tamil Nadu 635109, India

² 

Central Silk Board, Bengaluru, Karnataka 560068, India

Transcriptomics provides a link between the genome, proteome, and cellular phenotype; therefore, it is regarded as a unique approach to revealing the molecular basis of phenotypes. Despite the availability of huge datasets for a wide range of taxa in public databases, RNA-Seq data analysis and interpretation remain challenging. This is due to factors including a limited processing capability and the need for specialized coding expertise and expensive software. Here, we used publicly available RNA-Seq datasets and presented an in-depth description of each cutting-edge system/tool, especially Galaxy platform and TRAPID 2. We also conducted a downstream analysis of the authenticity of the taxonomic identity, the global transcriptome map/network, and the functional enrichment of overrepresented genes in silkworm (Bombyx mori L.) and mulberry (Morus spp.). We provided a flexible de novo assembly, annotations, and sufficient visualization to interpret the RNA-Seq analysis data. The current data-mining approach sheds light on the significant influence of silkworm black dilute (bd) mutants on melanogenesis and tyrosine degradation, which is mediated by dopachrome tautomerase (DTC). However, M. serrata revealed significant polyploid trade-offs, such as investments in biochemical expenditure, nucleic acid metabolism, the intrinsic activity of transposable elements, and a functional association with chloroplasts. Overall, the current data-mining method reduces costs and requires no prior coding expertise to comprehend the genetic foundation of phenotype variance. The present approach can also help to decode the genetic architecture of novel phenotypes, including trait-specific accessions, ploidy-associated traits, and the hybridity effect of not only Seri-genetic resources but also other model and non-model organisms.

4.4. Reconstruction of Transcriptome Datasets to Understand the Molecular Basis Behind the Advantageous Effect of Triploid over Diploid Mulberry (Morus spp.)

Tanya

Ahmed Sheik ¹, Raju Mondal ², Dr. Nishitha Naik V.¹

¹ 

CSB-Central Sericultural Germplasm Resources Centre, Central Silk Board, Hosur, Tamil Nadu 635109, India

² 

Mulberry Tissue Culture Lab, CSB-Central Sericultural Germplasm Resources Centre, Tamil Nadu 635109, India

Polyploidy is a key factor influencing traits such as chloroplast number, stomatal size, and leaf yield. In mulberry (Morus spp.), triploids are well known to be superior to other ploidies in terms of yield traits. However, the cellular and molecular processes driving these benefits remain unclear. Transcriptome analysis is a powerful tool used to understand the molecular mechanisms underlying various plant traits, which requires coding skills and high-performance computers that hinder the utilization of publicly available huge RNA-Seq datasets. To address this issue, repository RNA-Seq datasets were analyzed using Galaxy to decode the genetic basis of the superior functionality of triploid mulberry. Leaf transcriptome datasets of diploids and triploids were retrieved and analyzed in Galaxy, where the genome of M. indica K2 was used as a reference. A variety of tools, including Trimmomatic, HISAT2, Stringtie, StringtieMerge, Featurecount, DEseq2, and WGCNA, were employed to identify differentially expressed genes, which were further analyzed using iDEP tools. For data interpretation, a PCA, volcano plot, heatmap, dendrogram, and bubble diagram were generated. The analysis revealed that triploid mulberry germplasm yielded a considerable number of up- and downregulated genes compared to diploids. Notably, the upregulated genes were associated with photosynthesis, chlorophyll biosynthesis, and carbon fixation, which in turn relay the molecular basis of the advantageous effect of triploids over diploids. These transcriptome studies indicate that polyploidization enhances photosynthetic capacity and other metabolic attributes. Therefore, this methodology helps to decipher the molecular basis of the advantageous effect of triploids over diploids without coding skills and high-performance computers.

5. Session: Microbial Genetics and Genomics

5.1. Artemisia Californica Draft Leaf and Flower Transcriptome and Soil WGS Resources

• Savanah Senn ¹, Steven Carrell ², John Hsieh ¹, Daila Melendez^1,3, Karu Smith ¹, Meika Best ¹, Daniel Moran ¹, Ray A. Enke ⁴, Bruce Nash ⁵, Gerald Presley ⁶

¹ 

Los Angeles Pierce College Department of Agriculture Sciences, Plant Science program, Woodland Hills, CA 91371

² 

Oregon State University, Center for Quantitative Life Sciences, Corvallis, OR 97331

³ 

Oregon State University, Department of Horticulture, Corvallis, OR 97331

⁴ 

James Madison University, Department of Biology, Harrisonburg, Virginia, 22807

⁵ 

Cold Spring Harbor Laboratory, DNA Learning Center, Cold Spring Harbor, NY 11724

⁶ 

Oregon State University, Department of Wood Science and Engineering, Corvallis, OR 97331

Secondary metabolite production in Artemisia has been studied for its anti-parasitic, anti-tumor, and anti-inflammatory actions. Artemisia californica is a drought-resilient plant with medicinal value used traditionally as a topical for severe pain relief; flavonoids, stilbenes, sesquiterpene lactones, and terpenes are the main therapeutic components.

We aimed to create novel genomic resources, including a leaf & flower transcriptome, and soil WGS metagenomics from the A. californica rootzone. Snap-frozen plant tissues and rhizosphere soil were collected from the field and sent to BGIA for extraction, library preparation, and sequencing. Following QC, de novo assembly and quantification, transcripts were annotated with the best SPROT BLASTX and BLASTP hits. The completeness of the assemblies was assessed using BUSCO. Transcripts with TPM > 10 and length > 1000 were examined for evidence of candidate genes related to the production of the expected bioactive compounds.

Assembly summary statistics were computed. There were 212,818 total sequences with a total length of 2.07 × 10⁸. The average sequence length was 908; the length ranged between 193 and 13,630. The L50 value was 41,357, N50 was 1591, and N80 was 647. The Artemisia californica transcriptome was 91.4% complete. In the transcripts, there was evidence of sesquiterpene production, sesquiterpene lactones, and flavonoids. Beta-caryophyllene synthase was related to Artemisia; however, there was low similarity (62.70% ID; TPM = 14.86; length = 2889). Multiple sesquiterpenoids and triterpenoids appeared to be elevated. Chalcone synthase transcripts were 96.23% similar to Australasian lineages of Asteraceae.

There was evidence of a distinct rootzone microbial community. Geodermatophilus from Actinomycetes was well-represented. The Biobakery metagenomics output indicated that there are differences in taxonomy in soil samples from different plant species, although there were few differences in function.

Future studies should focus on Nanopore sequencing of plant RNA and mining for novel bacterial species in the Artemisia rootzone using WGS data. Further studies should include biochemical analysis to confirm the genomic findings.

5.2. Resistance Gene Profiles of Multidrug-Resistant Klebsiella spp. from Poultry Samples

• Vanessa Silva ^1,2,3, Catarina Freitas ², Jessica Ribeiro ^1,2,3, Pedro Pinto ⁴, Madalena Vieira-Pinto ^5,6, Patrícia Poeta ^1,2, Gilberto Igrejas ^1,3,7

¹ 

Associated Laboratory for Green Chemistry (LAQV-REQUIMTE), University NOVA of Lisboa, Lisboa, Caparica, Portugal

² 

Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

³ 

Department of Genetics and Biotechnology, Functional Genomics and Proteomics’ Unit, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal

⁴ 

Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal

⁵ 

Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

⁶ 

Veterinary and Animal Science Research Center (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

⁷ 

Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

The increasing prevalence of antibiotic-resistant bacteria, especially Klebsiella spp., represents a major threat to both human and veterinary medicine. This study aimed to isolate and characterize the antimicrobial resistance profiles of Klebsiella spp. from broilers and broiler bursitis.

A total of 210 samples were collected, including 70 from hens, 40 from free-range chickens, 40 from bursitis, 20 from males, 20 from roosters, and 20 from young chickens. Antimicrobial susceptibility testing was conducted using the disk diffusion method, as recommended by the EUCAST and CLSI guidelines, against 14 antibiotics. Additionally, PCR analysis revealed the presence of several resistance genes.

From the 210 samples, 51 isolates of Klebsiella spp. were obtained, including 20 from hens, 10 from young chickens, 8 from bursitis, 6 from free-range chickens, 4 from roosters, and 3 from males. Regarding the antimicrobial resistance, 57% of the isolates were classified as multidrug-resistant. The highest levels of resistance were observed for ampicillin (98.04%) and amoxicillin–clavulanic acid (86.27%). Resistance to nalidixic acid, trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin, and tetracycline was detected in 52.94%, 50.98%, 47.06%, 45.10%, and 23.53% of the isolates, respectively. None of the isolates showed resistance to imipenem, cefotaxime, or ceftazidime. The sul2 gene was detected in 65.39% of isolates resistant to trimethoprim-sulfamethoxazole, while sul3 was found in only 3.85%. The bla_TEM gene, associated with beta-lactam resistance, was present in 78% of isolates, while bla_SHV was detected in 4%. For tetracycline resistance, the tetA and tetB genes were found in 58.33% and 41.67% of the isolates, respectively.

This study highlights the significant antimicrobial resistance found in Klebsiella spp. isolated from poultry, underscoring the public health risks associated with the consumption of poultry products. More than half of the isolates were multidrug-resistant, which calls for ongoing surveillance and responsible antibiotic use in animal production to mitigate the spread of resistant strains.

5.3. Genomic Basis of Mycorrhizal Fungal Specificity in Plant–Fungal Symbioses

• Niraj Kumar Prajapati
• Department of Horticulture, School of Agricultural Sciences and Technology, Babasaheb Bhimrao Ambedkar University (A Central University), Lucknow (U.P.)–226025, India

Mycorrhizal symbioses, crucial for plant nutrition and ecosystem functioning, exhibit varying degrees of specificity between plant hosts and fungal symbionts. This study explores the genomic underpinnings of mycorrhizal fungal specificity, synthesizing recent advances in understanding the molecular mechanisms governing these intricate associations. Comparative genomic analyses of over 100 mycorrhizal fungal species have revealed that the loss of plant cell wall-degrading enzymes is a common feature, with ectomycorrhizal fungi retaining only 10–15% of these enzymes compared to their saprotrophic ancestors. This adaptation reflects the transition from saprotrophic to symbiotic lifestyles. Studies on effector proteins have identified several mycorrhiza-induced small secreted proteins (MiSSPs) that play key roles in symbiosis establishment. For instance, the MiSSP7 gene in Laccaria bicolor is essential for the colonization of Populus roots, highlighting the importance of effectors in host specificity. Genomic investigations have also uncovered expansions in certain gene families, such as those encoding for nutrient transporters, with some ectomycorrhizal fungi possessing up to three times as many ammonium transporter genes than their non-mycorrhizal relatives. Furthermore, the analysis of plant genomes has revealed that up to 5% of plant genes are differentially expressed during mycorrhizal colonization, with common symbiosis genes being conserved across 80–90% of land plants. This study underscores the complex genomic landscape underlying mycorrhizal specificity, emphasizing the co-evolution of plant and fungal genomes. Future research directions include the functional characterization of candidate genes and an exploration of the epigenetic mechanisms in symbiosis regulation.

5.4. Machine Learning-Driven Omics Approaches for Diagnostic Biomarker in Tuberculosis Based on Differential Gene Expression Data

• Javed Aalam, Rafat Parveen
• Department of Computer Science, Jamia Millia Islamia, New Delhi, India-110025

Background: Tuberculosis (TB) continues to remain a global health challenge, with 10.6 million cases and 1.3 million fatalities reported in 2022. Mycobacterium tuberculosis presents diagnostic difficulties because traditional procedures such as the Tuberculin Skin Test and IGRA are limited. MicroRNAs, including miR-18b and miR-378d, have been identified as prospective biomarkers for tuberculosis.

Method: This extensive review examines the use of OMICS technologies (genomics, transcriptomics, proteomics) with machine learning algorithms to identify and assess tuberculosis biomarkers. The recent literature from PubMed, Scopus, and IEEE Xplore was studied, concentrating on standard techniques such as KNN, SVM, and deep learning models.

Results: Machine learning techniques, especially deep learning, routinely attained elevated accuracy rates (often above 95%) in the classification of tuberculosis infections utilizing OMICS data. Biomarkers like miR-29a, miR-21, and 2-hydroxyglutarate (2-HG) have been recognized as promising diagnostic tools for TB. Additional biomarkers, including IL-8, IL-6, IFN-γ, and FCGR1A from transcriptomics, serum amyloid A (SAA), and lipoarabinomannan (LAM) from proteomics, provide more accurate insights into TB infection and progression.

Conclusions: The integration of machine learning with OMICS data offers a groundbreaking approach for tuberculosis diagnosis and biomarker discovery. Additional research is required to improve feature selection and refine machine learning models for clinical applications, potentially revolutionizing tuberculosis detection and treatment methods.

5.5. Occurrence of Genetic Determinants of Resistance to Colistin and Biofilm in Carbapenem-Resistant Acinetobacter Baumannii Isolated from Hospitalized Patients

• Michał Karasek
• Student Research Group at the Department of Pharmaceutical Microbiology, Medical University of Lublin, Lublin, Poland

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a priority pathogen, according to the World Health Organization (WHO), which is the most threatening to human health, being responsible for pneumonia, bacteraemia, urinary tract, skin, and soft tissue infections, with mortality rates approaching 35%. The aim of this study was to evaluate the occurrence of colistin resistance (mcr1-5) and biofilm genes (surA1, bap, ompA, luxR, epsA) in 26 CRAb isolates collected from hospitalized patients and Acinetobacter baumanii ATCC 19606 as the reference strain. Gene prevalence was determined by multiplex or single PCR (polymerase chain reaction), in a 25 µL mixture (12.5 μL of REDTaq^® ReadyMix™ PCR Reaction Mix, 1 μL of each primer, and 2 μL bacterial DNA). As a result, 92.3% (24/26) and 96.2% (25/26) of the isolates were mcr1- and mcr3-positive, respectively. None of the isolates possessed the mcr2, mcr4, and mcr5 genes. All CRAb isolates had the surA1, bap, ompA, and luxR genes, while 61.5% of isolates (16/26) harbored the epsA gene.

CRAb are highly pathogenic bacteria isolated from infections and are increasingly resistant to most available therapies. Every effort should be made to prevent the spread of these microorganisms, including analyzing the presence of new genetic determinants in the CRAb genome, and identifying biofilm-related genes whose expression inhibition could more effectively eradicate these pathogens.

5.6. Regional Variability of Spotted Fever Group Rickettsia Genospecies: Insights from Eight Regions in Russia

• Alexey V. Rakov, Tatiana A. Chekanova, Ketevan Petremgvdlishvili
• Laboratory for Natural Focal Infections Epidemiology, Central Research Institute of Epidemiology, 111123 Moscow, Russia

Introduction: In recent years, there has been growing interest in tick-borne infections, particularly tick-borne rickettsioses (TBRs). Assessing the genospecies composition and regional distribution of spotted fever group rickettsiae (SFGR) is crucial for optimizing the risk-based surveillance of TBR. However, the genospecies diversity of SFGR populations in the Russian Federation remains understudied. To address this, we studied ticks collected from vegetation, humans, and animals in eight regions of Russia to identify the SFGR genospecies, which may have significance for human morbidity.

Materials and Methods: From 2020 to 2024, we collected 2431 ticks from eight regions of Russia, representing Western Siberia (Altai Krai), North Caucasus (Karachay-Cherkessia), Southern Russia (Astrakhan Oblast), the Volga region (Samara Oblast), Central Russia (Moscow, Tula Oblast, Oryol Oblast), and Central Europe (Kaliningrad Oblast). We utilized commercial qPCR kits for SFGR screening and identified genospecies by Sanger sequencing partial genes for citrate synthase (gltA) and outer-membrane protein A (ompA), comparing the results with the GenBank database.

Results: Our findings revealed six distinct genospecies of SFGR across the eight regions of Russia; however, the diversity of these genospecies varied by region. The highest diversity (five genospecies: R. raoultii, R. slovaca, R. helvetica, R. monacensis, and R. aeschlimannii) was found in North Caucasus in ticks from three genera. In Western Siberia, we detected three genospecies—R. sibirica, R. raoultii, and R. helvetica—in two genera of ticks. In the Volga region, the genospecies R. slovaca and R. raoultii were found in Dermacentor ticks, while in Southern Russia, R. raoultii and R. aeschlimannii were identified in two different genera of ticks. Central Russia (three regions) and Europe showed a similar pattern, with R. raoultii in Dermacentor and R. helvetica in Ixodes.

Conclusions: Our study highlights significant regional variations in SFGR genospecies diversity in Russia, underscoring the necessity for ongoing research and monitoring for public health.