Search results for: altered methionine metabolism

Authors: Rabab Makki

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Bone mineral density and bone metabolism are affected by various factors such as genetic, endocrine, mechanical and nutritional. Our understanding of nutritional influences on bone health is limited because most studies have focused on calcium. This study investigated the dietary factors which are likely t contribute to Osteoporosis in Saudi post-menopausal women, and correlated it with BMD. This is a case controlled study involved 36 postmenopausal Saudi females selected from the Orthopedics and osteoporosis outpatient clinics, and 25 postmenopausal Saudi females as controls from the primary clinic of Military Hospital in Riyadh. The women were diagnosed as osteoporotic based on the BMD measurement at any site (left femur neck, right femur neck, left total hip or right total hip or spine). Both the controls and the Osteoporotics were over 50 years of age and BMI between 31-34 kg/m2 had 2nd degree obesity, and were not free from other problems such as diabetes, hypertension, etc. Subjects (osteoporotics and controls) were interviewed to called data on demographic characterstics, medical history, dietary intake anthropometry (height and weight) bone mineral density. Blood samples were collected from subjects (Osteoporotics and controls). Analysis of serum calcium, vitamin D, phosphate were done at the main laboratory at Military Hospital Riyadh, by the laboratory technician while BMD was determined at the department of Nuclear Medicine by an expert technician and results were interpreted by radiologist.Data on frequency of consumption of animal food (meat, eggs, poultry and fish) and diary foods (milk, yogurt, cheese) of osteoporotic was less than control. In spite of the low intake there was no association with BMD.In general, the vegetables and fruits were consumed less by the osteoporotics than control. The only fruit which had shown a significant positive correlation is banana with right and left hip BMD total probably due to high potassium and minerals content which likely to prevent bone resorption. Mataziz vegetables combination of wheat showed a significant positive correlation with the same site (total right and left hip). Both osteoporotics abd controls were consuming table sugar. (But the sweet intake showed a significant negative correlation with left neck femur BMD, suggesting sucrose increase urinary calcium loss. Both osteoporotic and controls were consuming Arabic coffee. A negative significant correlation between intake of Arabic coffee and BMD of right neck femur of osteoporosis patient was observed. It could be suggested that increased intake of fruits and vegetables, might promote bone density while high intake of coffee and sugars might affect bone density, no significant correlation was observed between BMD at any site and diary product. We can say the major risk factors are inadequate nutrition. Further studies are needed among Saudi population to confirm these results.

Keywords: osteoporosi, Saudia Arabia, Riyadh Armed Forces, postmenopausal women

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Authors: Li Yang

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Tourism is employed by many countries to facilitate socioeconomic development and to assist in the heritage preservation. An “ethnic culture boom” is currently driving the tourism industry in China. Ethnic minorities, commonly portrayed as primitive, colorful and exotic, have become a big tourist draw. Many cultural attractions have been built throughout China to meet the demands of domestic tourists. Sacred cultural heritage sites have been rehabilitated as a major component of ethnic tourism. The purpose of this study is to examine the interconnected consequences of tourism development and tourism-related leisure property development and, and to discuss, in a broader context, issues and considerations that are pertinent to the management and development of ethnic attractions. The role of real estate in tourism development and its sociocultural consequences are explored. An empirical research was conducted in Yiren Ancient Town (literally, "Ancient Town of Yi People") in Chuxiong City, Yunnan Province, China. Multiple research methods, including in-depth interviews, informal discussions, on-site observations, and secondary data review were employed to measure residents and tourism decision-makers’ perceptions of ethnic tourism and to explore the impacts of tourism on local community. Key informants from government officials, tourism developers and local communities were interviewed individually to gather what they think about benefits and costs of tourism, and what their concerns about and hopes for tourism development are. Yiren Ancient Town was constructed in classical Yi architecture style featuring tranquil garden scenery. Commercial streets, entertainment complexes, and accommodation facilities occupied the center of the town, creating culturally distinctive and visually stimulating places for tourists. A variety of activities are presented to visitors, including walking tours of the town, staged dance shows, musical performances, ethnic festivals and ceremonies, tasting minority food and wedding shows. This study reveals that tourism real estate has transformed the town from a traditional neighborhood into diverse real estate landscapes. Ethnic architecture, costumes, festivals and folk culture have been represented, altered and reinvented through the tourist gaze and mechanisms of cultural production. Tourism is now a new economic driver of the community providing opportunities for the creation of small businesses. There was a general appreciation in the community that tourism has created many employment opportunities, especially for self-employment. However, profit-seeking is a primary motivation for the government, developers, businesses, and other actors involved in the tourism development process. As the town has attracted an increasing number of visitors, commercialization and business competition are intense in the town. Many residents complained about elevated land prices, making the town and the surroundings comparatively high-value locales. Local community is also concerned about the decline of traditional ethnic culture and an erosion of the sense of identity and place. A balance is difficult to maintain between protection and development. The preservation of ethnic culture and heritage should be enhanced if long-term sustainable development of tourism is to occur and the loss of ethnic identities is to be avoided.

Keywords: ancient town, ethnic tourism, local community, real estate, China

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Authors: A. M. Plante, F. O’Halloran, A. L. McCarthy

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By 2050 it is projected that 2 billion of the global population will be more than 60 years old. Older adults have unique dietary requirements and aging is associated with physiological changes that affect appetite, sensory perception, metabolism, and digestion. Therefore, it is essential that foods recommended and designed for older adults promote healthy aging. To assess cheese as a functional food for the elderly, a range of commercial cheese products were selected and compared for their antioxidant properties. Cheese from various milk sources (bovine, goats, sheep) with different textures and fat content, including cheddar, feta, goats, brie, roquefort, halloumi, wensleydale and gouda, were initially digested with two different simulated in vitro gastrointestinal digestion (SGID) models. One SGID model represented a validated in vitro adult digestion system and the second model, an elderly SGID, was designed to consider the physiological changes associated with aging. The antioxidant potential of all cheese digestates was investigated using in vitro chemical-based antioxidant assays, (2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP) and total phenolic content (TPC)). All adult model digestates had high antioxidant activity across both DPPH ( > 70%) and FRAP ( > 700 µM Fe²⁺/kg.fw) assays. Following in vitro digestion using the elderly SGID model, full-fat red cheddar, low-fat white cheddar, roquefort, halloumi, wensleydale, and gouda digestates had significantly lower (p ≤ 0.05) DPPH radical scavenging properties compared to the adult model digestates. Full-fat white cheddar had higher DPPH radical scavenging activity following elderly SGID digestion compared to the adult model digestate, but the difference was not significant. All other cheese digestates from the elderly model were comparable to the digestates from the adult model in terms of radical scavenging activity. The FRAP of all elderly digestates were significantly lower (p ≤ 0.05) compared to the adult digestates. Goats cheese was significantly higher (p ≤ 0.05) in FRAP (718 µM Fe²/kg.fw) compared to all other digestates in the elderly model. TPC levels in the soft cheeses (feta, goats) and low-fat cheeses (red cheddar, white cheddar) were significantly lower (p ≤ 0.05) in the elderly digestates compared to the adult digestates. There was no significant difference in TPC levels, between the elderly and adult model for full-fat cheddar (red, white), roquefort, wensleydale, gouda, and brie digestates. Halloumi cheese was the only cheese that was significantly higher in TPC levels following elderly digestion compared to adult digestates. Low fat red cheddar had significantly higher (p ≤ 0.05) TPC levels compared to all other digestates for both adult and elderly digestive systems. Findings from this study demonstrate that aging has an impact on the bioactivity of cheese, as antioxidant activity and TPC levels were lower, following in vitro elderly digestion compared to the adult model. For older adults, soft cheese, particularly goats cheese, was associated with high radical scavenging and reducing power, while roquefort cheese had low antioxidant activity. Also, elderly digestates of halloumi and low-fat red cheddar were associated with high TPC levels. Cheese has potential as a functional food for the elderly, however, bioactivity can vary depending on the cheese matrix. Funding for this research was provided by the RISAM Scholarship Scheme, Cork Institute of Technology, Ireland.

Keywords: antioxidants, cheese, in-vitro digestion, older adults

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Authors: Pierre Mintom, Armel Assiene Agamou, Leslie Toukem, William Dakam, Christine Fernande Nyangono Biyegue

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Context: Hyperuricemia, the presence of high levels of uric acid in the blood, is a known precursor to the development of gout. Recent studies have suggested a strong association between hyperuricemia and disorders of lipoprotein metabolism, specifically hypercholesterolemia. Understanding the factors associated with these conditions in gout patients is essential for effective treatment and management. Research Aim: The objective of this study was to determine the prevalence of hyperuricemia, hypercholesterolemia, and the double burden of hyperuricemia-hypercholesterolemia in the gouty population. Additionally, the study aimed to identify the factors associated with these conditions. Methodology: The study utilized a database from a survey of 150 gouty patients recruited at the Laquintinie Hospital in Douala between August 2017 and February 2018. The database contained information on anthropometric parameters, biochemical markers, and the food and drugs consumed by the patients. Hyperuricemia and hypercholesterolemia were defined based on specific serum uric acid and total cholesterol thresholds, and the double burden was defined as the co-occurrence of hyperuricemia and hypercholesterolemia. Findings: The study found that the prevalence rates for hyperuricemia, hypercholesterolemia, and the double burden were 61.3%, 76%, and 50.7% respectively. Factors associated with these conditions included hypertriglyceridemia, atherogenicity index TC/HDL ratio, atherogenicity index LDL/HDL ratio, family history, and the consumption of specific foods and drinks. Theoretical Importance: The study highlights the strong association between hyperuricemia and dyslipidemia, providing important insights for guiding treatment strategies in gout patients. Additionally, it emphasizes the significance of nutritional education in managing these metabolic disorders, suggesting the need to address eating habits in gout patients. Data Collection and Analysis Procedures: Data was collected through surveys and medical records of gouty patients. Information on anthropometric parameters, biochemical markers, and dietary habits was recorded. Prevalence rates and associated factors were determined through statistical analysis, employing odds ratios to assess the risks. Question Addressed: The study aimed to address the prevalence rates and associated factors of hyperuricemia, hypercholesterolemia, and the double burden in gouty patients. It sought to understand the relationships between these conditions and determine their implications for treatment and nutritional education. Conclusion: Findings show that it’s exists an association between hyperuricemia and hypercholesterolemia in gout patients, thus creating a double burden. The findings underscore the importance of considering family history and eating habits in addressing the double burden of hyperuricemia-hypercholesterolemia. This study provides valuable insights for guiding treatment approaches and emphasizes the need for nutritional education in gout patients. This study specifically focussed on the sick population. A case–control study between gouty and non-gouty populations would be interesting to better compare and explain the results observed.

Keywords: gout, hyperuricemia, hypercholesterolemia, double burden

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Authors: Anuj Tyagi, Balwinder Singh, Naveen Kumar B. T., Niraj K. Singh

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Characterization of diverse microbial communities in specific environment plays a crucial role in the better understanding of their functional relationship with the ecosystem. It is now well established that gut microbiome of fish is not the simple replication of microbiota of surrounding local habitat, and extensive species, dietary, physiological and metabolic variations in fishes may have a significant impact on its composition. Moreover, overuse of antibiotics in human, veterinary and aquaculture medicine has led to rapid emergence and propagation of antibiotic resistance genes (ARGs) in the aquatic environment. Microbial communities harboring specific ARGs not only get a preferential edge during selective antibiotic exposure but also possess the significant risk of ARGs transfer to other non-resistance bacteria within the confined environments. This phenomenon may lead to the emergence of habitat-specific microbial resistomes and subsequent emergence of virulent antibiotic-resistant pathogens with severe fish and consumer health consequences. In this study, gut microbiota of freshwater carp (Labeo rohita) was investigated by shotgun metagenomics to understand its taxonomic composition and functional capabilities. Metagenomic DNA, extracted from the fish gut, was subjected to sequencing on Illumina NextSeq to generate paired-end (PE) 2 x 150 bp sequencing reads. After the QC of raw sequencing data by Trimmomatic, taxonomic analysis by Kraken2 taxonomic sequence classification system revealed the presence of 36 phyla, 326 families and 985 genera in the fish gut microbiome. At phylum level, Proteobacteria accounted for more than three-fourths of total bacterial populations followed by Actinobacteria (14%) and Cyanobacteria (3%). Commonly used probiotic bacteria (Bacillus, Lactobacillus, Streptococcus, and Lactococcus) were found to be very less prevalent in fish gut. After sequencing data assembly by MEGAHIT v1.1.2 assembler and PROKKA automated analysis pipeline, pathway analysis revealed the presence of 1,608 Metacyc pathways in the fish gut microbiome. Biosynthesis pathways were found to be the most dominant (51%) followed by degradation (39%), energy-metabolism (4%) and fermentation (2%). Almost one-third (33%) of biosynthesis pathways were involved in the synthesis of secondary metabolites. Metabolic pathways for the biosynthesis of 35 antibiotic types were also present, and these accounted for 5% of overall metabolic pathways in the fish gut microbiome. Fifty-one different types of antibiotic resistance genes (ARGs) belonging to 15 antimicrobial resistance (AMR) gene families and conferring resistance against 24 antibiotic types were detected in fish gut. More than 90% ARGs in fish gut microbiome were against beta-lactams (penicillins, cephalosporins, penems, and monobactams). Resistance against tetracycline, macrolides, fluoroquinolones, and phenicols ranged from 0.7% to 1.3%. Some of the ARGs for multi-drug resistance were also found to be located on sequences of plasmid origin. The presence of pathogenic bacteria and ARGs on plasmid sequences suggested the potential risk due to horizontal gene transfer in the confined gut environment.

Keywords: antibiotic resistance, fish gut, metabolic pathways, microbial diversity

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Authors: Charalabos Antonatos, Mariza Panoutsopoulou, Georgios K. Georgakilas, Evangelos Evangelou, Yiannis Vasilopoulos

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The extended tissue damage and severe clinical outcomes of autoimmune diseases, accompanied by the high annual costs to the overall health care system, highlight the need for an efficient therapy. Increasing knowledge over the pathophysiology of specific chronic inflammatory diseases, namely Psoriasis (PsO), Inflammatory Bowel Diseases (IBD) consisting of Crohn’s disease (CD) and Ulcerative colitis (UC), and Rheumatoid Arthritis (RA), has provided insights into the underlying mechanisms that lead to the maintenance of the inflammation, such as Tumor Necrosis Factor alpha (TNF-α). Hence, the anti-TNFα biological agents pose as an ideal therapeutic approach. Despite the efficacy of anti-TNFα agents, several clinical trials have shown that 20-40% of patients do not respond to treatment. Nowadays, high-throughput technologies have been recruited in order to elucidate the complex interactions in multifactorial phenotypes, with the most ubiquitous ones referring to transcriptome quantification analyses. In this context, a random effects meta-analysis of available gene expression cDNA microarray datasets was performed between responders and non-responders to anti-TNFα therapy in patients with IBD, PsO, and RA. Publicly available datasets were systematically searched from inception to 10th of November 2020 and selected for further analysis if they assessed the response to anti-TNFα therapy with clinical score indexes from inflamed biopsies. Specifically, 4 IBD (79 responders/72 non-responders), 3 PsO (40 responders/11 non-responders) and 2 RA (16 responders/6 non-responders) datasetswere selected. After the separate pre-processing of each dataset, 4 separate meta-analyses were conducted; three disease-specific and a single combined meta-analysis on the disease-specific results. The MetaVolcano R package (v.1.8.0) was utilized for a random-effects meta-analysis through theRestricted Maximum Likelihood (RELM) method. The top 1% of the most consistently perturbed genes in the included datasets was highlighted through the TopConfects approach while maintaining a 5% False Discovery Rate (FDR). Genes were considered as Differentialy Expressed (DEGs) as those with P ≤ 0.05, |log2(FC)| ≥ log2(1.25) and perturbed in at least 75% of the included datasets. Over-representation analysis was performed using Gene Ontology and Reactome Pathways for both up- and down-regulated genes in all 4 performed meta-analyses. Protein-Protein interaction networks were also incorporated in the subsequentanalyses with STRING v11.5 and Cytoscape v3.9. Disease-specific meta-analyses detected multiple distinct pro-inflammatory and immune-related down-regulated genes for each disease, such asNFKBIA, IL36, and IRAK1, respectively. Pathway analyses revealed unique and shared pathways between each disease, such as Neutrophil Degranulation and Signaling by Interleukins. The combined meta-analysis unveiled 436 DEGs, 86 out of which were up- and 350 down-regulated, confirming the aforementioned shared pathways and genes, as well as uncovering genes that participate in anti-inflammatory pathways, namely IL-10 signaling. The identification of key biological pathways and regulatory elements is imperative for the accurate prediction of the patient’s response to biological drugs. Meta-analysis of such gene expression data could aid the challenging approach to unravel the complex interactions implicated in the response to anti-TNFα therapy in patients with PsO, IBD, and RA, as well as distinguish gene clusters and pathways that are altered through this heterogeneous phenotype.

Keywords: anti-TNFα, autoimmune, meta-analysis, microarrays

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Authors: A. Moghadasein, M. Ghasemi, S. Fazelifar

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Aim: Chemerin is a novel adipokine that play an important role in regulating lipid metabolism and abiogenesis. Chemerin is dependent on autocrine and paracrine signals for the differentiation and maturation of fat cells; it also regulates glucose uptake in fat cells and stimulates lipolysis. It has been reported that in adipocytes, chemerin enhances the insulin-stimulated glucose and causes the phosphorylation of tyrosine in Insulin receptor substrate. According to the studies, Chemerin may increase insulin sensitivity in adipose tissue and is largely associated with Body mass index, triglycerides, and blood pressure in those with normal glucose tolerance. There is limited information available regarding the effect of exercise training on serum chemerin concentrations. The purpose of this study was to investigate the effect of endurance training on serum chemerin levels and lipids of plasma in overweight women. Methodology: This study was a quasi-experimental research with a pre-post test design. After required examination and verification of high pressure by the physician, 22 obese subjects (age: 35.64±5.55 yr, weight: 75.62±9.30 kg, body mass index: 32.4±1.6 kg/m2) were randomly assigned to aerobic training (n= 12) and control (n= 12) groups. Participants completed a questionnaire indicating the lack of sports history during the past six months, the lack of anti-hypertension drugs use, hormone therapy, cardiovascular problems, and complete stoppage of menstrual cycle. Aerobic training was performed 3 times weekly for 8 weeks. Resting levels of chemerin plasma, metabolic parameters were measured prior to and after the intervention. The control group did not participate in any training program. In this study, ethical considerations included the complete description of the objectives to the study participants, ensuring the confidentiality of their information. Kolmogorov-Smirnov and Levin test were used for determining the normal distribution of data and homogeneity of variances, respectively. Analyze of variance with repeated measure were used to investigate the changes in the intra-group and the differences in inter-group of variables. Statistical operations were performed using SPSS 16 and the significance level of the tests was considered at P < 0.05. Results: After an 8 week aerobic training, levels of chemerin plasma were significantly decreased in aerobic trained group when compared with their control groups (p < 0.05).Concurrently, levels of HDL-c were significantly decreased (p < 0.05) whereas, levels of cholesterol, TG and LDL-c, showed no significant changes (p > 0.05). No significant correlations between chemerin levels and weight loss were observed in subjects with overweight women. Conclusion: The present study demonstrated, 8 weeks aerobic training, reduced serum chemerin concentrations in overweight women. Whereas, aerobic training exercise programmers affected the lipid profile response of obese subjects differently. However further research is warranted in order to unravel the molecular mechanism for the range of responses and the role of serum chemerin.

Keywords: chemerin, aerobic training, lipid profile, obese women

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Authors: Tugba Altin

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In a time characterized by escalating climate change impacts, communities globally face extreme events with deep-reaching tangible and intangible consequences. At the intersection of these phenomena lies the profound impact on the cultural and emotional connections that individuals forge with their environments. This study casts a spotlight on the Lytton Creek Fire of 2021, showcasing it as an exemplar of both the visible destruction brought by such events and the more covert yet deeply impactful disturbances to place attachment (PA). Defined as the emotional and cognitive bond individuals form with their surroundings, PA is critical in comprehending how such catastrophic events reshape cultural identity and the bond with the land. Against the stark backdrop of the Lytton Creek Fire's devastation, the research seeks to unpack the multilayered dynamics of PA amidst the tangible wreckage and the intangible repercussions such as emotional distress and disrupted cultural landscapes. Delving deeper, it examines how affected populations renegotiate their affiliations with these drastically altered environments, grappling with both the tangible loss of their homes and the intangible challenges to solace, identity, and community cohesion. This exploration is instrumental in the broader climate change narrative, as it offers crucial insights into how these personal-place relationships can influence and shape climate adaptation and recovery strategies. Departing from traditional data collection methodologies, this study adopts an interpretive phenomenological approach enriched by hermeneutic insights and places the experiences of the Lytton community and its co-researchers at its core. Instead of conventional interviews, innovative methods like walking audio sessions and photo elicitation are employed. These techniques allow participants to immerse themselves back into the environment, reviving and voicing their memories and emotions in real-time. Walking audio captures reflections on spatial narratives after the trauma, whereas photo voices encapsulate the intangible emotions, presenting a visual representation of place-based experiences. Key findings emphasize the indispensability of addressing both the tangible and intangible traumas in community recovery efforts post-disaster. The profound changes to the cultural landscape and the subsequent shifts in PA underscore the need for holistic, culturally attuned, and emotionally insightful adaptation strategies. These strategies, rooted in the lived experiences and testimonies of the affected individuals, promise more resonant and effective recovery efforts. The research further contributes to climate change discourse, highlighting the intertwined pathways of tangible reconstruction and the essentiality of emotional and cultural rejuvenation. Furthermore, the use of participatory methodologies in this inquiry challenges traditional research paradigms, pointing to potential evolutionary shifts in qualitative research norms. Ultimately, this study underscores the need for a more integrative approach in addressing the aftermath of environmental disasters, ensuring that both physical and emotional rebuilding are given equal emphasis.

Keywords: place attachment, community recovery, disaster reponse, sensory responses, intangible traumas, visual methodologies

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Authors: S. Kudlacik-Kramarczyk, M. Kedzierska, B. Tyliszczak

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Recently, the continuous development of medicine and related sciences has been observed. Particular emphasis is directed on the development of biomaterials, i.e., non-toxic, biocompatible and biodegradable materials that may improve the effectiveness of treatment as well as the comfort of patients. This is particularly important in the case of cancer treatment. Currently, there are many methods of cancer treatment based primarily on chemotherapy and the surgical removal of the tumor, but it is worth noting that these therapies also cause many side effects. Among women, the most common cancer is breast cancer. It may be completely cured, but the consequence of treatment is partial or complete breast mastectomy and radiation therapy, which results in severe skin burns. The skin of the patient after radiation therapy is very burned, and therefore requires intensive care and high frequency of dressing changes. The traditional dressing adheres to the burn wounds and does not absorb adequate amount of exudate from injuries and the patient is forced to change the dressing every 2 hours. Therefore, the main purpose was to develop an innovative combination of dressing material with drug carriers that may be used in anti-cancer therapy. The innovation of this solution is the combination of these two products into one system, i.e., a transdermal system with the possibility of a controlled release of the drug- cytostatic. Besides, the possibility of modifying the hydrogel matrix with aloe vera juice provides this material with new features favorable from the point of view of healing processes of burn wounds resulting from the radiation therapy. In this study, hydrogel materials containing protein spheres with the active substance have been obtained as a result of photopolymerization process. The reaction mixture consisting of the protein (albumin) spheres incorporated with cytostatic, chitosan, adequate crosslinking agent and photoinitiator has been subjected to the UV radiation for 2 minutes. Prepared materials have been subjected to the numerous studies including the analysis of cytotoxicity using murine fibroblasts L929. Analysis was conducted based on the mitochondrial activity test (MTT reduction assay) which involves the determining the number of cells characterized by proper metabolism. Hydrogel materials obtained using different amount of crosslinking agents have been subjected to the cytotoxicity analysis. According to the standards, tested material is defined as cytotoxic when the viability of cells after 24 h incubation with this material is lower than 70%. In the research, hydrogel polymer materials containing protein spheres incorporated with the active substance, i.e. a cytostatic, have been developed. Such a dressing may support the treatment of cancer due to the content of the anti-cancer drug - cytostatic, and may also provide a soothing effect on the healing of the burn wounds resulted from the radiation therapy due to the content of aloe vera juice in the hydrogel matrix. Based on the conducted cytotoxicity studies, it may be concluded that the obtained materials do not adversely affect the tested cell lines, therefore they can be subjected to more advanced analyzes.

Keywords: hydrogel polymers, cytostatics, drug carriers, cytotoxicity

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Authors: Mustafa Metin Donma, Orkide Donma

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Childhood obesity is an important clinical problem because it may lead to chronic diseases during the adulthood period of the individual. Obesity is a metabolic disease associated with low-grade inflammation. The liver occurs at the center of metabolic pathways. Adropin, fibroblast growth factor-21 (FGF-21), and fetuin-A are hepatokines. Due to the immense participation of the liver in glucose metabolism, these liver-derived factors may be associated with insulin resistance (IR), which is a phenomenon discussed within the scope of obesity problems. The aim of this study is to determine the concentrations of adropin, FGF-21, and fetuin-A in childhood obesity, to point out possible differences between the obesity groups, and to investigate possible associations among these three hepatokines in obese and morbidly obese children. A total of one hundred and thirty-two children were included in the study. Two obese groups were constituted. The groups were matched in terms of mean ± SD values of ages. Body mass index values of obese and morbidly obese groups were 25.0 ± 3.5 kg/m² and 29.8 ± 5.7 kg/m², respectively. Anthropometric measurements including waist circumference, hip circumference, head circumference, and neck circumference were recorded. Informed consent forms were taken from the parents of the participants. The ethics committee of the institution approved the study protocol. Blood samples were obtained after overnight fasting. Routine biochemical tests, including glucose- and lipid-related parameters, were performed. Concentrations of the hepatokines (adropin, FGF-21, fetuin A) were determined by enzyme-linked immunosorbent assay. Insulin resistance indices such as homeostasis model assessment for IR (HOMA-IR), alanine transaminase-to aspartate transaminase ratio (ALT/AST), diagnostic obesity notation model assessment laboratory index, diagnostic obesity notation model assessment metabolic syndrome index as well as obesity indices such as diagnostic obesity notation model assessment-II index, and fat mass index were calculated using the previously derived formulas. Statistical evaluation of the study data as well as findings of the study was performed by SPSS for Windows. Statistical difference was accepted significant when p is smaller than 0.05. Statistically significant differences were found for insulin, triglyceride, high-density lipoprotein cholesterol levels of the groups. A significant increase was observed for FGF-21 concentrations in the morbidly obese group. Higher adropin and fetuin-A concentrations were observed in the same group in comparison with the values detected in the obese group (p > 0.05). There was no statistically significant difference between the ALT/AST values of the groups. In all of the remaining IR and obesity indices, significantly increased values were calculated for morbidly obese children. Significant correlations were detected between HOMA-IR and each of the hepatokines. The highest one was the association with fetuin-A (r=0.373, p=0.001). In conclusion, increased levels observed in adropin, FGF-21, and fetuin-A have shown that these hepatokines possess increasing potential going from obese to morbid obese state. Out of the correlations found with the IR index, the most affected hepatokine was fetuin-A, the parameter possibly used as the indicator of the advanced obesity stage.

Keywords: adropin, fetuin A, fibroblast growth factor-21, insulin resistance, pediatric obesity

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Authors: Srijata Mitra, Mobina Parveen, Pranab Roy, Narayan Chandra Chattopadhyay

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Chlorinated hydrocarbon can be a major pollution problem in groundwater as well as soil. Many people interact with these chemicals on daily accidentally or by professionally in the laboratory. One of the most common sources for Chlorinated hydrocarbon contamination of soil and groundwater are industrial effluents. The wide use and discharge of Trichloroethylene (TCE), a volatile chlorohydrocarbon from chemical industry, led to major water pollution in rural areas. TCE is an mainly used as an industrial metal degreaser in industries. Biotransformation of TCE to the potent carcinogen vinyl chloride (VC) by consortia of anaerobic bacteria might have role for the above purpose. For these reasons, the aim of current study was to isolate and characterized the genes involved in TCE metabolism and also to investigate the in silico study of those genes. To our knowledge, only one aromatic dioxygenase system, the toluene dioxygenase in Pseudomonas putida F1 has been shown to be involved in TCE degradation. This is first instance where Bacillus cereus group being used in biodegradation of trichloroethylene. A novel bacterial strain 2479 was isolated from oil depot site at Rajbandh, Durgapur (West Bengal, India) by enrichment culture technique. It was identified based on polyphasic approach and ribotyping. The bacterium was gram positive, rod shaped, endospore forming and capable of degrading trichloroethylene as the sole carbon source. On the basis of phylogenetic data and Fatty Acid Methyl Ester Analysis, strain 2479 should be placed within the genus Bacillus and species cereus. However, the present isolate (strain 2479) is unique and sharply different from the usual Bacillus strains in its biodegrading nature. Fujiwara test was done to estimate that the strain 2479 could degrade TCE efficiently. The gene for TCE biodegradation was PCR amplified from genomic DNA of Bacillus cereus 2479 by using todC1 gene specific primers. The 600bp amplicon was cloned into expression vector pUC I8 in the E. coli host XL1-Blue and expressed under the control of lac promoter and nucleotide sequence was determined. The gene sequence was deposited at NCBI under the Accession no. GU183105. In Silico approach involved predicting the physico-chemical properties of deduced Tce1 protein by using ProtParam tool. The tce1 gene contained 342 bp long ORF encoding 114 amino acids with a predicted molecular weight 12.6 kDa and the theoretical pI value of the polypeptide was 5.17, molecular formula: C559H886N152O165S8, total number of atoms: 1770, aliphatic index: 101.93, instability index: 28.60, Grand Average of Hydropathicity (GRAVY): 0.152. Three differentially expressed proteins (97.1, 40 and 30 kDa) were directly involved in TCE biodegradation, found to react immunologically to the antibodies raised against TCE inducible proteins in Western blot analysis. The present study suggested that cloned gene product (TCE1) was capable of degrading TCE as verified chemically.

Keywords: cloning, Bacillus cereus, in silico analysis, TCE

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Authors: Suliman Al-Fayoumi, Sherri Amberg, Huafeng Zhou, Jack W. Singer, James P. Dean

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Pacritinib is an oral kinase inhibitor with specificity for JAK2, FLT3, IRAK1, and CSF1R. In clinical studies, pacritinib was well tolerated with clinical activity in patients with myelofibrosis. The most frequent adverse events (AEs) observed with pacritinib are gastrointestinal (diarrhea, nausea, and vomiting; mostly grade 1-2 in severity) and typically resolve within 2 weeks. A human ADME mass balance study demonstrated that pacritinib is predominantly cleared via hepatic metabolism and biliary excretion (>85% of administered dose). The major hepatic metabolite identified, M1, is not thought to materially contribute to the pharmacological activity of pacritinib. Hepatic diseases are known to impair hepatic blood flow, drug-metabolizing enzymes, and biliary transport systems and may affect drug absorption, disposition, efficacy, and toxicity. This phase 1 study evaluated the pharmacokinetics (PK) and safety of pacritinib and the M1 metabolite in study subjects with mild, moderate, or severe hepatic impairment (HI) and matched healthy subjects with normal liver function to determine if pacritinib dosage adjustments are necessary for patients with varying degrees of hepatic insufficiency. Study participants (aged 18-85 y) were enrolled into 4 groups based on their degree of HI as defined by Child-Pugh Clinical Assessment Score: mild (n=8), moderate (n=8), severe (n=4), and healthy volunteers (n=8) matched for age, BMI, and sex. Individuals with concomitant renal dysfunction or progressive liver disease were excluded. A single 400 mg dose of pacritinib was administered to all participants. Blood samples were obtained for PK evaluation predose and at multiple time points postdose through 168 h. Key PK parameters evaluated included maximum plasma concentration (Cmax), time to Cmax (Tmax), area under the plasma concentration time curve (AUC) from hour zero to last measurable concentration (AUC0-t), AUC extrapolated to infinity (AUC0-∞), and apparent terminal elimination half-life (t1/2). Following treatment, pacritinib was quantifiable for all study participants at 1 h through 168 h postdose. Systemic pacritinib exposure was similar between healthy volunteers and individuals with mild HI. However, there was a significant difference between those with moderate and severe HI and healthy volunteers with respect to peak concentration (Cmax) and plasma exposure (AUC0-t, AUC0-∞). Mean Cmax decreased by 47% and 57% respectively in participants with moderate and severe HI vs matched healthy volunteers. Similarly, mean AUC0-t decreased by 36% and 45% and mean AUC0-∞ decreased by 46% and 48%, respectively in individuals with moderate and severe HI vs healthy volunteers. Mean t1/2 ranged from 51.5 to 74.9 h across all groups. The variability on exposure ranged from 17.8% to 51.8% across all groups. Systemic exposure of M1 was also significantly decreased in study participants with moderate or severe HI vs. healthy participants and individuals with mild HI. These changes were not significantly dissimilar from the inter-patient variability in these parameters observed in healthy volunteers. All AEs were grade 1-2 in severity. Diarrhea and headache were the only AEs reported in >1 participant (n=4 each). Based on these observations, it is unlikely that dosage adjustments would be warranted in patients with mild, moderate, or severe HI treated with pacritinib.

Keywords: pacritinib, myelofibrosis, hepatic impairment, pharmacokinetics

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Authors: Berengere Vire, Nicolas Salvetat, Yoann Lannay, Guillaume Marcellin, Siem Van Der Laan, Franck Molina, Dinah Weissmann

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Predicting suicidal behaviors is one of the most complex challenges of daily psychiatric practices. Today, suicide risk prediction using biological tools is not validated and is only based on subjective clinical reports of the at-risk individual. Therefore, there is a great need to identify biomarkers that would allow early identification of individuals at risk of suicide. Alterations of adenosine-to-inosine (A-to-I) RNA editing of neurotransmitter receptors and other proteins have been shown to be involved in etiology of different psychiatric disorders and linked to suicidal behavior. RNA editing is a co- or post-transcriptional process leading to a site-specific alteration in RNA sequences. It plays an important role in the epi transcriptomic regulation of RNA metabolism. On postmortem human brain tissue (prefrontal cortex) of depressed suicide victims, Alcediag found specific alterations of RNA editing activity on the mRNA coding for the serotonin 2C receptor (5-HT2cR). Additionally, an increase in expression levels of ADARs, the RNA editing enzymes, and modifications of RNA editing profiles of prime targets, such as phosphodiesterase 8A (PDE8A) mRNA, have also been observed. Interestingly, the PDE8A gene is located on chromosome 15q25.3, a genomic region that has recurrently been associated with the early-onset major depressive disorder (MDD). In the current study, we examined whether modifications in RNA editing profile of prime targets allow identifying disease-relevant blood biomarkers and evaluating suicide risk in patients. To address this question, we performed a clinical study to identify an RNA editing signature in blood of depressed patients with and without the history of suicide attempts. Patient’s samples were drawn in PAXgene tubes and analyzed on Alcediag’s proprietary RNA editing platform using next generation sequencing technology. In addition, gene expression analysis by quantitative PCR was performed. We generated a multivariate algorithm comprising various selected biomarkers to detect patients with a high risk to attempt suicide. We evaluated the diagnostic performance using the relative proportion of PDE8A mRNA editing at different sites and/or isoforms as well as the expression of PDE8A and the ADARs. The significance of these biomarkers for suicidality was evaluated using the area under the receiver-operating characteristic curve (AUC). The generated algorithm comprising the biomarkers was found to have strong diagnostic performances with high specificity and sensitivity. In conclusion, we developed tools to measure disease-specific biomarkers in blood samples of patients for identifying individuals at the greatest risk for future suicide attempts. This technology not only fosters patient management but is also suitable to predict the risk of drug-induced psychiatric side effects such as iatrogenic increase of suicidal ideas/behaviors.

Keywords: blood biomarker, next-generation-sequencing, RNA editing, suicide

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Authors: Eric Bang

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Interactions between receptors and ligands are crucial for many essential biological processes, including neurotransmission and metabolism. Ligand-receptor analyses that examine cell behavior and interactions often utilize cell type-specific RNA expressions from single-cell RNA sequencing (scRNA-seq) data. Using CellPhoneDB, a public repository consisting of ligands, receptors, and ligand-receptor interactions, the cell-cell interactions were explored in a specific scRNA-seq dataset from kidney tissue and portrayed the results with dot plots and heat maps. Depending on the type of cell, each ligand-receptor pair was aligned with the interacting cell type and calculated the positori probabilities of these associations, with corresponding P values reflecting average expression values between the triads and their significance. Using single-cell data (sample kidney cell references), genes in the dataset were cross-referenced with ones in the existing CellPhoneDB dataset. For example, a gene such as Pleiotrophin (PTN) present in the single-cell data also needed to be present in the CellPhoneDB dataset. Using the single-cell transcriptomics data via slide-seq and reference data, the CellPhoneDB program defines cell types and plots them in different formats, with the two main ones being dot plots and heat map plots. The dot plot displays derived measures of the cell to cell interaction scores and p values. For the dot plot, each row shows a ligand-receptor pair, and each column shows the two interacting cell types. CellPhoneDB defines interactions and interaction levels from the gene expression level, so since the p-value is on a -log10 scale, the larger dots represent more significant interactions. By performing an interaction analysis, a significant interaction was discovered for myeloid and T-cell ligand-receptor pairs, including those between Secreted Phosphoprotein 1 (SPP1) and Fibronectin 1 (FN1), which is consistent with previous findings. It was proposed that an effective protocol would involve a filtration step where cell types would be filtered out, depending on which ligand-receptor pair is activated in that part of the tissue, as well as the incorporation of the CellPhoneDB data in a streamlined workflow pipeline. The filtration step would be in the form of a Python script that expedites the manual process necessary for dataset filtration. Being in Python allows it to be integrated with the CellPhoneDB dataset for future workflow analysis. The manual process involves filtering cell types based on what ligand/receptor pair is activated in kidney cells. One limitation of this would be the fact that some pairings are activated in multiple cells at a time, so the manual manipulation of the data is reflected prior to analysis. Using the filtration script, accurate sorting is incorporated into the CellPhoneDB database rather than waiting until the output is produced and then subsequently applying spatial data. It was envisioned that this would reveal wherein the cell various ligands and receptors are interacting with different cell types, allowing for easier identification of which cells are being impacted and why, for the purpose of disease treatment. The hope is this new computational method utilizing spatially explicit ligand-receptor association data can be used to uncover previously unknown specific interactions within kidney tissue.

Keywords: bioinformatics, Ligands, kidney tissue, receptors, spatial transcriptome

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Authors: Ernest Moles, Patricia Urbán, María Belén Jiménez-Díaz, Sara Viera-Morilla, Iñigo Angulo-Barturen, Maria Antònia Busquets, Xavier Fernàndez-Busquets

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Bearing in mind the absence of an effective vaccine against malaria and its severe clinical manifestations causing nearly half a million deaths every year, this disease represents nowadays a major threat to life. Besides, the basic rationale followed by currently marketed antimalarial approaches is based on the administration of drugs on their own, promoting the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the parasitized erythrocyte high enough to kill the intracellular pathogen while minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the specific delivery of immunoliposome (iLP)-encapsulated antimalarials to Plasmodium falciparum-infected red blood cells (pRBCs). Unfortunately, this strategy has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here, we show that encapsulation efficiencies reaching >96% can be achieved for the weakly basic drugs chloroquine (CQ) and primaquine using the pH gradient active loading method in liposomes composed of neutrally charged, saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the intracellular delivery of drugs not affecting the erythrocytic metabolism. Using this strategy, we have obtained unprecedented nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Polyethylene glycol-coated liposomes conjugated with monoclonal antibodies specific for the erythrocyte surface protein glycophorin A (anti-GPA iLP) were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5 μM total lipid in the culture, with >95% of added iLPs retained into the cells. When exposed for only 15 min to P. falciparum in vitro cultures synchronized at early stages, free CQ had no significant effect over parasite viability up to 200 nM drug, whereas iLP-encapsulated 50 nM CQ completely arrested its growth. Furthermore, when assayed in vivo in P. falciparum-infected humanized mice, anti-GPA iLPs cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg. In comparison, free CQ administered at 1.75 mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement in drug antimalarial efficacy is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.

Keywords: immunoliposomal nanoparticles, malaria, prophylactic-therapeutic polyvalent activity, targeted drug delivery

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Authors: Shatarupa Thakurta Roy

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Along the limelight of mainstream academic practices in Indian art, exist a significant lot of habitual art practices that are mutually susceptible in their contemporary forms. Narrative folk paintings of regional India has successfully dispersed to its audience social messages through pulsating pictures and orations. The paper consists of images from narrative scroll paintings on ‘Ramayana’ theme from various neighboring states as well as districts in India, describing their subtle differences in style of execution, method, and use of material. Despite sharing commonness in the choice of subject matter, habitual and ceremonial Indian folk art in its formative phase thrived within isolated locations to yield in remarkable variety in the art styles. The differences in style took place district wise, cast wise and even gender wise. An open flow is only evident in the contemporary expressions as a result of substantial changes in social structures, mode of communicative devices, cross-cultural exposures and multimedia interactivities. To decipher the complex nature of popular cultural taste of contemporary India it is important to categorically identify its root in vernacular symbolism. The realization of modernity through European primitivism was rather elevated as a perplexed identity in Indian cultural margin in the light of nationalist and postcolonial ideology. To trace the guiding factor that has still managed to obtain ‘Indianness’ in today’s Indian art, researchers need evidences from the past that are yet to be listed in most instances. They are commonly created on ephemeral foundations. The artworks are also found in endangered state and hence, not counted much friendly for frequent handling. The museums are in dearth of proper technological guidelines to preserve them. Even though restoration activities are emerging in the country, the existing withered and damaged artworks are in threat to perish. An immediacy of digital achieving is therefore envisioned as an alternative to save this cultural legacy. The method of this study is, two folded. It primarily justifies the richness of the evidences by conducting categorical aesthetic analysis. The study is supported by comments on the stylistic variants, thematic aspects, and iconographic identities alongside its anthropological and anthropomorphic significance. Further, it explores the possible ways of cultural preservation to ensure cultural sustainability that includes technological intervention in the form of digital transformation as an altered paradigm for better accessibility to the available recourses. The study duly emphasizes on visual description in order to culturally interpret and judge the rare visual evidences following Feldman’s four-stepped method of formal analysis combined with thematic explanation. A habitual design that emerges and thrives within complex social circumstances may experience change placing its principle philosophy at risk by shuffling and altering with time. A tradition that respires in the modern setup struggles to maintain timeless values that operate its creative flow. Thus, the paper hypothesizes the survival and further growth of this practice within the dynamics of time and concludes in realization of the urgency to transform the implicitness of its knowledge into explicit records.

Keywords: aesthetic, identity, implicitness, paradigm

Procedia PDF Downloads 357

Authors: Antonela Matana, Dubravka Brdar, Vesela Torlak, Marijana Popovic, Ivana Gunjaca, Ozren Polasek, Vesna Boraska Perica, Maja Barbalic, Ante Punda, Caroline Hayward, Tatijana Zemunik

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Parathyroid hormone (PTH) plays a critical role in the regulation of bone mineral metabolism and calcium homeostasis. Higher PTH levels are associated with heart failure, hypertension, coronary artery disease, cardiovascular mortality and poorer bone health. A twin study estimated that 60% of the variation in PTH concentrations is genetically determined. Only one GWAS of PTH concentration has been reported to date. Identified loci explained 4.5% of the variance in circulating PTH, suggesting that additional genetic variants remain undiscovered. Therefore, the aim of this study was to identify novel genetic variants associated with PTH levels in a general population. We have performed a GWAS meta-analysis on 2596 individuals originating from three Croatian cohorts: City of Split and the Islands of Korčula and Vis, within a large-scale project of “10,001 Dalmatians”. A total of 7 411 206 variants, imputed using the 1000 Genomes reference panel, with minor allele frequency ≥ 1% and Rsq ≥ 0.5 were analyzed for the association. GWAS within each data set was performed under an additive model, controlling for age, gender and relatedness. Meta-analysis was conducted using the inverse-variance fixed-effects method. Furthermore, to identify sex-specific effects, we have conducted GWAS meta-analyses analyzing males and females separately. In addition, we have performed biological pathway analysis. Four SNPs, representing one locus, reached genome-wide significance. The most significant SNP was rs11099476 on chromosome 4 (P=1.15x10-8), which explained 1.14 % of the variance in PTH. The SNP is located near the protein-coding gene RASGEF1B. Additionally, we detected suggestive association with SNPs, rs77178854 located on chromosome 2 in the DPP10 gene (P=2.46x10-7) and rs481121 located on chromosome 1 (P=3.58x10-7) near the GRIK1 gene. One of the top hits detected in the main meta-analysis, intron variant rs77178854 located within DPP10 gene, reached genome-wide significance in females (P=2.21x10-9). No single locus was identified in the meta-analysis in males. Fifteen biological pathways were functionally enriched at a P<0.01, including muscle contraction, ion homeostasis and cardiac conduction as the most significant pathways. RASGEF1B is the guanine nucleotide exchange factor, known to be associated with height, bone density, and hip. DPP10 encodes a membrane protein that is a member of the serine proteases family, which binds specific voltage-gated potassium channels and alters their expression and biophysical properties. In conclusion, we identified 2 novel loci associated with PTH levels in a general population, providing us with further insights into the genetics of this complex trait.

Keywords: general population, genome-wide association analysis, parathyroid hormone, single nucleotide polymorphisms.

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Authors: Tugba Altin

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In an era marked by pronounced climate change consequences, communities are observed to confront traumatic events that yield both tangible and intangible repercussions. Such events not only cause discernible damage to the landscape but also deeply affect the intangible aspects, including emotional distress and disruptions to cultural landscapes. The Lytton Creek Fire of 2021 serves as a case in point. Beyond the visible destruction, the less overt but profoundly impactful disturbance to place attachment (PA) is scrutinized. PA, representing the emotional and cognitive bonds individuals establish with their environments, is crucial for understanding how such events impact cultural identity and connection to the land. The study underscores the significance of addressing both tangible and intangible traumas for holistic community recovery. As communities renegotiate their affiliations with altered environments, the cultural landscape emerges as instrumental in shaping place-based identities. This renewed understanding is pivotal for reshaping adaptation planning. The research advocates for adaptation strategies rooted in the lived experiences and testimonies of the affected populations. By incorporating both the tangible and intangible facets of trauma, planning efforts are suggested to be more culturally attuned and emotionally insightful, fostering true resonance with the affected communities. Through such a comprehensive lens, this study contributes enriching the climate change discourse, emphasizing the intertwined nature of tangible recovery and the imperative of emotional and cultural healing after environmental disasters. Following the pronounced aftermath of the Lytton Creek Fire in 2021, research aims to deeply understand its impact on place attachment (PA), encompassing the emotional and cognitive bonds individuals form with their environments. The interpretive phenomenological approach, enriched by a hermeneutic framework, is adopted, emphasizing the experiences of the Lytton community and co-researchers. Phenomenology informed the understanding of 'place' as the focal point of attachment, providing insights into its formation and evolution after traumatic events. Data collection departs from conventional methods. Instead of traditional interviews, walking audio sessions and photo elicitation methods are utilized. These allow co-researchers to immerse themselves in the environment, re-experience, and articulate memories and feelings in real-time. Walking audio facilitates reflections on spatial narratives post-trauma, while photo voices captured intangible emotions, enabling the visualization of place-based experiences. The analysis is collaborative, ensuring the co-researchers' experiences and interpretations are central. Emphasizing their agency in knowledge production, the process is rigorous, facilitated by the harmonious blend of interpretive phenomenology and hermeneutic insights. The findings underscore the need for adaptation and recovery efforts to address emotional traumas alongside tangible damages. By exploring PA post-disaster, the research not only fills a significant gap but advocates for an inclusive approach to community recovery. Furthermore, the participatory methodologies employed challenge traditional research paradigms, heralding potential shifts in qualitative research norms.

Keywords: wildfire recovery, place attachment, trauma recovery, cultural landscape, visual methodologies

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

Procedia PDF Downloads 219

Authors: Irina Deputat, Anatoly Gribanov, Yuliya Dzhos, Alexandra Nekhoroshkova, Tatyana Yemelianova, Irina Bolshevidtseva, Irina Deryabina, Yana Kereush, Larisa Startseva, Tatyana Bagretsova, Irina Ikonnikova

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In the modern world the number of elderly people increases. Preservation of functionality of an organism in the elderly becomes very important now. During aging the higher cortical functions such as feelings, perception, attention, memory, and ideation are gradual decrease. It is expressed in the rate of information processing reduction, volume of random access memory loss, ability to training and storing of new information decrease. Perspective directions in studying of aging neurophysiological parameters are brain imaging: computer electroencephalography, neuroenergy mapping of a brain, and also methods of studying of a neurodynamic brain processes. Research aim – to study features of a brain aging in elderly people by electroencephalogram (EEG) and the DC-potential level. We examined 130 people aged 55 - 74 years that did not have psychiatric disorders and chronic states in a decompensation stage. EEG was recorded with a 128-channel GES-300 system (USA). EEG recordings are collected while the participant sits at rest with their eyes closed for 3 minutes. For a quantitative assessment of EEG we used the spectral analysis. The range was analyzed on delta (0,5–3,5 Hz), a theta - (3,5–7,0 Hz), an alpha 1-(7,0–11,0 Hz) an alpha 2-(11–13,0 Hz), beta1-(13–16,5 Hz) and beta2-(16,5–20 Hz) ranges. In each frequency range spectral power was estimated. The 12-channel hardware-software diagnostic ‘Neuroenergometr-KM’ complex was applied for registration, processing and the analysis of a brain constant potentials level. The DC-potential level registered in monopolar leads. It is revealed that the EEG of elderly people differ in higher rates of spectral power in the range delta (р < 0,01) and a theta - (р < 0,05) rhythms, especially in frontal areas in aging. By results of the comparative analysis it is noted that elderly people 60-64 aged differ in higher values of spectral power alfa-2 range in the left frontal and central areas (р < 0,05) and also higher values beta-1 range in frontal and parieto-occipital areas (р < 0,05). Study of a brain constant potential level distribution revealed increase of total energy consumption on the main areas of a brain. In frontal leads we registered the lowest values of constant potential level. Perhaps it indicates decrease in an energy metabolism in this area and difficulties of executive functions. The comparative analysis of a potential difference on the main assignments testifies to unevenness of a lateralization of a brain functions at elderly people. The results of a potential difference between right and left hemispheres testify to prevalence of the left hemisphere activity. Thus, higher rates of functional activity of a cerebral cortex are peculiar to people of early advanced age (60-64 years) that points to higher reserve opportunities of central nervous system. By 70 years there are age changes of a cerebral power exchange and level of electrogenesis of a brain which reflect deterioration of a condition of homeostatic mechanisms of self-control and the program of processing of the perceptual data current flow.

Keywords: brain, DC-potential level, EEG, elderly people

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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: Nada Al-Qallaf, Aisha Al-Barood, Djamel Lekmine, Srinivasan Vedhapuri

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Kuwait Oil Company (KOC) under the supervision of Kuwait National Focal Point (KNFP) is planning to remediate 26 million (M) m3 of oil-contaminated soil in oil fields of Kuwait as a direct and indirect fallout of the Gulf War during 1990-1991. This project is funded by the United Nations Compensation Commission (UNCC) under the Kuwait Environmental Remediation Program (KERP). Oil-contamination of the soil occurred due to the destruction of the oil wells and spilled crude oil across the land surface and created ‘oil lakes’ in low lying land. Aerial fall-out from oil spray and combustion products from oil fires combined with the sand and gravel on the ground surface to form a layer of hardened ‘Tarcrete’. The unique fresh groundwater lenses present in the Raudhatain and Sabriya subsurface areas had been impacted by the discharge and/or spills of dissolved petroleum constituents. These fresh groundwater aquifers were used for drinking water purposes until 1990, prior to invasion. This has significantly damages altered the landscape, ecology and habitat of the flora and fauna and in Kuwait Desert. Under KERP, KOC is fully responsible for the planning and execution of the remediation and restoration projects in KOC oil fields. After the initial recommendation of UNCC to construct engineered landfills for containment and disposal of heavily contaminated soils, two landfills were constructed, one in North Kuwait and another in South East Kuwait of capacity 1.7 million m3 and 0.5 million m3 respectively. KOC further developed the Total Remediation Strategy in conjunction with KNFP and has obtained UNCC approval. The TRS comprises of elements such as Risk Based Approach (RBA), Bioremediation of low Contaminated Soil levels, Remediation Treatment Technologies, Sludge Disposal via Beneficial Recycling or Re-use and Engineered landfills for Containment of untreatable materials. Risk Based Assessment as a key component to avoid any unnecessary remedial works, where it can be demonstrated that human health and the environment are sufficiently protected in the absence of active remediation. This study demonstrates on the risks of tarcrete materials spread over areas 20 Km2 on the fresh Ground water lenses/catchment located beneath the Sabriyah and Raudhatain oil fields in North Kuwait. KOC’s primary objective is to provide justification of using RBA, to support a case with the Kuwait regulators to leave the tarcrete material in place, rather than seek to undertake large-scale removal and remediation. The large-scale coverage of the tarcrete in the oil fields and perception that the residual contamination associated with this source is present in an environmentally sensitive area essentially in ground water resource. As part of this assessment, conceptual site model (CSM) and complete risk-based and fate and transport modelling was carried out which includes derivation of site-specific assessment criteria (SSAC) and quantification of risk to identified waters resource receptors posed by tarcrete impacted areas. The outcome of this assessment was determined that the residual tarcrete deposits across the site area shall not create risks to fresh groundwater resources and the remedial action to remove and remediate the surficial tarcrete deposits is not warranted.

Keywords: conceptual site model, fresh groundwater, oil-contaminated soil, tarcrete, risk based assessment

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

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Authors: Keshari Shrestha, Philip Vatterott

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Introduction: Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a rare and potentially fatal drug-related syndrome. DRESS classically presents with a diffuse maculopapular rash, fevers, and eosinophilia more than three weeks after drug exposure. DRESS can present with multi-organ involvement, with liver damage being the most common and severe. Pulmonary involvement is a less common manifestation and is associated with poor clinical outcomes. Chest imaging is often nonspecific, and symptoms can range from mild cough to acute respiratory distress syndrome (ARDS) . This is a case of a 49-year-old female with a history of recent clostridium difficile colitis status post treatment with oral vancomycin who presented with rash, acute liver and kidney failure, as well as diffuse nodular alveolar lung opacities concerning for DRESS syndrome with multi-organ involvement. Clinical Course: This patient initially presented to an outside hospital with clostridium difficile colitis, acute liver injury, and acute kidney injury. She developed a desquamating maculopapular rash in the setting of recent oral vancomycin, meloxicam, and furosemide initiation. She was hospitalized on two additional occasions with worsening altered mental status, liver injury, and acute kidney injury and was initiated on intermittent hemodialysis. Notably, she was found to have systemic eosinophilia (4100 cells/microliter) several weeks prior. She was transferred to this institution for further management where she was found to have encephalopathy, jaundice, lower extremity edema, and diffuse bilateral rhonchorous breath sounds on pulmonary examination. The patient was started on methylprednisolone for suspected DRESS syndrome. She underwent an evaluation for alternative causes of her organ failure. Her workup included a negative infectious, autoimmune, metabolic, toxic, and malignant work-up. Abdominal computed tomography (CT) and ultrasound were remarkable for evidence of hepatic steatosis and possible cirrhotic morphology. Additionally, a chest CT demonstrated diffuse and symmetric nodular alveolar lung opacities with peripheral sparing not consistent with acute respiratory distress syndrome or edema. Ultimately, her condition continued to decline, and she required intubation on several occasions. On hospital day 25 she succumbed to distributive shock in the setting of probable sepsis and multi-organ failure. Discussion: DRESS syndrome occurs in 1 in 1,000 to 10,000 patients with a mortality rate of around 10%. Anti-convulsant, anti-bacterial, anti-viral, and sulfonamide drugs are the most common drugs implicated in the development of DRESS syndrome; however, the list of offending agents is extensive . The diagnosis of DRESS syndrome is made after excluding other causes of disease such as infectious and autoimmune etiologies. The RegiSCAR scoring system is used to diagnose DRESS syndrome with 2-3 points indicating possible disease, 4-5 probable disease, and >5 definite disease. This patient scored a 7 on the RegiSCAR scale for eosinophilia, rash, organ involvement, and exclusion of other causes (infectious and autoimmune). While the pharmacologic trigger in this case is unknown, it is speculated to be caused by vancomycin, meloxicam, or furosemide due to the favorable timeline of initiation. Despite aggressive treatment, DRESS syndrome can often be fatal. Because of this, early diagnosis and treatment of patients with suspected DRESS syndrome is imperative.

Keywords: drug reaction with eosinophilia and systemic symptoms, multi-organ failure, pulmonary involvement, renal failure

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Authors: Suikinai Nobre Santos, Ranko Gacesa, Paul F. Long, Itamar Soares de Melo

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Extremophilic microorganism are adapted to biotopes combining several stress factors (temperature, pressure, radiation, salinity and pH), which indicate the richness valuable resource for the exploitation of novel biotechnological processes and constitute unique models for investigations their biomolecules (1, 2). The above information encourages us investigate bioprospecting synthesized compounds by a noval actinomycete, designated thermotolerant Streptomyces caatingaensis CMAA 1322, isolated from sample soil tropical dry forest (Caatinga) in the Brazilian semiarid region (3-17°S and 35-45°W). This set of constrating physical and climatic factores provide the unique conditions and a diversity of well adapted species, interesting site for biotechnological purposes. Preliminary studies have shown the great potential in the production of cytotoxic, pesticidal and antimicrobial molecules (3). Thus, to extend knowledge of the genes clusters responsible for producing biosynthetic pathways of natural products in strain CMAA1322, whole-genome shotgun (WGS) DNA sequencing was performed using paired-end long sequencing with PacBio RS (Pacific Biosciences). Genomic DNA was extracted from a pure culture grown overnight on LB medium using the PureLink genomic DNA kit (Life Technologies). An approximately 3- to 20-kb-insert PacBio library was constructed and sequenced on an 8 single-molecule real-time (SMRT) cell, yielding 116,269 reads (average length, 7,446 bp), which were allocated into 18 contigs, with 142.11x coverage and N50 value of 20.548 bp (BioProject number PRJNA288757). The assembled data were analyzed by Rapid Annotations using Subsystems Technology (RAST) (4) the genome size was found to be 7.055.077 bp, comprising 6167 open reading frames (ORFs) and 413 subsystems. The G+C content was estimated to be 72 mol%. The closest-neighbors tool, available in RAST through functional comparison of the genome, revealed that strain CMAA1322 is more closely related to Streptomyces hygroscopicus ATCC 53653 (similarity score value, 537), S. violaceusniger Tu 4113 (score value, 483), S. avermitilis MA-4680 (score value, 475), S. albus J1074 (score value, 447). The Streptomyces sp. CMAA1322 genome contains 98 tRNA genes and 135 genes copies related to stress response, mainly osmotic stress (14), heat shock (16), oxidative stress (49). Functional annotation by antiSMASH version 3.0 (5) identified 41 clusters for secondary metabolites (including two clusters for lanthipeptides, ten clusters for nonribosomal peptide synthetases [NRPS], three clusters for siderophores, fourteen for polyketide synthetase [PKS], six clusters encoding a terpene, two clusters encoding a bacteriocin, and one cluster encoding a phenazine). Our work provide in comparative analyse of genome and extract produced (data no published) by lineage CMAA1322, revealing the potential of microorganisms accessed from extreme environments as Caatinga” to produce a wide range of biotechnological relevant compounds.

Keywords: caatinga, streptomyces, environmental stresses, biosynthetic pathways

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Authors: Ananya Barui, Provas Banerjee, Jyotirmoy Chatterjee

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Application of optical technology in medicine and biology has a long track-record. In this endeavor, OCT is able to attract both engineers and biologists to work together in the field of photonics for establishing a striking non-invasive imaging technology. In contrast to other in vivo imaging modalities like Raman imaging, confocal imaging, two-photon microscopy etc. which can perform in vivo imaging upto 100-200 micron depth due to limitation in numerical aperture or scattering, however, OCT can achieve high-resolution imaging upto few millimeters of tissue structures depending on their refractive index in different anatomical location. This tomographic system depends on interference of two light waves in an interferometer to produce a depth profile of specimen. In wound healing, frequent collection of biopsies for follow-up of repair process could be avoided by such imaging technique. Real time skin OCT (the optical biopsy) has efficacy in deeper and faster illumination of cutaneou tissue to acquire high resolution cross sectional images of their internal micro-structure. Swept Source-OCT (SS-OCT), a novel imaging technique, can generate high-speed depth profile (~ 2 mm) of wound at a sweeping rate of laser with micron level resolution and optimum coherent length of 5-6 mm. Normally multi-layered skin tissue depicts different optical properties along with variation in thickness, refractive index and composition (i.e. keratine layer, water, fat etc.) according to their anatomical location. For instance, stratum corneum, the upper-most and relatively dehydrated layer of epidermis reflects more light and produces more lucid and a sharp demarcation line with rest of the hydrated epidermal region. During wound healing or regeneration, optical properties of cutaneous tissue continuously altered with maturation of wound bed. More mature and less hydrated tissue component reflects more light and becomes visible as a brighter area in comparison to immature region which content higher amount water or fat that depicts as a darker area in OCT image. Non-healing wound possess prolonged inflammation and inhibits nascent proliferative stage. Accumulation of necrotic tissues also prevents the repair of non-healing wounds. Due to high resolution and potentiality to reflect the compositional aspects of tissues in terms of their optical properties, this tomographic method may facilitate in differentiating non-healing and acute wounds in addition to clinical observations. Non-invasive OCT offers better insight regarding specific biological status of tissue in health and pathological conditions, OCT images could be associated with histo-pathological ‘gold standard’. This correlated SS-OCT and microscopic evaluation of the wound edges can provide information regarding progressive healing and maturation of the epithelial components. In the context of searching analogy between two different imaging modalities, their relative performances in imaging of healing bed were estimated for probing an alternative approach. Present study validated utility of SS-OCT in revealing micro-anatomic structure in the healing bed with newer information. Exploring precise correspondence of OCT images features with histo-chemical findings related to epithelial integrity of the regenerated tissue could have great implication. It could establish the ‘optical biopsy’ as a potent non-invasive diagnostic tool for cutaneous pathology.

Keywords: histo-pathology, non invasive imaging, OCT, wound healing

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Authors: Maddalena Arigoni, Maria Luisa Ratto, Raffaele A. Calogero, Luca Alessandri

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The presence of tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Understanding the causes and progression of cancer requires research efforts aimed at characterizing heterogeneity, which can be facilitated by evolving single-cell sequencing technologies. However, analyzing single-cell data necessitates computational methods that often lack objective validation. Therefore, the establishment of benchmarking datasets is necessary to provide a controlled environment for validating bioinformatics tools in the field of single-cell oncology. Benchmarking bioinformatics tools for single-cell experiments can be costly due to the high expense involved. Therefore, datasets used for benchmarking are typically sourced from publicly available experiments, which often lack a comprehensive cell annotation. This limitation can affect the accuracy and effectiveness of such experiments as benchmarking tools. To address this issue, we introduce omics benchmark experiments designed to evaluate bioinformatics tools to depict the heterogeneity in single-cell tumor experiments. We conducted single-cell RNA sequencing on six lung cancer tumor cell lines that display resistant clones upon treatment of EGFR mutated tumors and are characterized by driver genes, namely ROS1, ALK, HER2, MET, KRAS, and BRAF. These driver genes are associated with downstream networks controlled by EGFR mutations, such as JAK-STAT, PI3K-AKT-mTOR, and MEK-ERK. The experiment also featured an EGFR-mutated cell line. Using 10XGenomics platform with cellplex technology, we analyzed the seven cell lines together with a pseudo-immunological microenvironment consisting of PBMC cells labeled with the Biolegend TotalSeq™-B Human Universal Cocktail (CITEseq). This technology allowed for independent labeling of each cell line and single-cell analysis of the pooled seven cell lines and the pseudo-microenvironment. The data generated from the aforementioned experiments are available as part of an online tool, which allows users to define cell heterogeneity and generates count tables as an output. The tool provides the cell line derivation for each cell and cell annotations for the pseudo-microenvironment based on CITEseq data by an experienced immunologist. Additionally, we created a range of pseudo-tumor tissues using different ratios of the aforementioned cells embedded in matrigel. These tissues were analyzed using 10XGenomics (FFPE samples) and Curio Bioscience (fresh frozen samples) platforms for spatial transcriptomics, further expanding the scope of our benchmark experiments. The benchmark experiments we conducted provide a unique opportunity to evaluate the performance of bioinformatics tools for detecting and characterizing tumor heterogeneity at the single-cell level. Overall, our experiments provide a controlled and standardized environment for assessing the accuracy and robustness of bioinformatics tools for studying tumor heterogeneity at the single-cell level, which can ultimately lead to more precise and effective cancer diagnosis and treatment.

Keywords: single cell omics, benchmark, spatial transcriptomics, CITEseq

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Authors: Zakia Fatima, Liu Lu, Donghao Li

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The analysis of biological fluid metabolites holds significant importance in various areas, such as medical research, food science, and public health. Investigating the levels and distribution of nutrients and their metabolites in biological samples allows researchers and healthcare professionals to determine nutritional status, find hypovitaminosis or hypervitaminosis, and monitor the effectiveness of interventions such as dietary supplementation. Moreover, analysis of nutrient metabolites provides insight into their metabolism, bioavailability, and physiological processes, aiding in the clarification of their health roles. Hence, the exploration of a distinct, efficient, eco-friendly, and simpler methodology is of great importance to evaluate the metabolic content of complex biological samples. In this work, a green and rapid analytical method based on an automated online two-dimensional microscale carbon fiber/activated carbon fiber fractionation system and time-of-flight mass spectrometry (2DμCFs-TOF-MS) was used to evaluate metabolites of urine samples after oral administration of food supplements. The automated 2DμCFs instrument consisted of a microcolumn system with bare carbon fibers and modified carbon fiber coatings. Carbon fibers and modified carbon fibers exhibit different surface characteristics and retain different compounds accordingly. Three kinds of mobile-phase solvents were used to elute the compounds of varied chemical heterogeneities. The 2DμCFs separation system has the ability to effectively separate different compounds based on their polarity and solubility characteristics. No complicated sample preparation method was used prior to analysis, which makes the strategy more eco-friendly, practical, and faster than traditional analysis methods. For optimum analysis results, mobile phase composition, flow rate, and sample diluent were optimized. Water-soluble vitamins, fat-soluble vitamins, and amino acids, as well as 22 vitamin metabolites and 11 vitamin metabolic pathway-related metabolites, were found in urine samples. All water-soluble vitamins except vitamin B12 and vitamin B9 were detected in urine samples. However, no fat-soluble vitamin was detected, and only one metabolite of Vitamin A was found. The comparison with a blank urine sample showed a considerable difference in metabolite content. For example, vitamin metabolites and three related metabolites were not detected in blank urine. The complete single-run screening was carried out in 5.5 minutes with the minimum consumption of toxic organic solvent (0.5 ml). The analytical method was evaluated in terms of greenness, with an analytical greenness (AGREE) score of 0.72. The method’s practicality has been investigated using the Blue Applicability Grade Index (BAGI) tool, obtaining a score of 77. The findings in this work illustrated that the 2DµCFs-TOF-MS approach could emerge as a fast, sustainable, practical, high-throughput, and promising analytical tool for screening and accurate detection of various metabolites, pharmaceuticals, and ingredients in dietary supplements as well as biological fluids.

Keywords: metabolite analysis, sustainability, carbon fibers, urine.

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Authors: Imdad Ullah Khan, Yongseok Yoon, Kyeung Uk Choi, Kwang Rae Jo, Namyul Kim, Eunbee Lee, Wan Hee Kim, Oh-Kyeong Kweon

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The primary spinal cord injury (SCI) causes mechanical damage to the neurons and blood vessels. It leads to secondary SCI, which activates multiple pathological pathways, which expand neuronal damage at the injury site. It is characterized by vascular disruption, ischemia, excitotoxicity, oxidation, inflammation, and apoptotic cell death. It causes nerve demyelination and disruption of axons, which perpetuate a loss of impulse conduction through the injured spinal cord. It also leads to the production of myelin inhibitory molecules, which with a concomitant formation of an astroglial scar, impede axonal regeneration. The pivotal role regarding the neuronal necrosis is played by oxidation and inflammation. During an early stage of spinal cord injury, there occurs an abundant expression of reactive oxygen species (ROS) due to defective mitochondrial metabolism and abundant migration of phagocytes (macrophages, neutrophils). ROS cause lipid peroxidation of the cell membrane, and cell death. Abundant migration of neutrophils, macrophages, and lymphocytes collectively produce pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1beta (IL-1β), matrix metalloproteinase, superoxide dismutase, and myeloperoxidases which synergize neuronal apoptosis. Therefore, it is crucial to control inflammation and oxidation injury to minimize the nerve cell death during secondary spinal cord injury. Therefore, in response to oxidation and inflammation, heme oxygenase-1 (HO-1) is induced by the resident cells to ameliorate the milieu. In the meanwhile, neurotrophic factors are induced to promote neuroregeneration. However, it seems that anti-stress enzyme (HO-1) and neurotrophic factor (BDNF) do not significantly combat the pathological events during secondary spinal cord injury. Therefore, optimum healing can be induced if anti-inflammatory and neurotrophic factors are administered in a higher amount through an exogenous source. During the first experiment, the inflammation and neuroregeneration were selectively targeted. HO-1 expressing MSCs (HO-1 MSCs) and BDNF expressing MSCs (BDNF MSC) were co-transplanted in one group (combination group) of dogs with subacute spinal cord injury to selectively control the expression of inflammatory cytokines by HO-1 and induce neuroregeneration by BDNF. We compared the combination group with the HO-1 MSCs group, BDNF MSCs group, and GFP MSCs group. We found that the combination group showed significant improvement in functional recovery. It showed increased expression of neural markers and growth-associated proteins (GAP-43) than in other groups, which depicts enhanced neuroregeneration/neural sparing due to reduced expression of pro-inflammatory cytokines such as TNF-alpha, IL-6 and COX-2; and increased expression of anti-inflammatory markers such as IL-10 and HO-1. Histopathological study revealed reduced intra-parenchymal fibrosis in the injured spinal cord segment in the combination group than in other groups. Thus it was concluded that selectively targeting the inflammation and neuronal growth with the combined use of HO-1 MSCs and BDNF MSCs more favorably promote healing of the SCI. HO-1 MSCs play a role in controlling the inflammation, which favors the BDNF induced neuroregeneration at the injured spinal cord segment of dogs.

Keywords: HO-1 MSCs, BDNF MSCs, neuroregeneration, inflammation, anti-inflammation, spinal cord injury, dogs

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Authors: Kyriaki Hatziagapiou, Konstantinos Bethanis, Olti Nikola, Elias Christoforides, Eleni Koniari, Eleni Kakouri, George Lambrou, Christina Kanaka-Gantenbein

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Glioblastoma multiforme (GBM) remains a serious health challenge, as current therapeutic modalities continue to yield unsatisfactory results, with the average survival rarely exceeding 1-2 years. Natural compounds still provide some of the most promising approaches for discovering new drugs. The non-psychotropic cannabidiol (CBD) deriving from Cannabis sativa L. provides such promise. CBD is endowed with anticancer, antioxidant, and genoprotective properties as established in vitro and in in vivo experiments. CBD’s selectivity towards cancer cells and its safe profile suggest its usage in cancer therapies. However, the bioavailability of oral CBD is low due to poor aqueous solubility, erratic gastrointestinal absorption, and significant first-pass metabolism, hampering its therapeutic potential and resulting in a variable pharmacokinetic profile. In this context, CBD can take great advantage of nanomedicine-based formulation strategies. Cyclodextrins (CDs) are cyclic oligosaccharides used in the pharmaceutical industry to incorporate apolar molecules inside their hydrophobic cavity, increasing their stability, water solubility, and bioavailability or decreasing their side effects. CBD-inclusion complexes with CDs could be a good strategy to improve its properties, like solubility and stability to harness its full therapeutic potential. The current research aims to study the potential cytotoxic effect of CBD and CBD-CDs complexes CBD-RMβCD (randomly methylated β-cyclodextrin) and CBD-HPβCD (hydroxypropyl-b-CD) on the A172 glioblastoma cell line. CBD is diluted in 10% DMSO, and CBD/CDs solutions are prepared by mixing solid CBD, solid CDs, and dH2O. For the biological assays, A172 cells are incubated at a range of concentrations of CBD, CBD-RMβCD and CBD-HPβCD, RMβCD, and HPβCD (0,03125-4 mg/ml) at 24, 48, and 72 hours. Analysis of cell viability after incubation with the compounds is performed with Alamar Blue viability assay. CBD’s dilution to DMSO 10% was inadequate, as crystals are observed; thus cytotoxicity experiments are not assessed. CBD’s solubility is enhanced in the presence of both CDs. CBD/CDs exert significant cytotoxicity in a dose and time-dependent manner (p < 0.005 for exposed cells to any concentration at 48, 72, and 96 hours versus cells not exposed); as their concentration and time of exposure increases, the reduction of resazurin to resofurin decreases, indicating a reduction in cell viability. The cytotoxic effect is more pronounced in cells exposed to CBD-HPβCD for all concentrations and time-points. RMβCD and HPβCD at the highest concentration of 4 mg/ml also exerted antitumor action per se since manifesting cell growth inhibition. The results of our study could afford the basis of research regarding the use of natural products and their inclusion complexes as anticancer agents and the shift to targeted therapy with higher efficacy and limited toxicity. Acknowledgments: The research is partly funded by ΙΚΥ (State Scholarships Foundation) – Post-doc Scholarships-Partnership Agreement 2014-2020.

Keywords: cannabidiol, cyclodextrins, glioblastoma, hydroxypropyl-b-Cyclodextrin, randomly-methylated-β-cyclodextrin

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