Search results for: biomarkers

Authors: Tamphasana Wairokpam

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Inflammatory myopathies (IMs) have long been recognised as a heterogenous family of myopathies with acute, subacute, and sometimes chronic presentation and are potentially treatable. Necrotizing autoimmune myopathies (NAM) are a relatively new subset of myopathies. Patients generally present with subacute onset of proximal myopathy and significantly elevated creatinine kinase (CK) levels. It is being increasingly recognised that there are limitations to the independent diagnostic utility of muscle biopsy. Immunohistochemistry tests may reveal important information in these cases. The traditional classification of IMs failed to recognise NAM as a separate entity and did not adequately emphasize the diversity of IMs. This review and case report on NAM aims to highlight the heterogeneity of this entity and focus on the distinct clinical presentation, biopsy findings, specific auto-antibodies implicated, and available treatment options with prognosis. This article is a meta-analysis of literatures on NAM and a case report illustrating the clinical course, investigation and biopsy findings, antibodies implicated, and management of a patient with NAM. The main databases used for the search were Pubmed, Google Scholar, and Cochrane Library. Altogether, 67 publications have been taken as references. Two biomarkers, anti-signal recognition protein (SRP) and anti- hydroxyl methylglutaryl-coenzyme A reductase (HMGCR) Abs, have been found to have an association with NAM in about 2/3rd of cases. Interestingly, anti-SRP associated NAM appears to be more aggressive in its clinical course when compared to its anti-HMGCR associated counterpart. Biopsy shows muscle fibre necrosis without inflammation. There are reports of statin-induced NAM where progression of myopathy has been seen even after discontinuation of statins, pointing towards an underlying immune mechanism. Diagnosisng NAM is essential as it requires more aggressive immunotherapy than other types of IMs. Most cases are refractory to corticosteroid monotherapy. Immunosuppressive therapy with other immunotherapeutic agents such as IVIg, rituximab, mycophenolate mofetil, azathioprine has been explored and found to have a role in the treatment of NAM. In conclusion,given the heterogeneity of NAM, it appears that NAM is not just a single entity but consists of many different forms, despite the similarities in presentation and its classification remains an evolving field. A thorough understanding of underlying mechanism and the clinical correlation with antibodies associated with NAM is essential for efficacious management and disease prognostication.

Keywords: inflammatory myopathies, necrotising autoimmune myopathies, anti-SRP antibody, anti-HMGCR antibody, statin induced myopathy

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Authors: Priya Kulkarni, Soumya Koppikar, Narendrakumar Wagh, Dhanshri Ingle, Onkar Lande, Abhay Harsulkar

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Cartilage is an extracellular matrix composed of aggrecan, which imparts it with a great tensile strength, stiffness and resilience. Disruption in cartilage metabolism leading to progressive degeneration is a characteristic feature of Osteoarthritis (OA). The process involves enzymatic depolymerisation of cartilage specific proteoglycan, releasing free glycosaminoglycan (GAG). This released GAG in synovial fluid (SF) of knee joint serves as a direct measure of cartilage loss, however, limited due to its invasive nature. Western Ontario and McMaster Universities Arthritis Index (WOMAC) is widely used for assessing pain, stiffness and physical-functions in OA patients. The scale is comprised of three subscales namely, pain, stiffness and physical-function, intends to measure patient’s perspective of disease severity as well as efficacy of prescribed treatment. Twenty SF samples obtained from OA patients were analysed for their GAG values in SF using DMMB based assay. LK 1.0 vernacular version was used to attain WOMAC scale. The results were evaluated using SAS University software (Edition 1.0) for statistical significance. All OA patients revealed higher GAG values compared to the control value of 78.4±30.1µg/ml (obtained from our non-OA patients). Average WOMAC calculated was 51.3 while pain, stiffness and function estimated were 9.7, 3.9 and 37.7, respectively. Interestingly, a strong statistical correlation was established between WOMAC function subscale and GAG (p = 0.0102). This subscale is based on day-to-day activities like stair-use, bending, walking, getting in/out of car, rising from bed. However, pain and stiffness subscale did not show correlation with any of the studied markers and endorsed the atypical inflammation in OA pathology. On one side, where knee pain showed poor correlation with GAG, it is often noted that radiography is insensitive to cartilage degenerative changes; thus OA remains undiagnosed for long. Moreover, active cartilage degradation phase remains elusive to both, patient and clinician. Through analysis of large number of OA patients we have established a close association of Kellgren-Lawrence grades and increased cartilage loss. A direct attempt to correlate WOMAC and radiographic progression of OA with various biomarkers has not been attempted so far. We found a good correlation in GAG levels in SF and the function subscale.

Keywords: cartilage, Glycosaminoglycan, synovial fluid, western ontario and McMaster Universities Arthritis Index

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Authors: Jae-Hyeon Kim, Michael Lee

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Intrinsic and acquired resistance limits the therapeutic benefits of oncogenic BRAF inhibitors in melanoma. MicroRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation. Thus, we investigated miRNA expression patterns in melanoma cell lines to identify candidate biomarkers for acquired resistance to BRAF inhibitor. Here, we used Affymetrix miRNA V3.0 microarray profiling platform to compare miRNA expression levels in three cell lines containing BRAF inhibitor-sensitive A375P BRAF V600E cells, their BRAF inhibitor-resistant counterparts (A375P/Mdr), and SK-MEL-2 BRAF-WT cells with intrinsic resistance to BRAF inhibitor. The miRNAs with at least a two-fold change in expression between BRAF inhibitor-sensitive and –resistant cell lines, were identified as differentially expressed. Averaged intensity measurements identified 138 and 217 miRNAs that were differentially expressed by 2 fold or more between: 1) A375P and A375P/Mdr; 2) A375P and SK-MEL-2, respectively. The hierarchical clustering revealed differences in miRNA expression profiles between BRAF inhibitor-sensitive and –resistant cell lines for miRNAs involved in intrinsic and acquired resistance to BRAF inhibitor. In particular, 43 miRNAs were identified whose expression was consistently altered in two BRAF inhibitor-resistant cell lines, regardless of intrinsic and acquired resistance. Twenty five miRNAs were consistently upregulated and 18 downregulated more than 2-fold. Although some discrepancies were detected when miRNA microarray data were compared with qPCR-measured expression levels, qRT-PCR for five miRNAs (miR-3617, miR-92a1, miR-1246, miR-1936-3p, and miR-17-3p) results showed excellent agreement with microarray experiments. To further investigate cellular functions of miRNAs, we examined effects on cell proliferation. Synthetic oligonucleotide miRNA mimics were transfected into three cell lines, and proliferation was quantified using a colorimetric assay. Of the 5 miRNAs tested, only miR-1246 altered cell proliferation of A375P/Mdr cells. The transfection of miR-1246 mimic strongly conferred PLX-4720 resistance to A375P/Mdr cells, implying that miR-1246 upregulation confers acquired resistance to BRAF inhibition. We also found that PLX-4720 caused much greater G2/M arrest in A375P/Mdr cells transfected with miR-1246mimic than that seen in scrambled RNA-transfected cells. Additionally, miR-1246 mimic partially caused a resistance to autophagy induction by PLX-4720. These results indicate that autophagy does play an essential death-promoting role inPLX-4720-induced cell death. Taken together, these results suggest that miRNA expression profiling in melanoma cells can provide valuable information for a network of BRAF inhibitor resistance-associated miRNAs.

Keywords: microRNA, BRAF inhibitor, drug resistance, autophagy

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Authors: Mohamed Malek Khenissi, Mohamed Montassar Ben Slama, Anis Belhaj Mohamed, Moncef Saidi

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Northern Tunisia is well known by its different and complex structural and geological zones that have been the result of a geodynamic history that extends from the early Mesozoic era to the actual period. One of these zones is the salt province, where the Halokinesis process is manifested by a number of NE/SW salt structures such as Jebel Ech-Cheid which represents masses of materials characterized by a high plasticity and low density. The salt masses extrusions that have been developed due to an extension that started from the late Triassic to late Cretaceous. The evolution of salt bodies within sedimentary basins have not only contributed to modify the architecture of the basin, but it also has certain geochemical effects which touch mainly source rocks that surround it. It has been demonstrated that the presence of salt structures within sedimentary basins can influence its temperature distribution and thermal history. Moreover, it has been creating heat flux anomalies that may affect the maturity of organic matter and the timing of hydrocarbon generation. Field samples of the Bahloul source rock (Cenomanan-Tunonian) were collected from different sights from all around Ech Cheid salt structure and evaluated using Rock-eval pyrolysis and GC/MS techniques in order to assess the degree of maturity evolution and the heat flux anomalies in the different zones analyze. The Total organic Carbon (TOC) values range between 1 to 9% and the (Tmax) ranges between 424 and 445°C, also the distribution of the source rock biomarkers both saturated and aromatic changes in a regular fashions with increasing maturity and this are shown in the chromatography results such as Ts/(Ts+Tm) ratios, 22S/(22S+22R) values for C31 homohopanes, ββ/(ββ+αα)20R and 20S/(20S+20R) ratios for C29 steranes which gives a consistent maturity indications and assessment of the field samples. These analyses are carried to interpret the maturity evolution and the heat flux around Ech Cheid salt structure through the geological history. These analyses also aim to demonstrate that the salt structure can have a direct effect on the geothermal gradient of the basin and on the maturity of the Bahloul Formation source rock. The organic matter has reached different stages of thermal maturity, but delineate a general increasing maturity trend. Our study confirms that the J. Ech Cheid salt body have on the first hand: a huge influence on the local distribution of anoxic depocentre at least within Cenomanian-Turonian time. In the second hand, the thermal anomaly near the salt mass has affected the maturity of Bahloul Formation.

Keywords: Bahloul formation, depocentre, GC/MS, rock-eval

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Authors: Alexander Heckman, Rohan Goswami, Zachi Attia, Paul Friedman, Peter Noseworthy, Demilade Adedinsewo, Pablo Moreno-Franco, Rickey Carter, Tathagat Narula

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Background: Postoperative atrial fibrillation (POAF) is associated with adverse health consequences, higher costs, and longer hospital stays. Utilizing existing predictive models that rely on clinical variables and circulating biomarkers, multiple societies have published recommendations on the treatment and prevention of POAF. Although reasonably practical, there is room for improvement and automation to help individualize treatment strategies and reduce associated complications. Methods and Results: In this retrospective cohort study of solid organ transplant recipients, we evaluated the diagnostic utility of a previously developed AI-based ECG prediction for silent AF on the development of POAF within 30 days of transplant. A total of 2261 non-cardiac transplant patients without a preexisting diagnosis of AF were found to have a 5.8% (133/2261) incidence of POAF. While there were no apparent sex differences in POAF incidence (5.8% males vs. 6.0% females, p=.80), there were differences by race and ethnicity (p<0.001 and 0.035, respectively). The incidence in white transplanted patients was 7.2% (117/1628), whereas the incidence in black patients was 1.4% (6/430). Lung transplant recipients had the highest incidence of postoperative AF (17.4%, 37/213), followed by liver (5.6%, 56/1002) and kidney (3.6%, 32/895) recipients. The AUROC in the sample was 0.62 (95% CI: 0.58-0.67). The relatively low discrimination may result from undiagnosed AF in the sample. In particular, 1,177 patients had at least 1 AI-ECG screen for AF pre-transplant above .10, a value slightly higher than the published threshold of 0.08. The incidence of POAF in the 1104 patients without an elevated prediction pre-transplant was lower (3.7% vs. 8.0%; p<0.001). While this supported the hypothesis that potentially undiagnosed AF may have contributed to the diagnosis of POAF, the utility of the existing AI-ECG screening algorithm remained modest. When the prediction for POAF was made using the first postoperative ECG in the sample without an elevated screen pre-transplant (n=1084 on account of n=20 missing postoperative ECG), the AUROC was 0.66 (95% CI: 0.57-0.75). While this discrimination is relatively low, at a threshold of 0.08, the AI-ECG algorithm had a 98% (95% CI: 97 – 99%) negative predictive value at a sensitivity of 66% (95% CI: 49-80%). Conclusions: This study's principal finding is that the incidence of POAF is rare, and a considerable fraction of the POAF cases may be latent and undiagnosed. The high negative predictive value of AI-ECG screening suggests utility for prioritizing monitoring and evaluation on transplant patients with a positive AI-ECG screening. Further development and refinement of a post-transplant-specific algorithm may be warranted further to enhance the diagnostic yield of the ECG-based screening.

Keywords: artificial intelligence, atrial fibrillation, cardiology, transplant, medicine, ECG, machine learning

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Authors: Danna Jia, Bin Li

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Background: Atopic dermatitis (AD) is a chronic and refractory inflammatory skin disease characterized by relapsing eczematous and pruritic skin lesions. The global prevalence of AD ranges from 1~ 20%, and its incidence rates are increasing. It affects individuals from infancy to adulthood, significantly impacting their daily lives and social activities. Despite its major health burden, the precise mechanisms underlying AD remain unknown. Understanding the genetic differences associated with AD is crucial for advancing diagnosis and targeted treatment development. This study aims to identify candidate genes of AD by using bioinformatics analysis. Methods: We conducted a comprehensive analysis of four pooled transcriptomic datasets (GSE16161, GSE32924, GSE130588, and GSE120721) obtained from the Gene Expression Omnibus (GEO) database. Differential gene expression analysis was performed using the R statistical language. The differentially expressed genes (DEGs) between AD patients and normal individuals were functionally analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Furthermore, a protein-protein interaction (PPI) network was constructed to identify candidate genes. Results: Among the patient-level gene expression datasets, we identified 114 shared DEGs, consisting of 53 upregulated genes and 61 downregulated genes. Functional analysis using GO and KEGG revealed that the DEGs were mainly associated with the negative regulation of transcription from RNA polymerase II promoter, membrane-related functions, protein binding, and the Human papillomavirus infection pathway. Through the PPI network analysis, we identified eight core genes: CD44, STAT1, HMMR, AURKA, MKI67, and SMARCA4. Conclusion: This study elucidates key genes associated with AD, providing potential targets for diagnosis and treatment. The identified genes have the potential to contribute to the understanding and management of AD. The bioinformatics analysis conducted in this study offers new insights and directions for further research on AD. Future studies can focus on validating the functional roles of these genes and exploring their therapeutic potential in AD. While these findings will require further verification as achieved with experiments involving in vivo and in vitro models, these results provided some initial insights into dysfunctional inflammatory and immune responses associated with AD. Such information offers the potential to develop novel therapeutic targets for use in preventing and treating AD.

Keywords: atopic dermatitis, bioinformatics, biomarkers, genes

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Authors: Ramin Mehdiabadi, Neda Menbari, Mohammad Nazir Menbari

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As a pressing public health concern, breast cancer stands as the predominant oncological diagnosis and principal cause of cancer-related mortality among women globally, accounting for 11.7% of new cancer incidences and 6.9% of cancer-related deaths. The annual figures indicate that approximately 230,480 women are diagnosed with breast cancer in the United States alone, with 39,520 succumbing to the disease. While developed economies have reported a deceleration in both incidence and mortality rates across various forms of cancer, including breast cancer, emerging and low-income economies manifest a contrary escalation, largely attributable to lifestyle-mediated risk factors such as tobacco usage, physical inactivity, and high caloric intake. Breast cancer is distinctly characterized by molecular heterogeneity, manifesting in specific subtypes delineated by biomarkers—Estrogen Receptors (ER), Progesterone Receptors (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). These subtypes, comprising Luminal A, Luminal B, HER2-enriched, triple-negative/basal-like, and normal-like, necessitate nuanced, subtype-specific therapeutic regimens, thereby challenging the applicability of generalized treatment protocols. Within this molecular complexity, the transcription factor Forkhead Box M1 (FoxM1) has garnered attention as a significant driver of cellular proliferation, tumorigenesis, metastatic progression, and treatment resistance in a spectrum of human malignancies, including breast cancer. Concurrently, microRNAs (miRs), specifically miR-216b-5p, have been identified as post-transcriptional gene expression regulators and potential tumor suppressors. The overarching objective of this academic investigation is to explicate the multifaceted interrelationship between FoxM1 and miR-216b-5p across the disparate molecular subtypes of breast cancer. Employing a methodologically rigorous, interdisciplinary research design that incorporates cutting-edge molecular biology techniques, sophisticated bioinformatics analytics, and exhaustive meta-analyses of extant clinical data, this scholarly endeavor aims to unveil novel biomarker-specific therapeutic pathways. By doing so, this research is positioned to make a seminal contribution to the advancement of personalized, efficacious, and minimally toxic treatment paradigms, thus profoundly impacting the global efforts to ameliorate the burden of breast cancer.

Keywords: breast cancer, fox m1, microRNAs, mir-216b-5p, gene expression

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Authors: Marisol Warthon-Medina, Jenny Plumb, Ayoub Aljawaldeh, Mark Roe, Ailsa Welch, Maria Glibetic, Paul M. Finglas

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Similar to Western Countries, the Eastern Mediterranean Region (EMR) also presents major public health issues associated with the increased consumption of sugar, fat, and salt. Therefore, one of the policies of the World Health Organization’s (WHO) EMR is to reduce the intake of salt, sugar, and fat (Saturated fatty acids, trans fatty acids) to address the risk of non-communicable diseases (i.e. diabetes, cardiovascular disease, cancer) and obesity. The project objective is to assess status and provide training and capacity development in the use of improved standardized methodologies for updated food composition data, dietary intake methods, use of suitable biomarkers of nutritional value and determine health outcomes in low and middle-income countries (LMIC). Training exchanges have been developed with clusters of countries created resulting from regional needs including Sudan, Egypt and Jordan; Tunisia, Morocco, and Mauritania; and other Middle Eastern countries. This capacity building will lead to the development and sustainability of up-to-date national and regional food composition databases in LMIC for use in dietary monitoring assessment in food and nutrient intakes. Workshops were organized to provide training and capacity development in the use of improved standardized methodologies for food composition and food intake. Training needs identified and short-term scientific missions organized for LMIC researchers including (1) training and knowledge exchange workshops, (2) short-term exchange of researchers, (3) development and application of protocols and (4) development of strategies to reduce sugar and fat intake. An initial training workshop, Morocco 2018 was attended by 25 participants from 10 EMR countries to review status and support development of regional food composition. 4 training exchanges are in progress. The use of improved standardized methodologies for food composition and dietary intake will produce robust measurements that will reinforce dietary monitoring and policy in LMIC. The capacity building from this project will lead to the development and sustainability of up-to-date national and regional food composition databases in EMR countries. Supported by the UK Medical Research Council, Global Challenges Research Fund, (MR/R019576/1), and the World Health Organization’s Eastern Mediterranean Region.

Keywords: dietary intake, food composition, low and middle-income countries, status.

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Authors: Elena Maria Scalisi, Roberta Pecoraro, Martina Contino, Sara Ignoto, Carmelo Iaria, Santi Concetto Pavone, Gino Sorbello, Loreto Di Donato, Maria Violetta Brundo

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5G Technology is evolving to satisfy a variety of service requirements that may allow high data-rate connections (1Gbps) and lower latency times than current (<1ms). In order to support a high data transmission speed and a high traffic service for eMBB (enhanced mobile broadband) use cases, 5G systems have the characteristic of using different frequency bands of the radio wave spectrum (700 MHz, 3.6-3.8 GHz and 26.5-27.5 GHz), thus taking advantage of higher frequencies than previous mobile radio generations (1G-4G). However, waves at higher frequencies have a lower capacity to propagate in free space and therefore, in order to guarantee the capillary coverage of the territory for high reliability applications, it will be necessary to install a large number of repeaters. Following the introduction of this new technology, there has been growing concern over the past few months about possible harmful effects on human health. The aim of this preliminary study is to evaluate possible short term effects induced by 5G-millimeter waves on embryonic development and early life stages of Danio rerio by Z-FET. We exposed developing zebrafish at frequency of 27 GHz, with a standard pyramidal horn antenna placed at 15 cm far from the samples holder ensuring an incident power density of 10 mW/cm2. During the exposure cycle, from 6 h post fertilization (hpf) to 96 hpf, we measured a different morphological endpoints every 24 hours. Zebrafish embryo toxicity test (Z-FET) is a short term test, carried out on fertilized eggs of zebrafish and it represents an effective alternative to acute test with adult fish (OECD, 2013). We have observed that 5G did not reveal significant impacts on mortality nor on morphology because exposed larvae showed a normal detachment of the tail, presence of heartbeat, well-organized somites, therefore hatching rate was lower than untreated larvae even at 48 h of exposure. Moreover, the immunohistochemical analysis performed on larvae showed a negativity to the HSP-70 expression used as a biomarkers. This is a preliminary study on evaluation of potential toxicity induced by 5G and it seems appropriate to underline the importance that further studies would take, aimed at clarifying the probable real risk of exposure to electromagnetic fields.

Keywords: Biomarker of exposure, embryonic development, 5G waves, zebrafish embryo toxicity test

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Authors: Iulianna Taritsa, Kuldeep Neote, Eric Fossel

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Lysosome-induced immunogenic cell death (LIICD) is a powerful mechanism of targeting cancer cells that kills circulating malignant cells and primes the host’s immune cells against future remission. Current immunotherapies for cancer are limited in preventing recurrence – a gap that can be bridged by training the immune system to recognize cancer neoantigens. Lysosomal leakage can be induced therapeutically to traffic antigens from dying cells to dendritic cells, which can later present those tumorigenic antigens to T cells. Previous research has shown that oxidative agents administered in the tumor microenvironment can initiate LIICD. We generated a fusion protein between an oxidative agent known as xanthine oxidase (XO) and a mini-antibody specific for EGFR/HER2-sensitive breast tumor cells. The anti-EGFR single domain antibody fragment is uniquely sourced from llama, which is functional without the presence of a light chain. These llama micro-antibodies have been shown to be better able to penetrate tissues and have improved physicochemical stability as compared to traditional monoclonal antibodies. We demonstrate that the fusion protein created is stable and can induce early markers of immunogenic cell death in an in vitro human breast cancer cell line (SkBr3). Specifically, we measured overall cell death, as well as surface-expressed calreticulin, extracellular ATP release, and HMGB1 production. These markers are consensus indicators of ICD. Flow cytometry, luminescence assays, and ELISA were used respectively to quantify biomarker levels between treated versus untreated cells. We also included a positive control group of SkBr3 cells dosed with doxorubicin (a known inducer of LIICD) and a negative control dosed with cisplatin (a known inducer of cell death, but not of the immunogenic variety). We looked at each marker at various time points after cancer cells were treated with the XO/antibody fusion protein, doxorubicin, and cisplatin. Upregulated biomarkers after treatment with the fusion protein indicate an immunogenic response. We thus show the potential for this fusion protein to induce an anticancer effect paired with an adaptive immune response against EGFR/HER2+ cells. Our research in human cell lines here provides evidence for the success of the same therapeutic method for patients and serves as the gateway to developing a new treatment approach against breast cancer.

Keywords: apoptosis, breast cancer, immunogenic cell death, lysosome

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Authors: B. Gonzalez-Yebra, H. Barajas, P. Palomares, M. Hernandez, O. Torres, M. Ayala, A. L. González, G. Vazquez-Ortiz, M. L. Guzman

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Leukemia arises by molecular alterations of the normal hematopoietic stem cell (HSC) transforming it into a leukemic stem cell (LSC) with high cell proliferation, self-renewal, and cell differentiation. Chronic myeloid leukemia (CML) originates from an LSC-leading to elevated proliferation of myeloid cells and acute lymphoblastic leukemia (ALL) originates from an LSC development leading to elevated proliferation of lymphoid cells. In both cases, LSC can be identified by multicolor flow cytometry using several antibodies. However, to date, LSC levels in peripheral blood (PB) are not established well enough in ALL and CML patients. On the other hand, the detection of the minimal residue disease (MRD) in leukemia is mainly based on the identification of the mRNA bcr-abl gene in CML patients and some other genes in ALL patients. There is no a properly biomarker to detect MDR in both types of leukemia. The objective of this study was to determine mRNA bcr-abl and the percentage of LSC in peripheral blood of patients with CML and ALL and identify a possible association between the amount of LSC in PB and clinical data. We included in this study 19 patients with Leukemia. A PB sample was collected per patient and leukocytes were obtained by Ficoll gradient. The immunophenotype for LSC CD34+ was done by flow cytometry analysis with CD33, CD2, CD14, CD16, CD64, HLA-DR, CD13, CD15, CD19, CD10, CD20, CD34, CD38, CD71, CD90, CD117, CD123 monoclonal antibodies. In addition, to identify the presence of the mRNA bcr-abl by RT-PCR, the RNA was isolated using TRIZOL reagent. Molecular (presence of mRNA bcr-abl and LSC CD34+) and clinical results were analyzed with descriptive statistics and a multiple regression analysis was performed to determine statistically significant association. In total, 19 patients (8 patients with ALL and 11 patients with CML) were analyzed, 9 patients with de novo leukemia (ALL = 6 and CML = 3) and 10 under treatment (ALL = 5 and CML = 5). The overall frequency of mRNA bcr-abl was 31% (6/19), and it was negative in ALL patients and positive in 80% in CML patients. On the other hand, LSC was determined in 16/19 leukemia patients (%LSC= 0.02-17.3). The Novo patients had higher percentage of LSC (0.26 to 17.3%) than patients under treatment (0 to 5.93%). The amount of LSC was significantly associated with the amount of LSC were: absence of treatment, the absence of splenomegaly, and a lower number of leukocytes, negative association for the clinical variables age, sex, blasts, and mRNA bcr-abl. In conclusion, patients with de novo leukemia had a higher percentage of circulating LSC than patients under treatment, and it was associated with clinical parameters as lack of treatment, absence of splenomegaly and a lower number of leukocytes. The mRNA bcr-abl detection was only possible in the series of patients with CML, and molecular detection of LSC could be identified in the peripheral blood of all leukemia patients, we believe the identification of circulating LSC may be used as biomarker for the detection of the MRD in leukemia patients.

Keywords: stem cells, leukemia, biomarkers, flow cytometry

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Authors: C. Miranda, S. Cunha, R. Soares, M. Maia, G. Igrejas, F. Silva, P. Poeta

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The worldwide inappropriate use of antibiotics has increased the emergence of antimicrobial-resistant microorganisms isolated from animals, humans, food, and the environment. To combat this complex and multifaceted problem is essential to know the prevalence in livestock animals and possible ways of transmission among animals and between these and humans. Enterococci species, in particular E. faecalis and E. faecium, are the most common nosocomial bacteria, causing infections in animals and humans. Thus, the aim of this study was to characterize resistance and virulence factors genes among two enterococci species isolated from calf colostrums in Portuguese dairy farms. The 55 enterococci isolates (44 E. faecalis and 11 E. faecium) were tested for the presence of the resistance genes for the following antibiotics: erythromicyn (ermA, ermB, and ermC), tetracycline (tetL, tetM, tetK, and tetO), quinupristin/dalfopristin (vatD and vatE) and vancomycin (vanB). Of which, 25 isolates (15 E. faecalis and 10 E. faecium) were tested until now for 8 virulence factors genes (esp, ace, gelE, agg, cpd, cylA, cylB, and cylLL). The resistance and virulence genes were performed by PCR, using specific primers and conditions. Negative and positive controls were used in all PCR assays. All enterococci isolates showed resistance to erythromicyn and tetracycline through the presence of the genes: ermB (n=29, 53%), ermC (n=10, 18%), tetL (n=49, 89%), tetM (n=39, 71%) and tetK (n=33, 60%). Only two (4%) E. faecalis isolates showed the presence of tetO gene. No resistance genes for vancomycin were found. The virulence genes detected in both species were cpd (n=17, 68%), agg (n=16, 64%), ace (n=15, 60%), esp (n=13, 52%), gelE (n=13, 52%) and cylLL (n=8, 32%). In general, each isolate showed at least three virulence genes. In three E. faecalis isolates was not found virulence genes and only E. faecalis isolates showed virulence genes for cylA (n=4, 16%) and cylB (n=6, 24%). In conclusion, these colostrum samples that were consumed by calves demonstrated the presence of antibiotic-resistant enterococci harbored virulence genes. This genotypic characterization is crucial to control the antibiotic-resistant bacteria through the implementation of restricts measures safeguarding public health. Acknowledgements: This work was funded by the R&D Project CAREBIO2 (Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics - towards innovative theragnostic biomarkers), with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

Keywords: antimicrobial resistance, calf, colostrums, enterococci

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Authors: Mateusz Bilski, Jakub Klas, Emilia Kowalczyk, Sylwia Koziej, Katarzyna Kulszo, Ludmiła Grzybowska- Szatkowska

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Background: Liver metastases are a common complication of primary solid tumors and sig-nificantly reduce patient survival. In the era of increasing diagnosis of oligometastatic disease and oligoprogression, methods of local treatment of metastases, i.e. MDT, are becoming more important. Implementation of such treatment can be considered for liver metastases, which are a common complication of primary solid tumors and significantly reduce patient survival. To date, the mainstay of treatment for oligometastatic disease has been surgical resection, but not all patients qualify for the procedure. As an alternative to surgical resection, radiotherapy techniques have become available, including stereotactic body radiation therapy (SBRT) or high-dose interstitial brachytherapy (iBT). iBT is an invasive method that emits very high doses of radiation from the inside of the tumor to the outside. This technique provides better tumor coverage than SBRT while having little impact on surrounding healthy tissue and elim-inates some concerns involving respiratory motion. Methods: We conducted a systematic re-view of the scientific literature on the use of brachytherapy in the treatment of liver metasta-ses from 2018 - 2023 using PubMed and ResearchGate browsers according to PRISMA rules. Results: From 111 articles, 18 publications containing information on 729 patients with liver metastases were selected. iBT has been shown to provide high rates of tumor control. Among 14 patients with 54 unresectable RCC liver metastases, after iBT LTC was 92.6% during a median follow-up of 10.2 months, PFS was 3.4 months. In analysis of 167 patients after treatment with a single fractional dose of 15-25 Gy with brachytherapy at 6- and 12-month follow-up, LRFS rates of 88,4-88.7% and 70.7 - 71,5%, PFS of 78.1 and 53.8%, and OS of 92.3 - 96.7% and 76,3% - 79.6%, respectively, were achieved. No serious complications were observed in all patients. Distant intrahepatic progression occurred later in patients with unre-sectable liver metastases after brachytherapy (PFS: 19.80 months) than in HCC patients (PFS: 13.50 months). A significant difference in LRFS between CRC patients (84.1% vs. 50.6%) and other histologies (92.4% vs. 92.4%) was noted, suggesting a higher treatment dose is necessary for CRC patients. The average target dose for metastatic colorectal cancer was 40 - 60 Gy (compared to 100 - 250 Gy for HCC). To better assess sensitivity to therapy and pre-dict side effects, it has been suggested that humoral mediators be evaluated. It was also shown that baseline levels of TNF-α, MCP-1 and VEGF, as well as NGF and CX3CL corre-lated with both tumor volume and radiation-induced liver damage, one of the most serious complications of iBT, indicating their potential role as biomarkers of therapy outcome. Con-clusions: The use of brachytherapy methods in the treatment of liver metastases of various cancers appears to be an interesting and relatively safe therapeutic method alternative to sur-gery. An important challenge remains the selection of an appropriate brachytherapy method and radiation dose for the corresponding initial tumor type from which the metastasis origi-nated.

Keywords: liver metastases, brachytherapy, CT-HDRBT, iBT

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Authors: Sofia Papadimitriou

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INTRODUCTION: The differential diagnosis between acute viral and bacterial infections is an important cost-effectiveness parameter at the stage of the treatment process in order to achieve the maximum benefits in therapeutic intervention by combining the minimum cost to ensure the proper use of antibiotics.The discovery of sensitive and robust molecular diagnostic tests in response to the role of the host in infections has enhanced the accurate diagnosis and differentiation of infections. METHOD: The study used a sample of six independent blood samples (total=756) which are associated with human proteins-proteins, each of which at the transcription stage expresses a different response in the host network between viral and bacterial infections.Τhe individual blood samples are subjected to a sequence of computer filters that identify a gene panel corresponding to an autonomous diagnostic score. The data set and the correspondence of the gene panel to the diagnostic patents a new Bangalore -Viral Bacterial (BL-VB). FINDING: We use a biomarker based on the blood of 10 genes(Panel-VB) that are an important prognostic value for the detection of viruses from bacterial infections with a weighted average AUROC of 0.97(95% CL:0.96-0.99) in eleven independent samples (sets n=898). We discovered a base with a patient score (VB 10 ) according to the table, which is a significant diagnostic value with a weighted average of AUROC 0.94(95% CL: 0.91-0.98) in 2996 patient samples from 56 public sets of data from 19 different countries. We also studied VB 10 in a new cohort of South India (BL-VB,n=56) and found 97% accuracy in confirmed cases of viral and bacterial infections. We found that VB 10 (a)accurately identifies the type of infection even in unspecified cases negative to the culture (b) shows its clinical condition recovery and (c) applies to all age groups, covering a wide range of acute bacterial and viral infectious, including non-specific pathogens. We applied our VB 10 rating to publicly available COVID 19 data and found that our rating diagnosed viral infection in patient samples. RESULTS: Τhe results of the study showed the diagnostic power of the biomarker VB 10 as a diagnostic test for the accurate diagnosis of acute infections in recovery conditions. We look forward to helping you make clinical decisions about prescribing antibiotics and integrating them into your policies management of antibiotic stewardship efforts. CONCLUSIONS: Overall, we are developing a new property of the RNA-based biomarker and a new blood test to differentiate between viral and bacterial infections to assist a physician in designing the optimal treatment regimen to contribute to the proper use of antibiotics and reduce the burden on antimicrobial resistance, AMR.

Keywords: acute infections, antimicrobial resistance, biomarker, blood transcriptome, systems biology, classifier diagnostic score

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Authors: G. C. Santini, C. Potrich, L. Lunelli, L. Vanzetti, S. Marasso, M. Cocuzza, C. Pederzolli

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The relevance of circulating miRNAs as non-invasive biomarkers for several pathologies is nowadays undoubtedly clear, as they have been found to have both diagnostic and prognostic value able to add fundamental information to patients’ clinical picture. The availability of these data, however, relies on a time-consuming process spanning from the sample collection and processing to the data analysis. In light of this, strategies which are able to ease this procedure are in high demand and considerable effort have been made in developing Lab-on-a-chip (LOC) devices able to speed up and standardise the bench work. In this context, a very promising polydimethylsiloxane (PDMS)-based microdevice which integrates the processing of the biological sample, i.e. purification of extracellular miRNAs, and reverse transcription was previously developed in our lab. In this study, we aimed at the improvement of the miRNA extraction performances of this micro device by increasing the ability of its surface to absorb extracellular miRNAs from biological samples. For this purpose, we focused on the modulation of two properties of the material: roughness and charge. PDMS surface roughness was modulated by casting with several templates (terminated with silicon oxide coated by a thin anti-adhesion aluminum layer), followed by a panel of curing conditions. Atomic force microscopy (AFM) was employed to estimate changes at the nanometric scale. To introduce modifications in surface charge we functionalized PDMS with different mixes of positively charged 3-aminopropyltrimethoxysilanes (APTMS) and neutral poly(ethylene glycol) silane (PEG). The surface chemical composition was characterized by X-ray photoelectron spectroscopy (XPS) and the number of exposed primary amines was quantified with the reagent sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (s-SDTB). As our final end point, the adsorption rate of all these different conditions was assessed by fluorescence microscopy by incubating a synthetic fluorescently-labeled miRNA. Our preliminary analysis identified casting on thermally grown silicon oxide, followed by a curing step at 85°C for 1 hour, as the most efficient technique to obtain a PDMS surface roughness in the nanometric scaleable to trap miRNA. In addition, functionalisation with 0.1% APTMS and 0.9% PEG was found to be a necessary step to significantly increase the amount of microRNA adsorbed on the surface, therefore, available for further steps as on-chip reverse transcription. These findings show a substantial improvement in the extraction efficiency of our PDMS microdevice, ultimately leading to an important step forward in the development of an innovative, easy-to-use and integrated system for the direct purification of less abundant circulating microRNAs.

Keywords: circulating miRNAs, diagnostics, Lab-on-a-chip, polydimethylsiloxane (PDMS)

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Authors: Seema Dhariwal, Kiran Maan, Ruchi Baghel, Apoorva Sharma, Poonam Rana

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Introduction: Traumatic brain injury (TBI) is defined as the temporary or permanent alteration in brain function and pathology caused by an external mechanical force. It represents the leading cause of mortality and morbidity among children and youth individuals. Various models of TBI in rodents have been developed in the laboratory to mimic the scenario of injury. Blast overpressure injury is common among civilians and military personnel, followed by accidents or explosive devices. In addition to this, the lateral Controlled cortical impact (CCI) model mimics the blunt, penetrating injury. Method: In the present study, we have developed two different mild TBI models using blast and CCI injury. In the blast model, helium gas was used to create an overpressure of 130 kPa (±5) via a shock tube, and CCI injury was induced with an impact depth of 1.5mm to create diffusive and focal injury, respectively. C57BL/6J male mice (10-12 weeks) were divided into three groups: (1) control, (2) Blast treated, (3) CCI treated, and were exposed to different injury models. Serum was collected on Day1 and day7, followed by biphasic extraction using MTBE/Methanol/Water. Prepared samples were separated on Charged Surface Hybrid (CSH) C18 column and acquired on UHPLC-Q-TOF-MS using ESI probe with inhouse optimized parameters and method. MS peak list was generated using Markerview TM. Data were normalized, Pareto-scaled, and log-transformed, followed by multivariate and univariate analysis in metaboanalyst. Result and discussion: Untargeted profiling of lipids generated extensive data features, which were annotated through LIPID MAPS® based on their m/z and were further confirmed based on their fragment pattern by LipidBlast. There is the final annotation of 269 features in the positive and 182 features in the negative mode of ionization. PCA and PLS-DA score plots showed clear segregation of injury groups to controls. Among various lipids in mild blast and CCI, five lipids (Glycerophospholipids {PC 30:2, PE O-33:3, PG 28:3;O3 and PS 36:1 } and fatty acyl { FA 21:3;O2}) were significantly altered in both injury groups at Day 1 and Day 7, and also had VIP score >1. Pathway analysis by Biopan has also shown hampered synthesis of Glycerolipids and Glycerophospholipiods, which coincides with earlier reports. It could be a direct result of alteration in the Acetylcholine signaling pathway in response to TBI. Understanding the role of a specific class of lipid metabolism, regulation and transport could be beneficial to TBI research since it could provide new targets and determine the best therapeutic intervention. This study demonstrates the potential lipid biomarkers which can be used for injury severity diagnosis and identification irrespective of injury type (diffusive or focal).

Keywords: LipidBlast, lipidomic biomarker, LIPID MAPS®, TBI

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Authors: Drago Bratkovic, Curtis Gravance, David Ketteridge, Ravi Krishnan, Michael Imperiale

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The mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases that have a common defect in the catabolism of glycosaminoglycans (GAGs). MPS I is the most common of the MPS diseases. Manifestations of MPS I include coarsening of facial features, corneal clouding, developmental delay, short stature, skeletal manifestations, hearing loss, cardiac valve disease, hepatosplenomegaly, and umbilical and inguinal hernias. Treatments for MPS I restore or activate the missing or deficient enzyme in the case of enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT). Pentosan polysulfate sodium (PPS) is a potential treatment to improve bone and joint manifestations of MPS I. The mechanisms of action of PPS that are relevant to the treatment of MPS I are the ability to: (i) Reduce systemic and accumulated GAG, (ii) Reduce inflammatory effects via the inhibition of NF-kB, resulting in the reduction in pro-inflammatory mediators. (iii) Reduce the expression of the pain mediator nerve growth factor in osteocytes from degenerating joints. (iv) Inhibit the cartilage degrading enzymes related to joint dysfunction in MPS I. PPS is being evaluated as an adjunctive therapy to ERT and/or HSCT in an open-label, single-centre, phase 2 study. Patients are ≥ 5 years of age with a diagnosis of MPS I and previously received HSCT and/or ERT. Three white, female, patients with MPS I-Hurler, ages 14, 15, and 19 years, and one, white male patient aged 15 years are enrolled. All were diagnosed at ≤2 years of age. All patients received HSCT ≤ 6 months after diagnosis. Two of the patients were treated with ERT prior to HSCT, and 1 patient received ERT commencing 3 months prior to HSCT. Two patients received 0.75mg/kg and 2 patients received 1.5mg/kg of PPS. PPS was well tolerated at doses of 0.75 and 1.5 mg/kg to 47 weeks of continuous dosing. Of the 19 adverse events (AEs), 2 were related to PPS. One AE was moderate (pre-syncope) and 1 was mild (injection site bruising), experienced in the same patient. All AEs were reported as mild or moderate. There have been no SAEs. One subject experienced a COVID-19 infection and PPS was interrupted. The MPS I signature GAG fragments, sulfated disaccharide and UA-HNAc S, tended to decrease in 3 patients from baseline through Week 25. Week 25 GAG data are pending for the 4th patient. Overall, most biomarkers (inflammatory, cartilage degeneration, and bone turnover) evaluated in the 3 patients with 25-week assessments have indicated either no change or a reduction in levels compared to baseline. In 3 patients, there was a trend toward improvement in the 2MWT from baseline to Week 48 with > 100% increase in 1 patient (01-201). In the 3 patients that had Week 48 assessments, patients and proxies reported improvement in PGIC, including “worthwhile difference” (n=1), or “made all the difference” (n=2).

Keywords: MPS I, pentosan polysulfate sodium, clinical study, 2MWT, QoL

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Authors: Ioannis Panagiotopoulos, Aristidis Anastasakis, Konstantinos Toutouzas, Ioannis Iakovou, Charalampos Vlachopoulos, Vasilis Voudris, Georgios Tziomalos, Konstantinos Tsioufis, Efstathios Kastritis, Alexandros Briassoulis, Kimon Stamatelopoulos, Alexios Antonopoulos, Paraskevi Exadaktylou, Evanthia Giannoula, Anastasia Katinioti, Maria Kalantzi, Evangelos Leontiadis, Eftychia Smparouni, Ioannis Malakos, Nikolaos Aravanis, Argyrios Doumas, Maria Koutelou

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Introduction: Cardiac amyloidosis from Transthyretin (ATTR-CA) is an infiltrative disease characterized by the deposition of pathological transthyretin complexes in the myocardium. This study describes the characteristics of patients diagnosed with ATTR-CA from 2019 until present at the Nuclear Medicine Department of Onassis Cardiac Surgery Center and AHEPA Hospital. These centers have extensive experience in amyloidosis and modern technological equipment for its diagnosis. Materials and Methods: Records of consecutive patients (N=73) diagnosed with any type of amyloidosis were collected, analyzed, and prospectively followed. The diagnosis of amyloidosis was made using specific myocardial scintigraphy with Tc-99m DPD. Demographic characteristics, including age, gender, marital status, height, and weight, were collected in a database. Clinical characteristics, such as amyloidosis type (ATTR and AL), serum biomarkers (BNP, troponin), electrocardiographic findings, ultrasound findings, NYHA class, aortic valve replacement, device implants, and medication history, were also collected. Some of the most significant results are presented. Results: A total of 73 cases (86% male) were diagnosed with amyloidosis over four years. The mean age at diagnosis was 82 years, and the main symptom was dyspnea. Most patients suffered from ATTR-CA (65 vs. 8 with AL). Out of all the ATTR-CA patients, 61 were diagnosed with wild-type and 2 with two rare mutations. Twenty-eight patients had systemic amyloidosis with extracardiac involvement, and 32 patients had a history of bilateral carpal tunnel syndrome. Four patients had already developed polyneuropathy, and the diagnosis was confirmed by DPD scintigraphy, which is known for its high sensitivity. Among patients with isolated cardiac involvement, only 6 had left ventricular ejection fraction below 40%. The majority of ATTR patients underwent tafamidis treatment immediately after diagnosis. Conclusion: In conclusion, the experiences shared by the two centers and the continuous exchange of information provide valuable insights into the diagnosis and management of cardiac amyloidosis. Clinical suspicion of amyloidosis and early diagnostic approach are crucial, given the availability of non-invasive techniques. Cardiac scintigraphy with DPD can confirm the presence of the disease without the need for a biopsy. The ultimate goal still remains continuous education and awareness of clinical cardiologists so that this systemic and treatable disease can be diagnosed and certified promptly and treatment can begin as soon as possible.

Keywords: amyloidosis, diagnosis, myocardial scintigraphy, Tc-99m DPD, transthyretin

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Authors: Mustafa M. Donma, Orkide Donma

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Zinc is a vital element required for growth and development. This fact makes zinc important, particularly for children. It maintains normal cellular structure and functions. This essential element appears to have protective effects against coronary artery disease and cardiomyopathy. Higher serum zinc levels are associated with lower risk of cardiovascular diseases (CVDs). There is a significant association between low serum zinc levels and heart failure. Zinc may be a potential biomarker of cardiovascular health. High sensitive cardiac troponin T (hs-cTnT) and cardiac myosin binding protein C (cMyBP-C) are new generation markers used for prediagnosis, diagnosis, and prognosis of CVDs. The aim of this study is to determine zinc as well as new generation cardiac markers profiles in children with normal body mass index (N-BMI), obese (OB), morbid obese (MO) children, and children with metabolic syndrome (MetS) findings. The association among them will also be investigated. Four study groups were constituted. The study protocol was approved by the institutional Ethics Committee of Tekirdag Namik Kemal University. Parents of the participants filled informed consent forms to participate in the study. Group 1 is composed of 44 children with N-BMI. Group 2 and Group 3 comprised 43 OB and 45 MO children, respectively. Forty-five MO children with MetS findings were included in Group 4. World Health Organization age- and sex-adjusted BMI percentile tables were used to constitute groups. These values were 15-85, 95-99, and above 99 for N-BMI, OB, and MO, respectively. Criteria for MetS findings were determined. Routine biochemical analyses, including zinc, were performed. High sensitive-cTnT and cMyBP-C concentrations were measured by kits based on enzyme-linked immunosorbent assay principle. Appropriate statistical tests within the scope of SPSS were used for the evaluation of the study data. p<0.05 was accepted as statistically significant. Four groups were matched for age and gender. Decreased zinc concentrations were measured in Groups 2, 3, and 4 compared to Group 1. Groups did not differ from one another in terms of hs-cTnT. There were statistically significant differences between cMyBP-C levels of MetS group and N-BMI as well as OB groups. There was an increasing trend going from N-BMI group to MetS group. There were statistically significant negative correlations between zinc and hs-cTnT as well as cMyBP-C concentrations in MetS group. In conclusion, inverse correlations detected between zinc and new generation cardiac markers (hs-TnT and cMyBP-C) have pointed out that decreased levels of this physiologically essential trace element accompany increased levels of hs-cTnT as well as cMyBP-C in children with MetS. This finding emphasizes that both zinc and these new generation cardiac markers may be evaluated as biomarkers of cardiovascular health during severe childhood obesity precipitated with MetS findings and also suggested as the messengers of the future risk in the adulthood periods of children with MetS.

Keywords: cardiac myosin binding protein-C, cardiovascular diseases, children, high sensitive cardiac troponin T, obesity

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Authors: Yu-Chi Liao, Nai-Wen Guo, Chun‑Hung Lee, Yung-Chin Lu, Cheng-Hung Ko

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Objective: The incidence of behavioral addictions, especially substance use disorders (SUDs), is gradually be taken seriously with various physical health problems. Mindfulness-based relapse prevention (MBRP) is a treatment option for promoting long-term health behavior change in recent years. MBRP is a structured protocol that integrates formal meditation practices with the cognitive-behavioral approach of relapse prevention treatment by teaching participants not to engage in reappraisal or savoring techniques. However, considering SUDs as a complex brain disease, questionnaires and symptom evaluation are not sufficient to evaluate the effect of MBRP. Neurophysiological biomarkers such as quantitative electroencephalogram (QEEG) may improve accurately represent the curative effects. This study attempted to find out the neurophysiological indicator of MBRP in various severity SUD involuntary clients. Participants and Methods: Thirteen participants (all males) completed 8-week mindfulness-based treatment provided by trained, licensed clinical psychologists. The behavioral data were from the Severity of Dependence Scale (SDS) and Negative Mood Regulation Scale (NMR) before and afterMBRP treatment. The QEEG data were simultaneously recorded with executive attention tasks, called comprehensive nonverbal attention test(CNAT). The two-way repeated-measures (treatment * severity) ANOVA and independent t-test were used for statistical analysis. Results: Thirteen participants regrouped into high substance dependence (HS) and low substance dependence (LS) by SDS cut-off. The HS group showed more SDS total score and lower gamma wave in the Go/No Go task of CNAT at pretest. Both groups showed the main effect that they had a lower frontal theta/beta ratio (TBR) during the simple reaction time task of CNAT. The main effect showed that the delay errors of CNAT were lower after MBRP. There was no other difference in CNAT between groups. However, after MBRP, compared to LS, the HS group have resonant progress in improving SDS and NMR scores. The neurophysiological index, the frontal TBR of the HS during the Go/No Go task of CNATdecreased than that of the LS group. Otherwise, the LS group’s gamma wave was a significant reduction on the Go/No Go task of CNAT. Conclusion: The QEEG data supports the MBRP can restore the prefrontal function of involuntary addicts and lower their errors in executive attention tasks. However, the improvement of MBRPfor the addict with high addiction severity is significantly more than that with low severity, including QEEG’s indicators and negative emotion regulation. Future directions include investigating the reasons for differences in efficacy among different severity of the addiction.

Keywords: mindfulness, involuntary clients, QEEG, emotion regulation

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Authors: Angelis P. Barlampas

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Learning Objectives: The purpose of this study is to inform briefly the reader about the applications of AI in chest radiology. Background: Currently, there are 190 FDA-approved radiology AI applications, with 42 (22%) pertaining specifically to thoracic radiology. Imaging findings OR Procedure details Aids of AI in chest radiology1: Detects and segments pulmonary nodules. Subtracts bone to provide an unobstructed view of the underlying lung parenchyma and provides further information on nodule characteristics, such as nodule location, nodule two-dimensional size or three dimensional (3D) volume, change in nodule size over time, attenuation data (i.e., mean, minimum, and/or maximum Hounsfield units [HU]), morphological assessments, or combinations of the above. Reclassifies indeterminate pulmonary nodules into low or high risk with higher accuracy than conventional risk models. Detects pleural effusion . Differentiates tension pneumothorax from nontension pneumothorax. Detects cardiomegaly, calcification, consolidation, mediastinal widening, atelectasis, fibrosis and pneumoperitoneum. Localises automatically vertebrae segments, labels ribs and detects rib fractures. Measures the distance from the tube tip to the carina and localizes both endotracheal tubes and central vascular lines. Detects consolidation and progression of parenchymal diseases such as pulmonary fibrosis or chronic obstructive pulmonary disease (COPD).Can evaluate lobar volumes. Identifies and labels pulmonary bronchi and vasculature and quantifies air-trapping. Offers emphysema evaluation. Provides functional respiratory imaging, whereby high-resolution CT images are post-processed to quantify airflow by lung region and may be used to quantify key biomarkers such as airway resistance, air-trapping, ventilation mapping, lung and lobar volume, and blood vessel and airway volume. Assesses the lung parenchyma by way of density evaluation. Provides percentages of tissues within defined attenuation (HU) ranges besides furnishing automated lung segmentation and lung volume information. Improves image quality for noisy images with built-in denoising function. Detects emphysema, a common condition seen in patients with history of smoking and hyperdense or opacified regions, thereby aiding in the diagnosis of certain pathologies, such as COVID-19 pneumonia. It aids in cardiac segmentation and calcium detection, aorta segmentation and diameter measurements, and vertebral body segmentation and density measurements. Conclusion: The future is yet to come, but AI already is a helpful tool for the daily practice in radiology. It is assumed, that the continuing progression of the computerized systems and the improvements in software algorithms , will redder AI into the second hand of the radiologist.

Keywords: artificial intelligence, chest imaging, nodule detection, automated diagnoses

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Authors: Sundru Manjulata Devi, Prakash M. Halami

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The immemorial use of fermented foods from vegetables, dairy and other biological sources are of great demand in India because of their health benefits. However, the diversity of Lactobacillus plantarum group (LPG) of vegetable origin has not been revealed yet, particularly with reference to their probiotic functionalities. In the present study, the different species of probiotic Lactobacillus plantarum group (LPG) i.e., L. plantarum subsp. plantarum MTCC 5422 (from fermented cereals), L. plantarum subsp. argentoratensis FG16 (from fermented bamboo shoot) and L. paraplantarum MTCC 9483 (from fermented gundruk) (as characterized by multiplex recA PCR assay) were considered to investigate their relative expression of MUB domains of mub gene (mucin binding protein) by Real time PCR. Initially, the allelic variation in the mub gene was assessed and found to encode three different variants (Type I, II and III). All the three types had 8, 9 and 10 MUB domains respectively (as analysed by Pfam database) and were found to be responsible for adhesion of bacteria to the host intestinal epithelial cells. These domains either get inserted or deleted during speciation or evolutionary events and lead to divergence. The reverse transcriptase qPCR analysis with mubLPF1+R1 primer pair supported variation in amplicon sizes with 300, 500 and 700 bp among different LPG strains. The relative expression of these MUB domains significantly unregulated in the presence of 1% mucin in overnight grown cultures. Simultaneously, the mub gene expressed efficiently by 7 fold in the culture L. paraplantarum MTCC 9483 with 10 MUB domains. An increase in the expression levels for L. plantarum subsp. plantarum MTCC 5422 and L. plantarum subsp. argentoratensis FG16 (MCC 2974) with 9 and 8 repetitive domains was around 4 and 2 fold, respectively. The detection and expression of an integrase (int) gene in the upstream region of mub gene reveals the excision and integration of these repetitive domains. Concurrently, an in vitro adhesion assay to mucin and exclusion of pathogens (such as Listeria monocytogenes and Micrococcus leuteus) was investigated and observed that the L. paraplantarum MTCC 9483 with more adhesion domains has more ability to adhere to mucin and inhibited the growth of pathogens. The production and expression of plantaricin-like bacteriocin (plnNC8 type) in MTCC 9483 suggests the pathogen inhibition. Hence, the expression of MUB domains can act as potential biomarkers in the screening of a novel probiotic LPG strain with adherence property. The present study provides a platform for an easy, rapid, less time consuming, low-cost methodology for the detection of potential probiotic bacteria. It was known that the traditional practices followed in the preparation of fermented bamboo shoots/gundruk/cereals of Indian foods contain different kinds of neutraceuticals for functional food and novel compounds with health promoting factors. In future, a detailed study of these food products can add more nutritive value, consumption and suitable for commercialization.

Keywords: adhesion gene, fermented foods, MUB domains, probiotics

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Authors: Alexandra Sargent, Sarah Ferris, Ioannis Theofanous

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The Abbott RealTime High Risk HPV test (RealTime HPV) is one of five assays clinically validated and approved by the English NHS Cervical Screening Programme (CSP) for HPV triage of low grade dyskaryosis and test-of-cure of treated Cervical Intraepithelial Neoplasia. The assay is a highly automated multiplex real-time PCR test for detecting 14 high risk (hr) HPV types, with simultaneous differentiation of HPV 16 and HPV 18 versus non-HPV 16/18 hrHPV. An endogenous internal control ensures sample cellularity, controls extraction efficiency and PCR inhibition. The original cervical specimen collected in SurePath (SP) liquid-based cytology (LBC) medium (BD Diagnostics) and the SP post-gradient cell pellets (SPG) after cytological processing are both CE marked for testing with the RealTime HPV test. During the 2011 NHSCSP validation of new tests only the original aliquot of SP LBC medium was investigated. Residual sample volume left after cytology slide preparation is low and may not always have sufficient volume for repeat HPV testing or for testing of other biomarkers that may be implemented in testing algorithms in the future. The SPG samples, however, have sufficient volumes to carry out additional testing and necessary laboratory validation procedures. This study investigates the correlation of RealTime HPV results of cervical specimens collected in SP LBC medium from women with low grade cytological abnormalities observed with matched pairs of original SP LBC medium and SP post-gradient cell pellets (SPG) after cytology processing. Matched pairs of SP and SPG samples from 750 women with borderline (N = 392) and mild (N = 351) cytology were available for this study. Both specimen types were processed and parallel tested for the presence of hrHPV with RealTime HPV according to the manufacturer´s instructions. HrHPV detection rates and concordance between test results from matched SP and SPGCP pairs were calculated. A total of 743 matched pairs with valid test results on both sample types were available for analysis. An overall-agreement of hrHPV test results of 97.5% (k: 0.95) was found with matched SP/SPG pairs and slightly lower concordance (96.9%; k: 0.94) was observed on 392 pairs from women with borderline cytology compared to 351 pairs from women with mild cytology (98.0%; k: 0.95). Partial typing results were highly concordant in matched SP/SPG pairs for HPV 16 (99.1%), HPV 18 (99.7%) and non-HPV16/18 hrHPV (97.0%), respectively. 19 matched pairs were found with discrepant results: 9 from women with borderline cytology and 4 from women with mild cytology were negative on SPG and positive on SP; 3 from women with borderline cytology and 3 from women with mild cytology were negative on SP and positive on SPG. Excellent correlation of hrHPV DNA test results was found between matched pairs of SP original fluid and post-gradient cell pellets from women with low grade cytological abnormalities tested with the Abbott RealTime High-Risk HPV assay, demonstrating robust performance of the test with both specimen types and reassuring the utility of the assay for cytology triage with both specimen types.

Keywords: Abbott realtime test, HPV, SurePath liquid based cytology, surepath post-gradient cell pellet

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Authors: Tristan R. Fraser, Christopher D. Steadman, Christopher J. Boos

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Myopericarditis is an inflammation syndrome characterised by clinical diagnostic criteria for pericarditis, such as chest pain, combined with evidence of myocardial involvement, such as elevation of biomarkers of myocardial damage, e.g., troponins. It can rarely be a complication of therapeutics used for dysregulated immune-mediated diseases such as inflammatory bowel disease (IBD), for example, mesalazine. The infrequency of mesalazine-induced myopericarditis adds to the challenge in its recognition. Rapid diagnosis and the early introduction of treatment are crucial. This case report follows a 24-year-old professional footballer with a past medical history of ulcerative colitis, recently started on mesalazine for disease control. Three weeks after mesalazine was initiated, he was admitted with fever, shortness of breath, and chest pain worse whilst supine and on deep inspiration, as well as elevated venous blood cardiac troponin T level (cTnT, 288ng/L; normal: <13ng/L). Myocarditis was confirmed on initial inpatient cardiac MRI, revealing the presence of florid myocarditis with preserved left ventricular systolic function and an ejection fraction of 67%. This was a longitudinal case study following the progress of a single individual with myopericarditis over four acute hospital admissions over nine weeks, with admissions ranging from two to five days. Parameters examined included clinical signs and symptoms, serum troponin, transthoracic echocardiogram, and cardiac MRI. Serial measurements of cardiac function, including cardiac MRI and transthoracic echocardiogram, showed progressive deterioration of cardiac function whilst mesalazine was continued. Prior to cessation of mesalazine, transthoracic echocardiography revealed a small global pericardial effusion of < 1cm and worsening left ventricular systolic function with an ejection fraction of 45%. After recognition of mesalazine as a potential cause and consequent cessation of the drug, symptoms resolved, with cardiac MRI performed as an outpatient showing resolution of myocardial oedema. The patient plans to make a return to competitive sport. Patients suffering from myopericarditis are advised to refrain from competitive sport for at least six months in order to reduce the risk of cardiac remodelling and sudden cardiac death. Additional considerations must be taken in individuals for whom competitive sport is an essential component of their livelihood, such as professional athletes. Myopericarditis is an uncommon, however potentially serious medical condition with a wide variety of aetiologies, including viral, autoimmune, and drug-related causes. Management is mainly supportive and relies on prompt recognition and removal of the aetiological process. Mesalazine-induced myopericarditis is a rare condition; as such increasing awareness of mesalazine as a precipitant of myopericarditis is vital for optimising the management of these patients.

Keywords: myopericarditis, mesalazine, inflammatory bowel disease, professional athlete

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Authors: Md. M. Hossain, Regan Roat, Jenica Christopherson, Colette Free, Zhiguang Guo

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Long noncoding RNA (lncRNA) mediated post-transcriptional gene regulation, and their epigenetic landscapes have been shown to be involved in many human diseases. However, their regulation in diabetes through governing islet’s β-cell function and survival needs to be elucidated. Due to the technical and ethical constraints, it is difficult to study their role in β-cell function and survival in human under in vivo condition. In this study, humanized mice have been developed through transplanting human pancreatic islet under the kidney capsule of NOD.SCID mice and induced β-cell death leading to diabetes condition to study lncRNA mediated regulation. For this, human islets from 3 donors (3000 IEQ, purity > 80%) were transplanted under the kidney capsule of STZ induced diabetic NOD.scid mice. After at least 2 weeks of normoglycecemia, lymphocytes from diabetic NOD mice were adoptively transferred and islet grafts were collected once blood glucose reached > 200 mg/dl. RNA from human donor islets, islet grafts from humanized mice with either adoptive lymphocyte transfer (ALT) or PBS control (CTL) were ribodepleted; barcoded fragment libraries were constructed and sequenced on the Ion Proton sequencer. lncRNA expression in isolated human islets, islet grafts from humanized mice with and without induced β-cell death and their regulation in human islets function in vitro under glucose challenge, cytokine mediated inflammation and induced apoptotic condition were investigated. Out of 3155 detected lncRNAs, 299 that highly expressed in islets were found to be significantly downregulated and 224 upregulated in ALT compared to CTL. Most of these are found to be collocated within 5 kb upstream and 1 kb downstream of 788 up- and 624 down-regulated mRNAs. Genomic Regions Enrichment of Annotations Analysis revealed deregulated and collocated genes are related to pancreas endocrine development; insulin synthesis, processing, and secretion; pancreatitis and diabetes. Many of them, that found to be located within enhancer domains for islet specific gene activity, are associated to the deregulation of known islet/βcell specific transcription factors and genes that are important for β-cell differentiation, identity, and function. RNA sequencing analysis revealed aberrant lncRNA expression which is associated to the deregulated mRNAs in β-cell function as well as in molecular pathways related to diabetes. A distinct set of candidate lncRNA isoforms were identified as highly enriched and specific to human islets, which are deregulated in human islets from donors with different BMIs and with type 2 diabetes. These RNAs show an interesting regulation in cultured human islets under glucose stimulation and with induced β-cell death by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the media of islets cultured with cytokines. Results of this study suggest that the islet specific lncRNAs are deregulated in human islet with β-cell death, hence important in diabetes. These lncRNAs might be important for human β-cell function and survival thus could be used as biomarkers and novel therapeutic targets for diabetes.

Keywords: β-cell, humanized mouse, pancreatic islet, LncRNAs

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Authors: M. Salvador, J. C. Martinez-Garcia, A. Moyano, M. C. Blanco-Lopez, M. Rivas

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Biosensors play a crucial role in the detection of molecules nowadays due to their advantages of user-friendliness, high selectivity, the analysis in real time and in-situ applications. Among them, Lateral Flow Immunoassays (LFIAs) are presented among technologies for point-of-care bioassays with outstanding characteristics such as affordability, portability and low-cost. They have been widely used for the detection of a vast range of biomarkers, which do not only include proteins but also nucleic acids and even whole cells. Although the LFIA has traditionally been a positive/negative test, tremendous efforts are being done to add to the method the quantifying capability based on the combination of suitable labels and a proper sensor. One of the most successful approaches involves the use of magnetic sensors for detection of magnetic labels. Bringing together the required characteristics mentioned before, our research group has developed a biosensor to detect biomolecules. Superparamagnetic nanoparticles (SPNPs) together with LFIAs play the fundamental roles. SPMNPs are detected by their interaction with a high-frequency current flowing on a printed micro track. By means of the instant and proportional variation of the impedance of this track provoked by the presence of the SPNPs, quantitative and rapid measurement of the number of particles can be obtained. This way of detection requires no external magnetic field application, which reduces the device complexity. On the other hand, the major limitations of LFIAs are that they are only qualitative or semiquantitative when traditional gold or latex nanoparticles are used as color labels. Moreover, the necessity of always-constant ambient conditions to get reproducible results, the exclusive detection of the nanoparticles on the surface of the membrane, and the short durability of the signal are drawbacks that can be advantageously overcome with the design of magnetically labeled LFIAs. The approach followed was to coat the SPIONs with a specific monoclonal antibody which targets the protein under consideration by chemical bonds. Then, a sandwich-type immunoassay was prepared by printing onto the nitrocellulose membrane strip a second antibody against a different epitope of the protein (test line) and an IgG antibody (control line). When the sample flows along the strip, the SPION-labeled proteins are immobilized at the test line, which provides magnetic signal as described before. Preliminary results using this practical combination for the detection and quantification of the Prostatic-Specific Antigen (PSA) shows the validity and consistency of the technique in the clinical range, where a PSA level of 4.0 ng/mL is the established upper normal limit. Moreover, a LOD of 0.25 ng/mL was calculated with a confident level of 3 according to the IUPAC Gold Book definition. Its versatility has also been proved with the detection of other biomolecules such as troponin I (cardiac injury biomarker) or histamine.

Keywords: biosensor, lateral flow immunoassays, point-of-care devices, superparamagnetic nanoparticles

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Authors: Izabela P. Vatanabe, Rafaela Peron, Patricia Manzine, Marcia R. Cominetti

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Introduction: Considering the increase in life expectancy of world population, there is an emerging concern in health services to allocate better care and care to elderly, through promotion, prevention and treatment of health. It has been observed that frailty syndrome is prevalent in elderly people worldwide and this complex and heterogeneous clinical syndrome consist of the presence of physical frailty associated with cognitive dysfunction, though in absence of dementia. This can be characterized by exhaustion, unintentional weight loss, decreased walking speed, weakness and low level of physical activity, in addition, each of these symptoms may be a predictor of adverse outcomes such as hospitalization, falls, functional decline, institutionalization, and death. Cognitive frailty is a recent concept in literature, which is defined as the presence of physical frailty associated with mild cognitive impairment (MCI) however in absence of dementia. This new concept has been considered as a subtype of frailty, which along with aging process and its interaction with physical frailty, accelerates functional declines and can result in poor quality of life of the elderly. MCI represents a risk factor for Alzheimer's disease (AD) in view of high conversion rate for this disease. Comorbidities and physical frailty are frequently found in AD patients and are closely related to heterogeneity and clinical manifestations of the disease. The decreased platelets ADAM10 levels in AD patients, compared to cognitively healthy subjects, matched by sex, age and education. Objective: Based on these previous results, this study aims to evaluate whether ADAM10 platelet levels of could act as a biomarker of cognitive frailty. Methods: The study was approved by Ethics Committee of Federal University of São Carlos (UFSCar) and conducted in the municipality of São Carlos, headquarters of Federal University of São Carlos (UFSCar). Biological samples of subjects were collected, analyzed and then stored in a biorepository. ADAM10 platelet levels were analyzed by western blotting technique in subjects with MCI and compared to subjects without cognitive impairment, both with and without presence of frailty. Statistical tests of association, regression and diagnostic accuracy were performed. Results: The results have shown that ADAM10/β-actin ratio is decreased in elderly individuals with cognitive frailty compared to non-frail and cognitively healthy controls. Previous studies performed by this research group, already mentioned above, demonstrated that this reduction is still higher in AD patients. Therefore, the ADAM10/β-actin ratio appears to be a potential biomarker for cognitive frailty. The results bring important contributions to an accurate diagnosis of cognitive frailty from the perspective of ADAM10 as a biomarker for this condition, however, more experiments are being conducted, using a high number of subjects, and will help to understand the role of ADAM10 as biomarker of cognitive frailty and contribute to the implementation of tools that work in the diagnosis of cognitive frailty. Such tools can be used in public policies for the diagnosis of cognitive frailty in the elderly, resulting in a more adequate planning for health teams and better quality of life for the elderly.

Keywords: ADAM10, biomarkers, cognitive frailty, elderly

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Authors: Nicholas Mavrogiannis, Francesca Crivellari, Zachary Gagnon

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Development of low-cost, rapid, sensitive and portable biosensing systems are important for the detection and prevention of disease in developing countries, biowarfare/antiterrorism applications, environmental monitoring, point-of-care diagnostic testing and for basic biological research. Currently, the most established commercially available and widespread assays for portable point of care detection and disease testing are paper-based dipstick and lateral flow test strips. These paper-based devices are often small, cheap and simple to operate. The last three decades in particular have seen an emergence in these assays in diagnostic settings for detection of pregnancy, HIV/AIDS, blood glucose, Influenza, urinary protein, cardiovascular disease, respiratory infections and blood chemistries. Such assays are widely available largely because they are inexpensive, lightweight, and portable, are simple to operate, and a few platforms are capable of multiplexed detection for a small number of sample targets. However, there is a critical need for sensitive, quantitative and multiplexed detection capabilities for point-of-care diagnostics and for the detection and prevention of disease in the developing world that cannot be satisfied by current state-of-the-art paper-based assays. For example, applications including the detection of cardiac and cancer biomarkers and biothreat applications require sensitive multiplexed detection of analytes in the nM and pM range, and cannot currently be satisfied with current inexpensive portable platforms due to their lack of sensitivity, quantitative capabilities and often unreliable performance. In this talk, inexpensive label-free biomolecular detection at liquid interfaces using a newly discovered electrokinetic phenomenon known as fluidic dielectrophoresis (fDEP) is demonstrated. The electrokinetic approach involves exploiting the electrical mismatches between two aqueous liquid streams forced to flow side-by-side in a microfluidic T-channel. In this system, one fluid stream is engineered to have a higher conductivity relative to its neighbor which has a higher permittivity. When a “low” frequency (< 1 MHz) alternating current (AC) electrical field is applied normal to this fluidic electrical interface the fluid stream with high conductivity displaces into the low conductive stream. Conversely, when a “high” frequency (20MHz) AC electric field is applied, the high permittivity stream deflects across the microfluidic channel. There is, however, a critical frequency sensitive to the electrical differences between each fluid phase – the fDEP crossover frequency – between these two events where no fluid deflection is observed, and the interface remains fixed when exposed to an external field. To perform biomolecular detection, two streams flow side-by-side in a microfluidic T-channel: one fluid stream with an analyte of choice and an adjacent stream with a specific receptor to the chosen target. The two fluid streams merge and the fDEP crossover frequency is measured at different axial positions down the resulting liquid

Keywords: biodetection, fluidic dielectrophoresis, interfacial polarization, liquid interface

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Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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Authors: Diego R. Boiarski, Camila M. Toigo, Thais M. Sobjak, Andrey F. P. Santos, Silvia Romao, Ana T. B. Guimaraes

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Anthropogenic activities promote changes in the community’s structures and decrease the species abundance of amphibians. Biological communities of fluvial systems are assemblies of organisms that have adapted to regional conditions, including the physical environment and food resources, and are further refined through interactions with other species. The aim of this study was to assess neurotoxic alterations and in the antioxidant system on tadpoles of Lithobates catesbeianus exposed to waters from Cascavel River, in the south of Brazil. A total of 420 L of water was collected from the Cascavel River, 140 L from each of the three different locations: Site 1 – headwater; Site 2 – stretch of the stream that runs through an urbanized area; Site 3 – a stretch from the rural area. Twelve tadpoles were acclimated in each aquarium (100 L of water) for seven days. The water from each aquarium was replaced with the ones sampled from the river, except the one from the control aquarium. After seven days, a portion of the liver was removed and conditioned for ChE, SOD, CAT and LPO analysis; other part of the tissue was conditioned for histological analysis. The statistical analysis performed was one-way ANOVA, followed by post-hoc Tukey-HSD test, and the multivariate principal components analysis. It was not observed any neurotoxic effect, but a slight increase in SOD activity and elevation of CAT activity in both urban and rural environment. A decrease in LPO reaction was detected, mainly among the tadpoles exposed to the waters from the rural area. The results of the present study demonstrate the alteration of the antioxidant system, as well as liver histopathologies in tadpoles exposed mainly to waters collected in urban and rural environments. These alterations may cause the reduction in the velocity of the metamorphosis process from the tadpoles. Further, were observed histological alterations, highlighting necrotic areas mainly among the animals exposed to urban waters. Those damages can lead to metabolic dysfunction, interfering with survival capacity, diminishing not only individual fitness but for the whole population. In the interpretation synthesis of all biomarkers, the cellular damage gradient is perceptible, characterized by the variables related to the antioxidant system, due to the flow direction of the stream. This result is indicative that along the course of the creek occurs dumping of organic material, which promoted an acute response upon tadpoles of L. catesbeianus. and it was also observed the difference in tissue damage between the experimental groups and the control group, the latter presenting histological alterations, but to a lesser degree than the animals exposed to the waters of the Cascavel river. These damages, caused by reactive oxygen species possibly resulting from the contamination by organic compounds, can lead the animals to a series of metabolic dysfunctions, interfering with its metamorphosis capacity. Interruption of metamorphosis may affect survival, which may impair its growth, development and reproduction, diminishing not only the fitness of each individual but in a long-term, to the entire population.

Keywords: American bullfrog, histopathology, oxidative stress, urban creeks pollution

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