Fresh Samples Research Articles

Repurposing of PSMA-targeted diagnostic and therapeutic agents for the detection and treatment of giant cell tumors of bone

Giant cell tumor of bone (GCTB) is a rare bone tumor often necessitating surgical intervention, radiation therapy, or treatment with bisphosphonates or denosumab. 99mTc-MDP bone scintigraphy for GCTB has limited specificity, and the relatively high uptake of 18F-FDG in GCTB makes it challenging to differentiate it from other benign bone tumors. More specific detection and treatment modalities for GCTB are needed to enhance patient monitoring and outcomes. Prostate Specific Membrane Antigen (PSMA) is present in the neovasculature of various tumors, yet unexplored in GCTB. PSMA-targeted imaging and radiotherapeutic agents Locametz and Pluvicto are a powerful theranostic pair for detecting and treating PSMA-positive metastatic tumors, including those in bone, and thus have considerable potential to be repurposed for GCTB. This study aimed to determine if the vasculature of GCTB was PSMA-positive and whether targeting it with PSMA-specific agents was feasible. Using bone core samples from 28 GCTB patients and 9 negative controls, we present the first robust detection of PSMA on the tumor vasculature of GCTB. To demonstrate the potential repurposed use of PSMA-targeted agents in detecting and treating GCTB, we used a PSMA-specific fluorescent probe (FAM-C6-1298) as a model for these radiopharmaceutical agents. Incubation of fresh GCTB tissue samples with FAM-C6-1298 showed increased fluorescence intensity compared to controls, indicating successful targeting of PSMA in GCTB tissue. In conclusion, our data established that PSMA is not only present in the tumor vasculature of GCTB patient tissue but can be effectively targeted with repurposed PSMA-specific radiopharmaceuticals for diagnosis and therapy.

Investigation of short-term chemical changes in stable ruthenium added to soil by using X-ray absorption fine-structure analysis.

Radioactive ruthenium may be accidentally released from spent nuclear fuel reprocessing plants to the surrounding environment. However, research on the chemical behavior of radioactive Ru in the environment is limited, and the complex chemical properties of this element complicate the application of extraction methods for the analyses of its chemical forms. To obtain basic information regarding the outcome of radioactive Ru in the environment, we investigated the changes in the form of stable Ru added to soil through X-ray absorption fine-structure (XAFS) analysis. This study uses ruthenium tetroxide (RuO4), ruthenium dioxide (RuO2), ruthenium nitrosyl nitrate (Ru(NO)(NO3)3) and ruthenium chloride (RuCl3) as test sources. These are added to ultrapure water, soil solution or fresh soil samples, which are analyzed using XAFS immediately or 5 days after Ru addition. The Ru K-edge X-ray absorption near edge structure spectra acquired immediately after Ru addition differed with respect to the source. The XAFS results suggest that RuO4 immediately changes to tetravalent form after deposition from air to soil. For RuCl3, the ionic structure in the vicinity of Ru is affected by the soil even if the valence does not change immediately. By contrast, RuO2 and Ru(NO)(NO3)3 are highly stable in soil. The results show that the chemical forms of RuO2 and Ru(NO)(NO3)3 added to the soil solution and soil are retained for 5 days, whereas those of RuCl3 and RuO4 are affected by the soil solution and soil within a short period. These results emphasize the need to focus on the chemical form of Ru deposits and the form change after Ru addition when investigating the environmental fate of radioactive Ru.

Bacterial community in fresh fruits and vegetables sold in streets and open-air markets of Dakar, Senegal.

In the context of food transition, the agrifood sector has experienced increasingly strong demand for horticultural products. Fresh fruits and vegetables are mainly vehicles of microorganisms, including pathogens that cause human infections. Food safety and compliance with health standards in the fruits and vegetable value chain are significant concerns and multiple challenges, given the prevalence of foodborne pathologies in Southern countries, particularly in Senegal. This study aimed to describe the bacterial community and assess the microbiological quality of a range of fruits and vegetables sold on street stalls and open-air markets in Dakar, the capital city of Senegal. Samples purchased in seven sites were analyzed to isolate Escherichia coli (E. coli), Salmonella spp. and Vibrio spp. Some primary samples with a positive microbiological culture were subjected to DNA extraction and subsequent metagenomic analysis. A total of 240 fresh fruit and vegetable samples were collected, including lettuce (n = 40), tomatoes (n = 40), mango slices (n = 40), onion slices (n = 40), mint leaves (n = 40), strawberries and grapes (each n = 20). Of these samples, 50.83% (122/240) and 60.41% (145/240) were contaminated with E. coli and Vibrio spp., respectively, while Salmonella was not isolated in any product. E. coli and Vibrio spp. contamination levels, 1.3 103 to 6 106 Colonies Forming Units per gram of sample (CFU/g) and 4.55 102 to 8.73 106CFU/g, respectively, were significantly above the thresholds acceptable for human consumption. The most contaminated samples were lettuce, with a prevalence of 98% (39/40) for E. coli and Vibrio spp. followed by mint leaves with 100% (40/40) and 93% (37/40) of the samples containing E. coli and Vibrio spp. respectively. Out of 46 samples sequenced, the metagenomic analysis revealed high contamination rates for E. coli, Vibrio spp. and Salmonella spp., with 100%, 67.39% and 93.47% of prevalence, respectively. On the other hand, the alpha diversity analysis shows high bacterial diversity in lettuce and mint leaf samples, while the beta diversity analysis highlighted the presence of two major clusters. Our results stress the need for a surveillance system that extends this investigation to a national scale while increasing the number of sampling sites and products analyzed.

Morphometric Characterization and Fillet Yield of Two Commercial Fish Species from Panyam Fish Farm Plateau State

A total of 50 individual samples of Parachanna obscura and Clarias gariepinus were collected fresh from Panyam fish farm with the aid of crush ice for preservation until assessment was carried out to determine the fillet yield of Parachanna obscura in comparism with Clarias gariepinus. The whole fresh fish samples were taken to the department of Fisheries and Aquaculture laboratory of the Federal University, of Agriculture, Makurdi for assessment. Their Total and Standard Lengths were recorded (in cm) using a Measuring Rule, while Total Body Weights were recorded (in grams) using an electronic weighing balance. Data were collected on: the Total Weight (g), Total Length (cm), Standard Length (cm), Visceral Weight (g), Head Length (cm), Weight of Bones (g), Weight of Fins (g) and Weight of Fillet (g) in comparism between the two fish species. Descriptive statistics for morphometric data were determined using Minitab 18® (Minitab 2017). Regression and correlation of variables was carried out in R (R Core Team 2020). Results of the mean measured parameters of Parachana obscura and Clarias gariepinus from Panyam Fish Farm Jos, showed that Total Length (42.66 ± 0.58cm), Standard Length (38.56 ± 0.29cm), Fin Weight (15.51 ± 0.37g), Visceral Weight (11.04 ± 0.17g), Head Weight (1.49 ± 4.01g), Bone weight (39.84 ± 0.80g) and Weight of fillet (89.02 ± 2.38g) of Parachana obscura were higher than the mean measured parameters of Clarias gariepinus. There was a significant difference (p= .05) among the measured parameters between P. obscura and C. gariepinus. The higher fillet percentage per body weight of P. obscura in comparism to C. gariepinus is a pointer to a relatively higher economic value for commercial purposes of this species. This assessment also provides information for further studies on this species to be built on, as there is obvious need for Nigeria’s aquaculture system to diversify from the prevalent catfish culture.

Abstract 4135082: Small dense low-density lipoprotein cholesterol and risk of peripheral artery disease

Background: High levels of small dense low-density lipoprotein (sdLDL) is a hallmark of dyslipidemia; however, studies on sdLDL cholesterol levels and risk of peripheral artery disease are sparse and results inconclusive. Hypothesis: We tested the hypothesis that higher levels of sdLDL cholesterol are associated with increased risk of peripheral artery disease in a primary prevention setting. Methods: We studied 31,036 individuals free of lipid-lowering therapy, ischemic stroke, and myocardial infarction at study entry in 2013-2017. All had fresh sample measurements of sdLDL cholesterol. During a median follow-up on 6.2 years, 155 were diagnosed with peripheral artery disease. The association was confirmed using ankle-brachial index (ABI) ≤ 0.9 as endpoint. Lastly, as comparison across different vascular beds risk estimates for myocardial infarction and ischemic stroke were calculated. Results: Higher levels of sdLDL cholesterol were associated with higher risk of peripheral artery disease and an ABI ≤ 0.9 illustrated by cubic splines multivariable adjusted for sex, systolic blood pressure, education, body mass index (BMI), diabetes, large buoyant LDL cholesterol, and age. Per 1 mmol/L (37 mg/dL) higher sdLDL cholesterol hazard ratio for peripheral artery disease was 2.01 (95% CI: 1.41-2.85) and odds ratio for an ABI ≤ 0.9 was 1.51 (95% CI: 1.07-2.14) in multivariable adjusted models. The cumulative incidence of peripheral artery disease was respectively 1.5%, 2.5%, and 3.5% for individuals having a sdLDL cholesterol in the 1 st -50 th , 51 st -90 th , and the 91 st -100 th percentiles at age 80. For the 91 st -100 th versus the 1 st -50 th percentile a hazard ratio on 2.51 (95% CI: 1.50-4.20) for peripheral artery disease, 2.18 (95% CI: 1.58-3.01) for myocardial infarction, and 1.85 (95% CI: 1.37-2.49) for ischemic stroke was found. Furthermore, the association of higher sdLDL cholesterol with increased risk of peripheral artery disease was robust in sensitivity analyses. Conclusion: Higher levels of sdLDL cholesterol were robustly associated with increased risk of peripheral artery disease in a primary prevention setting.

IMMU-03. EXPLORING CANCER-SPECIFIC T CELLS AND ANTIGENS IN GLIOBLASTOMA BASED ON SINGLE-CELL SEQUENCING OF CD8+ TILS

Abstract BACKGROUND The effectiveness of cancer immunotherapy against glioblastoma (GBM) remains limited. This study aims to identify cancer-specific antigens to develop antigen-based cancer immunotherapies for GBM. We investigated candidate tumor antigen-specific T cells in GBM by single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq), and we explored candidate antigens through genetic analysis of tumor tissues. METHODS Flow cytometry analysis was conducted on fresh tumor digest samples to evaluate tumor-infiltrating lymphocytes (TILs) of GBM. Single-cell RNA and TCR sequencing were performed on CD8+ T cells in TILs. Both data generated by Cell Ranger software were loaded into Seurat at R studio. The dimensional reduction for clustering was performed with uniform manifold approximation and projection (UMAP). Additionally, we performed bulk RNA sequencing (RNA-seq) and whole exome sequencing (WES) on tumor tissues. RESULTS Two out of 20 patients (10%) exhibited abundant TILs in GBM. From these two patients, approximately 20,000 CD8+ T cells in total were analyzed in single-cell analysis. Clustering based on UMAP revealed a predominance of clusters expressing exhaustion markers, with high TCR clonality centered on exhausted T cell (TEX) clusters, like our previous date of lung cancer TILs recognizing tumor antigens. Moreover, within the TEX clusters, the expression of CXCL13, often reported as an antigen-specific marker in recent years, was significantly higher in our data compared to two publicly available datasets. Subsequently, based on RNA-seq and WES, 61 candidate neoantigens were estimated using machine-learning prediction algorithms from NEC Corporation. Furthermore, 5 overexpressed cancer/testis antigens, and 3 bacterial species-derived microbial peptides using Kraken2 were selected as antigen candidates. CONCLUSIONS We identified prospective tumor antigen-specific T cell subsets with high CXCL13 expression from two GBM patients. We plan to identify cancer-specific T cells and antigens from these candidates by T cell activation assay.

TMOD-24. DEVELOPMENT OF A 3D ORGANOTYPIC EXPLANT PLATFORM TO STUDY ONCOLYTIC VIRUS TREATMENT RESPONSE IN GLIOBLASTOMA

Abstract BACKGROUND Oncolytic viruses (OVs) are a promising strategy to induce an anti-tumor immune response in the lymphocyte-depleted environment of glioblastoma (GBM), however the mechanism of their oncolytic function is not well understood. Immune-intact experimental systems would be highly valuable in dissecting immune versus direct viral contribution to oncolysis. Here, we report on the development of an immune-intact organotypic explant (OE) model, amenable to perturbations and T-cell activity monitoring. METHODS OEs were made from fresh tumor samples and cultured in groups of 4-5. CAN-3310 OVs and T-cells were injected into each OE under a dissecting microscope. Cultured OEs were either fixed for immunofluorescence (IF) or dissociated into single-cell suspensions for flow cytometry and 5’ single-cell RNA (scRNA) and TCR sequencing. RESULTS Cultured OEs maintain the cellular density of the original tumor in both short- and long-term cultures. Ki67 IF shows that tumor cells retain their proliferative potential, allowing for renewal and repopulation. The TME, mainly microglia and macrophages, is maintained throughout culture. IF imaging of OV-treated OEs, 7 days post-treatment, shows significant GFP positivity in tumor cells demonstrating the feasibility of using OEs as an ex vivo model with OV. Additionally, autologous T-cells from the patient’s blood can survive in OEs, completing the TME with the equivalent of circulating T-cells. Finally, scRNA-seq of the original tumor and cultured OEs showed that the different cell types were represented at all time points. Tumor cell meta-module analysis showed an increase in hypoxic signatures recapitulating features of in vivo tumor cell growth. CONCLUSIONS OEs maintain tumor architecture and TME. OE co-culture with autologous T-cells allows us to better understand the oncolytic mechanisms of OVs. Future endeavors will include identifying expanded T-cell clones to create an OV-specific TCR repertoire. This OE system provides a new tool as an ex vivo platform to dissect glioma responses to therapy in a purely human system.

An Outbreak of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in a German Boar Stud: A Retrospective Analysis of PRRSV Shedding in Boar Semen

The porcine reproductive and respiratory syndrome virus (PRRSV) causes high economical costs due to reduced productivity and losses in pig production. The virus can infect sow herds through various routes. One possible risk factor is the transmission of PRRSV through artificial insemination with infected boar semen. For these reasons, conventional boar studs should be closely monitored to detect an outbreak of PRRSV at an early stage. In the presented retrospective study, 2184 fresh semen samples from 336 boars were investigated (RT-qPCR) after an accidental PRRSV introduction to the herd. Hence, the different shedding profiles of PRRSV via semen resulted in 42.2% where no virus was detected, 2.0% intermittent shedding, and 8.4% permanent shedding. The duration of viral shedding varies from 2 days to 83 days post outbreak (on average 33 days). A significant impact of breed on PRRSV shedding duration could not be shown. Also, the distribution of the shedding profile does not follow a consistent mode, indicating that not every boar is shedding the virus via semen.

IMMU-08. PRECLINICAL EVALUATION OF GAL-1-SPECIFIC CAR-T CELL FUNCTION IN AN IN VITRO AND EX VIVO MODEL OF GLIOBLASTOMA

Abstract BACKGROUND Human glioblastoma (GBM), especially those exhibiting anti-VEGF resistance, overexpress the carbohydrate-binding protein galectin-1 (Gal-1). To develop novel anti-GBM approaches, we developed Gal-1 targeted CAR-T cells to investigate potential antitumor effect with in vitro and ex vivo slice culture models. METHODS Uniform GBM brain slice cultures were collected using NICO’s Myriad resection tool and their Automated Preservation System (APS), designed to obtain fresh brain tumor samples maintaining tumor cytoarchitecture and microenvironment. To prepare Gal-1 CAR-T cells, we used creative laboratory to utilize a third-generation Gal-1-CAR construct with high binding and codon optimized scFv-Gal-1 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB (Gal-1 scFv-CD28-4-1BB-CD3ζ). We then transfected into the T cells, from peripheral blood, mononuclear cells using a lentiviral system. The anti-tumor capabilities of CAR-T cells were evaluated by co-culturing with GBM cells and ex vivo APS-GBM slices. RESULTS The collected APS- GBM slices samples were clear of debris and blood product, not requiring post-surgical mechanical slices, and exhibited viability up to 12 days over traditionally prepared ex vivo cultures. With this, we found Gal-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotypic and biological attributes such as cell viability, potency, migration, and T cell activation. Gal-1 scFv-CD28-4-1BB-CD3ζ -CAR-T cells killed more than 80% of GBM tumor cells within 24 hours in Gal-1+ve, but not Gal-1-ve GBM cells in vitro. Additionally, these CAR-T cells selectively caused significant release of IL-2, IFN-γ and TNF-α only from Gal-1+ve co-cultures and induced significant Gal-1+ve GBM cell death in an ex vivo model without any general toxicity. CONCLUSION Based on in vitro cell culture and ex vivo APS-brain slice culture results, we conclude that the novel Gal-1 scFv-CD28-4-1BB-CD3ζ CAR-T cell therapy may offer an effective therapeutic option. Further pre-clinical and clinical studies need to be performed to validate this novel approach

TMOD-08. DETAILED GENERATION OF AN ORTHOTOPIC INTRACRANIAL GLIOBLASTOMA (GBM) PATIENT-DERIVED XENOGRAFT (PDX) MODEL FOR PRECLINICAL IMMUNOTHERAPEUTIC STUDIES

Abstract PURPOSE This study aimed to establish an orthotopic intracranial PDX mouse model using cryopreserved GBM tissues expanded in a heterotopic subcutaneous PDX model for immunotherapeutic studies. The model is designed to closely replicate human GBM, enabling the exploration of its pathophysiology and the development of effective immunotherapies for this type of cancer. METHODS Glioblastoma tissues from 16 fresh and cryopreserved samples were used to establish a heterotopic subcutaneous PDX mouse model for tumor expansion. Successful engraftments were then used to create an orthotopic intracranial PDX mouse model. The tumor formation rates and durations were estimated in both models. Tumor fidelity to the original GBM tumors was confirmed through immunohistology (GFAP, P53, and Ki67) and genetic analysis (whole exome sequencing, RNA sequencing, and methylome analysis). The therapeutic effects of immunotherapy (allogenic NK cells) in combination with Avastin and Irinotecan were tested in the orthotopic intracranial PDX model. RESULTS The orthotopic intracranial GBM mouse model achieved 100% engraftment from successful tumor growth in the subcutaneous PDX model. The overall success rate was 81.25% (85.71% for cryopreserved tissues), with a median process duration of 26.5 weeks (19 weeks for the subcutaneous and 7.5 weeks for intracranial establishment). No significant differences in tumor formation were observed within one year for cryopreserved tissue in the subcutaneous model. Molecular and genetic analysis confirmed similar tumor characteristics between the intracranial PDX tumors and the original patient tumors. Additionally, NK cells combined with Avastin and Irinotecan demonstrated efficacy in this model. CONCLUSIONS We successfully established an orthotopic intracranial PDX mouse model using cryopreserved GBM tissues, with no observed differences within one year. These models accurately mirror patients’ tumor characteristics, promising advancements in glioblastoma research and treatment, particularly for immunotherapy studies. Keywords: Glioblastoma (GBM), cryopreserved GBM tissues, patient-derived xenograft (PDX) model, NOD/SCID IL-12Rγnull (NSG) mice

Laboratory detection of donors implicated in transfusion-transmitted malaria.

Transfusion-transmitted malaria (TTM) is rare in non-endemic areas (non-EAs) but can potentially be fatal. This review analyzes the laboratory results of donors causing TTM in non-EAs, to assess the detectability of their Plasmodium infection by molecular or antibody tests. TTM cases in the United States, Canada, and Europe since 2010 were identified through a literature review. Authors and laboratories were contacted for missing details about sample types and laboratory methods. Results of Plasmodium polymerase chain reaction (PCR) and antibody tests were summarized. Twelve cases of TTM and one bone marrow transplant transmission were identified. Of the 13 source donors, 12 were tested by PCR, 10 were positive on at least one sample; the 2 negative donors were tested only on retained segments of blood refrigerated for several weeks. All donors were PCR positive on a fresh sample except one who was positive on a retained but not a fresh sample. These PCRs targeted Plasmodium DNA with sensitivities in the range of 1000-10,000 parasites/mL. Antibody EIA was positive in only three of seven donors tested. This review found that antibody EIAs failed to detect four of the seven TTM donors tested. DNA-based PCRs were able to detect Plasmodium infection in all donors tested except for two tested only on samples likely to have deteriorated from prolonged storage. Recently developed ribosomal RNA-based molecular donor screening assays are approximately 1000 fold more sensitive than these DNA-based PCRs, holding promise as a potential method to further reduce TTM.

Investigation of the bond between bamboo and ribbed steel reinforcements and the surrounding concrete by pull-out test

Traditional housing did not use expensive materials like steel reinforcement, yet some have outlived sandcrete or mud-brick housing. One means to build at a low cost is to supplement high-cost materials with low-cost types, which have comparable strength. This study compares the bond between bamboo (Bambusa vulgaris and Bambusa vittata) reinforcement as well as ribbed steel enforcement and the surrounding concrete by pull-out test. Bamboo specimens were split and cut into the dimensions of 600 mm × 20 mm ×10 mm. Sixty (60) samples of each of the two varieties were prepared, thirty (30) were coated with tar oil as waterproof agent to a length of 200 mm and embedded into fresh concrete samples and allowed to cure, while another 30 did not receive any coating. Thirty (30) specimens of 12.5 mm diameter of ribbed steel rods of length 60 mm were used as control Materials for the pull-out test were also embedded in the centre of 150 mm concrete cubes and left to cure for 28 days. The load required to pull the steel rods from concrete (5.49±0.22 kN) was greater than those for both coated Bambusa vulgaris and Bambusa vittata (1.67±0.11 kN – 1.67±0.12 kN) and uncoated Bambusa vulgaris and Bambusa vulgaris vittata (2.04±0.14 kN– 2.14±0.19 kN) bamboo. The differences were significant (p<0.05). The pull-out was also conducted in conformity with the Bureau of Indian Standard (IS) 2770: 1997.

Public health concern of antimicrobial resistance and virulence determinants in E. coli isolates from oysters in Egypt

The emergence of critical-priority E. coli, carrying a wide array of resistance and virulence factors through food sources, poses a significant challenge to public health. This study aimed to investigate the potential role of oysters sold in Egypt as a source for E. coli, identify their resistance and virulence-associated gene profiles, and assess associated zoonotic risks. A total of 33 pooled fresh oyster samples were obtained from various retail fish markets in Egypt and examined bacteriologically for the presence of E. coli. Antimicrobial resistance was performed by the disk-diffusion method, and the multiple antibiotic resistance index (MAR) was calculated. All isolates were screened for extended-spectrum beta-lactamase (ESBL) (blaTEM, blaSHV, blaCTX−M, and blaOXA−1), plasmid-mediated AmpC blaCMY−2, and carbapenemases (blaKPC, blaNDM, blaVIM, and blaOXA−48) genes by Polymerase chain reaction. Moreover, the presence of virulence-encoding genes was investigated. The virulent MDR strains were clustered using R with the pheatmap package. The prevalence of E. coli was 72.7% (24 out of 33), with 66.7% of the isolates classified as multi-drug resistant, and 75% exhibited MAR values exceeding the 0.2 threshold. Different antimicrobial sensitivity phenotypes and genotype profiles were identified in E. coli isolates. The most prevalent gene detected among all isolates was blaTEM (22/24, 91.7%). Notably, all non-ESBL producers were positive for blaCMY2. Carbapenem-resistant and carbapenem-intermediate strains were carbapenemase producers, with the predominance of the blaKPC gene (11/24, 45.8%). Remarkably, twelve out of sixteen virulence genes were identified, with papC (21/24, 87.5%) and sfa (16/24, 66.7%) genes being the most prevalent. Most isolates carry virulence genes primarily associated with extra-intestinal pathogenic E. coli (ExPEC) (87.5%) and enteropathogenic (EPEC) (70.8%) pathotypes. Four E. coli isolates exhibit cluster patterns. This study provides the first insight into the emergence of virulent MDR E. coli among oysters in Egypt. It underscores the potential role of oysters as a source for disseminating these strains within aquatic ecosystems, presenting a possible threat to public health.

Effect of various culinary treatments on the glycoalkaloid content of potato peel

Potatoes are one of the most consumed foods worldwide. During germination, glycoalkaloids (GAs) with potential bioactive properties are produced, which, at high concentrations, may have adverse health effects. Potato peels, a byproduct generated in large quantities but increasingly used in contemporary cuisine, contain significant levels of these GAs. This paper examines GA levels in potato peels after various culinary treatments (boiling, steaming, baking, air frying, conventional deep frying, and double frying) to provide new data and ensure food safety. An optimized extraction method using ultrasound-assisted extraction (UAE) was employed, followed by purification with mesostructured SBA-15 silica functionalized with octadecylsilane (C18) chains as sorbent in solid-phase extraction, prior to HPLC-DAD determination of α-solanine and α-chaconine levels in the samples. The method was validated, obtaining excellent recovery values (88 – 96 %) and low detection and quantification limits (0.3 and 1 mg/L, respectively). Upon application of the validated analytical method to the samples culinarily treated using the most common cooking techniques (boiling, steaming, baking, air frying, conventional deep frying, and double frying) at various times and temperatures, the obtained results demonstrated significant reductions (between 52 % and 84 %) in GAs content in the treated potato peels, compared to fresh samples. Statically significant degradation of these compounds was observed through baking at elevated times and temperatures (74 – 84 %), as well as both conventional deep frying and double frying (76 – 83 %).

Urban Pigeons as Reservoirs of Critical Pathogens: Improved protocol for sequencing pigeon faeces in disease monitoring

The spread of pathogens by animals is a serious issue around the world that causes a severe threat to human health. Feral pigeons (Columba livia forma domestica) that live in urban areas are zoonotic carriers of various pathogens that can be transmitted to humans by faecal contamination. This study aimed to detect the presence of bacterial, viral, and specifically fungal pathogens in pigeon faeces based on the World Health Organization's (WHO) priority pathogen list published in 2022. Fresh faecal samples were collected at Uppsala, Central Station, and Svandammen, the pigeons' most relevant gathering spots and feeding sites. Genomic DNA was directly extracted from these samples, and ScilifeLab performed High throughput sequencing through Oxford Nanopore Technologies(ONT) (PromethION). Metagenomic analysis revealed that most of the critically prioritized viral and bacterial pathogens listed by WHO were present in pigeon faeces. Regarding fungal pathogens, which were the main objective of this study, samples from both studied locations contained all critical, high and, medium-important fungal pathogens published in the WHO list, such as Aspergillus fumigatus, Candida albicans, Candida auris, Cryptococcus neoformans, Nakaseomyces glabratus, Candida tropicalis, and Cryptococcus gattii. These fungal pathogens pose the risk of invasive fungal diseases and severe infections in low-immunity individuals and vulnerable populations. The findings indicate the importance of conducting further research to comprehensively understand potential exposure to feral pigeons. Furthermore, keeping pigeons away from sensitive areas, such as hospitals, and implementing measures to control pigeon populations can significantly decrease the spread of pathogens.

Assessment of milling and the green biosolvents ethyl lactate and 2-methyltetrahydrofuran (2-methyloxolane) for the ultrasound-assisted extraction of carotenoids in common and phytoene-rich Dunaliella bardawil microalgae

This study aimed to evaluate the efficiency of green solvents (ethyl lactate and 2-methyltetrahydrofuran (MeTHF)) compared to conventional solvents (ethanol, methanol, and dimethyl sulfoxide) in the ultrasound-assisted extraction of carotenoids from Dunaliella bardawil. Two types of algae were evaluated, a control (common) and a phytoene-enriched algae obtained by treatment with norfluorazon. The extractions were performed on fresh, freeze-dried, and encapsulated samples, with and without ball-milling pre-treatment. The pre-treatment had no significant effect on carotenoid extraction for any of the solvents tested. The solvents that achieved the highest carotenoid yield from fresh samples were MeTHF, methanol, and ethanol (control: 2105.3, 2001.8, and 1919.1 μg/g, respectively; phytoene-rich: 3220.9, 3669.1, and 3275.0 μg/g, respectively). In freeze-dried samples, ethanol was most effective in the control strain (14337.1 μg/g), while methanol yielded the most in the phytoene-rich strain (8464.1 μg/g). For encapsulated samples, MeTHF and ethanol were the top performers (control: 421.0 and 394.0 μg/g, respectively; phytoene-rich: 186.9 and 166.4 μg/g, respectively). Overall, the green solvent MeTHF proved to be a promising alternative to traditional solvents in the food industry.

Comparison of two methods of extracting bull epididymal spermatozoa

Extraction of epididymal spermatozoa may be necessary to avoid losing valuable genetic material, for example, from individuals of rare breeds or endangered species, but the resulting sperm samples may be of poor quality. Two methods of extracting bull epididymal spermatozoa from slaughterhouse material were compared. The bulls were 16-23 months of age. Spermatozoa were extracted by making an incision one cm in length in the tail of the epididymis to allow the spermatozoa to flow out (method A), or by flushing the tail of epididymis (method B). The two methods were used for each bull, alternating between right and left epididymis, i.e. if method A was used for the left epididymis in Bull 1, it was used for the right epididymis in bull 2, etc. Sperm concentration in the extracted samples was adjusted to 69 × 106/mL in Andromed; the semen was packed in 0.25 mL straws. After cooling for two h at 5°C, the straws were placed 4 cm above liquid nitrogen for 20 min before transferring them to liquid nitrogen. Sperm motility, viability, reactive oxygen species, membrane integrity and DNA fragmentation were analysed in the fresh samples and again after thawing. The results for all parameters in fresh semen were not different between methods. Although sperm quality was lower in thawed samples than in fresh samples, there was no difference in sperm quality between the two extraction methods in the thawed samples. In conclusion, both methods are useful for the extraction of bull epididymal spermatozoa.

Seafood as a source of antibiotic resistant Enterococcus spp

Seafood is a crucial global food source, but its role as a reservoir for antibiotic-resistant pathogens is concerning. This study focuses on the prevalence of antibiotic resistance in retail seafood, using Enterococcus spp. as an indicator. Seven categories of raw seafood were collected from retail markets in Central Italy. Out of 422 samples, 288 tested positive for Enterococcus spp., resulting in a prevalence rate of 68.25% (CI95%: 63.66%-72.51%). The most common species were E. faecalis (48%) and E. faecium (17.4%). Enterococcus spp. was most frequently found in cephalopods, salmon, bivalves, and crustaceans than in sea bass and bream. The odds of Enterococcus spp. recovery were higher in frozen than in fresh samples, while there was no difference between farm-raised and wild-caught seafood. A subset of 102 isolates was selected for antibiotic resistance testing, showing the highest resistance rates to quinupristin/dalfopristin (52.94%) and tetracycline (27.45%). Over 70% of isolates were multidrug-resistant. Additionally, strains resistant to vancomycin and oxazolidinones, two last-resort antimicrobials, were detected. Vancomycin resistance was observed in E. casseliflavus and E. gallinarum, which are naturally resistant due to the vanC chromosomal cluster. The optrA gene, responsible for resistance to oxazolidinones and phenicols, was found in E. thailandicus and E. faecium, where it was located on to a transferable plasmid. In conclusion, this study confirms the widespread presence of Enterococcus spp. in retail seafood. The detection of multi-resistant isolates and resistance to last-resort antimicrobials highlights significant human health risks, stressing the importance of including Enterococcus spp. in seafood antibiotic resistance surveillance.

A comparative study concerning the sensory, physicochemical, bacteriological, nutritional quality, heavy metal content, and health risk assessment of some low-cost fish species

Investigating fish quality, nutritional content, and health risk assessment for heavy metals can provide important guidance on eating seafood and protecting human health. Accordingly, the current research aimed to assess the quality and safety of some extensively consumed fish and recognize more types of fish widely available in Egypt with reasonable costs, high nutritional values, good quality, and safe for human consumption. To achieve this goal, 100 fresh fish samples including red porgy, sardine, eel fish, grass carp, and common carp (20 from each) were taken from local fish markets in Giza and examined for chemical composition, bacteriological quality, deterioration criteria, sensory attributes, heavy metal contents, and health risk assessment. Among the examined fish, red porgy exhibited significantly higher protein and sensory scores along with significantly lower fat, bacterial count, pH, total volatile base nitrogen, and trimethylamine values in contrast to carp fish. Nevertheless, sardine and eel fish obtained the highest values of thiobarbituric acid, free fatty acid, and acid number versus red porgy. Surprisingly, the concentrations of lead in grass carp fish, mercury in all examined samples, as well as arsenic in common carp fish, were lower than the detectable limits. Despite the target hazard quotient and hazard index of all examined heavy metals being below 1 in all fish samples, it could not be confirmed that eating such fish causes acceptable non-carcinogenic health risks due to exposure from fish consumption does not account for the total daily exposure to the heavy metal from all dietary sources. Moreover, the carcinogenic risk of all examined heavy metals in fish samples was higher than 10-6 indicating a high risk of carcinogenicity which needs great efforts to be reduced to the safe limits. In conclusion, the current study provides beneficial data to Egyptian society for choosing seafood with high nutritional value, good quality, and affordable prices.

Investigation of CST7 and hsa-miR-4793-5p gene expression in breast cancer

Breast cancer (BC) presents as a worldwide challenge, known as the most frequently diagnosed cancer in women. In 2022, BC was diagnosed in 2.3 million women with 670,000 deaths globally. In this research, our objective was to examine the CST7 and has-miR-4793-5p gene expression in BC tumor tissues and adjacent normal tissues.Using GSE57897 gene expression data from 422 BC samples and 31 breast samples from healthy controls which was based on the Platform GPL18722 (spotted oligonucleotide Homo sapiens microRNA (miRNA) array) in the Gene Expression Omnibus (GEO) and compare with miRNAs with a conserved target location on CST7 mRNA were found using databases. The study population included 60 fresh BC tissue samples and adjacent normal tissues as control. The Quantitative Real-Time PCR was used to evaluate the expression levels of CST7 and has-miR-4793-5p in the breast tissues.The present study, found that CST7 and hsa-miR-4793-5p were significantly increased in tumoral tissues in compare to normal tissues. Further analysis revealed a remarkable association between CST7 and hsa-miR-4793-5p gene expression alteration. ROC curve analysis demonstrated high accuracy for CST7 expression in BC tumors. Comparison of gene expression between different stages and patient family history showed significant findings. Due to the high sensitivity and specificity of the expression changes of these two genes, they are suitable candidates for further investigations to be considered as part of a diagnosis and prognosis panel.