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Hepatocellular carcinoma (HCC) stands as the most prevalent form of primary liver cancer, predominantly affecting patients with chronic liver diseases such as hepatitis B or C-induced cirrhosis. Diagnosis typically involves blood tests (assessing liver functions and HCC biomarkers), imaging procedures such as Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), and liver biopsies requiring the removal of liver tissue for laboratory analysis. However, these diagnostic methods either entail lengthy lab processes, require expensive imaging equipment, or involve invasive techniques like liver biopsies. Hence, there exists a crucial need for rapid, cost-effective, and noninvasive techniques to characterize HCC, whether in serum or tissue samples. In this study, we developed a spiral sensor implemented on a printed circuit board (PCB) technology that utilizes impedance spectroscopy and applied it to 24 tissues and sera samples as proof of concept. This newly devised circuit has successfully characterized HCC and normal tissue and serum samples. Utilizing the distinct dielectric properties between HCC cells and serum samples versus the normal samples across a specific frequency range, the differentiation between normal and HCC samples is achieved. Moreover, the sensor effectively characterizes two HCC grades and distinguishes cirrhotic/non-cirrhotic samples from tissue specimens. In addition, the sensor distinguishes cirrhotic/non-cirrhotic samples from serum specimens. This pioneering study introduces Electrical Impedance Spectroscopy (EIS) spiral sensor for diagnosing HCC and liver cirrhosis in clinical serum—an innovative, low-cost, rapid (< 2 min), and precise PCB-based technology without elaborate sample preparation, offering a novel non-labeled screening approach for disease staging and liver conditions.

The world urgently needs new methods to quickly and efficiently detect mutated viruses. An RNA-AuNP-based colorimetric biosensor is a highly sensitive, specific, and cost-effective tool that enables rapid, visual detection of target molecules for applications in disease diagnostics, environmental monitoring, and forensic analysis. An RNA-AuNP-based colorimetric biosensor requires precise control over nanoparticle dispersion and aggregation, which can be achieved using temperature regulation. A novel on-chip microelectrode is proposed to induce and monitor the aggregation of RNA-attached gold nanoparticles (AuNPs) through Joule heating and impedance spectroscopy. The proposed platform is implemented based on printed circuit board (PCB) technology, which has many advantages, such as fast and easy design and fabrication, low power consumption, and low costs. Joule heating is the process in which the energy of an electric current is converted into heat as it flows through a resistance. Impedance spectroscopy is an analytical technique that measures a system’s electrical response to an applied AC signal across a range of frequencies, providing insights into a sample’s dielectric properties. The results validate that the fabricated microelectrode is capable of heating a 20 µL droplet to 75 °C within 30 s, utilizing a low power input of only 3.75 watts and successfully inducing a color change based on the presence of hepatitis C virus (HCV) RNA, while impedance readings are used to monitor the aggregation.

The objective of this study is to develop a facile tool for the absolute detection and quantification of nucleic acid transcripts, using a gold nanoparticle-based optical biosensor. Topoisomerase 1 (TOP1) and tyrosyl DNA phosphodiesterase 2 (TDP2) were among the nucleic acid transcripts of choice due to their role as genomic instability biomarkers and their implication in various cancers and neurologic disorders. This opens the door to develop a simple tool that can be used for diagnosing and monitoring treatment response for such diseases, overcoming the requirements for high cost, time, and complexity of the existing technologies for the absolute quantification of transcripts of interest. The TOP1 and TDP2 mRNA transcripts were first captured specifically using magnetic nanoparticles that were functionalized with TOP1- and TDP2-specific probes, respectively. The captured mRNA was then directly detected and quantified using the gold aggregating gold (GAG) assay, without the need for amplification as in existing technologies used for the quantification of transcripts. A linear correlation exists between the GAG assay and the qPCR for the quantification of the TOP1 and TDP2 mRNA transcripts (10 -10 copies). The detection limit of the GAG assay in mRNA quantification was up to 10 copies per reaction. Wild-type and TDP2-deficient cell lines confirmed the assay specificity and reproducibility in distinguishing between different transcripts. The GAG assay can be utilized as an inexpensive, rapid, simple, and sensitive tool for the absolute quantification of RNA for different applications, instead of the laborious, expensive, and sophisticated real-time PCR.

The current study had aimed to assess the long-term dietary supplementation with Melaleucae aetheroleum , tea tree essential oil (TTO). The impact on growth performance, biochemical indices, immune function, oxidant/antioxidant activity, gene expression, histopathology, and resistance against Aeromonas sobria in Nile tilapia ( Oreochromis niloticus ) was investigated. Four groups (with five replicates; G1 (control group, G2, G3, and G4) of Nile tilapia received diets enriched with TTO (doses of 0.0, 0.5, 1.0, and 2.0 mL/kg diet) for 60 days, then fish were challenged by A. sobria . Outcomes indicated an extensive elevation in growth metrics (final body weight, weight gain, SGR, feed intake and fish body protein). Similarly, the total blood protein, albumin, total globulin levels, Serum complement-3 levels, lysozyme activity, immunoglobulin M (IgM), nitric oxide, and phagocytic activity were significantly enhanced in all treatments, notably in the 2.0 mL TTO/kg fed groups compared to the control. Lower levels of urea, creatinine, AST, ALP, ALT, cortisol, glucose, triglycerides, cholesterol and body crude lipids were observed in the fish that were fed a 2.0 mL TTO/kg diet. Supplementing TTO at 2.0-mL /kg diet revealed the best results for elevating CAT, SOD, and GSH activities plus declining MDA value in hepatic homogenate. Additionally, dietary 2.0-mL TTO/kg showed the best outcomes for the intestinal morphometry plus maintaining the histological picture in spleen and liver. Concurrently, fish that were fed a 2.0 mL TTO/kg diet exhibited a substantial upregulation of TNF-α , IL-1β , IL-10 , TFG-β , IFN-γ and BCL-2 genes in the liver, while, caspase-3 , and BAX were downregulated. Furthermore, TTO-enriched diets enhanced the relative percentage survival post- A. sobria challenge plus enhanced the clinical picture in a dose-dependent manner. Taken together, the findings revealed that long-term exposure to dietary TTO fortified the physiological performance, oxidant/anti-oxidant stability, immune function, gene expression, histological picture, and resistance of Nile tilapia against A. sobria .

The Pseudomonas putida strain was primarily identified and tested in vitro against antibiotic sensitivity for several antibiotics using the disc diffusion method. This isolate was also tested against sensitivity to carvacrol oil (c) and formic acid (f). The genotyping of Pseudomonas spp. and virulotyping for P. putida isolate was carried out and verified by 16S rDNA-PCR amplification. Furthermore, we assessed the efficacy of carvacrol oil and formic acid in vivo for treatment of P. Putida infection. For the in vivo challenge, 180 fish (Nile tilapia, Oreochromis niloticus) were divided into six groups: (G1: control (unchallenged), G2: carvacrol prophylaxis (3 g/kg), G3: formic acid prophylaxis (5 mL/kg), G4: control positive (challenged), G5: carvacrol treatment (3 g/kg), and G6: formic acid treatment (5 mL/kg); 30 fish per group) with three replicates. Following the challenge, nitric oxide and lysozyme activity were measured as essential indicators for fish immunity. The antioxidant parameters including SOD and catalase were computed to reflect the antioxidant status. Furthermore, relative percent survival (RPS) and mortality percent were evaluated to indicate functional immunity. The findings of the antibiotic sensitivity test showed that ciprofloxacin exhibited the largest inhibition zone. Additionally, formic acid (f) displayed the greatest inhibition zone compared to carvacrol oil (c) and was more effective in stimulating the immune-antioxidant response compared to carvacrol oil. The tested exotoxin A (tox A), exoenzyme S (exo S), and the nan1 associated-virulence genes were identified in the P. putida isolate. Overall, the current study verified the virulence of P. putida and highlighted the promising role of dietary addition of formic acid for enhancing the immune-antioxidant indicators and for mitigating P. putida infection. Future studies could be devoted to this field.

CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls.

(1) Background: The coronavirus (COVID-19) pandemic is still a major global health problem, despite the development of several vaccines and diagnostic assays. Moreover, the broad symptoms, from none to severe pneumonia, and the various responses to vaccines and the assays, make infection control challenging. Therefore, there is an urgent need to develop non-invasive biomarkers to quickly determine the infection severity. Circulating RNAs have been proven to be potential biomarkers for a variety of diseases, including infectious ones. This study aimed to develop a genetic network related to cytokines, with clinical validation for early infection severity prediction. (2) Methods: Extensive analyses of in silico data have established a novel IL11RA molecular network (IL11RNA mRNA, LncRNAs RP11-773H22.4 and hsa-miR-4257). We used different databases to confirm its validity. The differential expression within the retrieved network was clinically validated using quantitative RT-PCR, along with routine assessment diagnostic markers (CRP, LDH, D-dimmer, procalcitonin, Ferritin), in100 infected subjects (mild and severe cases) and 100 healthy volunteers. (3) Results: IL11RNA mRNA and LncRNA RP11-773H22.4, and the IL11RA protein, were significantly upregulated, and there was concomitant downregulation of hsa-miR-4257, in infected patients, compared to the healthy controls, in concordance with the infection severity. (4) Conclusion: The in-silico data and clinical validation led to the identification of a potential RNA/protein signature network for novel predictive biomarkers, which is in agreement with ferritin and procalcitonin for determination of COVID-19 severity.

The present study aimed to identify a safe and novel approach using zinc oxide/copper oxide nanocomposites (AZ) to enhance growth parameters, immunity, and fight Sarcoptic mange in vitro and in vivo in rabbits. In vitro: the acaricidal activity of AZ was assessed at concentrations of AZ-25: 2.5% w/w AZ/molasses, AZ-125: 12.5% w/w AZ/molasses, and controls (normal saline, molasses, and Ivermectin) every hour for seven hours under a stereoscopic microscope. In vivo: involved 40 rabbits (10 replicates/group). G1 served as the control negative group (normal un-infected rabbits), G2 served as the control negative group (infected rabbits), the animals in the G3 group were given a combination of AZ (40 mg/kg body weight (BW)) and molasses (5 mg/mL), and G4 served as the control to the vehicle; receiving molasses 8 mL/kg BW twice weekly for 6 weeks. Blood, serum, and tissue samples were collected at the middle and the end of the trial. AZ was made using the sonication sol–gel method. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were performed to confirm the crystal structure, purity, particle size, and oxidation states. AZ showed immunostimulant, acaricidal, and antioxidant effects with normal tissue histological structure and low tissue residual levels. Additionally, there were improvements in blood interferon-gamma, immunoglobulin (Ig) M, IgG, phagocytic activity, phagocytic index, globulin, and total protein in the AZ group. The XRD patterns of AZ were coordinated by XRD reference codes Crystallography Open Database (COD): 9016326 for Tenorite (CuO) and by XRD reference COD: 9004179 for Zincite (ZnO). The CuO and ZnO crystal sizes were 21.87 Å and 24.89 Å, respectively. The XPS spectra indicated the presence of Cu as Cu (II) and Zn as ZnO.OH and ZnO. In conclusion, AZ exhibited antioxidant, acaricidal, and immunostimulant effects, with mild residues in the brain, liver, and kidney tissues, while maintaining a normal histological structure of tissues.

The current study is a pioneer trial to verify the effectiveness of Petroselinum crispum (parsley) nanoparticles (PNPs) for fortifying physiological and biochemical biomarkers in female African catfish postexposure to bisphenol A (BPA) toxicity. The aim of this experiment is to verify the influence of PNPs for mitigating serum hemato‐biochemical alterations as well as antioxidant enzyme, and hormonal changes induced by BPA toxicity in the hepatic and ovarian tissues. Fish were equally allocated into four groups: group I as a control without any treatments, group II received a commercial diet + PNPs (4 g/kg diet). In group III, fish were subjected to 1/10 LC50 (BPA) (1.43 µg/L), while in group IV, fish got 1/10 LC50 BPA (1.43 µg/L) + PNPs (4 g/kg diet) for 60 consecutive days. Exposure to BPA showed macrocytic hypochromic anemia and leukopenia, and a noticeable elevation in glucose, alanine amino‐transferase (ALT), aspartate aminotransferase (AST), ALP, urea, creatinine, cortisol, cholesterol, and testosterone (T) hormone. Furthermore, serum AchE, estradiol (E2), follicle‐stimulating hormone (FSH), luteinizing hormone (LH), globulin, albumin, and total proteins were significantly decreased in a BPA‐exposed group. Alternatively, activities of superoxide dismutase (SOD), lipid peroxidation (LPO), and catalase (CAT) were notably augmented in the hepatic tissue and ovaries of the BPA‐supplemented fish. While total antioxidant capacity (TAC) and reduced glutathione (GSH) levels decreased in the equal tissues of exposed fish. PNPs‐supplemented diets in combination with BPA alleviated its destructive effects on the tested parameters. In conclusion, the results proved that BPA is an endocrine hormonal disruptor that induces imbalances in blood profile, hepato–renal indicators, and stress parameters, besides the occurrence of oxidative damage and reproductive dysfunction. Interestingly, PNPs have a protective role in attenuating BPA toxicity and modulating all the measured biomarkers, as well as improving the fertility of female Clarias gariepinus .