A new label free spiral sensor using impedance spectroscopy to characterize hepatocellular carcinoma in tissue and serum samples.

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.

Exposure to oxyfluorfen-induced hematobiochemical alterations, oxidative stress, genotoxicity, and disruption of sex hormones in male African catfish and the potential to confront by Chlorella vulgaris.

The present study evaluated the effect of chronic exposure to oxyfluorfen (OXY) on different physiological responses of male African catfish, Clarias gariepinus, and the ameliorative effect of Chlorella vulgaris. The fish (160 ± 5.10 g) were exposed to 1/20 LC50 of OXY (0.58 mg/L) for 60 consecutive days with or without co-administration of C. vulgaris (25 g/kg diet) in triplicate groups. The results revealed that chronic exposure to a sublethal level of OXY induced severe anemia and leukopenia. OXY-exposed fish experienced hypoproteinemia, marked lower AchE levels, and a significant increase in glucose, liver, and kidney function biomarkers. The DNA fragmentation of the liver increased by 15 % in fish compared to the control. On the other hand, lipid peroxidation, superoxide dismutase, and catalase activities were markedly increased in the liver and testes homogenates of the OXY-exposed fish. Meanwhile, total antioxidant capacity and glutathione S-transferase levels declined in the same tissues. Exposure to OXY induced a significant reduction in testosterone and luteinizing hormone levels and a significant increase in follicle stimulating hormone and estradiol. Meanwhile, C. vulgaris dietary supplementation succeeded in alleviating the negative impact of OXY on hematobiochemical parameters and restoring the antioxidant balance in the liver and testes. Furthermore, it ameliorated endocrine disruption and repaired sex hormone levels. In conclusion, exposure to OXY could induce systemic stress, oxidative stress, and endocrine disruption in male C. gariepinus. The dietary supplementation of C. vulgaris could be a potential protective strategy against the toxicity of OXY.

Age-related changes in testicular morphometry and function in Egyptian donkeys.

This study aimed to assess the effects of age on testicular morphometry and function in donkeys. Testes and epididymides of 57 donkeys were harvested immediately after slaughtering. The donkeys were grouped: young (1-4 years old, n = 13); adult (5-15 years old, n = 25) and aged (>15 years old, n = 19). Each testis and epididymis were weighed separately. Testicular volume was calculated. Epididymal sperm was harvested by retrograde flushing method, and sperm parameters were evaluated. The testicular parenchyma was immunolabelled for BAX and COX2. Adult and aged donkeys had greater testicular weight and volume than young (p < .05). Epididymal sperm concentration, motility and viability were greater (p < .05) in adults and aged (931.8 ± 39.3 and 858.2 ± 33.2 × 106 /ml) than in young animals (316.3 ± 72.8 × 106 /ml). Aged donkeys had a higher percentage of morphological sperm defects than the other categories (p < .05). Histological examination revealed the presence of age-related degenerative changes in testicular tissue of donkeys. Aged donkeys had higher COX2 protein expression than adult and young donkeys. BAX protein was overly expressed in adults than aged or young animals. In conclusion, advancement of age affects the testicular morphometry and function in donkeys.

In Silico Identification and Clinical Validation of a Novel Long Non-Coding RNA/mRNA/miRNA Molecular Network for Potential Biomarkers for Discriminating SARS CoV-2 Infection Severity.

(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.

Effect of body condition score on the testicular biometrical measures, semen characteristics and testosterone level in jackass.

This study was undertaken to evaluate the effect of body condition score (BCS) on testicle and epididymis biometrics, semen characteristics and testosterone level in Egyptian Jack. This study was conducted on 50 mature Jacks divided according to their body condition score into four groups: Poor (G1), moderate (G2), good (G3) and fat (G4). The complete testis was collected immediately after execution in the Giza Zoo abattoir; then, the epididymis was carefully dissected at the testicular junction. Biometrical measures including length, weight and volume were determined for the right and left testis and epididymis. Also, epididymal sperm was collected from all examined animas and evaluated for sperm concentration, progressive motility, viability and sperm abnormalities. Serum samples were collected for determination of total testosterone level. Results showed that the body condition score of the examined animal affects their biometrical measure of testicles and epididymis. There is a significant decrease (p < .05) in biometrical measures for the testicles and epididymis, sperm concentration, motility, viability and testosterone level in poor BCS animals (G1). The highest values were recorded in Good BCS (G3) Jacks. Conclusion: Jacks with good BCS (G3) should be selected for breeding activity in donkey.

Integration of transcriptomes analysis with spectral signature of total RNA for generation of affordable remote sensing of Hepatocellular carcinoma in serum clinical specimens.

Hepatocellular carcinoma (HCC) is a major global health problem with about 841,000 new cases and 782,000 deaths annually, due to lacking early biomarker/s, and centralized diagnosis. Transcriptomes research despite its infancy has proved excellence in its implementation in identifying a coherent specific cancer RNAs differential expression. However, results are sometimes overlapped by other cancer types which negatively affecting specificity, plus the high cost of the equipment used. Hyperspectral imaging (HSI) is an advanced tool with unique, spectroscopic features, is an emerging tool that has widely been used in cancer detection. Herein, a pilot study has been performed for HCC diagnosis, by exploiting HIS properties and the analysis of the transcriptome for the development of non-invasive remote HCC sensing. HSI data cube images of the sera extracted total RNA have been analyzed in HCC, normal subject, liver benign tumor, and chronic HCV with cirrhotic/non-cirrhotic liver groups. Data analyses have revealed a specific spectral signature for all groups and can be easily discriminated; at the computed optimum wavelength. Moreover, we have developed a simple setup based on a commercial laser pointer for sample illumination and a Smartphone CCD camera, with HSI consistent data output. We hypothesized that RNA differential expression and its spatial organization/folding are the key players in the obtained spectral signatures. To the best of our knowledge, we are the first to use HSI for sensing cancer based on total RNA in serum, using a Smartphone CCD camera/laser pointer. The proposed biosensor is simple, rapid (2 min), and affordable with specificity and sensitivity of more than 98% and high accuracy.

A novel HCV electrochemical biosensor based on a polyaniline@Ni-MOF nanocomposite.

Hepatitis-C virus ribonucleic acid (HCV-RNA) recognition and quantification based on real-time polymerase chain reaction (RT-PCR) is key to infection control, management, and response to treatment due to its specificity, sensitivity, and quantification capabilities. However, the high cost, time requirements, and need for sophisticated laboratory infrastructure have limited the use of this method in rapid screening, blood banks, and point-of-care testing (POCT). In this work, a novel label-free electrochemical biosensor constructed using a polyaniline@nickel metal-organic framework (Ni-MOF) nanocomposite was developed for direct detection of unamplified HCV nucleic acid. A robust biosensor was fabricated using smooth layer-by-layer deposition of the polyaniline@Ni-MOF nanocomposite, deoxyribonucleic acid (DNA) probe, and bovine serum albumin (BSA) onto a glassy carbon electrode (GCE) and was subsequently monitored real-time via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The sensitivity and specificity of the newly developed biosensor were specifically examined using the EIS approach. The results revealed that the novel biosensor is highly efficient in quantitative sensing of the HCV target in the presence of nonspecific nucleic acids over the range of 1 fM-100 nM with a detection limit of 0.75 fM (at a S/N ratio of 3). To the best of the authors' knowledge, the proposed biosensor is superior to other MOF platforms. These research findings are expected to have a positive influence on the quantitative detection of HCV RNA and other nucleic acids by offering exceptional accuracy and cost effectiveness, especially in low resource countries. Moreover, this biosensor could be simply adopted for full automation and used in point-of-care testing.

Nucleic acids biosensors based on metal-organic framework (MOF): Paving the way to clinical laboratory diagnosis.

Development of ultra-sensitive, high specific and cost-effective nucleic acids (NAs) biosensors is critical for early diagnosis of cancer, genetic diseases and follows up response to treatment. Metal-organic frameworks (MOFs) as sensing materials underwent significant development in recent years due to their unique merits, such as structural diversity, tunable pore scale, large surface area, remarkable adsorption affinities, and good thermal stability. MOFs have shown potential contribution in nucleic acids biosensors research. Herein, a comprehensive overview on NAs biosensors state of the art based on MOFs has been discussed extensively, including different MOFs platforms sensing strategies (fluorescence, electrochemistry, electrochemiluminescence, and colorimetric techniques), their analytical performance and figures of merit in clinical diagnostics, with the future perspective in introducing MOFs in clinical laboratory diagnostics. Moreover, the different MOFs synthesis methods have been highlighted to serve as a guide for the researchers in selecting the appropriate platform that suits their research needs, and applications.

Gold nanoparticles - an optical biosensor for RNA quantification for cancer and neurologic disorders diagnosis.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: A linear correlation exists between the GAG assay and the qPCR for the quantification of the TOP1 and TDP2 mRNA transcripts (101-104 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. CONCLUSION: 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.

Gold aggregating gold: A novel nanoparticle biosensor approach for the direct quantification of hepatitis C virus RNA in clinical samples.

The affordable and reliable detection of Hepatitis C Virus (HCV) RNA is a cornerstone in the management and control of infection, affecting approximately 3% of the global population. However, the existing technologies are expensive, labor intensive and time consuming, posing significant limitations to their wide-scale exploitation, particularly in economically deprived populations. Here, we utilized the unique optical and physicochemical properties of gold nanoparticles (AuNPs) to develop a novel assay platform shown to be rapid and robust in sensing and quantifying unamplified HCV RNA in clinical samples. The assay is based on inducing aggregation of citrate AuNPs decorated with a specific nucleic acid probe. Two types of cationic AuNPs, cysteamine and CTAB capped, were compared to achieve maximum assay performance. The technology is simple, rapid, cost effective and quantitative with 93.3% sensitivity, high specificity and detection limit of 4.57IU/µl. Finally, our data suggest that RNA folding impact the aggregation behavior of the functionalized AuNPs, with broader applications in other nucleic acid detection technologies.

The prognostic value of histidine-rich glycoprotein RNA in breast tissue using unmodified gold nanoparticles assay.

The aim of is this study is to explore the role of tissue histidine-rich glycoprotein (HRG) RNA as a promising clinically useful biomarker for breast cancer patients prognosis using nanogold assay. Expression of the HRG RNA was assessed by gold nanoparticles and conventional RT-PCR after purification by magnetic nanoparticles in breast tissue samples. The study included 120 patients, 60 of which were histologically proven breast carcinoma cases, 30 had benign breast lesions and 30 were healthy individuals who had undergone reductive plastic surgery. ER, PR and HER2 status were also investigated. The prognostic significance of tissue HRG RNA expression in breast cancer was explored. The magnetic nanoparticles coated with specific thiol modified oligonucleotide probe were used successfully in purification of HRG RNA from breast tissue total RNAs with satisfactory yield. The developed HRG AuNPs assay had a sensitivity and a specificity of 90 %, and a detection limit of 1.5 nmol/l. The concordance rate between the HRG AuNPs assay with RT-PCR after RNA purification using magnetic nanoparticles was 93.3 %. The median follow-up period was 60 months. Among traditional prognostic biomarkers, HRG was a significant independent prognostic marker in relapse-free survival (RFS). HRG RNA is an independent prognostic marker for breast cancer and can be detected using gold NPs assay, which is rapid, sensitive, specific, inexpensive to extend the value for breast cancer prognosis.

Direct detection of unamplified hepatoma upregulated protein RNA in urine using gold nanoparticles for bladder cancer diagnosis.

OBJECTIVE: To develop a gold nanoparticle (AuNP) assay for direct detection of unamplified HURP RNA in urine. DESIGN AND METHODS: HURP RNA was extracted from urine samples (50 bladder carcinoma patients, 25 benign bladder lesions, and 25 controls) and further purified using magnetic nanoparticles (MNPs), functionalized with HURP RNA-specific oligonucleotides, and then detected by RT-PCR or gold nanoparticles. RESULTS: The developed HURP RNA AuNP assay has a sensitivity and a specificity of 88.5% and 94%, respectively, and a detection limit of 2.4 nmol/L. The concordance between the HURP AuNP assay with RT-PCR after RNA purification using functionalized MNPs was 97%. CONCLUSIONS: The developed colorimetric HURP RNA AuNP assay is sensitive, simple, and can aid noninvasive diagnosis of bladder cancer.

Detection of unamplified HCV RNA in serum using a novel two metallic nanoparticle platform.

BACKGROUND: The unique properties of metallic nanoparticles have enabled their utilization in biosensing applications. A novel assay for the detection of hepatitis C virus (HCV) RNA in serum specimens has been developed using magnetic nanoparticles and unmodified cationic gold nanoparticles (AuNPs). METHODS: HCV RNA was extracted using magnetic nanoparticles functionalized with an oligonucleotide specific to HCV RNA. Extracted RNA is reacted with oligonucleotide sequence specific for HCV RNA in presence of unmodified cationic AuNPs. In positive samples, AuNPs are aligned onto the phosphate backbone of the RNA and their aggregation changes the solution color from red to blue. In the absence of target, solution color remains red. The assay has been tested on 50 serum clinical samples (25 HCV positive and 25 controls). RESULTS: The dual nanoparticles assay detected HCV RNA in serum and generated comparable results to real-time PCR. The assay had specificity and a sensitivity of 96% and 96.5%, respectively, and a detection limit of 15 IU/mL. CONCLUSIONS: The developed colorimetric dual nanoparticles HCV RNA assay is simple and inexpensive and can be used for rapid detection of unamplified HCV RNA in serum. Similar sensing platforms can be developed to detect other nucleic acid targets.

Influence of "glow discharge plasma" as an external stimulus on the self-assembly, morphology and binding affinity of gold nanoparticle-streptavidin conjugates.

In this study, we investigate the influence of glow discharge plasma (GDP) on the self-assembly, morphology and binding affinity of streptavidin coated gold nanoparticles (Au-NP-SV) and biotinylated antibody (bAb) adsorbed on a highly oriented pyrolytic graphite (HOPG) substrate. Atomic force microscope (AFM) was used to image the pre- and post-GDP treated samples. The analysis of the AFM images showed a considerable change in the aggregation and morphology of Au-NP-conjugates after treatment with GDP. To our knowledge, this is the first report on using GDP to enhance and speed-up the aggregation (sintering) of adsorbed NP biomolecular conjugates. These results show a promising route that could be generalized for other NPs and their conjugates. It can also be considered as an alternative and cheap aggregation method for controlling the binding affinity of biomolecular species on different surfaces with interesting applications.

Direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles.

BACKGROUND: Gold nanoparticles (AuNPs) exhibit a unique phenomenon known as Surface Plasmon Resonance, which is responsible for their intense red color. This color changes to blue upon aggregation of AuNPs. OBJECTIVE: This work aims to develop a rapid, simple and cheap assay for direct detection of unamplified HCV RNA extracted from clinical samples using unmodified AuNPs. METHODS: Serum samples were collected from healthy volunteers (n=45) and chronic HCV patients (n=30). Extracted RNA, hybridization buffer containing PBS, and a primer targeting the 5'UTR of HCV were mixed. The mixture was denatured, annealed, and then cooled to room temperature for 10 min followed by addition of AuNPs. RESULTS: Salt, primer, AuNPs concentrations and annealing temperature and time were all optimized. In HCV positive specimens, the color of the solution changed from red to blue within 1 min. The assay has a sensitivity of 92%, a specificity of 88.9%, and a detection limit of 50 copies/reaction. CONCLUSIONS: To our knowledge, this is the first assay that allows the detection of unamplified HCV RNA in clinical specimens using unmodified AuNPs. The developed assay is highly sensitive, has a turnaround time of 30 min, and eliminates the need for thermal cycling and detection instruments.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy.

AIM: To investigate the effects of mutations in domain III of the hepatitis C virus (HCV) internal ribosome entry sequences (IRES) on the response of chronic HCV genotype 4a patients to interferon therapy. METHODS: HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain III was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustalW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing. RESULTS: Analysis of RNA secondary structure revealed that insertions in domain III altered Watson-Crick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain III were statistically more prevalent in sustained viral response patients (SVR, n = 14) as compared to breakthrough (BT, n = 5) patients. CONCLUSION: The influence of mutations within domain III on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain III.