Single-Molecule Analysis of SARS-CoV-2 Double-Stranded Polynucleotides Using Solid-State Nanopore with AI-Assisted Detection and Classification: Implications for Understanding Disease Severity.

This study utilized solid-state nanopores, combined with artificial intelligence (AI), to analyze the double-stranded polynucleotides encoding angiotensin-converting enzyme 2, receptor-binding domain, and N protein, important parts of SARS-CoV-2 infection. By examining ionic current signals during DNA translocation, we revealed the dynamic interactions and structural characteristics of these nucleotide sequences and also quantified their abundance. Nanopores of sizes 3 and 10 nm were efficiently fabricated and characterized, ensuring an optimal experimental approach. Our results showed a clear relationship between DNA capture rates and concentration, proving our method's effectiveness. Notably, longer DNA sequences had higher capture rates, suggesting their importance for potential disease marker analysis. The 3 nm nanopore demonstrated superior performance in our DNA analysis. Using dwell time measurements and excluded currents, we were able to distinguish the longer DNA fragments, paving the way for a DNA length-based analysis. Overall, our research underscores the potential of nanopore technology, enhanced with AI, in analyzing COVID-19-related DNA and its implications for understanding disease severity. This provides insight into innovative diagnostic and treatment strategies.

Molecularly Imprinted Polymer-Amyloid Fibril-Based Electrochemical Biosensor for Ultrasensitive Detection of Tryptophan.

A tryptophan (Trp) sensor was investigated based on electrochemical impedance spectroscopy (EIS) of a molecularly imprinted polymer on a lysozyme amyloid fibril (MIP-AF). The MIP-AF was composed of aniline as a monomer chemically polymerized in the presence of a Trp template molecule onto the AF surface. After extracting the template molecule, the MIP-AF had cavities with a high affinity for the Trp molecules. The obtained MIP-AF demonstrated rapid Trp adsorption and substantial binding capacity (50 µM mg-1). Trp determination was studied using non-Faradaic EIS by drop drying the MIP-AF on the working electrode of a screen-printed electrode. The MIP-AF provided a large linear range (10 pM-80 µM), a low detection limit (8 pM), and high selectivity for Trp determination. Furthermore, the proposed method also indicates that the MIP-AF can be used to determine Trp in real samples such as milk and cancer cell media.

Molecular dynamics study on the effects of charged amino acid distribution under low pH condition to the unfolding of hen egg white lysozyme and formation of beta strands.

Aggregation of unfolded or misfolded proteins into amyloid fibrils can cause various diseases in humans. However, the fibrils synthesized in vitro can be developed toward useful biomaterials under some physicochemical conditions. In this study, atomistic molecular dynamics simulations were performed to address the mechanism of beta-sheet formation of the unfolded hen egg-white lysozyme (HEWL) under a high temperature and low pH. Simulations of the protonated HEWL at pH 2 and the non-protonated HEWL at pH 7 were performed at the highly elevated temperature of 450 K to accelerate the unfolding, followed by the 333 K temperature to emulate some previous in vitro studies. The simulations showed that HEWL unfolded faster, and higher beta-strand contents were observed at pH 2. In addition, one of the simulation replicas at pH 2 showed that the beta-strand forming sequence was consistent with the 'K-peptide', proposed as the core region for amyloidosis in previous experimental studies. Beta-strand formation mechanisms at the earlier stage of amyloidosis were explained in terms of the radial distribution of the amino acids. The separation between groups of positively charged sidechains from the hydrophobic core corresponded to the clustering of the hydrophobic residues and beta-strand formation.

In Vivo Analgesic, Anti-Inflammatory, and Anti-Diabetic Screening of Bacopa monnieri-Synthesized Copper Oxide Nanoparticles.

In this work, an ecofriendly approach for biogenic production of copper oxide nanoparticles (CuO-NPs) was proposed by utilizing the Bacopa monnieri leaf extract as a reducing and stabilizing agent. The synthesis of CuO-NPs was instantly confirmed by a shift in the color of the copper solution from blue to dark gray. The use of UV-visible spectroscopy revealed a strong narrow peak at 535 nm, confirming the existence of monoclinic-shaped nanoparticles. The average size of CuO-NPs was 34.4 nm, according to scanning electron microscopy and transmission electron microscopy studies. The pristine crystalline nature of CuO-NPs was confirmed by X-ray diffraction. The monoclinic form of CuO-NPs with a crystallite size of 22 nm was determined by the sharp narrow peaks corresponding to 273, 541, 698, 684, and 366 Bragg's planes at different 2θ values. The presence of different reducing metabolites on the surface of CuO was shown by Fourier transform infrared analysis. The biological efficacy of CuO-NPs was tested against Helicobacter felis, Helicobacter suis, Helicobacter salomonis. and Helicobacter bizzozeronii. H. suis was the most susceptible strain with an inhibition zone of 15.84 ± 0.89 mm at 5 mg/mL of NPs, while the most tolerant strain was H. bizzozeronii with a 13.11 ± 0.83 mm of inhibition zone. In in vivo analgesic activity, CuO-NPs showed superior efficiency compared to controls. The maximum latency time observed was 7.14 ± 0.12 s at a dose level of 400 mg/kg after 90 min, followed by 5.21 ± 0.29 s at 400 mg/kg after 60 min, demonstrating 65 and 61% of analgesia, respectively. Diclofenac sodium was used as a standard with a latency time of 8.6 ± 0.23 s. The results observed in the rat paw edema assays showed a significant inhibitory activity of the plant-mediated CuO-NPs. The percentage inhibition of edema was 74% after 48 h for the group treated with CuO-NPs compared to the control group treated with diclofenac (100 mg/kg) with 24% edema inhibition. The solution of CuO-NPs produced 82% inhibition of edema after 21 days when compared with that of the standard drug diclofenac (73%). CuO-NPs vividly lowered glucose levels in STZ-induced diabetic mice, according to our findings. Blood glucose levels were reduced by about 33.66 and 32.19% in CuO-NP and (CuO-NP + insulin) groups of mice, respectively. From the abovementioned calculations, we can easily conclude that B. monnieri-synthesized CuO-NPs will be a potential antibacterial, anti-diabetic, and anti-inflammatory agent on in vivo and in vitro basis.

Changes in Hematological Parameters with Pegylated Interferon in Chronic Hepatitis C Virus Infected Patients.

The liver is one of the most common sites of cancer in the world, hepatocellular carcinoma (HCC) predominating. HCC is the sixth most common cancer and the third leading cause of cancer related death overall. Hepatitis C is a major risk factor and HCV is a rapid spreading virus which has become a problem globally, including in Pakistan. Interferon alpha therapy is used against HCV disease to regulate cell reproduction and to boost the immune system. In minute amounts interferon alpha is produced naturally by the immune system in HCV patients in response to hepatitis C virus and binds to receptors in the target cells and starts transcription of 20-30 genes due to which it develops an antiviral influence. Interferon is also administered artificially to overcome HCV disease and remove the biological effect of the virus from the infected site. The use of interferon or Peg-IFN plus Ribavirin treatment is also associated with adverse effects on body. For the current study, a convenient sample of 156 HCV positive patients of both males and females were taken. To collect blood CP and ALT, a reduction of level data and other important information were collected from the patients at regular intervals. Findings were 11.4 % in the red blood cells (RBC), 9.64 % in the total leukocyte count (WBC), 8.4 % in the hemoglobin levels (HB), 30.3 % in the platelet (Plt) count in both sexes. There was significant reduction in ALT levels due to Pegylated interferon plus ribavirin therapy. Hence strict haemotological monitoring of blood CP and ALT levels is necessary at regular intervals to reduce severe side effects which may lead to morbidity and mortality.

Incidence of Active HCV infection amongst Blood Donors of Mardan District, Pakistan.

Hepatitis C is an ailment of liver caused by hepatitis C virus (HCV) infection. About 3% of the world population is infected by this virus. HCV infection is a leading reason for liver cirrhosis and therefore a major source of hepatocellular carcinoma. The study focused on the incidence of active HCV infection in blood donors of Mardan district of KPK, Pakistan. A total of 5318 blood donors were inspected for the presence of anti-HCV antibodies and HCV-RNA using ICT (immune-chromatographic test), ELISA and RT-PCR at Mardan Medical Complex (MMC), Mardan. Out of these, 157 (2.95%) were positive by ICT, 60 (1.12%) by ELISA and 56 (1.05%) for HCV-RNA. The frequency of active HCV infectivity amongst the blood donors from district Mardan, KPK Pakistan was 1.05 %. Application of strict measures during blood donor selection and use of proper screening assays such as ELISA in place of ICT devices can give a more accurate picture so that the incidence of this viral infection in HCV negative blood recipients can be reduced.

PAIgG and PAIgM levels in secondary dengue virus infections lead to thrombocytopenia in patients from KP, Pakistan

Objective:To understand the impact of platelet associated immunoglobulin G (PAIgG)/ platelet associated immunoglobulin M (PAIgM) on severity of dengue virus infection leading to thrombocytopenia. Methods: In this study we examined a total of 52 patients who were having secondary infection of dengue in acute phase by using competitive ELISA. Results: A decrease in the platelet count was observed at the acute phase of infection while all along the recovery stage the count of platelet was significantly increased. A significant decrease was observed inPAIgG andPAIgM in these subjects. Inverse correlation was found between platelets count andPAIgG/PAIgM among the subjects studied. In the platelets elution from ten subjects, anti-dengue virus immunoglobulin G and immunoglobulin M were observed.PAIgG andPAIgM with inclined levels were higher in dengue hemorrhagic fever than the classical dengue fever. In the development of dengue hemorrhagic feverPAIgM inclined level was independently associated with high specificity, showing a possible indication of dengue hemorrhagic fever. Conclusions: This study suggests that in secondary dengue virus infection, thePAIgGand PAIgM levels, and the activity of anti-dengue virus play key roles, both in the development and severity of the disease.

Weight loss in HCV patients can be used as a surrogate marker for evaluation of interferon (IFN-α) treatment efficacy - a prospective pilot study.

The aim of the study was to investigate whether weight loss followed the same pattern in HCV patients ('responders' and 'non-responders) after interferon (IFN-α) treatment. A total of 20 male HCV positive patients (mean age 33.1±9.9) in Peshawar, Pakistan participated in this study. They were initially tested as HCV positive, and were given IFN/Ribavarin treatment for 6 months. Changes in body weight (BW), lean body mass (LBM) and body fat (BF) were monitored on monthly basis. End to treatment response (ETR) was established by a final undetectable HCV RNA in serum at the end of therapy and the patients were categorized as either 'responders' or 'non-responders'. The results show a total of 12 out of 20 patients as 'responders' (60%). All patients lost weight and the mean weight loss in 'responders' and 'non-responders' was 6.2 (±1.5) and 5.8 (±1.4) Kg, respectively. There was a significant difference in the mean change in BW, LBM, and BF between 'responders' and 'non-responders' during the last 3 month period only. This suggests that difference in drug response in HCV starts from month 4 and onwards (i.e. during the last 3 months). In conclusion, weight trends during treatment should be monitored as weight loss may be used as a surrogate marker for ERT to the current standard of care.

The effects and underlying mechanism of interferon therapy on body weight and body composition.

Body weight changes in HCV patients on interferon therapy are well documented. However, the underlying mechanism involved in these changes is poorly understood and rarely reported. The main objectives of this review are to 1) discuss changes in body weight and other compartments of body composition, particularly, body fat, and 2) to discuss the underlying mechanism for these changes. The literature review suggests weight loss (12-29%) as a function of interferon therapy is common, affecting up to 90% of HCV patients. Whilst, loss in weight means proportionate loss in other body compartments (lean body mass and body fat, in particular) data on changes in segmented body composition are fragmentary. The possible mechanisms underlying weight loss or changes in other body composition have been reported and these include suppressed appetite due to induction of TNF by IFN, a decrease in serum leptin level, and importantly mitochondrial damage induced by the therapy. It is, therefore, suggested that close monitoring of chronic HCV patients receiving PEG-IFN and/or ribavirin for side effects of these drugs, particularly those related to weight loss, is vitally important from clinical point of view.

Higher estimates of daily dietary net endogenous acid production (NEAP) in the elderly as compared to the young in a healthy, free-living elderly population of Pakistan.

Dietary intake has been shown to influence the acid-base balance in human subjects; however, this phenomenon is poorly understood and rarely reported for the least well-studied segment of older people in a developing country. The aims of the present study were to: (1) quantify estimates of daily net endogenous acid production (NEAP) (mEq/d) in a sample of otherwise healthy elderly aged 50 years and above; and (2) compare NEAP between the elderly and young to determine the effects of aging, which could contribute to changes in the acid-base balance. Analyses were carried out among 526 elderly and 131 young participants (aged 50-80 and 23-28 years, respectively), all of whom were free of discernible disease, nonsmokers, and not on any chronic medication. Selected anthropometric factors were measured and 24-hour dietary recall was recorded. We used two measures to characterize dietary acid load: (1) NEAP estimated as the dietary potential renal acid load plus organic acid excretion, the latter as a multiple of estimated body surface area; and (2) estimated NEAP based on protein and K. For the young and elderly, the ranges of NEAP were 12.1-67.8 mEq/d and 2.0-78.3 mEq/d, respectively. Regardless of the method used, the mean dietary acid-base balance (NEAP) was significantly higher for the elderly than the young (P = 0.0035 for NEAP [elderly, 44.1 mEq/d versus young 40.1 mEq/d]; and P = 0.0035 for the protein:potassium ratio [elderly, 1.4 mEq/d versus young 1.1 mEq/d]). A positive and significant correlation was found between NEAP and energy, protein, and phosphorus (P < 0.05 for all trends). The findings from this study provide evidence of the relatively higher production of NEAP in older people, possibly as an effect of higher consumption of certain acid-producing foods by the elderly.