Advancements in SARS-CoV-2 detection: Navigating the molecular landscape and diagnostic technologies.

According to information from the World Health Organization, the world has experienced about 430 million cases of COVID-19, a world-wide health crisis caused by the SARS-CoV-2 virus. This outbreak, originating from China in 2019, has led to nearly 6 million deaths worldwide. As the number of confirmed infections continues to rise, the need for cutting-edge techniques that can detect SARS-CoV-2 infections early and accurately has become more critical. To address this, the Federal Drug Administration (FDA) has issued emergency use authorizations (EUAs) for a wide range of diagnostic tools. These include tests based on detecting nucleic acids and antigen-antibody reactions. The quantitative real-time reverse transcription PCR (qRT-PCR) assay stands out as the gold standard for early virus detection. However, despite its accuracy, qRT-PCR has limitations, such as complex testing protocols and a risk of false negatives, which drive the continuous improvement in nucleic acid and serological testing approaches. The emergence of highly contagious variants of the coronavirus, such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), has increased the need for tests that can specifically identify these mutations. This article explores both nucleic acid-based and antigen-antibody serological assays, assessing the performance of recently approved FDA tests and those documented in scientific research, especially in identifying new coronavirus strains.

Bakuchiol inhibits Pseudomonas aeruginosa's quorum sensing-dependent biofilm formation by selectively inhibiting its transcriptional activator protein LasR.

In the present study, we characterized Bakuchiol (Bak) as a new potent quorum sensing (QS) inhibitor against Pseudomonas aeruginosa biofilm formation. Upon extensive in vitro investigations, Bak was found to suppress the P. aeruginosa biofilm formation (75.5 % inhibition) and its associated virulence factor e.g., pyocyanin and rhamnolipids (% of inhibition = 71.5 % and 66.9 %, respectively). Upon LuxR-type receptors assay, Bak was found to selectively inhibit P. aeruginosa's LasR in a dose-dependent manner. Further in-depth molecular investigations (e.g., sedimentation velocity and thermal shift assays) revealed that Bak destabilized LasR upon binding and disrupted its functioning quaternary structure (i.e., the functioning dimeric form). The subsequent modeling and molecular dynamics (MD) simulations explained in more molecular detail how Bak interacts with LasR and how it can induce its dimeric form disruption. In conclusion, our study identified Bak as a potent and specific LasR antagonist that should be widely used as a chemical probe of QS in P. aeruginosa, offering new insights into LasR antagonism processes. The new findings shed light on the cryptic world of LuxR-type QS in this important opportunistic pathogen.

Bio-organic fertilizers promote yield, chemical composition, and antioxidant and antimicrobial activities of essential oil in fennel (Foeniculum vulgare) seeds.

The aromatic fennel plant (Foeniculum vulgare Miller) is cultivated worldwide due to its high nutritional and medicinal values. The aim of the current study was to determine the effect of the application of bio-organic fertilization (BOF), farmyard manure (FM) or poultry manure (PM), either individually or combined with Lactobacillus plantarum (LP) and/or Lactococcus lactis (LL) on the yield, chemical composition, and antioxidative and antimicrobial activities of fennel seed essential oil (FSEO). In general, PM + LP + LL and FM + LP + LL showed the best results compared to any of the applications of BOF. Among the seventeen identified FSEO components, trans-anethole (78.90 and 91.4%), fenchone (3.35 and 10.10%), limonene (2.94 and 8.62%), and estragole (0.50 and 4.29%) were highly abundant in PM + LP + LL and FM + LP + LL, respectively. In addition, PM + LP + LL and FM + LP + LL exhibited the lowest half-maximal inhibitory concentration (IC50) values of 8.11 and 9.01 µg mL-1, respectively, compared to L-ascorbic acid (IC50 = 35.90 µg mL-1). We also observed a significant (P > 0.05) difference in the free radical scavenging activity of FSEO in the triple treatments. The in vitro study using FSEO obtained from PM + LP + LL or FM + LP + LL showed the largest inhibition zones against all tested Gram positive and Gram negative bacterial strains as well as pathogenic fungi. This suggests that the triple application has suppressive effects against a wide range of foodborne bacterial and fungal pathogens. This study provides the first in-depth analysis of Egyptian fennel seeds processed utilizing BOF treatments, yielding high-quality FSEO that could be used in industrial applications.

Biogenic silver nanoparticles eradicate of Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (MRSA) isolated from the sputum of COVID-19 patients.

In recent investigations, secondary bacterial infections were found to be strongly related to mortality in COVID-19 patients. In addition, Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria played an important role in the series of bacterial infections that accompany infection in COVID-19. The objective of the present study was to investigate the ability of biosynthesized silver nanoparticles from strawberries (Fragaria ananassa L.) leaf extract without a chemical catalyst to inhibit Gram-negative P. aeruginosa and Gram-positive Staph aureus isolated from COVID-19 patient's sputum. A wide range of measurements was performed on the synthesized AgNPs, including UV-vis, SEM, TEM, EDX, DLS, ζ -potential, XRD, and FTIR. UV-Visible spectral showed the absorbance at the wavelength 398 nm with an increase in the color intensity of the mixture after 8 h passed at the time of preparation confirming the high stability of the FA-AgNPs in the dark at room temperature. SEM and TEM measurements confirmed AgNPs with size ranges of ∼40-∼50 nm, whereas the DLS study confirmed their average hydrodynamic size as ∼53 nm. Furthermore, Ag NPs. EDX analysis showed the presence of the following elements: oxygen (40.46%), and silver (59.54%). Biosynthesized FA-AgNPs (ζ = -17.5 ± 3.1 mV) showed concentration-dependent antimicrobial activity for 48 h in both pathogenic strains. MTT tests showed concentration-dependent and line-specific effects of FA-AgNPs on cancer MCF-7 and normal liver WRL-68 cell cultures. According to the results, synthetic FA-AgNPs obtained through an environmentally friendly biological process are inexpensive and may inhibit the growth of bacteria isolated from COVID-19 patients.

Limoniastrum monopetalum-Mediated Nanoparticles and Biomedicines: In Silico Study and Molecular Prediction of Biomolecules.

An in silico approach applying computer-simulated models helps enhance biomedicines by sightseeing the pharmacology of potential therapeutics. Currently, an in silico study combined with in vitro assays investigated the antimicrobial ability of Limoniastrum monopetalum and silver nanoparticles (AgNPs) fabricated by its aid. AgNPs mediated by L. monopetalum were characterized using FTIR, TEM, SEM, and DLS. L. monopetalum metabolites were detected by QTOF-LCMS and assessed using an in silico study for pharmacological properties. The antibacterial ability of an L. monopetalum extract and AgNPs was investigated. PASS Online predictions and the swissADME web server were used for antibacterial activity and potential molecular target metabolites, respectively. Spherical AgNPs with a 68.79 nm average size diameter were obtained. Twelve biomolecules (ferulic acid, trihydroxy-octadecenoic acid, catechin, pinoresinol, gallic acid, myricetin, 6-hydroxyluteolin, 6,7-dihydroxy-5-methoxy 7-O-ß-d-glucopyranoside, methyl gallate, isorhamnetin, chlorogenic acid, 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 6-O-(6-deoxy-ß-l-mannopyranosyl)-ß-d-glucopyranoside) were identified. The L. monopetalum extract and AgNPs displayed antibacterial effects. The computational study suggested that L. Monopetalum metabolites could hold promising antibacterial activity with minimal toxicity and an acceptable pharmaceutical profile. The in silico approach indicated that metabolites 8 and 12 have the highest antibacterial activity, and swissADME web server results suggested the CA II enzyme as a potential molecular target for both metabolites. Novel therapeutic agents could be discovered using in silico molecular target prediction combined with in vitro studies. Among L. Monopetalum metabolites, metabolite 12 could serve as a starting point for potential antibacterial treatment for several human bacterial infections.

Pioglitazone Ameliorates Hippocampal Neurodegeneration, Disturbances in Glucose Metabolism and AKT/mTOR Signaling Pathways in Pentyelenetetrazole-Kindled Mice.

Disturbance of glucose metabolism, nerve growth factor (NGF) and m-TOR signaling have been associated with the pathophysiology of epilepsy. Pioglitazone (PGZ) is an anti-diabetic drug that shows a protective effect in neurodegenerative diseases including epilepsy; however, its exact mechanism is not fully elucidated. The present study aimed to investigate the potential neuroprotective effect of PGZ in pentylenetetrazole (PTZ) kindled seizure in mice. Swiss male albino mice were randomly distributed into four groups, each having six mice. Group 1 was considered the control. Epilepsy was induced by PTZ (35 mg/kg i.p.) thrice a week for a total of 15 injections in all other groups. Group 2 was considered the untreated PTZ group while Group 3 and Group 4 were treated by PGZ prior to PTZ injection at two dose levels (5 and 10 mg/kg p.o., respectively). Seizure activity was evaluated after each PTZ injection according to the Fischer and Kittner scoring system. At the end of the experiment, animals were sacrificed under deep anesthesia and the hippocampus was isolated for analysis of glucose transporters by RT-PCR, nerve growth factor (NGF) by ELISA and mTOR by western blotting, in addition to histopathological investigation. The PTZ-treated group showed a significant rise in seizure score, NGF and m-TOR hyperactivation, along with histological abnormalities compared to the control group. Treatment with PGZ demonstrated a significant decrease in NGF, seizure score, m-TOR, GLUT-1 and GLUT-3 in comparison to the PTZ group. In addition, improvement of histological features was observed in both PGZ treated groups. These findings suggest that PGZ provides its neuroprotective effect through modulating m-TOR signaling, glucose metabolism and NGF levels.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Highly branched poly(ß-amino ester)s for gene delivery in hereditary skin diseases.

Non-viral gene therapy for hereditary skin diseases is an attractive prospect. However, research efforts dedicated to this area are rare. Taking advantage of the branched structural possibilities of polymeric vectors, we have developed a gene delivery platform for the treatment of an incurable monogenic skin disease - recessive dystrophic epidermolysis bullosa (RDEB) - based on highly branched poly(ß-amino ester)s (HPAEs). The screening of HPAEs and optimization of therapeutic gene constructs, together with evaluation of the combined system for gene transfection, were comprehensively reviewed. The successful restoration of type VII collagen (C7) expression both in vitro and in vivo highlights HPAEs as a promising generation of polymeric vectors for RDEB gene therapy into the clinic. Considering that the treatment of patients with genetic cutaneous disorders, such as other subtypes of epidermolysis bullosa, pachyonychia congenita, ichthyosis and Netherton syndrome, remains challenging, the success of HPAEs in RDEB treatment indicates that the development of viable polymeric gene delivery vectors could potentially expedite the translation of gene therapy for these diseases from bench to bedside.

Cartilage-Derived Progenitor Cell-Laden Injectable Hydrogel-An Approach for Cartilage Tissue Regeneration.

Cartilage-derived progenitor cells (CPCs) with the capability of self-renewal and multilineage differentiation have been identified as a suitable cell source for cartilage tissue regeneration. Despite decades of development in cell-delivery techniques, improved approaches are still required to maintain cell viability, provide a supportive environment, and implement appropriate cues to guide cartilage regeneration. This research work develops an injectable in situ gelation system as a cell carrier for CPCs to overcome cell-delivery drawbacks. The hydrogel was fabricated through a thiol-ene Michael addition reaction by cross-linking thiol-functionalized hyaluronic acid and hyperbranched poly(ethylene glycol) multi-acrylate macromer. The sol-gel transition, mechanical properties, microstructure, and degradation profile of the hydrogels were evaluated to ensure physical support, cell migration, and nutrient exchange within the system. Encapsulated CPCs maintained a high level of cell viability and proliferation property. Reverse transcription-quantitative real-time polymerase chain reaction confirmed that the extracellular matrix (ECM) secretion was enhanced under chondrogenic conditions. Moreover, the downregulated inflammation gene expression indicated the anti-inflammation ability of encapsulated CPCs. The study demonstrates that this rapid in situ forming hydrogel has excellent potential as a CPC delivery carrier by accelerating ECM production and retaining the phenotype and function of encapsulated CPCs.

Efficient and Robust Highly Branched Poly(ß-amino ester)/Minicircle COL7A1 Polymeric Nanoparticles for Gene Delivery to Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe congenital skin fragility disease caused by COL7A1 mutations that result in type VII collagen (C7) deficiency. Herein, we report a synergistic polyplex system that can efficiently restore C7 expression in RDEB keratinocytes. A highly branched multifunctional poly(ß-amino ester) (HPAE), termed as HC32-122, was optimized systematically as the high-performance gene delivery vector for keratinocytes, achieving much higher transfection capability than polyethylenimine, SuperFect, and Lipofectamine 2000 without inducing obvious cytotoxicity. Concurrently, a 12 kb length minicircle DNA encoding ∼9 kb full-length COL7A1 (MCC7) devoid of bacterial sequence was biosynthesized as the therapeutic gene. Combining the highly potent polymer and the miniaturized gene structure, HC32-122/MCC7 polyplexes achieve 96.4% cellular uptake efficiency, 4019-fold COL7A1 mRNA enhancement, and robust recombinant C7 expression. Structure-property investigations reveal that HC32-122 can effectively condense MCC7 to form small, uniform, compact, and positively charged spherical nanoparticles with high DNA release flexibility. Moreover, formulation study shows that sucrose is conductive to lyophilized HC32-122/DNA polyplexes for maintaining the transfection capability. Direct frozen polyplexes can maintain full gene transfection capability after one-year storage. High efficiency, biocompatibility, facile manipulation, and long-term stability make the HC32-122/MCC7 system a promising bench-to-bed candidate for treating the debilitating RDEB.

Highly branched poly(ß-amino ester) delivery of minicircle DNA for transfection of neurodegenerative disease related cells.

Current therapies for most neurodegenerative disorders are only symptomatic in nature and do not change the course of the disease. Gene therapy plays an important role in disease modifying therapeutic strategies. Herein, we have designed and optimized a series of highly branched poly(ß-amino ester)s (HPAEs) containing biodegradable disulfide units in the HPAE backbone (HPAESS) and guanidine moieties (HPAESG) at the extremities. The optimized polymers are used to deliver minicircle DNA to multipotent adipose derived stem cells (ADSCs) and astrocytes, and high transfection efficiency is achieved (77% in human ADSCs and 52% in primary astrocytes) whilst preserving over 90% cell viability. Furthermore, the top-performing candidate mediates high levels of nerve growth factor (NGF) secretion from astrocytes, causing neurite outgrowth from a model neuron cell line. This synergistic gene delivery system provides a viable method for highly efficient non-viral transfection of ADSCs and astrocytes.

Manipulation of Transgene Expression in Fibroblast Cells by a Multifunctional Linear-Branched Hybrid Poly(ß-Amino Ester) Synthesized through an Oligomer Combination Approach.

Delivery of functional genetic materials into fibroblast cells to manipulate the transgene expression is of great significance in skin gene therapy. Despite numerous polymeric gene delivery systems having been developed, highly safe and efficient fibroblast gene transfection has not yet been achieved. Here, through a new linear oligomer combination strategy, linear poly(ß-amino ester) oligomers are connected by the branching units, forming a new type of poly(ß-amino ester). This new multifunctional linear-branched hybrid poly(ß-amino ester) (LBPAE) shows high-performance fibroblast gene transfection. In human primary dermal fibroblasts (HPDFs) and mouse embryo fibroblasts (3T3s), ultrahigh transgene expression is achieved by LBPAE: up to 3292-fold enhancement in Gaussia luciferase (Gluc) expression and nearly 100% of green fluorescence protein expression are detected. Concurrently, LBPAE is of high in vitro biocompatibility. In depth mechanistic studies reveal that versatile LBPAE can navigate multiple extra- and intracellular barriers involved in the fibroblast gene transfection. More importantly, LBPAE can effectively deliver minicircle DNA encoding  COL7A1 gene (a large and functional gene construct) to substantially upregulate the expression of type VII collagen (C7) in HPDFs, demonstrating its great potential in the treatment of C7-deficiency related genodermatoses such as recessive dystrophic epidermolysis bullosa.

Highly Branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) for High Performance Gene Transfection.

The top-performing linear poly(ß-amino ester) (LPAE), poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32), has demonstrated gene transfection efficiency comparable to viral-mediated gene delivery. Herein, we report the synthesis of a series of highly branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (HC32) and explore how the branching structure influences the performance of C32 in gene transfection. HC32 were synthesized by an "A2 + B3 + C2" Michal addition strategy. Gaussia luciferase (Gluciferase) and green fluorescent protein (GFP) coding plasmid DNA were used as reporter genes and the gene transfection efficiency was evaluated in human cervical cancer cell line (HeLa) and human recessive dystrophic epidermolysis bullosa keratinocyte (RDEBK) cells. We found that the optimal branching structure led to a much higher gene transfection efficiency in comparison to its linear counterpart and commercial reagents, while preserving high cell viability in both cell types. The branching strategy affected DNA binding, proton buffering capacity and degradation of polymers as well as size, zeta potential, stability, and DNA release rate of polyplexes significantly. Polymer degradation and DNA release rate played pivotal parts in achieving the high gene transfection efficiency of HC32-103 polymers, providing new insights for the development of poly(ß-amino ester)s-based gene delivery vectors.

Star Poly(ß-amino esters) Obtained from the Combination of Linear Poly(ß-amino esters) and Polyethylenimine.

Composed of a three-dimensional structure with a central core and multiple radiating linear "arms", star polymers represent a significant type of branched macromolecular architectures. Due to the spatially defined core-shell-periphery architecture, star polymers have demonstrated their superiority in a variety of biomedical applications such as drug/gene delivery, molecular imaging, antibacterial agents, and so on. In this paper, we report the successful synthesis of a new type of star-shape poly(ß-amino esters) with low molecular weight PEI as core and linear PAE (LPAE) as arms. This new star-PAE exhibits low cytotoxicity and high gene transfection efficacy. Star-PAE achieved between 264-fold and 14781-fold higher gene transfection efficiency of primary rat adipose derived mesenchymal stem cells in comparison with studies performed with the individual PEI and LPAE, respectively. The results suggest that star-PAE is a promising nonviral gene delivery vector.