Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Tetrahedral framework nucleic acids/hyaluronic acid-methacrylic anhydride hybrid hydrogel with antimicrobial and anti-inflammatory properties for infected wound healing.

Bacterial resistance and excessive inflammation are common issues that hinder wound healing. Antimicrobial peptides (AMPs) offer a promising and versatile antibacterial option compared to traditional antibiotics, with additional anti-inflammatory properties. However, the applications of AMPs are limited by their antimicrobial effects and stability against bacterial degradation. TFNAs are regarded as a promising drug delivery platform that could enhance the antibacterial properties and stability of nanodrugs. Therefore, in this study, a composite hydrogel (HAMA/t-GL13K) was prepared via the photocross-linking method, in which tFNAs carry GL13K. The hydrogel was injectable, biocompatible, and could be instantly photocured. It exhibited broad-spectrum antibacterial and anti-inflammatory properties by inhibiting the expression of inflammatory factors and scavenging ROS. Thereby, the hydrogel inhibited bacterial infection, shortened the wound healing time of skin defects in infected skin full-thickness defect wound models and reduced scarring. The constructed HAMA/tFNA-AMPs hydrogels exhibit the potential for clinical use in treating microbial infections and promoting wound healing.

Intensity and retention time prediction improves the rescoring of protein-nucleic acid cross-links.

In protein-RNA cross-linking mass spectrometry, UV or chemical cross-linking introduces stable bonds between amino acids and nucleic acids in protein-RNA complexes that are then analyzed and detected in mass spectra. This analytical tool delivers valuable information about RNA-protein interactions and RNA docking sites in proteins, both in vitro and in vivo. The identification of cross-linked peptides with oligonucleotides of different length leads to a combinatorial increase in search space. We demonstrate that the peptide retention time prediction tasks can be transferred to the task of cross-linked peptide retention time prediction using a simple amino acid composition encoding, yielding improved identification rates when the prediction error is included in rescoring. For the more challenging task of including fragment intensity prediction of cross-linked peptides in the rescoring, we obtain, on average, a similar improvement. Further improvement in the encoding and fine-tuning of retention time and intensity prediction models might lead to further gains, and merit further research.

Clinical significance of viral markers testing by ELISA and Individual Donation Nucleic Acid Testing (ID-NAT) for blood screening in blood bank: Single center study in Egypt.

Prevention of transfusion-transmitted viral infections and insurance of safe blood transfusion are the main goals of all blood banks worldwide. Despite the high sensitivity and specificity of currently used enzyme linked immunosorbent assay (ELISA) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) testing, viral transmission could still occur during the window period. Introducing viral individual donation nucleic acid testing (ID-NAT) can greatly decrease such risk providing an additional layer in securing blood transfusion. We aimed to assess the clinical significance of viral markers testing by ELISA and ID-NAT for blood screening in the Blood Bank of Suez Canal University Hospital. We studied all donations (2132) collected during a two-months period. Blood donor samples were screened by ELISA and ID-NAT tests for HBV, HCV, and HIV. Serological testing results for HCV by ELISA revealed 2,122 (99.5 %) negative donations compared to 2,131 (99.95 %) negative donations by ID-NAT testing. Of the positive ELISA samples, only one was NAT positive. For HBV ELISA testing, 2,115 (99.2 %) donations were negative, also by ID-NAT testing 2,115 (99.2 %) donations were HBV DNA negative. Out of the negative ELISA samples, two samples were ID-NAT reactive donors which were missed by serology assay being in the window period. HIV ELISA testing revealed negative 2,130 (99.9 %) donations while ID-NAT testing showed 2,131 (99.95 %) negative donations and one positive donation. In conclusion, this is the first study carried out in the Suez Canal and Sinai region, Egypt to assess the importance of ID-NAT implementation. The introduction of ID-NAT in blood banks is an effective method for increasing safety of the blood transfusion.

Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.

CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.

Design and analysis of self-priming extension DNA hairpin probe for miRNA detection based on a unified dynamic programming framework.

MicroRNAs (miRNAs) are potential biomarkers for cancer diagnosis and prognosis, methods for detecting miRNAs with high sensitivity, selectivity, and stability are urgently needed. Various nucleic acid probes that have traditionally been for this purpose suffer several drawbacks, including inefficient signal-to-noise ratios and intensities, high cost, and time-consuming method establishment. Computing tools used for investigating the thermodynamics of DNA hybridization reactions can accurately predict the secondary structure of DNA and the interactions between DNA molecules. Herein, NUPACK was used to design a series of nucleic acid probes and develop a phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) signal amplification strategy, which enabled the ultrasensitive detection of miR-200a in serum samples. The free and binding energies of the DNA detection probes calculated using NUPACK, as well as the biological experimental results, were considered synthetically to select the best sequence and experimental conditions. A unified dynamic programming framework, NUPACK analysis and the experimental data, were complementary and improved the designed model in all respects. Our study demonstrates the feasibility of using computer technology such as NUPACK to simplify the experimental process and provide intuitive results.

Improved Catalytic Activity of Spherical Nucleic Acid Enzymes by Hybridization Chain Reaction and Its Application for Sensitive Analysis of Aflatoxin B1.

Conventional spherical nucleic acid enzymes (SNAzymes), made with gold nanoparticle (AuNPs) cores and DNA shells, are widely applied in bioanalysis owing to their excellent physicochemical properties. Albeit important, the crowded catalytic units (such as G-quadruplex, G4) on the limited AuNPs surface inevitably influence their catalytic activities. Herin, a hybridization chain reaction (HCR) is employed as a means to expand the quantity and spaces of G4 enzymes for their catalytic ability enhancement. Through systematic investigations, we found that when an incomplete G4 sequence was linked at the sticky ends of the hairpins with split modes (3:1 and 2:2), this would significantly decrease the HCR hybridization capability due to increased steric hindrance. In contrast, the HCR hybridization capability was remarkably enhanced after the complete G4 sequence was directly modified at the non-sticky end of the hairpins, ascribed to the steric hindrance avoided. Accordingly, the improved SNAzymes using HCR were applied for the determination of AFB1 in food samples as a proof-of-concept, which exhibited outstanding performance (detection limit, 0.08 ng/mL). Importantly, our strategy provided a new insight for the catalytic activity improvement in SNAzymes using G4 as a signaling molecule.

Biomolecular Condensates: Structure, Functions, Methods of Research.

The term "biomolecular condensates" is used to describe membraneless compartments in eukaryotic cells, accumulating proteins and nucleic acids. Biomolecular condensates are formed as a result of liquid-liquid phase separation (LLPS). Often, they demonstrate properties of liquid-like droplets or gel-like aggregates; however, some of them may appear to have a more complex structure and high-order organization. Membraneless microcompartments are involved in diverse processes both in cytoplasm and in nucleus, among them ribosome biogenesis, regulation of gene expression, cell signaling, and stress response. Condensates properties and structure could be highly dynamic and are affected by various internal and external factors, e.g., concentration and interactions of components, solution temperature, pH, osmolarity, etc. In this review, we discuss variety of biomolecular condensates and their functions in live cells, describe their structure variants, highlight domain and primary sequence organization of the constituent proteins and nucleic acids. Finally, we describe current advances in methods that characterize structure, properties, morphology, and dynamics of biomolecular condensates in vitro and in vivo.

Photophysics of a nucleic acid-protein crosslinking model strongly depends on solvation dynamics: an experimental and theoretical study.

We present a combined experimental and theoretical study of the photophysics of 5-benzyluracil (5BU) in methanol, which is a model system for interactions between nucleic acids and proteins. A molecular dynamics study of 5BU in solution through efficient DFT-based hybrid ab initio potentials revealed a remarkable conformational flexibility - allowing the population of two main conformers - as well as specific solute-solvent interactions, which both appear as relevant factors for the observed 5BU optical absorption properties. The simulated absorption spectrum, calculated on such an ensemble, enabled a molecular interpretation of the experimental UV-Vis lowest energy band, which is also involved in the induced photo-reactivity upon irradiation. In particular, the first two excited states (mainly involving the uracil moiety) both contribute to the 5BU lowest energy absorption. Moreover, as a key finding, the nature and brightness of such electronic transitions are strongly influenced by 5BU conformation and the microsolvation of its heteroatoms.

A quest for cytosolic sequons and their functions.

Evolution shapes protein sequences for their functions. Here, we studied the moonlighting functions of the N-linked sequon NXS/T, where X is not P, in human nucleocytosolic proteins. By comparing membrane and secreted proteins in which sequons are well known for N-glycosylation, we discovered that cyto-sequons can participate in nucleic acid binding, particularly in zinc finger proteins. Our global studies further discovered that sequon occurrence is largely proportional to protein length. The contribution of sequons to protein functions, including both N-glycosylation and nucleic acid binding, can be regulated through their density as well as the biased usage between NXS and NXT. In proteins where other PTMs or structural features are rich, such as phosphorylation, transmembrane ɑ-helices, and disulfide bridges, sequon occurrence is scarce. The information acquired here should help understand the relationship between protein sequence and function and assist future protein design and engineering.

Controllable and reusable seesaw circuit based on nicking endonucleases.

Seesaw circuits are essential for molecular computing and biosensing. However, a notable limitation of seesaw circuits lies in the irreversible depletion of components, precluding the attainment of system recovery and rendering nucleic acid circuits non-reusable. We developed a brand-new method for creating controllable and reusable seesaw circuits. By using the nicking endonucleases Nt.BbvCI and Nt.Alwi, we removed "functional components" while keeping the "skeletal components" for recurrent usage. T-inputs were introduced, increasing the signal-to-noise ratio of AND logic from 2.68 to 11.33 and demonstrating compatibility. We identified the logic switching feature and verified that it does not impair circuit performance. We also built intricate logic circuits, such as OR-AND gate, to demonstrate the versatility of our methodology. This controllable reusability extends the applications of nanotechnology and bioengineering, enhancing the practicality and efficiency of these circuits across various domains.

Tumor-associated antigen-specific cell imaging based on upconversion luminescence and nucleic acid rolling circle amplification.

Tumor-associated antigen (TAA)-based diagnosis has gained prominence for early tumor screening, treatment monitoring, prognostic assessment, and minimal residual disease detection. However, limitations such as low sensitivity and difficulty in extracting non-specific binding membrane proteins still exist in traditional detection methods. Upconversion luminescence (UCL) exhibits unique physical and chemical properties under wavelength near-infrared light excitation. Rolling circle amplification (RCA) is an efficient DNA amplification technique with amplification factors as high as 105. Therefore, the above two excellent techniques can be employed for highly accurate imaging analysis of tumor cells. Herein, we developed a novel nanoplatform for TAA-specific cell imaging based on UCL and RCA technology. An aptamer-primer complex selectively binds to Mucin 1 (MUC1), one of TAA on cell surface, to trigger RCA reaction, generating a large number of repetitive sequences. These sequences provide lots of binding sites for complementary signal probes, producing UCL from lanthanide-doped upconversion nanoparticles (UCNPs) after releasing quencher group. The experimental results demonstrate the specific attachment of upconversion nanomaterials to cancer cells which express a high level of MUC1, indicating the potential of UCNPs and RCA in tumor imaging.

Synthesis of cationic liposome nanoparticles using a thin film dispersed hydration and extrusion method.

Liposome nanoparticles can carry a wide range of therapeutic molecules including small molecules and nucleic acid-based therapeutics. Potential benefits include translocation across physiological barriers, reduced systemic toxicity, and enhanced pharmacokinetic parameters such as absorption, distribution, selective release and optimal elimination kinetics. Liposome nanoparticles can be generated with a wide range of natural and synthetic lipid-based molecules that confer desirable properties depending on the desired therapeutic application Nel et al (2023), Large (2021), Elkhoury (2020). This protocol article seeks to detail the procedures involved in the production of cationic liposomes using thin-film dispersed hydration method with an estimated uniform size of 60-70 nm for targeted drug administration in tumor cells, by modifying the previous one also published by the same authors cited here. The method was carrying out using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl (DOTAP, 2 mg) as cationic lipid and cholesterol (0.5 mg) in a molar ratio of 7:3 respectively. The liposomal suspension was obtained and its physical, chemical and biological properties were determined. A two-step extrusion process, using 100 nm and 50 nm polycarbonate membranes, was carried. The results demonstrate generation of liposome nanoparticles with a size of 60-70 nm stable for at least 16 weeks and with an encapsulation efficiency of approximately 81% using Doxorubicin.

DAMPs and DAMP-sensing receptors in inflammation and diseases.

Damage-associated molecular patterns (DAMPs) are endogenous danger molecules produced in cellular damage or stress, and they can activate the innate immune system. DAMPs contain multiple types of molecules, including nucleic acids, proteins, ions, glycans, and metabolites. Although these endogenous molecules do not trigger immune response under steady-state condition, they may undergo changes in distribution, physical or chemical property, or concentration upon cellular damage or stress, and then they become DAMPs that can be sensed by innate immune receptors to induce inflammatory response. Thus, DAMPs play an important role in inflammation and inflammatory diseases. In this review, we summarize the conversion of homeostatic molecules into DAMPs; the diverse nature and classification, cellular origin, and sensing of DAMPs; and their role in inflammation and related diseases. Furthermore, we discuss the clinical strategies to treat DAMP-associated diseases via targeting DAMP-sensing receptors.

TPMA: A two pointers meta-alignment tool to ensemble different multiple nucleic acid sequence alignments.

Accurate multiple sequence alignment (MSA) is imperative for the comprehensive analysis of biological sequences. However, a notable challenge arises as no single MSA tool consistently outperforms its counterparts across diverse datasets. Users often have to try multiple MSA tools to achieve optimal alignment results, which can be time-consuming and memory-intensive. While the overall accuracy of certain MSA results may be lower, there could be local regions with the highest alignment scores, prompting researchers to seek a tool capable of merging these locally optimal results from multiple initial alignments into a globally optimal alignment. In this study, we introduce Two Pointers Meta-Alignment (TPMA), a novel tool designed for the integration of nucleic acid sequence alignments. TPMA employs two pointers to partition the initial alignments into blocks containing identical sequence fragments. It selects blocks with the high sum of pairs (SP) scores to concatenate them into an alignment with an overall SP score superior to that of the initial alignments. Through tests on simulated and real datasets, the experimental results consistently demonstrate that TPMA outperforms M-Coffee in terms of aSP, Q, and total column (TC) scores across most datasets. Even in cases where TPMA's scores are comparable to M-Coffee, TPMA exhibits significantly lower running time and memory consumption. Furthermore, we comprehensively assessed all the MSA tools used in the experiments, considering accuracy, time, and memory consumption. We propose accurate and fast combination strategies for small and large datasets, which streamline the user tool selection process and facilitate large-scale dataset integration. The dataset and source code of TPMA are available on GitHub (https://github.com/malabz/TPMA).

Diagnóstico por amplificación isotérmica de ácidos nucleicos. Oportunidad para la farmacia comunitaria / Diagnosis by isothermal amplification of nucleic acids. Opportunity for community pharmacy

Esta revisión se centra en describir nuevos sistemas de diagnóstico molecular de tipo POC disponibles en el mercado que pueden implementarse fácilmente en farmacias comunitarias y tienen el potencial de ampliar la cartera de servicios farmacéuticos y hacer una contribución significativa a la mejora de la salud pública.El conocimiento de nuevas técnicas de diagnóstico molecular distintas de la PCR es relativamente desconocido. Sin embargo, las opciones disponibles son diversas y han alcanzado suficiente madurez tecnológica para su uso a gran escala. La pandemia de SARS-CoV-2 ha sacado al mercado pruebas de diagnóstico que, en algunos casos, se han utilizado exclusivamente en investigación durante décadas.La tecnología isotérmica de amplificación de ácidos nucleicos sigue evolucionando y es probable que en los próximos años seamos testigos de un aumento exponencial de su uso, así como del desarrollo de nuevas mejoras que simplifiquen y reduzcan aún más el coste de cada ensayo.Igualmente, no podemos obviar el hecho de que durante la pandemia de COVID-19, el público se ha habituado a autodiagnosticarse a través de canales de distribución masiva como las farmacias comunitarias, lo que puede abrir el sector a otras enfermedades —como las de transmisión sexual o salud animal—, el control de alimentos, la contaminación del agua y del aire (hongos) o la presencia de alérgenos.El conocimiento de estas nuevas tecnologías es esencial estrategia de vigilancia tecnológica e inteligencia competitiva del sector farmacéutico.(AU)

A responsive disulfide bond switch aptamer prodrug exhibiting enhanced stability and anticancer efficacy.

Aptamers have shown significant potential in treating diverse diseases. However, challenges such as stability and drug delivery limited their clinical application. In this paper, the development of AS1411 prodrug-type aptamers for tumor treatment was introduced. A Short oligonucleotide was introduced at the end of the AS1411 sequence with a disulfide bond as responsive switch. The results indicated that the aptamer prodrugs not only enhanced the stability of the aptamer against nuclease activity but also facilitated binding to serum albumin. Furthermore, in the reducing microenvironment of tumor cells, disulfide bonds triggered drug release, resulting in superior therapeutic effects in vitro and in vivo compared to original drugs. This paper proposes a novel approach for optimizing the structure of nucleic acid drugs, that promises to protect other oligonucleotides or secondary structures, thus opening up new possibilities for nucleic acid drug design.

Treatment for Covid-19 with SARS-CoV-2 neutralizing antibody BRII-196(Ambavirumab) plus BRII-198(Lomisivir): a retrospective cohort study.

BACKGROUND: Monoclonal antibody therapy for Covid-19 springs up all over the world and get some efficiency. This research aims to explore the treating effect of BRII-196(Ambavirumab) plus BRII-198(Lomisivir) on Covid-19. METHODS: In this retrospective cohort research, patients received standard care or plus BRII-196 /BRII-198 monoclonal antibodies. General comparison of clinical indexes and prognosis between Antibody Group and Control Group was made. Further, according to the antibody using time and patients' condition, subgroups included Early antibody group, Late antibody group, Mild Antibody Group, Mild Control Group, Severe Antibody Group and Severe Control Group. RESULTS: Length of stay(LOS) and interval of Covid-19 nucleic acid from positive to negative of Antibody Group were 12.0(IQR 9.0-15.0) and 14.0(IQR 10.0-16.0) days, less than those(13.0 (IQR 11.0-18.0) and 15.0 (IQR 12.8-17.0) days) of Control Group(p = 0.004, p = 0.004). LOS(median 10days) of Early Antibody Group was the shortest, significantly shorter than that of Control Group (median 13days)(p < 0.001). Interval(median 12days) of Covid-19 nucleic acid from positive to negative of Early Antibody Group also was significantly shorter than that of Control Group(median 15days) and Late Antibody Group(median 14days)(p = 0.001, p = 0.042). LOS(median 12days) and interval(median 13days) of Covid-19 nucleic acid from positive to negative of Mild Antibody Group was shorter than that of Mild Control Group(median 13days; median 14.5days)(p = 0.018, p = 0.033). CONCLUSION: The neutralizing antibody therapy, BRII-196 plus BRII-198 could shorten LOS and interval of Covid-19 nucleic acid from positive to negative. However, it didn't show efficacy for improving clinical outcomes among severe or critical cases.

Metal-Organic Framework-Mediated Delivery of Nucleic Acid across Intact Plant Cells.

Plant synthetic biology is applied in sustainable agriculture, clean energy, and biopharmaceuticals, addressing crop improvement, pest resistance, and plant-based vaccine production by introducing exogenous genes into plants. This technique faces challenges delivering genes due to plant cell walls and intact cell membranes. Novel approaches are required to address this challenge, such as utilizing nanomaterials known for their efficiency and biocompatibility in gene delivery. This work investigates metal-organic frameworks (MOFs) for gene delivery in intact plant cells by infiltration. Hence, small-sized ZIF-8 nanoparticles (below 20 nm) were synthesized and demonstrated effective DNA/RNA delivery into Nicotiana benthamiana leaves and Arabidopsis thaliana roots, presenting a promising and simplified method for gene delivery in intact plant cells. We further demonstrate that small-sized ZIF-8 nanoparticles protect RNA from RNase degradation and successfully silence an endogenous gene by delivering siRNA in N. benthamiana leaves.

Secreted novel AID/APOBEC-like deaminase 1 (SNAD1) - a new important player in fish immunology.

The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.