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We present an intelligent photothermal therapy agents by functionalizing gold nanoparticles with specific nucleic acid sequences. Hairpin nucleic acids are modified to the nanoparticles, forming AuNPs-1 and AuNPs-2. Upon infiltrating cancer cells, these nanoparticles undergo catalytic hairpin assembly in the presence of target miRNA, leading to aggregation and subsequent photothermal conversion. Under near-infrared laser irradiation, aggregated gold nanoparticles exhibit efficient photothermal conversion, selectively damaging cancer cells. This approach offers heightened selectivity, as nanoparticles only aggregate in environments with cancer biomarkers present, sparing normal cells. Cytotoxicity assays confirm minimal toxicity to normal cells. In vivo studies on mice bearing solid tumors validate the system's efficacy in tumor regression. Overall, this study highlights the potential of nucleic acid-functionalized gold nanoparticles in intelligent and selective cancer photothermal therapy, offering insights for targeted diagnosis and treatment development. [ABSTRACT FROM AUTHOR]

mRNA vaccines were highly effective in response to the COVID-19 pandemic, making them an attractive platform to address cancers and other infectious diseases. Many new mRNA vaccines in development are multivalent, which represents a difficulty for the standard assays commonly used to characterize the critical quality attributes of monovalent formulations. Here, we present a multiplexed analytical tool with nucleic acid microarray technology using the VaxArray platform that measures the identity and quantity of mono- and multivalent mixtures of naked mRNA and mRNA encapsulated in lipid nanoparticle formulations in under 2 h without any additional preparation steps, such as extraction or RT-PCR. Using a quadrivalent mixture of encapsulated mRNA constructs that encode for four unique proteins in a vaccine formulation, the VaxArray mRNA assay was demonstrated to be highly specific for each mRNA with sensitivity < 1 µg/mL. The quantification of individual mRNAs within the lipid nanoparticle mixture resulted in a precision of ≤10% RSD and an accuracy of 100 ± 9%. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) results from a lack of activity of the lysosomal enzyme α-Galactosidase A (α-Gal A), leading to the accumulation of glycosphingolipids in several different cell types. Protein supplementation by pDNA or mRNA delivery presents a promising strategy to tackle the underlying genetic defect in FD. Protein-coding nucleic acids in FD can be either delivered to the most affected sites by the disease, including heart, kidney and brain, or to specialized organs that can act as a production factory of the enzyme, such as the liver. Lipid-based systems are currently at the top of the ranking of non-viral nucleic acid delivery systems, and their versatility allows the linking to the surface of a wide range of molecules to control their biodistribution after intravenous administration. This systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement guidelines and provides an overview and discussion of the targeting ligands that have been employed so far to actively vectorize intravenously administered non-viral vectors based on lipid carriers to clinically relevant organs in the treatment of FD, for protein-coding nucleic acid (pDNA and mRNA) supplementation. Among the thirty-two studies included, the majority focus on targeting the liver and brain. The targeting of the heart has been reported to a lesser degree, whereas no articles addressing kidney-targeting have been recorded. Although a great effort has been made to develop organ-specific nucleic acid delivery systems, the design of active-targeted carriers with high quality, good clinical translation, and large-scale manufacturing capacity is still challenging. [ABSTRACT FROM AUTHOR]

Purpose: Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors. Methods: SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively. Results:C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 gx gs−1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%CDW). Total nucleic acid content of the yeasts was similar (6.8%CDW and 6.3%CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80. Conclusion: This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae. [ABSTRACT FROM AUTHOR]

Abstract Mycoplasma bovis (M. bovis) is capable of causing a range of diseases in cattle, encompassing calf pneumonia, arthritis, conjunctivitis, meningitis, and mastitis. It is widely recognized as one of the predominant pathogens posing a significant threat to the global cattle industry. Therefore, accurate and sensitive methods are urgently needed to detect M. bovis. This study aims to detect M. bovis by combining colloidal gold with biotin-labeled oligonucleotides to improve detection sensitivity and form a chromogenic detection probe based on signal amplification technology. Here, we developed a sensitive and specific polymerase chain reaction-lateral flow dipstick assay (PCR-LFD) strip for efficient nucleic acid detection of M. bovis. A pair of specific primers with 5’ ends labeled with biotin and digoxigenin probes was designed for PCR experiments. Colloidal gold particles-labeled anti-digoxigenin IgG coated gold-labeled test strip was prepared, streptavidin was used as the detection probe, and nitrocellulose membrane coated goat anti-mouse IgG was used as the control line. Our results showed that the detection limit of the PCR-LFD was 89 fg/µL for the M. bovis DNA. The results from the test strip were highly consistent with those from real-time qPCR. This assay were highly specific for M. bovis, as there were no cross-reactions with other microorganisms tested and the detection sensitivity of the test was also relatively high (97.67%). The novel strips present a promising tool for the cost-effective and sensitive diagnosis of M. bovis.

The principal etiological agent responsible for dental caries is Streptococcus mutans (S. mutans). The Moringa oleifera (M. oleifera) possesses antioxidant and antibacterial properties that function through the response to oxidative stress, which affects bacterial cell metabolism. This research examined M. oleifera impact on S. mutans growth, toxicity, glucan-binding protein (GBP) expression, and nucleic acid structure. Methods included spectrophotometry for growth analysis, enzyme-linked immunosorbent assay for GBP quantification, the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) MTT assay for cytotoxicity, Fourier transform infrared for nucleic acid changes, and docking simulation for ligand-receptor affinity. Results showed that M. oleifera significantly inhibited S. mutans growth at all concentrations over 24 and 48 h (optical density

Calcium phosphates (CaPs) and their substituted derivatives encompass a large number of compounds with a vast presence in nature that have aroused a great interest for decades. In particular, hydroxyapatite (HAp, Ca10(OH)2(PO4)6) is the most abundant CaP mineral and is significant in the biological world, at least in part due to being a major compound in bones and teeth. HAp exhibits excellent properties, such as safety, stability, hardness, biocompatibility, and osteoconductivity, among others. Even some of its drawbacks, such as its fragility, can be redirected thanks to another essential feature: its great versatility. This is based on the compound's tendency to undergo substitutions of its constituent ions and to incorporate or anchor new molecules on its surface and pores. Thus, its affinity for biomolecules makes it an optimal compound for multiple applications, mainly, but not only, in biological and biomedical fields. The present review provides a chemical and structural context to explain the affinity of HAp for biomolecules such as proteins and nucleic acids to generate hybrid materials. A size-dependent criterium of increasing complexity is applied, ranging from amino acids/nucleobases to the corresponding macromolecules. The incorporation of metal ions or metal complexes into these functionalized compounds is also discussed. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are the most clinically advanced drug delivery system for nucleic acid therapeutics, exemplified by the success of the COVID-19 mRNA vaccines. However, their clinical use is currently limited to hepatic diseases and vaccines due to their tendency to accumulate in the liver upon intravenous administration. To fully leverage their potential, it is essential to understand and address their liver tropism, while also developing strategies to enhance delivery to tissues beyond the liver. Ensuring that these therapeutics reach their target cells while avoiding off-target cells is essential for both their efficacy and safety. There are three potential targeting strategies—passive, active, and endogenous—which can be used individually or in combination to target nonhepatic tissues. In this review, we delve into the recent advancements in LNP engineering for delivering nucleic acid beyond the liver. [ABSTRACT FROM AUTHOR]

Mycoplasma bovis (M. bovis) is capable of causing a range of diseases in cattle, encompassing calf pneumonia, arthritis, conjunctivitis, meningitis, and mastitis. It is widely recognized as one of the predominant pathogens posing a significant threat to the global cattle industry. Therefore, accurate and sensitive methods are urgently needed to detect M. bovis. This study aims to detect M. bovis by combining colloidal gold with biotin-labeled oligonucleotides to improve detection sensitivity and form a chromogenic detection probe based on signal amplification technology. Here, we developed a sensitive and specific polymerase chain reaction-lateral flow dipstick assay (PCR-LFD) strip for efficient nucleic acid detection of M. bovis. A pair of specific primers with 5' ends labeled with biotin and digoxigenin probes was designed for PCR experiments. Colloidal gold particles-labeled anti-digoxigenin IgG coated gold-labeled test strip was prepared, streptavidin was used as the detection probe, and nitrocellulose membrane coated goat anti-mouse IgG was used as the control line. Our results showed that the detection limit of the PCR-LFD was 89 fg/µL for the M. bovis DNA. The results from the test strip were highly consistent with those from real-time qPCR. This assay were highly specific for M. bovis, as there were no cross-reactions with other microorganisms tested and the detection sensitivity of the test was also relatively high (97.67%). The novel strips present a promising tool for the cost-effective and sensitive diagnosis of M. bovis. [ABSTRACT FROM AUTHOR]

Immunotherapy combats tumors by enhancing the body's immune surveillance and clearance of tumor cells. Various nucleic acid drugs can be used in immunotherapy, such as DNA expressing cytokines, mRNA tumor vaccines, small interfering RNAs (siRNA) knocking down immunosuppressive molecules, and oligonucleotides that can be used as immune adjuvants. Nucleic acid drugs, which are prone to nuclease degradation in the circulation and find it difficult to enter the target cells, typically necessitate developing appropriate vectors for effective in vivo delivery. Biomimetic drug delivery systems, derived from viruses, bacteria, and cells, can protect the cargos from degradation and clearance, and deliver them to the target cells to ensure safety. Moreover, they can activate the immune system through their endogenous activities and active components, thereby improving the efficacy of antitumor immunotherapeutic nucleic acid drugs. In this review, biomimetic nucleic acid delivery systems for relieving a tumor immunosuppressive microenvironment are introduced. Their immune activation mechanisms, including upregulating the proinflammatory cytokines, serving as tumor vaccines, inhibiting immune checkpoints, and modulating intratumoral immune cells, are elaborated. The advantages and disadvantages, as well as possible directions for their clinical translation, are summarized at last. [ABSTRACT FROM AUTHOR]

Collecting and preserving biological samples in the field, particularly in remote areas in tropical forests, prior to laboratory analysis is challenging. Blood samples in many cases are used for nucleic acid‐based species determination, genomics or pathogen research. In most cases, maintaining a cold chain is impossible and samples remain at ambient temperature for extended periods of time before controlled storage conditions become available. Dried blood spot (DBS) storage, blood stored on cellulose‐based paper, has been widely applied to facilitate sample collection and preservation in the field for decades. However, it is unclear how long‐term storage on this substrate affects nucleic acid concentration and integrity. We analysed nucleic acid quality from DBS stored on Whatman filter paper no. 3 and FTA cards for up to 15 years in comparison to cold‐chain stored samples using four nucleic acid extraction methods. We examined the ability to identify viral sequences from samples of 12 free‐ranging primates in the Amazon forest, using targeted hybridization capture, and determined if mitochondrial genomes could be retrieved. The results suggest that even after extended periods of storage, DBS will be suitable for some genomic applications but may be of limited use for viral pathogen research, particularly RNA viruses. [ABSTRACT FROM AUTHOR]

Purpose: This study evaluated whether a nanostructured lipid carrier (NLC) delivery system could safely and accurately deliver nucleic acids to the cell nucleus using the enhanced green fluorescent protein (EGFP)-C1 plasmid model. Methods: The NLC was formulated using the emulsification method and equipped for cationic lipid-mediated transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which interacts electrostatically with nucleic acid. The NLC attributes, including size, polydispersity index, and zeta potential, were assessed by dynamic light scattering (DLS). The morphological structure was analyzed using transmission electron microscopy. Entrapment efficiency was evaluated by a direct method. Cellular uptake mechanisms of pEGFP-C1-NLC and the ability of pEGFP-C1 to penetrate the nucleus of TM4 cells to express EGFP were observed using confocal microscopy. Results: pEGFP-C1-NLC exhibited particle sizes in the range 56-88 nm with a particle charge range of -6.0 to+1.3 mV. The polydispersity index<0.5 showed good size uniformity, and entrapment efficiency of pEGFP-C1in the NLC was 92.06±2.295%. The NLC formulation was internalized predominantly via caveolae-mediated endocytosis, as indicated by EGFP expression following successful delivery of pEGFP by the NLC into the cells. Conclusion: NLC formulation could deliver genetic material to the nucleus and could be considered a gene therapy candidate for spermatogenesis. [ABSTRACT FROM AUTHOR]

In this study water-soluble N-Acetyl Cysteine Capped-Cadmium Telluride QDs (NAC/CdTe nanocrystals) using N-acetyl cysteine as a stabilizer were prepared to investigate the utility of quantum dots (QDs) in distinguishing damaged DNA, (extracted from blood samples of leukaemia patients), from intact DNA (extracted from blood samples of healthy individuals) to be used for biosensing application. Based on the optical characterization of the prepared QDs, the XRD results revealed the formation of the NAC-CdTe-QDs with a grain size of 7.1nm. Whereas, the SEM test showed that the spherical size of the NAC-CdTe-QDs lies within 11~33nm. NACCdTe-QDs have superior PL emission properties at of 550nm and UV-Vis absorption peak at 300nm. The energy gap measurement through PL and UV-Vis was found to be 2.2eV and 2.3 eV, respectively. The interaction between the synthesized QDs and the extracted genomic DNA (both cancer damaged DNA and healthy undamaged DNA) was analysed optically, and compared to the normal reference DNA. The results showed a shift in the maximum fluorescence emission intensities (observed at 540nm nm for a damaged sample and 535 for a reference cell). Based on the obtained fluorescence results, the present study reached the conclusion that the prepared core/shell QDs could be employed as probes for diagnosing genetically disrupted DNA that is associated with malignant diseases from healthy DNA. [ABSTRACT FROM AUTHOR]

The review describes the fundamental electrochemical properties of nucleic acids manifested on solid electrodes, with an emphasis on the spatial structure of macromolecules. The formation of the double helix impedes the contact of the electroactive groups of the nitrogenous bases with the electrode surface, resulting in the disappearance of the analytical signal of deoxyribonucleic acid (DNA). The insufficient electroactivity of the double-stranded DNA is overcome by introducing electrochemically active fragments into the nucleic acid sequence through the polymerase incorporation of chemically modified nucleotides. Currently, an extensive range of artificial nucleotides has been synthesized, which contain various electroactive groups capable of both oxidation and reduction on electrode surfaces at different potentials. Artificial modified nucleotides must exhibit high electrochemical activity while also serving as good substrates for enzymes (polymerases) involved in nucleic acid amplification reactions. Introducing modified nucleotides instead of natural ones into polymerase reactions represents a compromise between the number of labels inserted in one amplicon and the length and quantity of the resulting products. Modified nucleotides find application in the detection of gene mutations and single-nucleotide polymorphisms, nucleic acid sequencing, determination of protein and peptide concentrations, and the detection of pathogenic viruses and bacteria. With the advancement of isothermal amplification methods, the development, synthesis, and investigation of artificial nucleotides have become highly relevant for creating new off-laboratory electrochemical nucleic acid analyzers. [ABSTRACT FROM AUTHOR]

Increasing global consumption of protein over the last five decades, coupled with concerns about the impact on emissions of animal‐based protein production, has created interest in alternative protein sources. Microbial proteins (MPs), derived through the fermentation of agro‐industrial byproducts, present a promising option. This review assesses a century of advancements in this domain. We conducted a comprehensive review and meta‐analysis, examining 347 relevant research papers to identify trends, technological advancements, and key influencing factors in the production of MP. The analysis covered the types of feedstocks and microbes, fermentation methods, and the implications of nucleic acid content on the food‐grade quality of proteins. A conditional inference tree model and Bayesian factor were used to ascertain the impact of various parameters on protein content. Out of all the studied parameters, such as type of feedstock (lignocellulose, free sugars, gases, and others), type of fermentation (solid, liquid, gas), type of microbe (bacteria, fungi, yeast, and mix), and operating parameters (temperature, time, and pH), the type of fermentation and microbe were identified as the largest influences on protein content. Gas and liquid fermentation demonstrated higher protein content, averaging 52% and 42%, respectively. Among microbes, bacterial species produced a higher protein content of 51%. The suitable operating parameters, such as pH, time, and temperature, were also identified for different microbes. The results point to opportunities for continued innovation in feedstock, microbes, and regulatory alignment to fully realize the potential of MP in contributing to global food security and sustainability goals. [ABSTRACT FROM AUTHOR]

Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

The early diagnosis of cancer is vital for effective treatment and improved prognosis. Tumor biomarkers, which can be used for the early diagnosis, treatment, and prognostic evaluation of cancer, have emerged as a topic of intense research interest in recent years. Nucleic acid, as a type of tumor biomarker, contains vital genetic information, which is of great significance for the occurrence and development of cancer. Currently, living cell nucleic acid probes, which enable the in situ imaging and dynamic monitoring of nucleic acids, have become a rapidly developing field. This review focuses on living cell nucleic acid probes that can be used for the early diagnosis of tumors. We describe the fundamental design of the probe in terms of three units and focus on the roles of different nanomaterials in probe delivery. [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation (LLPS) has been recognized as a universal biological phenomenon. It plays an important role in life activities. LLPS is induced by weak interactions between intrinsically disordered regions or low complex domains. Nucleic acids are widely present in cells, and shown to be closely related to LLPS. Their structure and electronegativity provide the excellent platforms for the formation of phase‐separated condensates. In this review, we summarize the interconnected regulation between nucleic acids and LLPS demonstrated in in vivo and in vitro studies. Beside homogeneous and single‐phase condensates, complicated and multicompartment LLPS induced by nucleic acids is discussed as well. Recent advances about nucleic‐acid‐induced LLPS as a new pathogenic mechanism and drug design direction are highlighted, especially virus‐mediated disease treatment and prevention. [ABSTRACT FROM AUTHOR]

Plant viruses are famed for serious economic losses to most of the agricultural and horticultural crops all around the world. The losses caused by plant viruses are estimated to be several billion bucks per year and are therefore a major threat to global crop production and food security. Traditional diagnostic methods based on biological and physical properties of virus such as appearance of symptoms, bioassay on indicator plants, vector borne and virus particle morphology by electron microscopy are cumbersome, time-consuming and expensive. Recognition of disease based on the symptoms indicated by virus affected plants, can invariably be the first process in several cases, however it's confusing additionally as a result of the symptoms are changeable and depends on several factors like abiotic conditions. There are several sensitive and effective diagnostic methods developed during the last four decades based on virus protein e.g., immunosorbent electron microscopy (ISEM), enzyme-linked immunosorbent assay (ELISA) and nucleic acid e.g., nucleic acid hybridization, isothermal and thermostable PCRs, for plant virus detection, each having its own advantage and disadvantage. This review provides an update on the progress made on the techniques that are useful for early, sensitive, accurate and reliable detection and diagnosis of plant viruses. [ABSTRACT FROM AUTHOR]

Dissection of the matter into its constituents leads us to the smallest particles that we know. These particles form a material structure that is determined by the electromagnetic field generated and carried by those particles. Changes in any of the two major constituents leads to changes in that material system, be it a living organism or a lifeless object. The latter statement carries the mystery of life that is born from a continuous and programmed series of system changes fuelled by an energy source with a yet unknown functioning mechanism. The present work is a theoretical approach towards the understanding and potential discovery of the aforementioned, not-yet-known cellular energetic mechanism. Understanding the energetic basis of intracellular biochemistry is equally important in human and animal therapeutics. Additionally, as all such discoveries offer novel solutions in various fields of the global industry, the final outcome of this theoretical work also brings about the idea of a new discovery in electronics industry. [ABSTRACT FROM AUTHOR]

Small molecule drugs can be used to target nucleic acids (NA) to regulate biological processes. Computational modeling methods, such as molecular docking or scoring functions, are commonly employed to facilitate drug design. However, the accuracy of the scoring function in predicting the closest-to-native docking pose is often suboptimal. To overcome this problem, a machine learning model, RmsdXNA, was developed to predict the root-mean-square-deviation (RMSD) of ligand docking poses in NA complexes. The versatility of RmsdXNA has been demonstrated by its successful application to various complexes involving different types of NA receptors and ligands, including metal complexes and short peptides. The predicted RMSD by RmsdXNA was strongly correlated with the actual RMSD of the docked poses. RmsdXNA also outperformed the rDock scoring function in ranking and identifying closest-to-native docking poses across different structural groups and on the testing dataset. Using experimental validated results conducted on polyadenylated nuclear element for nuclear expression triplex, RmsdXNA demonstrated better screening power for the RNA-small molecule complex compared to rDock. Molecular dynamics simulations were subsequently employed to validate the binding of top-scoring ligand candidates selected by RmsdXNA and rDock on MALAT1. The results showed that RmsdXNA has a higher success rate in identifying promising ligands that can bind well to the receptor. The development of an accurate docking score for a NA–ligand complex can aid in drug discovery and development advancements. The code to use RmsdXNA is available at the GitHub repository https://github.com/laiheng001/RmsdXNA. [ABSTRACT FROM AUTHOR]

Background: Most respiratory viruses can cause serious lower respiratory diseases at any age. Therefore, timely and accurate identification of respiratory viruses has become even more important. This study focused on the development of rapid nucleic acid testing techniques for common respiratory infectious diseases in the Chinese population. Methods: Multiplex fluorescent quantitative polymerase chain reaction (PCR) assays were developed and validated for the detection of respiratory pathogens including the novel coronavirus (SARS-CoV-2), influenza A virus (FluA), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). Results: The assays demonstrated high specificity and sensitivity, allowing for the simultaneous detection of multiple pathogens in a single reaction. These techniques offer a rapid and reliable method for screening, diagnosis, and monitoring of respiratory pathogens. Conclusion: The implementation of these techniques might contribute to effective control and prevention measures, leading to improved patient care and public health outcomes in China. Further research and validation are needed to optimize and expand the application of these techniques to a wider range of respiratory pathogens and to enhance their utility in clinical and public health settings. [ABSTRACT FROM AUTHOR]

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization. [ABSTRACT FROM AUTHOR]

In this study, NAC-capped CdTe/CdS/ZnS core/double shell QDs were synthesized in an aqueous medium to investigate their utility in distinguishing normal DNA from mutated DNA extracted from biological samples. Following the interaction between the synthesized QDs with DNA extracted from leukemia cases (represents damaged DNA) and that of healthy donors (represents undamaged DNA), differential fluorescent emission maxima and intensities were observed. It was found that damaged DNA from leukemic cells DNA-QDs conjugates at 585 nm while intact DNA (from healthy subjects) DNA–QDs conjugates at 574 nm. The obtained results from the optical analyses indicate that the prepared QDs could be utilized as probe for detecting disrupted DNA that is associated with a number of diseases including malignancies. Additionally, the manufactured NAC-CdTe core with CdS shell and ZnS shell QDs were further characterized by high-resolution transmission using field emission scanning electron microscopy (FESEM), energy dispersive X-ray fluorescence (EDX), X-ray diffraction (XRD), infrared spectrum (IR), UV-vis absorbance, photoluminescence (PL) and absorbency intensity using the fully automatic ELISA. The XRD results revealed the formation of NAC-CdTe/CdS/ZnS QDs with a grain size of 5.7 nm. While EDX assay emphasizes the compound content of Cd, S, Zn and Te elements. Whereas SEM test's findings propose the spherical size of NAC- CdTe/CdS/ZnS QDs within the range of 10–40 nm. The demonstrated mono-dispersed lattice structure of NAC-CdTe core with CdS shell and ZnS shell QDs has superior PL emission properties at λ e m i of ∼ 600 nm and UV-Vis absorption bands at 350 nm. Overall, this study suggests that the synthesized QDs could be employed in developing optical biosensors for a variety of biomedical applications to improve early detection of diseases marked by damaged DNA profile including cancers. [ABSTRACT FROM AUTHOR]

In recent years, the prevalence of diabetic wounds has significantly increased, posing a substantial medical challenge due to their propensity for infection and delayed healing. These wounds not only increase mortality rates but also lead to amputations and severe mobility issues. To address this, advancements in bioactive molecules such as genes, growth factors, proteins, peptides, stem cells, and exosomes into targeted gene therapies have emerged as a preferred strategy among researchers. Additionally, the integration of photothermal therapy (PTT), nucleic acid, and gene therapy, along with 3D printing technology and the layer-by-layer (LBL) self-assembly approach, shows promise in diabetic wound treatment. Effective delivery of small interfering RNA (siRNA) relies on gene vectors. This review provides an in-depth exploration of the pathophysiological characteristics observed in diabetic wounds, encompassing diminished angiogenesis, heightened levels of reactive oxygen species, and impaired immune function. It further examines advancements in nucleic acid delivery, targeted gene therapy, advanced drug delivery systems, layer-by-layer (LBL) techniques, negative pressure wound therapy (NPWT), 3D printing, hyperbaric oxygen therapy, and ongoing clinical trials. Through the integration of recent research insights, this review presents innovative strategies aimed at augmenting the multifaceted management of diabetic wounds, thus paving the way for enhanced therapeutic outcomes in the future.

In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). In vitro studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future in vivo applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both in vitro diagnostics and cancer molecular imaging with prospects for future theranostic applications.

While their broad utility in various chemistry fields were well recognized for decades, fluoroalcohols have recently emerged as a unique solvent system for bioconjugation development. This review describes examples and roles of fluoroalcohols such as trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) for chemical modification of biomolecules such as polypeptides, nucleic acids, and saccharides. Many chemical modification processes were facilitated by notable functions of those fluoroalcohols such as a proton shuttle, reversible adduct formation with reactive species, and compatibility with electrochemistry/photochemistry. The usefulness of the fluoroalcohol solvents can be even promoted by its combination with a different solvent system for reaction enhancement and protein stabilization. The collection of the various chemical transformations in this review is an indication of the rapid growth of the solvent-assisted bioconjugation field.

In contrast to conventional therapies that require repeated dosing, gene therapy can treat diseases by correcting defective genes after a single transfection and achieving cascade amplification, and has been widely studied in clinical settings. However, nucleic acid drugs are prone to catabolism and inactivation. A variety of nucleic acid drug vectors have been developed to protect the target gene against nuclease degradation and increase the transformation efficiency and safety of gene therapy. In addition, gene therapy is often combined with chemotherapy, phototherapy, magnetic therapy, ultrasound, and other therapeutic modalities to improve the therapeutic effect. This review systematically introduces ribonucleic acid (RNA) interference technology, antisense oligonucleotides, and clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing. It also introduces the commonly used nucleic acid drug vectors, including viral vectors (adenovirus, retrovirus, etc.), organic vectors (lipids, polymers, etc.), and inorganic vectors (MOFs, carbon nanotubes, mesoporous silica, etc.). Then, we describe the combined gene therapy modalities and the pathways of action and report the recent applications in solid tumors of the combined gene therapy. Finally, the challenges of gene therapy in solid tumor treatment are introduced, and the prospect of application in this field is presented.

Abstract Nucleic acids are not only essential biomolecules that drive critical life processes such as growth, development, reproduction, inheritance, and mutation, but also serve as significant markers for disease diagnosis, pathogen identification, and cancer screening. Nevertheless, several challenges have hindered the widespread use of nucleic acids in biomedicine, such as susceptibility to degradation, limited cellular uptake efficiency, potential toxicity, and uncontrollable activity. Photo‐regulation offers an effective solution to address these challenges. It allows for the precise control of nucleic acid structure and function and enhances the stability and safety of their application in biomedicine. In this review, we systematically review the structural characteristics of the three primary photosensitive groups commonly used in the regulation of nucleic acid molecules (i.e., photocleavable molecules, photoisomerization molecules, and photo‐crosslinking molecules) under light irradiation. Subsequently, recent research advances in the development and application of photo‐modulation strategies based on these photosensitive molecules in antisense oligonucleotides, RNA interference, nucleic acid amplification, and CRISPR/Cas systems are outlined. Finally, we discuss the challenges faced in the widespread application of these photo‐regulatory strategies and outline potential future directions for their development.

To systematically elucidate how metal-binding antimicrobial peptide SIF4 exerts its antimicrobial activity by targeting DNA topoisomerase without destroying the cytoplasmic membrane, Escherichia coli was used as a model strain to investigate the binding mode of SIF4 with its genomic DNA and the impact of SIF4 on DNA topoisomerase I and II activities and intracellular nucleic acid biosynthesis. Results showed that SIF4 could bind to genomic DNA in a manner similar to ethidium bromide (EB) intercalation with strong inhibitory effect on topoisomerase I but weak effect on topoisomerase II, and catalyzed RNA transcription to exert antimicrobial activity by interfering with the unwinding of negative DNA supercoils and RNA polymerase binding. It was also found that the biosynthesis of intracellular DNA and RNA was inhibited to different degrees after 12 h treatment with SIF4, which exhibited a good dose-effect relationship. There was no significant difference in the amounts of intracellular DNA and RNA between the 1/2 minimum inhibitory concentration (MIC) group and the control group (P > 0.05), but there was a significant difference between the MIC and 2 MIC groups and the control group (P < 0.05). Our results may provide theoretical support for the application of SIF4 in the biocontrol of foodborne E. coli.

mRNA vaccines were highly effective in response to the COVID-19 pandemic, making them an attractive platform to address cancers and other infectious diseases. Many new mRNA vaccines in development are multivalent, which represents a difficulty for the standard assays commonly used to characterize the critical quality attributes of monovalent formulations. Here, we present a multiplexed analytical tool with nucleic acid microarray technology using the VaxArray platform that measures the identity and quantity of mono- and multivalent mixtures of naked mRNA and mRNA encapsulated in lipid nanoparticle formulations in under 2 h without any additional preparation steps, such as extraction or RT-PCR. Using a quadrivalent mixture of encapsulated mRNA constructs that encode for four unique proteins in a vaccine formulation, the VaxArray mRNA assay was demonstrated to be highly specific for each mRNA with sensitivity < 1 µg/mL. The quantification of individual mRNAs within the lipid nanoparticle mixture resulted in a precision of ≤10% RSD and an accuracy of 100 ± 9%.

The success of genomic sequencing is impossible without the development of information technologies and mathematical methods for data processing to establish various features in the analyzed objects (nucleic acids) and trends in their changes. The volume of experimental data in the research of the genome has grown significantly, and new methods and algorithms are required for their processing. The primary stage of processing the data of devices for genomic parallel sequencing is the evaluation of the parameters of images obtained from video cameras in the form of electrical signals. The next stage of processing is the construction of a sequence of nucleotides according to algorithms that depend on the principle of operation of the device for sequencing nucleic acids. When performing this stage, algorithms for evaluating quality indicators for all individual readings (reads) are important. One of the ways to assess quality is to use algorithms based on the k-measure analysis methodology. The calculation of the number of occurrences of k-measures during the experiment on the parallel sequencing system makes it possible to assess the reliability of the analysis. In this article, algorithms for processing genetic analyzer data are considered. [ABSTRACT FROM AUTHOR]

Ovarian cancer continues to be a difficult medical issue that affects millions of individuals worldwide. Important platforms for cancer immunotherapy include checkpoint inhibitors, chimeric antigen receptor T cells, bispecific antibodies, cancer vaccines, and other cell‐based treatments. To avoid numerous infectious illnesses, conventional vaccinations based on synthetic peptides, recombinant subunit vaccines, and live attenuated and inactivated pathogens are frequently utilized. Vaccine manufacturing processes, however, are not entirely safe and carry a significant danger of contaminating living microorganisms. As a result, the creation of substitute vaccinations is required for both viral and noninfectious illnesses, including cancer. Recently, there has been testing of nucleic acid vaccines, or NAVs, as a cancer therapeutic. Tumor antigens (TAs) are genetically encoded by DNA and mRNA vaccines, which the host uses to trigger immune responses against ovarian cancer cells that exhibit the TAs. Despite being straightforward, safe, and easy to produce, NAVs are not currently thought to be an ideal replacement for peptide vaccines. Some obstacles to this strategy include selecting the appropriate therapeutic agents (TAs), inadequate immunogenicity, and the immunosuppressive characteristic of ovarian cancer. We focus on strategies that have been employed to increase NAVs' effectiveness in the fight against ovarian cancer in this review. [ABSTRACT FROM AUTHOR]

The development of nucleic acid‐based drugs holds great promise for therapeutic applications, but their effective delivery into cells is hindered by poor cellular membrane permeability and inherent instability. To overcome these challenges, delivery vehicles are required to protect and deliver nucleic acids efficiently. Silica nanoparticles (SiNPs) have emerged as promising nanovectors and recently bioregulators for gene delivery due to their unique advantages. In this review, a summary of recent advancements in the design of SiNPs for nucleic acid delivery and their applications is provided, mainly according to the specific type of nucleic acids. First, the structural characteristics and working mechanisms of various types of nucleic acids are introduced and classified according to their functions. Subsequently, for each nucleic acid type, the use of SiNPs for enhancing delivery performance and their biomedical applications are summarized. The tailored design of SiNPs for selected type of nucleic acid delivery will be highlighted considering the characteristics of nucleic acids. Lastly, the limitations in current research and personal perspectives on future directions in this field are presented. It is expected this opportune review will provide insights into a burgeoning research area for the development of next‐generation SiNP‐based nucleic acid delivery systems. [ABSTRACT FROM AUTHOR]

Background and Aim: Management of chronic hepatitis delta (CHD) requires reliable tests for HDV RNA quantification. The aim of the study was to compare two extraction methods for the quantification of HDV RNA in untreated and bulevirtide (BLV)‐treated CHD patients. Methods: Frozen sera from untreated and BLV‐treated CHD patients were tested in a single‐centre study for HDV RNA levels (Robogene 2.0, Roboscreen GmbH, Leipzig, Germany; LOD 6 IU/mL) with two extraction methods: manual (INSTANT Virus RNA/DNA kit; Roboscreen GmbH, Leipzig, Germany) versus automated (EZ1 DSP Virus Kit; Qiagen, Hilden, Germany). BLV‐treated patients were sampled at baseline and during therapy. Results: Two hundred sixty‐four sera collected from 157 CHD (139 untreated, 18 BLV‐treated) patients were analysed: age 51 (28–78), 59% males, 90% of European origin, 60% cirrhotics, ALT 85 (17–889) U/L, HBsAg 3.8 (1.7–4.6) Log IU/mL, 81% HBV DNA undetectable, 98% HDV genotype 1. Median HDV RNA was 4.53 (.70–8.10) versus 3.77 (.70–6.93) Log IU/mL by manual versus automated extraction (p <.0001). Manual extraction reported similar HDV RNA levels in 31 (20%) patients, higher in 119 (76%) [+.5 and +1 log10 in 60; > +1 log10 in 59] and lower in 7 (4%). Among 18 BLV‐treated patients, rates of HDV RNA < LOD significantly differed between the two assays at Weeks 16 and 24 (0% vs. 22%, p =.02; 11% vs. 44%, p =.03), but not at later timepoints. By contrast, virological response rates were similar. Conclusions: Quantification of HDV RNA by Robogene 2.0 is influenced by the extraction method, the manual extraction being 1 Log more sensitive. [ABSTRACT FROM AUTHOR]

RNA G‐quadruplexes (D‐rG4s) are prevalent in the transcriptome and play crucial regulatory roles in various biological processes. Recently, L‐RNA aptamers have been reported to recognize functional rG4s with a strong binding affinity and specificity. However, owing to the poor cell penetration capacity of L‐RNA aptamers, their biological applications are currently limited. Herein, we rationally design an L‐RNA aptamer‐peptide conjugate, Tamra_Ahx_R8_L‐Apt.4‐1c, which can efficiently translocate into the cytosol and target the rG4 of interest. Notably, we demonstrate diverse regulatory roles of Tamra_Ahx_R8_L‐Apt.4‐1c on rG4 motif present in different regions of mRNAs and further expand the application in different cell lines. Our novel and biocompatible conjugate enhances the cellular uptake of the L‐RNA aptamer, and our robust strategy enables non‐canonical RNA structures to be targeted by L‐RNA aptamers for gene control in cells. [ABSTRACT FROM AUTHOR]

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5′-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide. [ABSTRACT FROM AUTHOR]

Simple Summary: This study presents a comprehensive analysis of the global prevalence of bovine parainfluenza-3 virus (BPI3V) in cattle, a significant pathogen in the bovine respiratory disease complex (BRDC). By systematically reviewing and meta-analysing data from 71 articles across 32 countries, the study reveals variations in BPI3V prevalence based on different detection methods, including antibodies, antigen and nucleic acid detection, and virus isolation. The findings indicate a similar prevalence of BPI3V in cattle with BRDC compared to that in the general cattle population. This research underscores the need for dedicated BPI3V studies to better understand its role within BRDC and inform targeted disease management strategies. Bovine parainfluenza-3 virus (BPI3V) is an important respiratory pathogen in cattle, contributing to syndromes in the bovine respiratory disease complex (BRDC). Despite its significance, the understanding of its prevalence remains fragmented, especially within the larger framework of BRDC. This systematic review and meta-analysis aimed to determine the global prevalence of BPI3V in cattle using varied detection methods and to highlight associated risk factors. Of 2187 initially retrieved articles, 71 were selected for analysis, covering 32 countries. Depending on the detection method employed, the meta-analysis revealed significant variations in BPI3V prevalence. In the general cattle population, the highest prevalence was observed using the antibody detection method, with a proportion of 0.64. In contrast, in cattle with BRDC, a prevalence of 0.75 was observed. For the antigen detection method, a prevalence of 0.15 was observed, exclusively in cattle with BRDC. In nucleic acid detection, a prevalence of 0.05 or 0.10 was observed in the general and BRDC cattle populations, respectively. In virus isolation methods, a prevalence of 0.05 or 0.04 was observed in the general and BRDC cattle populations, respectively. These findings highlight the differences in the detection ability of different methods in identifying BPI3V. Other factors, such as country, study year, coinfections, farm size, the presence of respiratory signs, sex, and body weight, may also affect the prevalence. Most studies were anchored within broader BRDC investigations or aimed at detecting other diseases, indicating a potential under-representation of focused BPI3V research. BPI3V plays an important role in BRDC, with its prevalence varying significantly based on the detection methodology. To further understand its unique role within BRDC and pave the way for targeted interventions, there is an evident need for independent, dedicated research on BPI3V. [ABSTRACT FROM AUTHOR]

Objectives Nanotechnology, the manipulation of matter at the anatomical and molecular levels, has paved the way for the creation of materials with unique and enhanced properties. One of its significant applications is in the realm of nano-biotechnology, where various subfields, such as nanostructures, play a crucial role in fields like biotechnology and structural and cellular genomics. Nano-biotechnology offers a groundbreaking approach to research by allowing scientists to introduce chemicals and components into cells and fabricate novel materials through innovative techniques like assembling. Particularly noteworthy is its contribution to the in vivo administration of Nucleic Acid (NA) therapeutics. Advanced nanoscale biotechnology strategies have played a pivotal role in this domain by providing precise control over crucial parameters such as dimension, charge, drug loading, and response to external signals of delivery transporters. The integration of cutting-edge nanoscale biotechnology in the field of cancer immunotherapy holds significant promise for overcoming obstacles in NA treatment. This involves addressing challenges related to the clinical and preclinical research of different nanocarriers. This article serves as a concise overview, delving into the characteristics and encountered difficulties associated with various nanocarriers during clinical and preclinical investigations. Materials and methods This paper discusses a range of NA methods and elucidates how state-of-the-art nanoscale biotechnology actively supports and enhances NA treatment. By manipulating the physical and chemical attributes of delivery transporters, nanotechnology allows for the optimization of therapeutic outcomes, making it an invaluable tool in the advancement of medical research. Conclusions In conclusion, the merger of nanotechnology and biotechnology, specifically in the field of nano)biotechnology, opens up new avenues for scientific exploration and medical advancements. The precision and versatility offered by nanoscale techniques hold immense potential in revolutionizing the landscape of NA therapeutics, paving the way for more effective and targeted treatments. The ongoing progress in cutting-edge Nanobiotechnology (N-Bio-Tech) exemplifies the relentless pursuit of solutions to some of the most significant challenges in medical science, particularly in the realm of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine. [ABSTRACT FROM AUTHOR]

Context and results: Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance spectroscopy methods has been successfully used to predict the structures of nucleic acids. These methods measure the distances between spin labels yielding distance equations that are solved using numerical algorithms to provide one or several structural predictions. In this work, the minimum number of SDSL distance measurements and distance measurement types required to predict a unique nucleic acid structure were investigated. Our results indicate that at least six distance measurements should be obtained given that the distance measurements do not connect one SDSL on one arm with more than three SDSLs on the other arm. Moreover, there may be a preference for 1-to-1 SLs distance measurements rather than 1-to-many SLs as the latter was linked to undefined structures discussed in this study. Methods: Pairs of double-helical arms of nucleic acid were simulated using the finite element software Pro/ENGINEER (PTC Inc., Boston, MA). In each simulation, a specific SDSL distance measurement plan was adopted and the resulting structure was tested for movability. Immovable structures indicate that this plan will potentially result in a unique structural prediction of the nucleic acid. All the possible plans for SDSL distance measurements were investigated either by direct measurement or by extrapolation. [ABSTRACT FROM AUTHOR]

With the rapid development of clinical diagnosis and treatment, many traditional and conventional in vitro diagnosis technologies are unable to meet the demands of clinical medicine development. In this situation, nanomaterials are rapidly developing and widely used in the field of in vitro diagnosis. Nanomaterials have distinct size-dependent physical or chemical properties, and their optical, magnetic, electrical, thermal, and biological properties can be modulated at the nanoscale by changing their size, shape, chemical composition, and surface functional groups, particularly because they have a larger specific surface area than macromaterials. They provide an amount of space to modify different molecules on their surface, allowing them to detect small substances, nucleic acids, proteins, and microorganisms. Combining nanomaterials with in vitro diagnosis is expected to result in lower detection limits, higher sensitivity, and stronger selectivity. In this review, we will discuss the classfication and properties of some common nanomaterials, as well as their applications in protein, nucleic acids, and other aspect detection and analysis for in vitro diagnosis, especially on aging-related nanodiagnostics. Finally, it is summarized with guidelines for in vitro diagnosis.

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn’t allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.

Abstract Background It is increasingly recognized that conventional food production systems are not able to meet the globally increasing protein needs, resulting in overexploitation and depletion of resources, and environmental degradation. In this context, microbial biomass has emerged as a promising sustainable protein alternative. Nevertheless, often no consideration is given on the fact that the cultivation conditions affect the composition of microbial cells, and hence their quality and nutritional value. Apart from the properties and nutritional quality of the produced microbial food (ingredient), this can also impact its sustainability. To qualitatively assess these aspects, here, we investigated the link between substrate availability, growth rate, cell composition and size of Cupriavidus necator and Komagataella phaffii. Results Biomass with decreased nucleic acid and increased protein content was produced at low growth rates. Conversely, high rates resulted in larger cells, which could enable more efficient biomass harvesting. The proteome allocation varied across the different growth rates, with more ribosomal proteins at higher rates, which could potentially affect the techno-functional properties of the biomass. Considering the distinct amino acid profiles established for the different cellular components, variations in their abundance impacts the product quality leading to higher cysteine and phenylalanine content at low growth rates. Therefore, we hint that costly external amino acid supplementations that are often required to meet the nutritional needs could be avoided by carefully applying conditions that enable targeted growth rates. Conclusion In summary, we demonstrate tradeoffs between nutritional quality and production rate, and we discuss the microbial biomass properties that vary according to the growth conditions.

Calcium phosphates (CaPs) and their substituted derivatives encompass a large number of compounds with a vast presence in nature that have aroused a great interest for decades. In particular, hydroxyapatite (HAp, Ca10(OH)2(PO4)6) is the most abundant CaP mineral and is significant in the biological world, at least in part due to being a major compound in bones and teeth. HAp exhibits excellent properties, such as safety, stability, hardness, biocompatibility, and osteoconductivity, among others. Even some of its drawbacks, such as its fragility, can be redirected thanks to another essential feature: its great versatility. This is based on the compound’s tendency to undergo substitutions of its constituent ions and to incorporate or anchor new molecules on its surface and pores. Thus, its affinity for biomolecules makes it an optimal compound for multiple applications, mainly, but not only, in biological and biomedical fields. The present review provides a chemical and structural context to explain the affinity of HAp for biomolecules such as proteins and nucleic acids to generate hybrid materials. A size-dependent criterium of increasing complexity is applied, ranging from amino acids/nucleobases to the corresponding macromolecules. The incorporation of metal ions or metal complexes into these functionalized compounds is also discussed.

Immunotherapy combats tumors by enhancing the body’s immune surveillance and clearance of tumor cells. Various nucleic acid drugs can be used in immunotherapy, such as DNA expressing cytokines, mRNA tumor vaccines, small interfering RNAs (siRNA) knocking down immunosuppressive molecules, and oligonucleotides that can be used as immune adjuvants. Nucleic acid drugs, which are prone to nuclease degradation in the circulation and find it difficult to enter the target cells, typically necessitate developing appropriate vectors for effective in vivo delivery. Biomimetic drug delivery systems, derived from viruses, bacteria, and cells, can protect the cargos from degradation and clearance, and deliver them to the target cells to ensure safety. Moreover, they can activate the immune system through their endogenous activities and active components, thereby improving the efficacy of antitumor immunotherapeutic nucleic acid drugs. In this review, biomimetic nucleic acid delivery systems for relieving a tumor immunosuppressive microenvironment are introduced. Their immune activation mechanisms, including upregulating the proinflammatory cytokines, serving as tumor vaccines, inhibiting immune checkpoints, and modulating intratumoral immune cells, are elaborated. The advantages and disadvantages, as well as possible directions for their clinical translation, are summarized at last.

Abstract Targeted protein degradation (TPD) is an emerging therapeutic strategy with the potential to modulate disease-associated proteins that have previously been considered undruggable, by employing the host destruction machinery. The exploration and discovery of cellular degradation pathways, including but not limited to proteasomes and lysosome pathways as well as their degraders, is an area of active research. Since the concept of proteolysis-targeting chimeras (PROTACs) was introduced in 2001, the paradigm of TPD has been greatly expanded and moved from academia to industry for clinical translation, with small-molecule TPD being particularly represented. As an indispensable part of TPD, biological TPD (bioTPD) technologies including peptide-, fusion protein-, antibody-, nucleic acid-based bioTPD and others have also emerged and undergone significant advancement in recent years, demonstrating unique and promising activities beyond those of conventional small-molecule TPD. In this review, we provide an overview of recent advances in bioTPD technologies, summarize their compositional features and potential applications, and briefly discuss their drawbacks. Moreover, we present some strategies to improve the delivery efficacy of bioTPD, addressing their challenges in further clinical development.

Intervertebral disc degeneration (IVDD) is a leading cause of low back pain. The strategy of using functional materials to deliver nucleic acids provides a powerful tool for ameliorating IVDD. However, the immunogenicity of nucleic acid vectors and the poor mechanical properties of functional materials greatly limit their effects. Herein, antagomir-204–3p (AM) shows low immunogenicity and effectively inhibits the apoptosis of nucleus pulposus cells. Moreover, a high-strength biohydrogel based on zinc-oxidized sodium alginate-gelatin (ZOG) is designed as a multifunctional nucleic acid delivery platform. ZOG loaded with AM (ZOGA) exhibits great hygroscopicity, antibacterial activity, biocompatibility, and biodegradability. Moreover, ZOGA can be cross-linked with nucleus pulposus tissue to form a high-strength collagen network that improves the mechanical properties of the intervertebral disc (IVD). In addition, ZOGA provides an advantageous microenvironment for genetic expression in which AM can play an efficient role in maintaining the metabolic balance of the extracellular matrix. The results of the radiological and histological analyses demonstrate that ZOGA restores the height of the IVD, retains moisture in the IVD, and maintains the tissue structure. The ZOGA platform shows the sustained release of nucleic acids and has the potential for application to ameliorate IVDD, opening a path for future studies related to IVD.

CRISPR/Cas systems have become integral parts of nucleic acid detection apparatus and biosensors. Various CRISPR/Cas systems such as CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, CRISPR/Cas14 and CRISPR/Cas3 utilize different mechanisms to detect or differentiate biological activities and nucleotide sequences. Usually, CRISPR/Cas-based nucleic acid detection systems are combined with polymerase chain reaction, loop-mediated isothermal amplification, recombinase polymerase amplification and transcriptional technologies for effective diagnostics. Premised on these, many CRISPR/Cas-based nucleic acid biosensors have been developed to detect nucleic acids of viral and bacterial pathogens in clinical samples, as well as other applications in life sciences including biosecurity, food safety and environmental assessment. Additionally, CRISPR/Cas-based nucleic acid detection systems have showed better specificity compared with other molecular diagnostic methods. In this review, we give an overview of various CRISPR/Cas-based nucleic acid detection methods and highlight some advances in their development and components. We also discourse some operational challenges as well as advantages and disadvantages of various systems. Finally, important considerations are offered for the improvement of CRISPR/Cas-based nucleic acid testing.