Search Results (318)

Cardiovascular diseases such as myocardial infarction or limb ischemia are characterized by regression of blood vessels. Local delivery of growth factors (GFs) involved in angiogenesis such as fibroblast blast growth factor-2 (FGF-2) has been shown to trigger collateral neovasculature and might lead to a therapeutic strategy. In vivo, heparin, a sulfated polysaccharide present in abundance in the extracellular matrix (ECM), has been shown to function as a local reservoir for FGF-2 by binding FGF-2 and other morphogens and it plays a role in the evolution of GF gradients. To access injectable biomaterials that can mimic such natural electrostatic interactions between soluble signals and macromolecules and mechanically tunable environments, the backbone of agarose, a thermogelling marine–algae-derived polysaccharide, was modified with sulfate, phosphate, and carboxylic moieties and the interaction and release of FGF-2 from these functionalized hydrogels was assessed by ELISA in vitro and CAM assay in ovo. Our findings show that FGF-2 remains active after release, and FGF-2 release profiles can be influenced by sulfated and phosphorylated agarose, and in turn, promote varied blood vessel formation kinetics. These modified agaroses offer a simple approach to mimicking electrostatic interactions experienced by GFs in the extracellular environment and provide a platform to probe the role of these interactions in the modulation of growth factor activity and may find utility as an injectable gel for promoting angiogenesis and as bioinks in 3D bioprinting. Full article

Background: The advent of next-generation sequencing (NGS) has revolutionized the analysis of genetic data, enabling rapid identification of pathogenic variants in patients with inborn errors of immunity (IEI). Sometimes, the use of NGS-based technologies is associated with challenges in the evaluation of the clinical significance of novel genetic variants. Methods: In silico prediction tools, such as SpliceAI neural network, are often used as a first-tier approach for the primary examination of genetic variants of uncertain clinical significance. Such tools allow us to parse through genetic data and emphasize potential splice-altering variants. Further variant assessment requires precise RNA assessment by agarose gel electrophoresis and/or cDNA Sanger sequencing. Results: We found two novel heterozygous variants in the coding region of the LYST gene (c.10104G>T, c.10894A>G) in an individual with a typical clinical presentation of Chediak–Higashi syndrome (CHS). The SpliceAI neural network predicted both variants as probably splice-altering. cDNA assessment by agarose gel electrophoresis revealed the presence of abnormally shortened splicing products in each variant’s case, and cDNA Sanger sequencing demonstrated that c.10104G>T and c.10894A>G substitutions resulted in a shortening of the 44 and 49 exons by 41 and 47 bp, respectively. Both mutations probably lead to a frameshift and the formation of a premature termination codon. This, in turn, may disrupt the structure and/or function of the LYST protein. Conclusions: We identified two novel variants in the LYST gene, predicted to be deleterious by the SpliceAI neural network. Agarose gel cDNA electrophoresis and cDNA Sanger sequencing allowed us to verify inappropriate splicing patterns and establish these variants as disease-causing. Full article

Clostridioides difficile is a common etiological factor of hospital infections, which, in extreme cases, can lead to the death of patients. Most strains belonging to this bacterium species synthesize very dangerous toxins: toxin A (TcdA) and B (TcdB) and binary toxin (CDT). The aim of this study was to assess the suitability of agarose gel electrophoresis separation of multiplex PCR amplicons to investigate the toxinogenic potential of C. difficile strains. Additionally, the frequency of C. difficile toxin genes and the genotypes of toxin-producing strains were determined. Ninety-nine C. difficile strains were used in the detection of the presence of genes encoding all of these toxins using the multiplex PCR method. In 85 (85.9%) strains, the presence of tcdA genes encoding enterotoxin A was detected. In turn, in 66 (66.7%) isolates, the gene encoding toxin B (tcdB) was present. The lowest number of strains tested was positive for genes encoding a binary toxin. Only 31 (31.3%) strains possessed the cdtB gene and 22 (22.2%) contained both genes for the binary toxin subunits (the cdtB and cdtA genes). A relatively large number of the strains tested had genes encoding toxins, whose presence may result in a severe course of disease. Therefore, the accurate diagnosis of patients, including the detection of all known C. difficile toxin genes, is very important. The multiplex PCR method allows for the quick and accurate determination of whether the tested strains of this bacterium contain toxin genes. Agarose gel electrophoresis is a useful tool for visualizing amplification products, allowing one to confirm the presence of specific C. difficile toxin genes as well as investigate their dissemination for epidemiological purposes. Full article

Antimicrobial resistance (AMR) is a critical global public health problem. Many bacterial pathogens use biofilm formation as their main pathogenicity mechanism, a practical tactic for surviving in natural settings and colonized host tissues. Research using ruthenium(II) complexes has demonstrated antibacterial action linked to photodynamic therapy, an alternate method of microbial control. Thus, in this work, the photosensitive nitro complex [RuCl(NO₂)(dppb)(4,4-Mebipy)] (I) was prepared and the X-ray structure was determined. Then, we investigated the antibacterial and antibiofilm activities, antibiotic-associated effects, and cytotoxicity. The results showed that complex I exhibited promising antimicrobial activity with MIC values ranging from 4 to 256 µg/mL and MBC from 4 to 32 µg/mL. The antimicrobial activity of this nitro complex was significantly enhanced with blue light irradiation, as confirmed by agarose gel electrophoresis of the pBR322 DNA, which must be related to the DNA cleavage promoted by the photorelease of NO. A synergistic effect against Staphylococcus aureus and Staphylococcus epidermidis strains was observed when combined with ampicillin, which exhibited FICI values from 0.186 to 0.311. Interestingly, complex I associated with tetracycline showed a synergistic effect only on Escherichia coli. Regarding biofilms, the irradiated complex I showed antibacterial activity against biofilm formation and mature biofilms. Furthermore, SEM and confocal analyses revealed changes in cell morphology and damage to the wall and plasma membrane. Complex I presented a percentage of hemolysis between 2 and 4%, and no cytotoxic effect was observed against murine dermal fibroblasts. In conclusion, the photoactivated ruthenium(II) complex showed antibacterial and antibiofilm activity against relevant bacteria. Full article

Objectives: The aim of this study was to describe the phenotypic and molecular characteristics of Acinetobacter baumannii isolates carrying resistance genes to beta-lactams and carbapenems in six Peruvian public hospital centers. Materials and methods: The susceptibility of bacterial isolates was determined using the automated MicroScan system, with interpretation according to the M100 S30 CLSI 2020. Resistance genes were identified by conventional polymerase chain reaction (PCR), and PCR products were visualized by 1% agarose gel electrophoresis. Results: Nine strains (TRU1, PM1, PM2, CUS1, CUS2, CUS3, CAL1, CAL2 and CAL3) out of a total of 21 strains in the study were reactivated, showing resistance of 77.8% to imipenem, ciprofloxacin and cefepime, followed by 66.7% resistance to meropenem and ceftazidime, indicating marked multidrug resistance. In addition, the detection of the group A beta-lactamase genes blaCTX-M and blaTEM was confirmed, showing co-resistance in strains CUS1, CUS2 and CUS3, despite their unusual presence in this pathogen, also determined by the presence of the group D carbapenemase blaOXA in strain CUS3, the only strain to show co-resistance of the three groups. Conclusion: The prevalence of Acinetobacter baumannii resistant to extended-spectrum beta-lactamases and carbapenemases in Peruvian public centers represents a critical challenge for the treatment of infections. Rigorous surveillance, infection control strategies, and the development of alternative therapies are urgently needed to address this growing bacterial resistance. Full article

Hydrogels like agarose have long been used as sieving media for the electrophoresis-based analysis of biopolymers. During gelation, the individual agarose strands tend to form hydrogen-bond mediated double-helical structures, allowing thermal reversibility and adjustable pore sizes for molecular sieving applications. The addition of tetrahydroxyborate to the agarose matrix results in transitional chemical cross-linking, offering an additional pore size adjusting option. Separation of SDS-proteins during gel electrophoresis is an activated process defined by the interplay between viscosity, gelation/cross-link formation/distortion, and sample conformation. In this paper, the subunits of a therapeutic monoclonal antibody were separated by capillary SDS agarose gel electrophoresis at different temperatures. The viscosity of the separation matrix was also measured at all temperatures. In both instances, Arrhenius plots were used to obtain the activation energy values. It was counterintuitively found that larger SDS–protein complexes required lower activation energies while their low-molecular-weight counterparts needed higher activation energy for their electromigration through the sieving matrix. As a first approximation, we considered this phenomenon the result of the electric force-driven distortion of the millisecond range lifetime reticulations by the larger and consequently more heavily charged electromigrating molecules. In the meantime, the sieving properties of the gel were still maintained, i.e., they allowed for the size-based separation of the sample components, proving the existence of the reticulations. Information about the activation energy sheds light on the possible deformation of the sieving matrix and the solute molecules. In addition, the activation energy requirement study helped in optimizing the separation temperature, e.g., with our sample mixture, the highest resolution was obtained for the high-molecular-weight fragments, i.e., between the non-glycosylated heavy chain and heavy-chain subunits at 25 °C (lower E_a requirement), while 55 °C was optimal for the lower-molecular-weight light chain and non-glycosylated heavy chain pair (lower E_a requirement). Future research directions and possible applications are also proposed. Full article

Inhalation of aerosolized uranium is recognized as a principal mode of exposure, posing significant risks of damage to the lungs, kidneys, and other vital organs. To enhance nuclide elimination from the body, chelating agents are employed; however, single-component chelators often exhibit limited spectral activity and low effectiveness, resulting in toxicologically relevant concentrations. We have developed a composite chelating agent composed of 3,4,3-Li(1,2-HOPO), DFP, and HEDP in optimized ratios, demonstrating marked improvements in eliminating inhaled uranium. The selection of these components was initially guided by an agarose gel dynamics method, focusing on uranium binding and removal efficacy. Optimization of the formula was conducted through response surface methodology in a cellular model. The compound’s ability to enhance survival rates in mice subjected to acute uranium inhalation was confirmed, showing a dose-dependent improvement in survival in severely affected mice. Comparative assessments indicated that this multifaceted chelating agent substantially surpasses the uranium tissue clearance achieved by individual chelating agents. Full article

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers. Full article

The previously available coding region for the spiny dogfish (Squalus acanthias) AQP3-2 gene was amplified from cDNAs using PCR. Agarose gel electrophoresis gave a band of the AQP3-2 coding region, as well as multiple smaller splice variant bands. The main AQP3-2 band and the largest and most fluorescently intense pair of these splice variant bands were cloned and sequenced. Amplifications were performed on a range of tissue cDNAs, but AQP3-2 was only expressed in the kidney and brain. Quantitative PCR amplifications using pre-existing kidney cDNA from an environmental salinity acclimation experiment showed that the abundance of mRNA from both the main AQP3-2 transcript and the largest splice variant (Splice Variant 1) was lower in 120% seawater (SW) acclimated fish, although only the values for Splice Variant 1 were statistically significant. A custom-made affinity-purified rabbit polyclonal AQP3-2 antibody was produced, and this gave four bands of around the correct sizes (which were 27 and 32 kDa) for the complete AQP3-2 and Splice Variant 1 proteins. Two of the bands may have been N-glycosylated forms of these proteins. Other bands were also present on the Western blot. No bands were present when the antibody was pre-blocked by the peptide antigen. In tissue sections of the dogfish kidney, immunohistochemical localization experiments showed that AQP3-2 was expressed in the early distal tubule (EDT) and late distal tubule (LDT) nephron segments. The results suggest that AQP3-2 may be involved in cell volume regulation in the EDT and water and urea absorption in the LDT nephron segment. Full article

Rice (Oryza sativa) plays a pivotal role in global food security. Understanding the genetics of rice cultivation is crucial, particularly for traits such as viviparous germination, which significantly influences germination and yield. Our research aimed to elucidate the genetic and molecular mechanisms by which the Sdr4 gene influences viviparous germination and to develop novel molecular markers for this gene to enhance breeding strategies against viviparous germination. In all, 683 rice cultivars and 100 F₂ plants were used for viviparous germination and genetic analysis using KASP (Kompetitive Allele-Specific PCR) and agarose gel-based markers related to viviparous germination tolerance. We developed and used a polymorphic agarose gel-based marker and a KASP marker targeting the Sdr4 gene. A genetic analysis of field-grown rice cultivars and the F₂ population revealed that the two markers on Sdr4 were functional for the genomic selection of SNPs and InDels related to dormancy. The Pearson correlation coefficient (r = 0.74, p-value = 3.31 × 10⁻⁸) between the Sdr4-IND KASP marker genotype and viviparous germination rate demonstrated a significant positive correlation, supporting the marker’s utility for selecting rice varieties with diminished viviparous germination. This insight serves as a critical theoretical foundation for breeding strategies for developing early-maturing rice varieties with enhanced resistance to viviparous germination, addressing pivotal challenges in rice cultivation, and ensuring food security. Full article

Concatemeric viral DNA is packaged into bacteriophage P22 procapsids via a headful packaging mechanism mediated by a molecular machine consisting of small (gp3) and large (gp2) terminase subunits. Although a negative stain reconstruction exists for the terminase holoenzyme, it is not clear how this complex binds the dodecameric portal protein located at a 5-fold mismatch vertex. Herein, we describe new assemblies for the holoenzyme. Both native mass spectrometry and transmission electron microscopy reveal that the P22 terminase complex adopts three main assemblies, which include a nonameric S-terminase bound to two L-terminase 1(gp3)₉:2(gp2), two nonameric S-terminase bound to five L-terminase 2(gp3)₉:5(gp2), and three nonameric S-terminase bound to seven L-terminase 3(gp3)₉:7(gp2). Native agarose gel electrophoresis shows that the terminase complex interacts with procapsids with mild crosslinking. These results herein illustrate the P22 terminase complex can adopt a variety of conformations and assembly states. Full article

Background and Objectives: Alzheimer’s dementia is a progressive neurodegenerative disease that affects memory abilities due to genetic and environmental factors. A well-known gene that influences the risk of Alzheimer’s disease is the apolipoprotein E (APOE) gene. The APOE gene is involved in the production of a protein that helps transport cholesterol and other types of fat in the bloodstream. Problems in this process are thought to contribute to the development of Alzheimer’s disease. APOE comes in several forms, which are called alleles (ε2, ε3, ε4). Materials and Methods: Therefore, our study aims to identify those subjects with a higher genetic risk through the polymorphism of the APOE gene, using a population screening in patients with a clinical diagnosis of AD in a region of Spain, Castilla y León, as potential biomarkers and to identify individuals at increased genetic risk by polymorphism of the APOE gene. An observational case-control study was conducted in Castilla y León (Spain). Saliva samples were collected and the ApoE gene was analyzed by PCR and agarose gel electrophoresis, respecting ethical criteria. Results: In the Alzheimer’s population in Castilla y León, a high prevalence of ApoE3 (74%) was found, followed by ApoE4 (22%); in addition, a higher presence of the ε4 allele was found in the Alzheimer’s disease (AD) group than in the control group. It was also observed that the ε2/ε2 genotype was not found in any individual with AD but was found in healthy subjects and that the opposite was observed for the ε4/ε4 genotype. The odds ratio (OR) indicated a risk four times greater of having AD if having the ε4 allele. Conclusions: The demonstrated relation between the different isoforms and the likelihood of developing AD has led to its consideration as a biomarker and a potential pre-symptomatic therapy. The molecular mechanisms that confer a disruptive and protective role to ApoE4 and ApoE2, respectively, are still being studied. Full article

Nanomedicine has introduced strategies that provide precise diagnosis and treatment with fewer side effects than traditional therapies. Treatments for neurodegenerative disorders, including Parkinson’s disease, are palliative, necessitating an innovative delivery system with a curative function. This study investigated a solid lipid nanoparticle (SLNP) system’s ability to bind and safely deliver siRNA in vitro. SLNPS were formulated using sphingomyelin and cholesterol, with Ginkgo biloba leaf extract (GBE) incorporated to enhance biocompatibility and neuroprotection. Poly-L-lysine (PLL) functionalization ensured successful siRNA binding, safe transport, and protection from nuclease degradation. SLNPs were physicochemically characterized, with binding and protection of siRNA assessed using agarose gels. Cytotoxicity, apoptotic induction, and cellular uptake studies were undertaken in the human neuroblastoma (SH-SY5Y) and embryonic kidney (HEK293) cells. The GBE-PLL-SLNPs had an average size of 93.2 nm and demonstrated enhanced binding and protection of the siRNA from enzyme digestion, with minimal cytotoxicity in HEK293 (<10%) and SH-SY5Y cells (<15%). Caspase 3/7 activity was significantly reduced in both cells, while efficient cellular uptake was noted. The present study provided a solid basis as a proof of principle study for future applications of the potential therapeutic in vitro, promising to address the unmet medical needs of patients with neurological disorders. Full article

Objective: The study aimed to optimize protocols for the joint extraction of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from 0.025 × 10⁶ CFU of Candida albicans, targeting to overcome the challenges in the extraction of these genetic materials. Materials and methods: From this, treated silicon carbide (SiC) granules were added to fungal samples from methods 1, 2, and 3 obtained from aliquots of BHI or Sabouraud medium to cause cell lysis and enable the isolation of these macromolecules by phenol and chloroform. The concentration and integrity of the extracted nucleic acids were analyzed, respectively, by spectrophotometry using the A₂₆₀/A₂₈₀ ratios and 1% agarose gel electrophoresis. Results: Therefore, method 3 is the one that most comprises samples considered pure of both DNA and RNA, simultaneously. Furthermore, the presence of intact RNAs corresponding to the base pair size such as 5.8 S rRNA and tRNA was verified during electrophoresis, considering the particularities of RNA, which makes it very unstable and easily degraded. Conclusions: Thus, it results in a faster and simpler method in addition to obtain promising results using minimal amounts of biological sample and offering a valuable alternative for small laboratories to work with molecular biology. Full article

Treponema pallidum subspp. pallidum is a spirochaete bacterium that causes syphilis, one of the most common sexually transmitted diseases. Syphilis progresses through four distinct stages, each characterized by specific symptoms, namely primary, secondary, latent, and late (tertiary) syphilis. Serology has been considered the primary diagnostic approach. However, it is plagued by problems such as the limited specificity of nontreponemal tests and the inadequate correlation of treponemal tests with disease activity. In this study, we focused on the development of a loop-mediated isothermal amplification assay utilizing hydroxy naphthol blue (LAMP-HNB) for the diagnosis of T. pallidum subspp. pallidum. Specifically, this study seeks to determine the analytical sensitivity (limit of detection; LOD) and analytical specificity. Four hundred clinical serum samples were analyzed for diagnostic sensitivity, specificity, and predictive value, and each technique’s 95% confidence intervals (95% CI, p < 0.05) were evaluated. The limit of detection for polymerase chain reaction with agarose gel electrophoresis (PCR-AGE), the loop-mediated isothermal amplification assay combined with agarose gel electrophoresis (LAMP-AGE), and LAMP-HNB were 116 pg/µL, 11.6 pg/µL, and 11.6 pg/ µL, respectively. Analytical specificity examinations indicated the absence of cross-reactivity with Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy human serum in PCR-AGE, LAMP-AGE, and LAMP-HNB. The diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV), and negative predictive value (NPV) for PCR-AGE were 100.00 (100.00)%, 94.50 (94.40–94.60)%, 94.79 (94.69–94.88)%, and 100.00 (100.00)%, respectively. While, for LAMP-AGE and LAMP-HNB, they were 100.00 (100.00)%, 91.00 (90.87–91.13)%, 91.74 (91.63–91.86)%, and 100.00 (100.00)%, respectively. The LAMP-HNB test is simple, rapid, highly sensitive, and highly specific, without requiring expensive equipment. In the future, the LAMP-HNB assay may develop into a single-step diagnostic process, enabling the use as point-of-care testing for the diagnosis, prevention, and management of syphilis infection. Full article