Various Types Of Biological Samples Research Articles

Advances in DNA Extraction Techniques: A Comprehensive Review of Methods and Applications

The extraction of DNA is a fundamental process in molecular biology, underpinning a wide range of applications from genetic research to forensic science and medical diagnostics. This review aims to explore the latest advancements in DNA extraction techniques, highlighting their principles, applications, and suitability for various types of biological samples. Traditional DNA extraction methods, such as phenol-chloroform and ethanol precipitation, have served as the backbone of DNA isolation for decades. However, these techniques often involve hazardous chemicals and can be time-consuming. Recent advancements have focused on developing safer, faster, and more efficient methods, with a focus on automation and scalability. Innovations such as magnetic bead-based extraction, silica column purification, and specialized kits have greatly simplified the process, allowing for high-throughput applications in clinical and research settings. In addition to these technical improvements, new approaches have emerged to address specific challenges, such as extracting DNA from degraded or trace samples, as seen in forensic investigations, or isolating cell-free DNA for non-invasive prenatal testing and cancer diagnostics. Moreover, microfluidic devices and lab-on-a-chip technologies are transforming the landscape by enabling DNA extraction from minute samples with minimal reagent consumption. This review discusses the principles underlying these advanced techniques, their benefits and limitations, and the specific contexts in which they excel. It also considers future trends, including further automation, integration with sequencing platforms, and the potential for point-of-care applications. By examining the current state of DNA extraction technology, this review aims to provide researchers and practitioners with a comprehensive guide to the best methods for their specific needs.

Analysis Of Admissibility Of Dna Based Forensic Evidence

The primary objective of the study is to understand the nature and characteristics of DNA based forensic evidence and to analyse grounds that affects its admissibility in the Indian Courts. This research paper analyses various types of biological samples from which DNA can be extracted to undertake DNA profiling. It analyses standard operating procedure for extraction, testing and preservation of the sample including its handling. The study doctrinally analyses various judgements of the superior courts to highlight the grounds accepted by the courts that affect the admissibility of DNA based forensic evidence for criminal trial read in conjunction of relevant statutes. The data for the research has been obtained through both primary and secondary sources in the form reported judgements on court websites, statutes in official gazettes, research papers published in reputed journal both online and offline, including edited books. The primary research objective this research paper aims to achieve is: -
 
 To develop understanding of DNA based forensic evidence.
 
 To identify principles affecting admissibility of DNA based forensic evidence.

Comparison of two molecular barcodes for the study of equine strongylid communities with amplicon sequencing.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

Paraquat and Diquat: Recent Updates on Their Pretreatment and Analysis Methods since 2010 in Biological Samples.

Paraquat (PQ) and diquat (DQ) are quaternary ammonium herbicides which have been used worldwide for controlling the growth of weeds on land and in water. However, PQ and DQ are well known to be toxic. PQ is especially toxic to humans. Moreover, there is no specific antidote for PQ poisoning. The main treatment for PQ poisoning is hemoperfusion to reduce the PQ concentration in blood. Therefore, it is essential to be able to detect PQ and DQ concentrations in biological samples. This critical review summarizes the articles published from 2010 to 2022 and can help researchers to understand the development of the sample treatment and analytical methods for the determination of PQ and DQ in various types of biological samples. The sample preparation includes liquid-liquid extraction, solid-phase extraction based on different novel materials, microextration methods, and other methods. Analytical methods for quantifying PQ and DQ, such as different chromatography and spectroscopy methods, electrochemical methods, and immunological methods, are illustrated and compared. We focus on the latest advances in PQ and DQ treatment and the application of new technologies for these analyses. In our opinion, tandem mass spectrometry is a good choice for the determination of PQ and DQ, due to its high sensitivity, high selectivity, and high accuracy. As far as we are concerned, the best LOD of 4 pg/mL for PQ in serum can be obtained.

M6A-SAC-seq for quantitative whole transcriptome m6A profiling.

N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative m6A sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively. Then the library can be prepared from various types of RNA samples using a ligation-based strategy, with m6A modifications being labeled by the enzyme and cofactor. Detailed instructions on ensuing data analysis are also included in this protocol. The method generates highly reproducible results, uncovering 31,233-129,263 sites using as little as 2 ng of poly A+ RNA. These identified sites correspond well with previous m6A profiling results, covering over 65% of peaks detected by the antibody-based approaches. Compared with other currently available methods, this method can be applied to various types of biological samples, including fresh and frozen tissues as well as formalin-fixed paraffin-embedded samples, providing a quantitative method to uncover new insights into m6A biology. The protocol requires basic expertise in molecular biology, recombinant protein expression and organic synthesis. The whole protocol can be done in 15 days, with the library preparation taking 5 days.

Design and development of smartphone-based imaging platform using electroluminescence illumination

Smartphone-based imaging platforms have shown great prospects as compared to the traditional optical microscopes. With their continuous development, they seem to have a wide variety of biomedical applications. In this work, a smartphone-based imaging platform was designed, developed, and fabricated using an electroluminescence display panel (EDP) as the source of illumination. The EDP is found to have homogenous intensities across the panel and helped in customizing the illumination with high image quality and contrast of the sample under study. The incorporation of polymeric BLIPS lens to the smartphone camera further increased the stability in image acquisition, easy to align the optical components as well as cost-effective. The current development can visualize various types of biological samples with high magnification.

Green Synthesis-Based Magnetic Diatoms for Biological Applications

Here, we report the green synthesis of silica-based materials for the treatment of biological samples such as bacteria enrichment and exosome isolation. The synthesized materials were nanosized, recyclable, and magnetically separable using an eco-friendly hydrothermal synthesis approach. Fe3O4 formation on the surface of biosilica porous diatomite via precipitation was used to create a magnetic diatom (mag-DE) through a bottom-up approach, which was low cost, robust, nontoxic, and eco-friendly. Following the addition of l-arginine-containing amino groups, the mag-DE showed broad absorbance in the visible light spectrum (UV–vis), and the amine functional groups of 3-aminopropyl triethoxysilane on the DE via the amidine bond were confirmed using Fourier transform infrared analysis. Further studies were conducted by conjugating the mag-DE with concanavalin A (ConA), a known micro-antimicrobial agent for bacterial enrichment, as well as with a specific antibody for exosome isolation. The large-volume samples obtained were easy to use, and the materials could enrich a minimum of 50 colony-forming units (CFUs) of Salmonella and also showed a promising ability to capture another bacterial species. In addition, the modified synthetic mag-DE could be used for exosome isolation using affinity purification. Functionalized mag-DE could be magnetically isolated in a rapid and more efficient manner than using the conventional method, which can be altered for exosome isolation in research and clinical applications. This study presents the unique physical and chemical properties of green synthesized mag-DE, whose surface can be easily modified. Thus, it creates a novel potential strategy for several desirable applications for various types of biological samples.

Biorepositories (biobanks) of human body fluids and materials as archives for tracing early infections of COVID-19.

Identifying the individuals and geographical regions witnessing early infections or outbreaks of SARS-CoV-2 and its variants is helpful for studying the early epidemiology or even the origin of the novel coronavirus. Here, we put forward a strategy that can potentially contribute to this goal. Human body fluids and biological materials collected before the COVID-19 pandemic may serve as archives for retrospective testing of early human infections before the recent outbreaks. These have been routinely donated, collected, and archived, creating biorepositories or “biobanks” for clinical or research purposes. SARS-CoV-2 genetic materials and its antibodies have been confirmed in various types of biological samples from COVID-19 patients, including blood, sperm, umbilical cord blood, lung, heart, kidney and so on, making these biological archives as candidates for detecting early COVID-19 infections. Unlike sewage-based epidemiology which only provides information on the geographical aspect, viruses identified in archived human biological samples provide direct links to individuals, from whom a wealth of personal information including their profession, hobbies and activities, travel history, and previous exposure to wildlife can all be retrieved. By analyzing the patterns and links in the behavior of those early infected individuals, it is possible to trace the origin of the virus, for instance, in certain wild animals or local environments.

A Universal Model for the Log-Normal Distribution of Elasticity in Polymeric Gels and Its Relevance to Mechanical Signature of Biological Tissues.

Simple SummaryMechanical properties of biological tissues are increasingly recognized as important in biology. Atomic force microscopy (AFM) is one of the main tools used to assess elastic properties of various types of biological samples. It has been noted that elasticity values frequently follow a log-normal distribution. We propose in this communication a physical model explaining this fact, and we propose that distribution-type analysis could increase the information obtained from AFM studies on biological tissues.The mechanosensitivity of cells has recently been identified as a process that could greatly influence a cell’s fate. To understand the interaction between cells and their surrounding extracellular matrix, the characterization of the mechanical properties of natural polymeric gels is needed. Atomic force microscopy (AFM) is one of the leading tools used to characterize mechanically biological tissues. It appears that the elasticity (elastic modulus) values obtained by AFM presents a log-normal distribution. Despite its ubiquity, the log-normal distribution concerning the elastic modulus of biological tissues does not have a clear explanation. In this paper, we propose a physical mechanism based on the weak universality of critical exponents in the percolation process leading to gelation. Following this, we discuss the relevance of this model for mechanical signatures of biological tissues.

Putrified tissue analysis: A challenge for Forensic toxicologists

The postmortem toxicology helps in determining the cause of death and information on events immediately before death. Forensic toxicologists receive various types of biological samples with putrefaction process which pose a greater challenge in chemical analysis. Interpretation of analytical toxicology results must incorporate pharmacokinetics and toxicology of the agents in question, the circumstances under which death occurred including the mechanism of exposure. The aim of present study is to describe the type of biological samples submitted for toxicological analysis in forensic toxicology section and factors associated in their analysis.

Design, synthesis and application of fluorogenic probe for detecting l-asparaginase in serum samples

Fluorescence-based in vitro assays are highly sensitive, selective and convenient to use, which is suitable for qualitative and quantitative detection of various types of biological samples. Herein, we designed and synthesized a novel fluorogenic probe TPAN-Asn for in vitro l-asparaginase detection. TPAN-Asn exhibited selective and robust response to l-asparaginase over various anions, cations and amino acids. More importantly, we have demonstrated that TPAN-Asn is able to accurately quantify the amount of l-asparaginase in patient serum samples. These results suggest that TPAN-Asn holds great potential in the benchmarking and analysis of l-asparaginase-based cancer therapy.

The impact of chimerism on DNA-based human identification from skin surface cells of post-allogenic hematopoietic stem cell transplantation (HCST) patients

The use of biological traces recovered from touched or handled items increased with the advance of the forensic analysis system. Thus, DNA profiles obtained from touch DNA became a useful tool in forensic investigation. However, a chimeric person with more than one chromosomal population can be challenging for a forensic analyst. We investigated the genetic profile in blood, buccal swab, and skin swabs from twenty-four recipients aged 21–63 years who underwent a matched sibling allogeneic hematopoietic stem cell transplantation with no sign of skin graft versus host disease. Autosomal short tandem repeats genotyping was performed to evaluate chimerism status at 15 loci along with gender marker Amelogenin. According to our results, donor chimerism was detected in all recipient's blood samples, while in buccal swabs, five recipients showed no presence of donor-derived cells in their genotype. Epithelial cells swabbed from hand fingertips were not devoid of donor-derived cells since all recipients showed high chimerism (39.69%–96.66%) in their genotypes. A significant change in chimerism was seen among various types of biological samples (p<0.05). No correlations were observed between chimerism and recipient age, gender, or time after transplant (p> 0.05). The loci D21S11, D8S1179, and FGA were the most informative, whereas D13S317, Vwa, and TOPX were the least informative STR markers. We concluded that touch DNA from a person who has undergone a successful allogeneic HSCTs should not be considered as reliable evidence for human identifications. Therefore, necessary precautions must be taken to avoid false identification and miscarriage of justice.

Database-Assisted Globally Optimized Targeted Mass Spectrometry (dGOT-MS): Broad and Reliable Metabolomics Analysis with Enhanced Identification.

Targeted mass spectrometry (MS) is an important measurement approach in metabolomics with strong analytical performance, given its specificity, sensitivity, and quantitative capacity. However, traditional targeted-MS relies heavily on chemical standards for the development of various detection panels; thus, its metabolite coverage is often limited to those well-known and commercially available compounds. To address this fundamental gap, we previously developed a novel approach [ H. Gu et al. Anal. Chem. 2015 , 87 , 12355 - 12362 ], globally optimized targeted (GOT)-MS, which enables reliable metabolic analysis with broad coverage using a single triple quadrupole instrument. In the present study, we further developed and optimized an innovative targeted MS approach, database-assisted globally optimized targeted (dGOT)-MS, which utilizes the HMDB and METLIN databases to significantly improve both identification and metabolite coverage. As it is well-known, these metabolomics databases have a comprehensive collection of metabolites and their tandem MS spectra; therefore, in this study, multiple reaction monitoring transitions (MRMs) were directly obtained from the databases and, after optimizing MS parameters for those MRMs, 927 metabolites were measured from a plasma aqueous extract sample with high reliability by dGOT-MS. Of these, 310 were confirmed using pure chemical standards while the rest were annotated by identification level using database entries. Furthermore, using breast cancer diagnosis as a proof-of-principle metabolomics application, we showed dGOT-MS to significantly outperform a traditional large-scale targeted MS assay for potential biomarker discovery. In principle, dGOT-MS is able to cover all metabolites (including lipids) that have been characterized in these comprehensive metabolomics databases from various types of biological samples. Therefore, dGOT-MS opens a novel avenue for MS measurements and may play an important role in many future metabolomics studies.

Isothermal calorimetry for monitoring of grape juice fermentation with yeasts immobilized on nylon-6 nanofibrous membranes

Isothermal microcalorimetry is often used to monitor microbiological activity and growth in various types of biological samples. Usually, it is useful to convert the isothermal microcalorimetry data into biologically meaningful data such as growth rate, lag phase or maximum growth. In this work, isothermal calorimetry was applied for monitoring of grape juice fermentation by Saccharomyces cerevisiae immobilized in nylon-6 nanofibrous membranes (NFM) prepared by electrospinning in comparison with freely dispersed yeasts. The resulting heat flow can be fitted using various approaches applying graphical, exponential, Gompertz, modified logistic and Michaelis–Menten models. Advantages and drawbacks of each approach for monitoring of the immobilized yeasts have been discussed in relation to the S. cerevisiae yeasts. Analysis of the fitting parameters showed that immobilized yeasts had the highest growth rate and maximum growth in comparison with freely dispersed yeasts. Parallel measurements of sugar and oxygen consumption rates for immobilized and free yeasts cells confirmed the calorimetric data. The results of this work demonstrate practical approach to analysis and modeling of calorimetric data in food analysis and advantages of application of NFM as solid support for yeast immobilization.

Cross-sectional study of Treponema pallidum PCR in diagnosis of primary and secondary syphilis.

Syphilis remains a major challenge and a complex diagnosis. We aim to evaluate the role of polymerase chain reaction (PCR) in Treponema pallidum (Tp) detection in various types of biological samples in the diagnosis of early syphilis. We conducted a cross-sectional study including all attendees of the STI clinic with clinical suspicion of early syphilis. One or more specimens for the detection of Tp by PCR testing were collected. The overall sensitivity of Tp PCR test was 82.61% (95% CI: 68.6-92.2%). Tp PCR test had sensitivity of 84.6% (95% CI: 54.6-98.1%) in primary syphilis cases and 81.8% (95% CI: 64.5-93%) in secondary syphilis cases. PCR test performance was independent of HIV status. Tp PCR test is a fast and reliable method for the detection of Tp in skin lesions of early syphilis, and it is a powerful tool in clinical settings.

Tools for building de novo transcriptome assembly

The availability of RNA-Seq method allows researchers to capture the spatial or temporal profile of transcriptomes from various types of biological samples. The transcriptome data from a species can be analyzed in the context of its sequenced genomes or closely related genome to score biological sample-specific transcript isoforms, novel transcribed regions and to refine gene models including identification of new genes, in addition to the differential gene expression analysis. However, many plant species of importance currently lack a sequenced genome or a closely related reference genome and thus, rely on the de novo methods for generating transcript models and transcriptome assemblies. Here we describe various tools used for de novo transcriptome assembly and discuss the data management practices and standards.

Salmonella infection and carriage in reptiles in a zoological collection.

OBJECTIVE To identify important subspecies and serovars of Salmonella enterica in a captive reptile population and clinically relevant risk factors for and signs of illness in Salmonella-positive reptiles. DESIGN Retrospective cross-sectional study. ANIMALS 11 crocodilians (4 samples), 78 snakes (91 samples), 59 lizards (57 samples), and 34 chelonians (23 samples) at the Bronx Zoo from 2000 through 2012. PROCEDURES Data pertaining to various types of biological samples obtained from reptiles with positive Salmonella culture results and the reptiles themselves were analyzed to determine period prevalence of and risk factors for various Salmonella-related outcomes. RESULTS Serovar distribution differences were identified for sample type, reptile phylogenetic family, and reptile origin and health. Salmonella enterica subsp enterica was the most common subspecies in Salmonella cultures (78/175 [45%]), identified across all reptilian taxa. Salmonella enterica subsp diarizonae was also common (42/175 [24%]) and was recovered almost exclusively from snakes (n = 33), many of which had been clinically ill (17). Clinically ill reptiles provided 37% (64) of Salmonella cultures. Factors associated with an increased risk of illness in reptiles with a positive culture result were carnivorous diet and prior confiscation. Snakes had a higher risk of illness than other reptile groups, whereas lizards had a lower risk. Bony changes, dermatitis, and anorexia were the most common clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE This study provided new information on Salmonella infection or carriage and associated clinical disease in reptiles. Associations identified between serovars or subspecies and reptile groups or clinical disease can guide management of Salmonella-positive captive reptiles.

Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)*

Targeted high-resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared with the reference method of selected reaction monitoring performed on triple quadrupole instruments. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data acquisition scheme enabled the analysis of moderate-to-large peptide numbers while retaining a high performance level. This new method, called internal standard triggered-parallel reaction monitoring (IS-PRM), relies on added internal standards and the on-the-fly adjustment of acquisition parameters to drive in real-time measurement of endogenous peptides. The acquisition time management was designed to maximize the effective time devoted to measure the analytes in a time-scheduled targeted experiment. The data acquisition scheme alternates between two PRM modes: a fast low-resolution "watch mode" and a "quantitative mode" using optimized parameters ensuring data quality. The IS-PRM method exhibited a highly effective use of the instrument time. Applied to the analysis of large peptide sets (up to 600) in complex samples, the method showed an unprecedented combination of scale and analytical performance, with limits of quantification in the low amol range. The successful analysis of various types of biological samples augurs a broad applicability of the method, which is likely to benefit a wide range of proteomics experiments.

Focus on photonics, biomedical optics, and microscopy

Focus on photonics, biomedical optics, and microscopy

Capillary electrophoresis-mass spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) has proven to be useful for metabolomics studies. Charged metabolites are first separated by CE based on charge and size and are subsequently selectively detected using MS. The major advantages of CE-MS are its high resolution and the fact that almost any charged species can be analyzed by two methods, both cationic and anionic. This technique can readily be applied to various types of biological samples originating from bacteria, plants, mammals, and body fluids. This chapter highlights detailed practical procedures for using this technology.