Successful Amplification Research Articles

Development of a loop-mediated isothermal amplification method for the rapid detection of Clostridium botulinum serotypes E and F.

Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) found in a wide variety of foods cause poisoning in humans with high mortality rates. Mouse bioassays (MBAs), the gold standard method for BoNT detection, have a low detection limit; however, require experienced personnel and take a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAATs) with primers targeting species-specific genes. In this study, for each serotype E and F, six LAMP primers were designed based on multiple sequence alignments of the conserved regions of the bont/E and bont/F genes collected from 180 serotype E strains and 23 serotype F strains published in NCBI. In silico PCR amplification with the outer primer pairs showed successful amplification of the target fragments. To validate the LAMP method, we constructed two synthetic plasmids containing the target sequences extended approximately 10-50bp to both ends. The specificity of the primers was further evaluated using six different Clostridium species and eight strains belonging to other common food poisoning-related bacterial species. Employing the synthetic plasmids, the optimal temperatures and limits of detection (LODs) were determined for bont/E (63°C, LOD ≤ 101 copies/reaction) and bont/F (65°C, LOD ≤ 102 copies/reaction) within 30min. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved the coverage from 15 to 45%. For future directions, applications of the established method, especially with the degenerate primers, could be used as an alternative assay for the rapid and sensitive detection of C. botulinum.

Binding of MAP3773c Protein of Mycobacterium avium subsp. paratuberculosis in the Mouse Ferroportin1 Coding Region

Mycobacterium avium subsp. paratuberculosis (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene in murine macrophage J774 A.1. We conducted a sequence alignment to identify potential interaction sites for MAP3773c. Following this, we used in silico analysis to predict binding interactions, complemented by electrophoretic mobility shift assay (EMSA) to confirm in vitro binding of MAP3773c. The map3773c gene was cloned into the pcDNA3.1 vector, with subsequent expression analysis carried out via Western blotting and real-time PCR. Chromatin immunoprecipitation (CHiP) assays were performed on transfected macrophages to confirm binding in the native chromatin context. Our in silico and in vitro analysis indicated that MAP3773c interacts with two binding motifs within the FPN1 coding region. The ChiP results provided additional validation, demonstrating the binding of MAP3773c to the FPN1 chromatin through successful amplification of the associated chromatin fragment via PCR. Our study demonstrated that MAP3773c binds to FPN1 and provides insight into the role of MAP3773c and its effect on host iron transport.

Cross-amplification of microsatellite markers on the endangered tree species Castanopsis argentea from other Castanopsis species: a valuable tool for genetic diversity analysis

Castanopsis argentea is an endangered tree naturally distributed in Java and Sumatera Island and considered a keystone species in the submontane forest ecosystem. A laboratory study was conducted to test the cross-amplification potential of seven pairs of Castanopsis cuspidata var. sieboldii microsatellite markers to be used for Castanopsis argentea. The rate of cross-amplification success, average number of alleles per locus (NA ), observed heterozygosity (HO ), expected heterozygosity (HE ), and fixation index (FIS ) were determined, and cross-amplification potential was investigated. The rate of amplification success was 85.71% (6 out of 7 markers were successful amplified), NA was 7.67, HO ranged from 0.467 to 0.923, HE ranged from 0.441 to 0.745, and the fixation index (FIS ) ranged from −0.413 to 0.278. Results indicate the high transferability of C. cuspidata var. sieboldii microsatellite markers to C. argentea. Six markers were suitable for genetic diversity and population genetic studies on C. argentea.

Discovery of Simple Sequence Repeat Markers in the Endemic Durian Kura-Kura (Durio testudinarius Becc.)

Durio testudinarius, known as Durian Kura-Kura, is a native Bornean durian species in the Malvaceae family with fruit at the base of the main stem. Recent advances in genomic-based next-generation sequencing are being used to conserve germplasm and enhance plant breeding. This method rapidly and cost-effectively sequences plant genomes, allowing for simple sequence repeat markers to be constructed in silico. This study aimed to design and evaluate novel, simple sequence repeat markers utilizing next-generation sequencing microsatellite data of D. testudinarius. A total of 20 sequences containing di-, tri-, tetra-, and penta-nucleotide motif repeats were selected and designed to generate primers using Primer3 online software. The PCR results of the twenty primers tested on D. testudinarius and 19 genomic DNA samples of species from the Malvales order showed successful amplification. These newly developed simple sequence repeat markers are crucial for genetic population analyses, determining population structure, and enhancing plant breeding programs and conservation strategies for D. testudinarius and its relative's germplasm resources.

Three-Level Chirality Transfer and Amplification in Liquid Crystal Supramolecular Assembly for Achieving Full-Color and White Circularly Polarized Luminescence.

Chiral liquid crystal supramolecular assembly provides an ideal strategy for constructing excellent circularly polarized luminescence (CPL) materials. However, the chirality transfer in chiral liquid crystals normally occurs at two levels from the configurational chirality to the supramolecular phase chirality. The more precise and more levels of chirality transmission are fascinating but remain challenging. The present work reports the first success of three-level chirality transfer and amplification from configurationally point chirality of small molecules to conformationally helical chirality of helical polymers and finally to supramolecular phase chirality of cholesteric liquid crystals composed of chiral nonfluorescent polymers (P46) and nematic liquid crystals. Noticeably, the helical twisting power of P46 is five-fold larger than its monomer. Full-color and white CPL with maximum luminescence dissymmetry factor up to 1.54 and photoluminescence quantum yield up to 63.8% are realized utilizing helical supramolecular assembly combined with selective reflection mechanism. Also significantly, the electrically stimuli-responsive CPL switching device as well as anti-counterfeiting security, information encryption, and chiral logic gate applications are developed. This study deepens the understanding of chirality transfer and amplification across different hierarchical levels.

Comprehensive Evaluation of Cryptic Juglans Genotypes: Insight from Molecular Markers and Phylogenetic Analysis.

Background/Objectives: The current research work aimed to evaluate the cryptic walnut genotypes of the Hazara region in Pakistan by using DNA barcoding and phylogenetic analysis. Methods: Based on morphological traits such as nut size, nut shape, and the number of leaflets, five genotypes were chosen and samples were collected for the current study. For molecular analysis, gDNA was isolated from the fresh leaves, and the five most effective angiosperm-specific markers, ITS2, rbcLa, rbcLc, rpoC1, and UBE3, were utilized. Based on amplification, sequencing, and identification success rates, ITS2 and UBE3 were recorded as the most efficient markers followed by rbcLa, rbcLc, and rpoC1. Results: During phylogenetic analysis, the query genotype-1 based on ITS2 and genotype-2 based on UBE3 clustered with (KF454101.1-Juglans regia) and (KC870919.1-J. regia) with bootstraps of 56 and 100, respectively. Genotype-3 based on rbcla clustered in a major clade with J. regia L., cultivars (MN397935.1 J. regia 'Vina') and (MN397934.1-J. regia 'Serr'), (MN397933.1 J. regia 'Pedro'), (MN397932.1 J. regia 'Lara'), (MN397931.1 J. regia 'Howard'), and (MN397930.1 J. regia 'Hartley') with bootstrap of 100. Meanwhile, genotype-4 and genotype-5 based on rbclc and rpoC1 clustered with (MN397935.1 J. regia 'Vina') and (MN397934.1 J. regia 'Serr'), across the database sequences. To clarify the taxonomic status of cryptic walnut genotypes, it is necessary to combine diverse DNA barcodes. The results of ITS2 and UBE3, followed by rbcL barcoding markers, are promising taxonomic tools for cryptic walnut genotypes in Pakistan. Conclusions: It has been determined that the genotypes of walnuts in the study area are both J. regia L. and its cultivars and that the accuracy of discrimination regarding the genus Juglans L. is greater than 90%. The reported DNA barcodes are recommended for the correct identification and genetic evaluation of Juglans taxa and its population.

Petrous bones versus tooth cementum for genetic analysis of aged skeletal remains.

A proper sampling strategy is important to obtain sufficient DNA for successful identification of aged skeletal remains. The petrous bone is the highest DNA-yielding bone in the human body. Because DNA extraction from the petrous bone is very destructive, the demand for other DNA sources is significant. When investigating aged skeletal remains, teeth are usually preserved, and recent studies have shown that DNA in teeth can be best preserved in the dental cementum that surrounds the surface of the tooth root. To extract DNA from the surface of the tooth root, a nondestructive method without grinding was used. Petrous bones and teeth from 60 archaeological adult skeletons were analyzed. The DNA yield, degree of DNA degradation, and STR typing success were compared, and the results showed higher DNA yield and higher amplification success in petrous bones, despite higher degradation of petrous bones' DNA. The greater success of petrous bones is associated with poorly preserved DNA in a quarter of the teeth analyzed. When teeth with badly preserved DNA were excluded from the statistical analysis, no differences in the success of STR loci amplification were observed even if DNA yield was higher in petrous bones, which can be explained by greater degradation of petrous bones' DNA. When teeth are well preserved, they can be used for genetically analyzing aged skeletal remains instead of petrous bones, and a rapid nondestructive extraction method can be applied to shorten the identification process and to physically preserve the biological specimen.

Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Two Clarireedia spp., the Causal Agent of Dollar Spot of Turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

Genetic Diversity of Indonesian Pineapple (Ananas comosus (L.) Merr.) Cultivars Based on ISSR Markers

Pineapple (Ananas comosus (L.) Merr.) is the third-most important tropical fruit traded and widely cultivated in Indonesia with various cultivars. This study identifies genetic diversity and determines the phenetic relationship of nine pineapple accessions based on inter-simple sequence repeat (ISSR) markers. Four ISSR primers were utilized for genetic diversity analysis and diversity relationships using POPGENE 1.31 and MVSP 3.2 to form a dendrogram. The results showed that nine pineapple accessions revealed the successful amplification of 50 DNA bands, 46 polymorphic, with a percentage average of 89.38%. The calculation result of the effective alleles (Ne), Nei’s gene diversity (h), Shannon information index (I), and polymorphism information content (PIC) showed that ISSR 1 had the highest value and ISSR 16 had the lowest value. The average of the Ne value was 1.44; the average of h was 0.28, which indicated low genetic variation; the average of (I) was 0.43, which indicated that not all groups had the same frequency; the averages of the PIC value of 0.28 which showed that the four ISSR primers used were somewhat informative. The results of the phenetic relationship based on ISSR molecular markers showed two clusters that separated the accession of ‘Spanish’ from the accession of ‘Queen’ and ‘Cayenne.’ This resulting study showed that ISSR analysis was suitable for studying genetic diversity among pineapple cultivars.

Use of a novel combination of multiplex PCR and DNA barcoding in assessing authenticity of ginseng products

Ginseng (Panax sp.) is a medicinal plant used for its purported health benefits, primarily in East Asian countries. The COVID-19 pandemic led to a significant increase in sales of dietary supplements, including ginseng supplements, to purportedly support immune health and provide other health benefits. However, this heightened demand has subsequently increased the risk of adulteration in these dietary supplements. This study aimed to determine the efficacy of a novel combination of DNA barcoding and multiplex PCR to identify species in ginseng supplements. A total of 50 commercial ginseng supplements containing Panax ginseng, Panax quinquefolius, or Panax notoginseng were obtained from an online vendor. Two composite samples per product were subjected to DNA extraction followed by multiplex PCR and DNA barcoding with three genetic targets (i.e., rbcL, matK, and ITS2). Of the three DNA barcoding markers, ITS2 had the highest amplification success at 74%, and matK had the highest sequencing success at 60%. DNA barcoding alone identified species in 68% of products, while multiplex PCR had an overall 60% identification rate. The combination of both methods resulted in a 72% species identification rate. In addition to improving the overall identification rate, the combination of both methods allowed for greater species resolution and the detection of undeclared plant species. Future research should explore combining DNA-based methods with chemical-based approaches to improve ginseng detection capabilities and enable the quantification of undeclared adulterants.

Active diagnostic ingredients (ADIs) for PCR: A mini-bioreactor producing dNTPs with silica immobilized R5-kinases.

Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2'-deoxynucleoside 5'-triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self-immobilizing R5-silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5-kinase mini-bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in Plasmodium malariae NAATs, a high linear correlation was shown between the Ct value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini-bioreactor investigated as a proof-of-principle for work-flow integration with NAAT in low-resource research and diagnostics labs.

Extraction of Genomic DNA from Soil Samples by Polyethylene Glycol-Modified Magnetic Particles via Isopropanol Promotion and Ca2+ Mediation.

Obtaining reliable and informative DNA data from soil samples is challenging due to the presence of interfering substances and typically low DNA yields. In this work, we prepared poly(ethylene glycol)-modified magnetic particles (PEG@Fe3O4) for DNA purification. The particles leverage the facilitative effect of calcium ions (Ca2+), which act as bridges between DNA and PEG@Fe3O4 by coordinating with the phosphate groups of DNA and the hydroxyl groups on the particles. The addition of 2-propanol further enhances this Ca2+-mediated DNA adsorption by inducing a conformational change from the B-form to the more compact A-form of DNA. PEG@Fe3O4 demonstrates a DNA adsorption capacity of 144.6 mg g-1. When applied to the extraction of genomic DNA from soil samples, PEG@Fe3O4 outperforms commercial kits and traditional phenol-chloroform extraction methods in terms of DNA yield and purity. Furthermore, we developed a 16-channel automated DNA extraction device to streamline the process and reduce the extraction time. The successful amplification of target bacterial and fungal amplicons underscores the potential of this automated, device-assisted method for studying soil microbial diversity.

Development of a microfluidic SNP assay for lineage discrimination in the endangered hazel dormouse

The application of Genotyping-by-Sequencing (GBS) approaches is often restricted in wildlife monitoring and conservation genetics, as those fields often rely on noninvasively collected samples with low DNA content. Here we selected a subset of informative single-nucleotide polymorphisms (SNPs) from genome-wide data for lineage discrimination of a locally endangered Eurasian rodent, the hazel dormouse (Muscardinus avellanarius), and designed a microfluidic 96 SNP genotyping assay suitable for noninvasively collected samples. Analyses of 43 samples from different European countries confirmed successful discrimination of the Eastern and Western lineage and local substructure within those lineages, proving the suitability of the developed panel for identifying evolutionary significant units and conservation units. Application with 94 hair and scat samples collected in a recent monitoring study on the hazel dormouse in Southern Germany resulted in > 99.5% amplification success showing the applicability of the new tool in genetic wildlife monitoring and conservation studies.

Application of microsatellites in population genetic studies of reindeer (Rangifer tarandus) (review)

Over a few past decades, theoretical, analytical, and methodological advances in genetics have revolutionized population genetic research, providing a better understanding of evolutionary processes and the history of populations and species. Methodologically, this progress is largely due to the invention of polymerase chain reaction technology and the introduction of microsatellite DNA markers. This review discusses trends in the use of microsatellite markers as effective tools for solving a wide range of issues in population genetics, conservation and evolutionary biology of the only species of the genus Rangifer – reindeer. Based on the analysis of both experimental and review publications (78 sources) of the scientific teams of the Russian Federation, Canada, the United States of America, Ireland, Japan, China, Norway the first works on the successful amplification of reindeer microsatellites have been summarized. There has been demonstrated the significance of the data of markers for studying intra- and inter-population diversity, differentiation, genetic relationships, the impact of anthropogenic factors on genetic diversity and genetic isolation of populations, as well as for reconstructing the evolutionary history of the various reindeer forms.

Molecular and pathological screening of the current circulation of fowlpox and pigeon pox virus in backyard birds

Avian pox is a disease that has devastating impacts on both poultry and wild bird species. Avian pox is caused by various strains of avipoxviruses (APV). Nevertheless, the virus has been detected in pigeons and chickens that are raised in backyard areas, leading to substantial financial damage for small-scale producers. There is a lack of comprehensive information regarding the transmission of APV among birds in the backyards and residential areas. Hence, the present investigation closely monitored and observed APV in domesticated birds residing in backyard areas, with the aim of impeding the transmission of the virus to nearby poultry farms. In 2023, a total of fifty backyard flocks were surveyed for the presence of avian pox disease. Sixteen backyards (14 pigeons and 2 chickens) exhibited warty nodular lesions on their heads and nonfeathered body parts. APV was confirmed in nodular lesions by polymerase chain reaction (PCR) amplification and genetic sequencing. All samples from the lesions showed successful amplification of the p4b locus (core protein p4b). Four confirmed samples were tested for pathogenicity on the chicken embryo chorioallantoic membrane (CAM). Histopathological examination revealed ballooning degeneration and numerous intracytoplasmic inclusion bodies (Bollinger bodies) in the ectoderm of the infected CAM. Phylogenetic analysis revealed that the strains clustered into main clade A, with 11 in subclade A2 and 5 in subclade A1. Amino acid identity showed 100% similarity between the vaccine (fowlpox/VSVRI/Egypt) and some detected strains (PP537574 and PP537575). In addition, the PP537576.1 to PP537580.1 and PP537582.1 to PP537585.1 had 2-point mutations compared to the fowlpox/VSVRI/Egypt vaccine. The overall finding of low biosecurity levels in the investigated backyard birds emphasizes the significance of establishing sanitary measures and control vectors to reduce virus transmission routes and disease severity. In conclusion, it is necessary to emphasize the tracking of APV in backyard birds. Concurrently, we advised enhancing hygiene protocols, vector management, and subsequent vaccination to restrict the occurrence of APV outbreaks and prevent their transmission to neighboring poultry farms. Furthermore, it is crucial to incorporate molecular studies in order to enhance the vaccine seeds for disease management.

Synthesis and optical properties of tyramine-functionalized boron dipyrromethene dyes for cell bioimaging

Tyramine signaling amplification (TSA) technology is generally applied in immunofluorescence, enzyme-linked immunoassays, in situ hybridization techniques, etc. Successful amplification of fluoresence signals cannot be achieved without excellent fluorescent dyes. BODIPY fluorophore is an ideal probe for cell fluorescence imaging, but pristine BODIPY cannot be direct used in the TSA system. In the paper, the new red-shifted tyramide-conjugated BODIPY (BDP-B/C/D) was synthesized via the Knoevenagel condensation reaction, which based on the tyramide-conjugated BODIPY (BDP-A). The synthesized dyes were combined with tyramine to obtain which could be used as a fluorescent substrate for enzymatic reaction of TSA. By using the selected substrate (BDP-C) in TSA, we found it to be more sensitive than the commercial dye 594 styramide for the detection of low-abundance antigen proteins.

Rapid Detection of Staphylococcus Epidermidis Using Electric-Double-Layer (EDL) Field-Effect Transistor (FET) Biosensors with Loop-Mediated Isothermal Amplification

Staphylococcus epidermidis is a Gram-positive bacterium which commonly grows in human skin, nasal passages, respiratory tract, intestinal tract, air, and water. Although it is harmless to healthy individuals, immunocompromised patients are at risk of infection, which can lead to severe symptoms. With the increased reliance on antibiotics in hospitals, the antimicrobial resistance of Staphylococcus epidermidis has also risen, pointing out a challenge in clinical treatment.Therefore, the diagnosis of this bacterium has gained significant attention. The aim of this experiment is to detect bloodstream infections (BSIs) in the shortest possible time. The study is intended to be implemented in hospitals to provide a more convenient method for medical professionals to conduct screenings. The approach involves screening for the most common disease sequence, ssDNA, followed by sequential detection of corresponding cDNA, plasmids, and bacteria. To detect bacteria with the least amount of bacteerial load, a novel isothermal amplification method called Loop-mediated isothermal amplification (LAMP) is employed to increase the concentration of DNA of BSIs bacteria before measurement. The overall time required for this process doesn’t exceed one day, significantly increasing speed for the detection of blood-borne bacteria in hospitals. The core mechanism of this platform involves the use of electric-double-layer-gated field-effect-transistors (EDL-gated FETs) to amplify the gate voltage changes generated when the ssDNA binds with complementary DNA. These voltage changes are then converted into numerical values for analysis and interpretation.Currently, the research results show that this biomedical sensor can detect concentrations ranging from 1fM to 1pM. With the assistance of LAMP, it has successfully achieved a concentration of approximately 1yM (10-24 M) and detected with EDL-gated FETs. The experiment was also carried out step by step on simulated clinical specimens. The experiments have found successful serum nucleic acid amplification methods that provide excellent detection limits. Compared with other biomedical sensors in the past, it not only has lower detection limits but also brings features such as faster, more convenient, and more portable.Keywords: EDL, FET, Staphylococcus epidermidis, Biosensor, LAMP Figure 1

Optimizing single-tube nested PCR for enhanced genotyping of single cells and in vitro produced bovine embryos

Single-tube nested PCR (STnPCR) is a technique that improves nested PCR, reducing potential contamination and false-positive results, enhancing the amplification sensitivity. Despite being commonly used for the detection of microorganisms, STnPCR can be a valuable tool for bovine genotyping, encompassing essential targets as ROSA26 and TSPY, pivotal in the fields of animal reproduction, genetic improvement, and transgenic research. The objective of this study was to improve and innovate STnPCR for gene detection in cattle. We aimed to detect the ROSA26 and TSPY genes using low-concentration DNA samples, including single cells, small cell groups (one to five cells), in vitro-produced embryos, and bovine tissue samples. Moreover, we refined STnPCR for gene detection in up to single cells by conducting sensitivity testing with different concentration ratios of internal and external primers. Successful amplification of the ROSA26 and TSPY genes was achieved across all tested primer concentrations, even in single cells, with more consistent results observed at lower primer concentrations. Additionally, simultaneous gene amplification was achieved through STnPCR multiplexing, representing the first study of multiplex STnPCR in cattle. These outcomes not only confirm its effectiveness in detecting genetic markers for animal genetic improvement and transgenic elements but also pave the way for its widespread adoption in reproductive studies in bovines.

Searching for alternative high DNA-yielding bone types for DNA analysis of aged skeletal remains

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Niacinamide Antimicrobial Efficacy and Its Mode of Action via Microbial Cell Cycle Arrest.

Niacinamide is a versatile compound widely used in the personal care industry for its ample skin benefits. As a precursor to nicotinamide adenine dinucleotide (NAD+), essential for ATP production and a substrate for poly-ADP-ribose polymerase-1 (PARP-1), studies have highlighted its roles in DNA repair, cellular stress mechanisms, and anti-aging benefits. Niacinamide was also studied for its antimicrobial activity, particularly in the context of host-infection via host immune response, yet its direct antimicrobial activity and the mechanisms of action remain unclear. Its multifunctionality makes it an appealing bioactive molecule for skincare products as well as a potential preservative solution. This study explores niacinamide's antimicrobial mode of action against four common cosmetic pathogens. Our findings indicate that niacinamide is causing microbial cell cycle arrest; while cells were found to increase their volume and length under treatment to prepare for cell division, complete separation into two daughter cells was prevented. Fluorescence microscopy revealed expanded chromatin, alongside a decreased RNA expression of the DNA-binding protein gene, dps. Finally, niacinamide was found to directly interact with DNA, hindering successful amplification. These unprecedented findings allowed us to add a newly rationalized preservative facete to the wide range of niacinamide multi-functionality.