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The larval excretions/secretions (ES) of blowflies contain proteolytic enzymes and bacteria that assist with tissue breakdown. Decomposition fluid (DF) contains organic and inorganic waste products from cell death. This study investigated if human DNA recovery from blood was impacted by exposure to ES and DF over time. Lucilia sericata ES were collected daily from 50 larvae, and all available DF was collected from two fetal piglets left to decompose for 2 weeks. Daily for 3–5 days, 28 μL‐30 μL of ES, DF, or a 1:1 mixture of the fluids was added to 30 μL of blood on cotton. Three bloodstains per treatment were sampled every 12 h up to 3 days and at 1 and 2 weeks after initial addition of fluid. No PCR inhibition was detected, but DNA degradation increased over time, primarily in samples exposed to ES and ES/DF mixtures. The amount of DNA recovered decreased over time, but generally more DNA was recovered from DF samples than other samples. Full profiles, or partial profiles suitable for routine database searching (14–39 alleles), were generated from all DF and ES samples and at least one mixture sample at all timepoints. Partial profiles of between 1 and 13 alleles were obtained from all other mixture samples, except one mixture sample which generated no profile. These findings indicate bloodstain evidence recovered from maggot‐infested and/or decomposing bodies may generate forensically useful DNA evidence and should be analyzed as quickly as possible after collection or stored appropriately to prevent further degradation. [ABSTRACT FROM AUTHOR]

Bombyx mori cecropin A (Bmcecropin A) has antibacterial, antiviral, anti‐filamentous fungal and tumour cell inhibition activities and is considered a potential succedaneum for antibiotics. We clarified the antibacterial mechanism and structure–activity relationships and then directed the structure–activity optimization of Bmcecropin A. Firstly, we found Bmcecropin A shows a strong binding force and permeability to cell membranes like a detergent; Bmcecropin A could competitively bind to the cell membrane with the cell membrane‐specific dye DiI, then damaged the membrane for the access of DiI into the cytoplasm and leading to the leakage of electrolyte and proteins. Secondly, we found Bmcopropin A could also bind to and degrade DNA; furthermore, DNA library polymerase chain reaction (PCR) results indicated that Bmcecropin A inhibited DNA replication by non‐specific binding. In addition, we have identified C‐terminus amidation and serine‐lysine‐ glycine (SLG) amino acids of Bmcecropin A played critical roles in the membrane damage and DNA degradation. Based on the above results, we designed a mutant of Bmcecropin A (E9 to H, D17 to K, K33 to A), which showed higher antibacterial activity, thermostability and pH stability than ampicillin but no haemolytic activity. Finally, we speculated that Bmcecropin A damaged the cell membrane through a carpet model and drew the schematic diagram of its antibacterial mechanism, based on the antibacterial mechanism and the three‐dimensional configuration. These findings yield insights into the mechanism of antimicrobial peptide–pathogen interaction and beneficial for the development of new antibiotics. [ABSTRACT FROM AUTHOR]

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction (PCR) is an important tool for exogenous gene acquisition and recombinants identification. There exist two problems when using Serratia marcescens as a template for PCR amplification: amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable. The aim of the present work was to elucidate the reasons for this. By mixing PCR products amplified from Escherichia coli DH5α with S. marcescens supernatant or pellet, we found that the DNA‐degrading substance in S. marcescens is thermally resistant and present both intracellularly and extracellularly. We then determined that it is protein, and most likely S. marcescens nuclease, that degrades PCR products since the addition of SDS and EDTA can effectively inhibit or block the degradation of PCR products. By knocking out the S. marcescens nuclease encoding gene, nucA, we confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. This work is the first to show that the S. marcescens nuclease is temporarily and partially inhibited by high temperatures during PCR and recovers rapidly at room temperature after PCR. Significance statement: Amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable when using Serratia marcescens as a template for PCR amplification. The present work confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. It has been reported that S. marcescens nuclease is only active at temperatures from 0°C to 42°C, and there is no literature to date indicating the thermal stability of S. marcescens nuclease activity. There is even an article reporting that S. marcescens nuclease molecules are thermally degraded at temperatures above 40°C. This work is the first to show that the S. marcescens nuclease is thermally resistant. [ABSTRACT FROM AUTHOR]

Little is known about the underlying mechanisms that contribute to the persistence and degradation of DNA within soil. The goals of this study are to determine the duration of mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) persistence in soils enriched by surface-level human decomposition and to better understand the contribution of environmental factors. The surface-level decomposition of three human cadavers was documented over 11 weeks. Based on quantitative PCR results, we found nuDNA to persist in soils six weeks post-placement, while mtDNA was recoverable for the entire 11-week decomposition period. Principle components analyses and Spearman's rank correlations revealed that (1) time, (2) total body score, and (3) weekly average air temperature were significantly correlated with concentrations of nuDNA and mtDNA in soil, suggesting these factors play a role in the degradation of DNA in soils. [ABSTRACT FROM AUTHOR]

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis. [ABSTRACT FROM AUTHOR]

Abstract Background DNA (deoxy-ribonucleic acid) is a fundamental molecule housing genetic information crucial for forensic casework. However, its integrity is compromised over time due to degradation, affecting living and deceased organisms. Understanding the factors and mechanisms of DNA degradation is vital across scientific disciplines. Main body DNA degradation is a dynamic process influenced by factors like temperature, humidity, and ultraviolet radiation. The post-mortem interval affects organisms differently, and mechanisms such as hydrolysis, oxidation, and depurination impact DNA structural integrity. In forensic casework, DNA degradation poses challenges because degraded DNA samples can be difficult to analyze. Despite these challenges, DNA degradation has become an invaluable asset in forensic science. Fragmented DNA aids in historical identification and archaeological investigations. Additionally, DNA degradation helps estimate the time since death, assisting investigators in criminal timelines. Forensic experts use degradation patterns to deduce environmental conditions affecting a body, aiding crime scene reconstruction. In criminal investigations, advancements in DNA recovery, like next-generation sequencing, enable the analysis of severely degraded samples, enhancing the identification of suspects and victims. Conclusion DNA degradation, despite its challenges, is a potent tool in forensic science. Understanding the factors and mechanisms influencing DNA degradation is essential for its effective utilization in criminal casework. With ongoing advancements in forensic techniques and technologies, the reliability and utility of degraded DNA analysis are steadily increasing, helping to bring resolution to complex criminal cases and uncovering hidden clues in forensic investigations.

The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets ("[Auto]/[Y]"). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = −0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the "[Auto]/[D]" values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays. [ABSTRACT FROM AUTHOR]

Generating a DNA profile using autosomal Short Tandem Repeat (STR) DNA allele from skeletal remains is a crucial step in the identification process for mass disaster and unidentified person situations. The capacity to extract substantial amounts of relevant DNA from bones, which were highly durable biological components that can withstand soil elements and climatic conditions over long periods of time, would be advantageous. 70 femur male bone samples were collected from two different environmental mass graves (Al-Mahaweel massgrave) in Babil governorate and (Al-Saqlawiya massgrave) in Al-Anbar governorate in Iraq. The DNA from bone samples extracts were assessed by capillary electrophoresis and a standardized set of short tandem repeat (STR) loci are analyzed using Mutiplex 21 STR loci System to allow human identification by matching the profile of the dead individuals with their relatives. From these two different sites; soil samples were collected to analyze and study the different environmental factors and find a relation between the degree of preservation or degradation with these factors like temperature, humidity, salinity, PH and texture of soil and find how these factors can affect on pattern of degradation of DNA profiles. The average of the percentage of the detected alleles in each locus in the two sites; were 87% for Al-Mahaweel site and 32% for Al-Saqlawiya site. we found that clay soil, low salt concentration, PH slightly alkaline and low humidity for Al-Mahaweel site; all participate in DNA preservation while the chalky and very high salt concentration of Al-Saqlawiya site soil; all participate in DNA degradation. Predicting the effect of these factors on the quality of the DNA profiles can be helpful in finding better solutions for many complications that may face the process of analyzing samples and finding sufficient informative profiles. [ABSTRACT FROM AUTHOR]

Background: DNA (deoxy-ribonucleic acid) is a fundamental molecule housing genetic information crucial for forensic casework. However, its integrity is compromised over time due to degradation, affecting living and deceased organisms. Understanding the factors and mechanisms of DNA degradation is vital across scientific disciplines. Main body: DNA degradation is a dynamic process influenced by factors like temperature, humidity, and ultraviolet radiation. The post-mortem interval affects organisms differently, and mechanisms such as hydrolysis, oxidation, and depurination impact DNA structural integrity. In forensic casework, DNA degradation poses challenges because degraded DNA samples can be difficult to analyze. Despite these challenges, DNA degradation has become an invaluable asset in forensic science. Fragmented DNA aids in historical identification and archaeological investigations. Additionally, DNA degradation helps estimate the time since death, assisting investigators in criminal timelines. Forensic experts use degradation patterns to deduce environmental conditions affecting a body, aiding crime scene reconstruction. In criminal investigations, advancements in DNA recovery, like next-generation sequencing, enable the analysis of severely degraded samples, enhancing the identification of suspects and victims. Conclusion: DNA degradation, despite its challenges, is a potent tool in forensic science. Understanding the factors and mechanisms influencing DNA degradation is essential for its effective utilization in criminal casework. With ongoing advancements in forensic techniques and technologies, the reliability and utility of degraded DNA analysis are steadily increasing, helping to bring resolution to complex criminal cases and uncovering hidden clues in forensic investigations. [ABSTRACT FROM AUTHOR]

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Abstract Wetlands are ecologically and culturally significant ecosystems that are experiencing biodiversity declines globally. Biomonitoring techniques that use environmental DNA (eDNA) to detect and monitor biodiversity are well established in lake, riverine, and marine ecosystems. However, their use in wetlands requires further development due to the presence of sediments that block eDNA filters to limit water filtration, alongside a lack of standardized methodology. In this study, we examined eDNA dynamics to understand spatiotemporal biodiversity patterns in an Aotearoa New Zealand wetland and to optimize their application to wetland‐specific challenges. We sampled four sites across Opuatia Wetland at three time points during an austral spring. We conducted conventional taxonomic surveys, tested three different filter sizes (1.2 μm, 5 μm, and semi‐quantitative dacron filters), and assessed our ability to detect foreign DNA (from kea; Nestor notabilis) at different time points and distances post‐release. We found significant differences in DNA sequence composition across time and space, and when using different sized filters. eDNA data generally complemented (versus replaced) conventional survey and identification methods, with certain species only detected by one method or the other. Taxonomic resolution of conventional sampling and identification methods often exceeded that of eDNA. Foreign DNA was detectable 10 m from its release point for up to 1 week post‐release. Our results provide new considerations for future eDNA research in wetland environments, where rapid biomonitoring techniques are needed to support conservation and preservation.

Abstract Sampling is one of the most important and fundamental processes in field biology. Among many sampling methods in entomology, window traps have long been considered one of the most effective and standardized methods. Unfortunately, the DNA quality of samples collected by window traps is typically low due to incursion by water, mainly via rainfall. Water cannot be avoided in long‐term surveys, even with a roof, due to wind and condensation. As we move into the genomics era, this shortfall in DNA quality has become problematic for molecular biomonitoring, and solutions are urgently needed. In this study, we developed the Water‐Exclusion Trap (WET), a 3D printable long‐term storable bottom collector for window or pitfall traps. The collection medium in WETs does not dilute because water is excluded and evaporation is limited, maximizing DNA preservation. Our field tests show that the WET outperforms conventional traps in preserving DNA quality. Insect samples preserved in WETs in the field had significantly higher PCR success rates and DNA Integrity Numbers compared to conventional traps. The WET addresses a critical challenge in the use of window traps, DNA degradation caused by water intrusion, and represents a significant advancement in fieldwork for genomic research in entomology. We provide the 3D printing template of the WET, enabling immediate, free use for all. Furthermore, it can be easily customized for different environments and purposes, and can be adapted to a variety of other traps, including pitfall traps, funnel traps and more.

Generating degraded DNA samples can be useful for forensic DNA research as this can mimic the condition of crime scene samples and results from such samples. Methods which use quick and cost-effective ultraviolet light (UV) exposure can be utilized to degrade DNA but are not well documented. This study aimed to establish a procedure and define the parameters required to generate Short Tandem Repeat (STR) profiles at different degradation levels. UV exposure times between 0 and 60 s were tested using genomic and control DNA. Sample eluates were quantified with QuantifilerTM Trio pre- and post-UV exposure, and select samples were profiled using PowerPlex® 21. Quantification data and STR profiles were analysed to identify metrics which indicated degradation severity. Samples were observed to be degraded by UV exposure as evidenced by established DNA degradation markers. No specific metric was identified to reliably determine the severity of sample degradation, but it was noted that UV exposure between 5 and 10 s resulted in mild degradation, between 15 and 25 s generated moderate degradation, and between 45 and 60 s resulted in severe degradation in STR profiles. This study indicates that quantification before and after UV exposure may be useful to estimate the degree of degradation via one quantification target. [ABSTRACT FROM AUTHOR]

Sampling is one of the most important and fundamental processes in field biology. Among many sampling methods in entomology, window traps have long been considered one of the most effective and standardized methods. Unfortunately, the DNA quality of samples collected by window traps is typically low due to incursion by water, mainly via rainfall. Water cannot be avoided in long‐term surveys, even with a roof, due to wind and condensation. As we move into the genomics era, this shortfall in DNA quality has become problematic for molecular biomonitoring, and solutions are urgently needed.In this study, we developed the Water‐Exclusion Trap (WET), a 3D printable long‐term storable bottom collector for window or pitfall traps. The collection medium in WETs does not dilute because water is excluded and evaporation is limited, maximizing DNA preservation.Our field tests show that the WET outperforms conventional traps in preserving DNA quality. Insect samples preserved in WETs in the field had significantly higher PCR success rates and DNA Integrity Numbers compared to conventional traps.The WET addresses a critical challenge in the use of window traps, DNA degradation caused by water intrusion, and represents a significant advancement in fieldwork for genomic research in entomology. We provide the 3D printing template of the WET, enabling immediate, free use for all. Furthermore, it can be easily customized for different environments and purposes, and can be adapted to a variety of other traps, including pitfall traps, funnel traps and more. [ABSTRACT FROM AUTHOR]

Within anatomical willed body programs and skeletal collections, whole bodies and their disassociated limbs and organs are identified and tracked. However, if these tracking mechanisms fail, DNA recovered from the formalin‐fixed tissues/organs could provide an additional layer of quality assurance. Embalming fluids preserve biological tissues; however, they also damage, fragment, and cross‐link DNA and protein molecules. This project investigated the success of STR‐typing from various soft tissue and bone samples that were fixed with embalming solutions with a range of formaldehyde concentrations. Formalin‐fixed samples dissected from five cadavers, including skin, muscle, bone, heart, and kidney were used in Phase 1 of this study. In Phase 2, an additional 57 tissue samples from various embalmed organs and body parts were collected to demonstrate long‐term fixation and direct applicability within a body donor program. DNA was extracted from the samples using the QIAamp® FFPE Tissue Kit (QIAGEN), quantified with the Investigator® QuantiPlex® Pro RGQ qPCR Kit (QIAGEN), and amplified using the Investigator® 24plex and 26plex QS Kits and the Investigator® DIPplex Kit (QIAGEN). The results show the DNA was severely damaged, degraded, and often in low amounts (after one year post‐embalming). Sampling from skin and muscle tissues embalmed with ~2.5%–5% formaldehyde solutions appears to be the best strategy for identification, while also maintaining the preservation of the tissues. The results of this project can provide informative data when determining which genotyping strategy may be best suited for the identification, re‐association, and establishment of a database for the provenance of formalin‐fixed human remains. [ABSTRACT FROM AUTHOR]

Mislabeling of hamburgers with undeclared chicken has frequently been reported. A fast TaqMan real-time PCR assay was developed to estimate chicken percentage in raw and heat-treated hamburgers in the current work. The calibration curve was plotted with the normalization approach (ΔCt; the difference calculation between chicken-cytb and eukaryotic-18S rRNA assays) for five reference chicken mixtures (100–0.01%) using a duplex TaqMan-qPCR protocol. The R2 and efficiency values of the normalized curve were 0.9993 and ~ 100%, respectively. In the validation plan, analysis of model samples (raw, oven-cooked, and autoclaved) under repeatability conditions represented the bias values within the range of ± 25% and RSD values < 25%, except for a few autoclaved model samples. Therefore, this developed and validated model could be considered a useful and reliable method for estimating chicken species (with a sensitivity of 0.01%) in raw and cooked hamburgers. [ABSTRACT FROM AUTHOR]

Different types of disasters, whether natural or human in character, lead to the significant loss of human lives. In the latter case, the quick action of identification of corpses and human remains is mandatory. There are a variety of protocols to identify victims; however, genetics is one of the tools that allows an exact identification of the victim. However, several factors may interfere with this identification, from the biological samples' degradation not allowing the analysis of nuclear information, to failure to dispose of biological samples from family members. Access to certain family members could be a determinant of the proper choice of genetic markers that allow the identification of the victim, or his/her inclusion in a given genetic maternal or paternal lineage. New advances in the field of genetics are soon expected to allow for the identification of victims from disasters with only their biological postmortem samples; it may also be possible to draw a robot portrait of a victim's most likely physical characteristics. In all cases, genetics is the only modern tool with universal character and can be used in essentially all biological samples, giving and identification of more or less accurate statistical character, depending on whether nuclear or lineage markers are used. [ABSTRACT FROM AUTHOR]

Objectives DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. Methods We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. Results A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72 , P <.0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). Conclusions To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio. [ABSTRACT FROM AUTHOR]

Lethal bronzing (LB) and huanglongbing (HLB) are harmful plant diseases causing significant economic losses in Florida agriculture. Both diseases are caused by bacteria that are transmitted by Hemipteran insect vectors. Accurate detection of pathogens within insect vectors can help provide a better understanding of disease epidemiology. Monitoring of the vector of LB is done primarily using sticky traps within palm canopies. However, it is unknown how long pathogen and vector DNA remain intact under field conditions. If significant DNA degradation takes place over the course of days or weeks, there is a possibility of false negatives occurring when detecting pathogens from these surveys. This study determined how long Haplaxius crudus Van Duzee (Hemiptera: Cixiidae) and LB DNA could remain detectable on sticky traps under field conditions in Florida in winter and summer, using PCR and qPCR. Additionally, this study compared the DNA degradation of Diaphorina citri Kuwayama (Hemiptera: Liviidae) and Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB. The results showed that DNA concentration and amplification rate declined as time on sticky traps increased. Degradation varied between different target genes. The amplification rate of insect genes from sticky trap samples suggests that sticky traps should be changed weekly in summer, and every 2 wk in winter for accurate H. crudus detection. Traps should be changed every 4 days for phytoplasma detection. Traps can be changed monthly for accurate D. citri and CLas detection. Based on these results, standard monitoring protocols can be implemented to more accurately detect vectors and pathogens. [ABSTRACT FROM AUTHOR]

Abstract Extra‐organismal DNA (eoDNA) from material left behind by organisms (noninvasive DNA, e.g., feces, hair) or from environmental samples (eDNA, e.g., water, soil) is a valuable source of genetic information. However, the relatively low quality and quantity of eoDNA, which can be further degraded by environmental factors, results in reduced amplification and sequencing success. This is often compensated for through cost‐ and time‐intensive replications of genotyping/sequencing procedures. Therefore, system‐ and site‐specific quantifications of environmental degradation are needed to maximize sampling efficiency (e.g., fewer replicates, shorter sampling durations), and to improve species detection and abundance estimates. Using 10 environmentally diverse bat roosts as a case study, we developed a robust modeling pipeline to quantify the environmental factors degrading eoDNA, predict eoDNA quality, and estimate sampling‐site‐specific ideal exposure duration. Maximum humidity was the strongest eoDNA‐degrading factor, followed by exposure duration and then maximum temperature. We also found a positive effect when hottest days occurred later. The strength of this effect fell between the strength of the effects of exposure duration and maximum temperature. With those predictors and information on sampling period (before or after offspring were born), we reliably predicted mean eoDNA quality per sampling visit at new sites with a mean squared error of 0.0349. Site‐specific simulations revealed that reducing exposure duration to 2–8 days could substantially improve eoDNA quality for future sampling. Our pipeline identified high humidity and temperature as strong drivers of eoDNA degradation even in the absence of rain and direct sunlight. Furthermore, we outline the pipeline's utility for other systems and study goals, such as estimating sample age, improving eDNA‐based species detection, and increasing the accuracy of abundance estimates.

DNA barcoding is widely used in toxic species authentication, but due to serious DNA degradation of forensic materials, the application of full-length barcode sequences in food poisoning diagnosis is greatly limited. Nucleotide signature, a shorter specific molecular marker, derived from traditional DNA barcoding has been proposed as an emerging tool of toxic species detection in deeply processed materials. In this study, to resolve the frequent food poisoning accidents with unknown origin, we envisioned developing a nucleotide signature data set of common poisonous organisms and combining high-throughput sequencing (HTS) to reveal the poisoning cause. Ninety-three individuals and 1093 DNA barcode sequences of twelve common poisonous plants, fish, mushrooms and their related species were collected. Through sequence alignment and screening, the nucleotide signatures were respectively developed and validated as their specific molecular markers. The sequence length varied from 19 bp to 38 bp. These fragments were conserved within the same species or genera, and the specificity between related species has been also demonstrated. To further evaluate the application potential of nucleotide signature in forensic diagnosis, simulated forensic specimens (SFS) containing different poisonous ingredients were sequenced by HTS with PCR-free libraries. As a result, the nucleotide signature was successfully captured from original HTS data without assembly and annotation, accompanied by a high detection sensitivity of 0.1 ng/µl in mixture system. Therefore, this method was suitable for the assay of forensic materials with serious DNA degradation. The present study undoubtedly provides a new perspective and strong support for the detection of toxic ingredients and the diagnosis of food poisoning.

Microplastics (MPs) affect both marine and terrestrial biota worldwide for their harmful effects, which range from physical cell damage to physiological deterioration. In this research, microplastics were quantified from gills, liver and muscle of demersal Benguela hakes Merluccius polli (n = 94), caught by commercial trawling from northwest African waters. Plastic polymers were identified using Fourier Transformed-infraRed spectroscopy (FT-iR). Fulton’s k condition factor and the degree of DNA degradation in liver were measured. None of the individuals were free of MPs, whose concentration ranged from 0.18 particles/g in muscle to 0.6 in liver. Four hazardous polymers were identified: 2-ethoxyethylmethacrylate, polyester, polyethylene terephthalate, and poly-acrylics. MP concentration in liver was correlated negatively with the condition factor, suggesting physiological damage. Positive association of MP concentration and liver DNA degradation was explained from cell breakage during trawl hauls during decompression, suggesting an additional way of MPs harm in organisms inhabiting at great depth. This is the first report of potential MPs-driven damage in this species; more studies are recommended to understand the impact of MP pollution on demersal species.

PCR-MPS is an emerging tool for the analysis of low-quality DNA samples. In this study, we used PCR-MPS to analyse 32 challenging bone DNA samples from three Second World War victims, which previously yielded no results in conventional STR PCR-CE typing. The Identity Panel was used with 27 cycles of PCR. Despite that we only had an average of 6.8 pg of degraded DNA as template, 30 out of 32 libraries (93.8%) produced sequencing data for about 63/90 autosomal markers per sample. Out of the 30 libraries, 14 (46.7%) yielded single source genetic profiles in agreement with the biological identity of the donor, whereas 12 cases (40.0%) resulted in SNP profiles that did not match or were mixed. The misleading outcomes for those 12 cases were likely due to hidden exogenous human contamination, as shown by the higher frequencies of allelic imbalance, unusual high frequencies of allelic drop-ins, high heterozygosity levels in the consensus profiles generated from challenging samples, and traces of amplified molecular products in four out of eight extraction negative controls. Even if the source and the time of the contamination were not identified, it is likely that it occurred along the multi-step bone processing workflow. Our results suggest that only positive identification by statistical tools (e.g. likelihood ratio) should be accepted as reliable; oppositely, the results leading to exclusion should be treated as inconclusive because of potential contamination issues. Finally, strategies are discussed for monitoring the workflow of extremely challenging bone samples in PCR-MPS experiments with an increased number of PCR cycles. [ABSTRACT FROM AUTHOR]

For centuries, wolf depredation on livestock has triggered human–wildlife conflicts throughout Europe. Free-ranging dogs, however, are increasingly abundant and may also act as predators of livestock herds. This calls for combined efforts aimed at identifying the causes of depredation events and optimizing the procedures towards predators' identification. Here, we analysed 56 livestock depredation events in central Portugal, an area where conflict between wolves and human populations takes place. We estimated the mean minimum sampling effort (number of swabs) required to detect at least one potential predator and examined how extrinsic factors (i.e. time, meteorological conditions and vegetation cover) drive sample degradation and predator identification success. Free-ranging dogs were the only putative predator detected in most attacks (66%). Results indicate that a minimum of three swabs are needed to detect at least one predator, but using at least four would substantially increase the detection rate. We found that the longer the interval between an attack and sample collection and the higher the local humidity, the lower is the probability of identification success. On the other hand, higher temperatures and precipitation levels seem to be associated with a higher probability of success. The unexpected positive effect of precipitation may be linked to specific environmental contexts (i.e. higher precipitation levels in colder weather may still favour sample conservation). As identification success depends on time and weather conditions, the time span between a depredation event and sample collection should be reduced whenever possible, and sufficient samples should be collected to ensure an adequate detection success. [ABSTRACT FROM AUTHOR]

The evaluation of the integrity and quantity of DNA extracted from archaeological human remains is a fundamental step before using the latest generation sequencing techniques in the study of evolutionary processes. Ancient DNA is highly fragmented and chemically modified; therefore, the present study aims to identify indices that can allow the identification of potentially amplifiable and sequenceable DNA samples, reducing failures and research costs. Ancient DNA was extracted from five human bone remains from the archaeological site of Amiternum L'Aquila, Italy dating back to the 9th–12th century and was compared with standard DNA fragmented by sonication. Given the different degradation kinetics of mitochondrial DNA compared to nuclear DNA, the mitochondrially encoded 12s RNA and 18s ribosomal RNA genes were taken into consideration; fragments of various sizes were amplified in qPCR and the size distribution was thoroughly investigated. DNA damage degree was evaluated by calculating damage frequency (λ) and the ratio between the amount of the different fragments and that of the smallest fragment (Q). The results demonstrate that both indices were found to be suitable for identifying, among the samples tested, those less damaged and suitable for post-extraction analysis; mitochondrial DNA is more damaged than nuclear, in fact, amplicons up to 152 bp and 253 bp, respectively are obtained. [ABSTRACT FROM AUTHOR]

The investigation of DNA preservation in archaeological human remains has opened up unprecedented opportunities for understanding the genetic history of our ancestors. This article explores the methods employed to preserve and extract DNA from ancient samples, along with the challenges and limitations encountered in the process. Various preservation techniques, such as cold storage and desiccation, are utilized to maximize DNA preservation. However, DNA degradation and contamination remain significant challenges. The extraction and analysis of ancient DNA involve specialized techniques such as PCR and whole-genome sequencing. The insights gained from ancient DNA analysis have revolutionized our understanding of migration patterns, genetic diversity, and the origins of specific traits. As technology advances, new methods offer the potential for even more detailed genetic information. Interdisciplinary collaborations and continued research hold promise for further unraveling the mysteries of our collective human history. [ABSTRACT FROM AUTHOR]

Abstract Insect declines are a global issue with significant ecological and economic ramifications. Yet, we have a poor understanding of the genomic impact these losses can have. Genome‐wide data from historical specimens have the potential to provide baselines of population genetic measures to study population change, with natural history collections representing large repositories of such specimens. However, an initial challenge in conducting historical DNA data analyses is to understand how molecular preservation varies between specimens. Here, we highlight how Next‐Generation Sequencing methods developed for studying archaeological samples can be applied to determine DNA preservation from only a single leg taken from entomological museum specimens, some of which are more than a century old. An analysis of genome‐wide data from a set of 113 red‐tailed bumblebee Bombus lapidarius specimens, from five British museum collections, was used to quantify DNA preservation over time. Additionally, to improve our analysis and further enable future research, we generated a novel assembly of the red‐tailed bumblebee genome. Our approach shows that museum entomological specimens are comprised of short DNA fragments with mean lengths below 100 base pairs (BP), suggesting a rapid and large‐scale post‐mortem reduction in DNA fragment size. After this initial decline, however, we find a relatively consistent rate of DNA decay in our dataset, and estimate a mean reduction in fragment length of 1.9 bp per decade. The proportion of quality filtered reads mapping to our assembled reference genome was around 50%, and decreased by 1.1% per decade. We demonstrate that historical insects have significant potential to act as sources of DNA to create valuable genetic baselines. The relatively consistent rate of DNA degradation, both across collections and through time, mean that population‐level analyses—for example for conservation or evolutionary studies—are entirely feasible, as long as the degraded nature of DNA is accounted for.

Abstract Background Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. Method We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a − 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. Results We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. Conclusions These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera. Graphical Abstract

To regulate the degradation of transgenic DNA and lay theoretical foundations for the rational utilization of genetically modified (GM) products, variations in copy numbers and structural characteristics of DNA from GM soybean event GTS 40-3-2 during soybean protein concentrate (SPC) preparation were evaluated. Results showed that defatting and the first ethanol extraction were key procedures inducing DNA degradation. After these two procedures, copy numbers of the lectin and cp4 epsps targets decreased by more than 4 × 108, occupying 36.88–49.30% of the total copy numbers from raw soybean. Atomic force microscopy images visually revealed the degradation of DNA that thinned and shortened during SPC preparation. Circular dichroism spectra suggested a lower helicity of DNA from defatted soybean kernel flour and a conformation transition of DNA from B-type to A-type after ethanol extraction. The fluorescence intensity of DNA decreased during SPC preparation, verifying the DNA damage along this preparation chain. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the potentiality of a mineral and antioxidant-rich methanolic extract of the red marine alga Falkenbergia rufolanosa (FRE) against methyl-thiophanate (MT)-induced toxicity in adult rats. The animals were allocated into four groups: controls, MT (300 mg/kg), MT + FRE, and FRE-treated group for 7 days. Our results demonstrated severe mineral perturbations due to MT treatment, especially in calcium and phosphorus levels in plasma, urine, and bone. Similarly, the hematological analysis revealed increased red blood cells, platelets, and white blood cells associated with striking genotoxicity. Interestingly, a significant rise in lipid peroxidation and advanced oxidation protein products level in erythrocytes and bone were noted. Meanwhile, a depletion of the antioxidant status in both tissues occurred. These biochemical alterations were in harmony with DNA degradation and histological variation in bone and blood. In the other trend, data showed that treatment with alga improved MT-induced hematotoxicity, genotoxicity, and oxidative stress in the blood and bone. Osteo-mineral metabolism and bone histo-architecture were also noted. In conclusion, these findings demonstrated that the red alga Falkenbergia rufolanosa is a potent source of antioxidant and antibacterial agents, as revealed by the in vitro analysis. [ABSTRACT FROM AUTHOR]

• Immunohistochemical staining intensity is higher in adequately fixated tumor areas. • Immunohistochemical staining intensity is different within tumors, suggesting heterogeneous immunoreactivity. • Independent of tumor fixation, DNA fragments rarely exceeded a length of 300 base pairs. • The pathologist should be aware of the quality of tumor fixation during the interpretation of immunohistochemistry. The objective is to assess the impact of the quality of tissue fixation in surgical pathology on immunohistochemical (IHC) staining and DNA degradation. Twenty-five non-small cell lung cancer (NSCLC) resection specimens were analyzed. After resection, all tumors were processed according to the protocols in our center. In haematoxylin and eosin (H&E) stained tissue slides, adequately- and inadequately fixed tumor areas were microscopically demarcated, based on basement membrane detachment. In 10 IHC stains ALK (clone 5A4), PD-L (clone 22C3), CAM5.2, CK7, c-Met, KER-MNF116, NapsinA, p40, ROS1, TTF1) the immunoreactivity in H-scores was determined in adequately- and inadequately fixed, and necrotic tumor areas. From the same areas DNA was isolated, and DNA fragmentation in base pairs (bp) was measured. H-scores were significantly higher in H&E adequately fixed tumor areas in IHC stains KER-MNF116 (H-score 256 vs 15, p =0.001) and p40 (H-score 293 vs 248, p =0.028). All other stains showed a trend towards higher immunoreactivity in H&E adequately fixed areas. Independent of H&E adequatelty- or inadequately fixed areas, all IHC stains showed significant different IHC staining intensity within tumors, suggesting heterogeneous immunoreactivity (H-scores: PD-L1 123 vs 6, p = 0.001; CAM5.2 242 vs 101, p =<0.001; CK7 242 vs 128, p =<0.001; c-MET 99 vs 20, p =<0.001; KER-MNF116 281 vs 120, p =<0.001; Napsin A 268 vs 130, p = 0.005; p40 292 vs 166, p = 0.008; TTF1 199 vs 63, p =<0.001). DNA fragments rarely exceeded a length of 300 bp, independent of adequate fixation. However, DNA fragments of 300 and 400 bp had higher concentrations in tumors with short fixation delay (<6 h vs >16 h) and short fixation time (<24 h vs >24 h). Impaired tissue fixation of resected lung tumors results in decreased IHC staining intensity in some parts of the tumor. This may impact the reliability of IHC analysis. [ABSTRACT FROM AUTHOR]

In this article, we describe multiple analytical strategies that were first developed for forensic purposes, on a set of three bone samples collected in 2011. We analyzed a single bone sample (patella) collected from the artificially mummified body of the Baron Pasquale Revoltella (1795–1869), as well two femurs which allegedly belonged to the Baron's mother (Domenica Privato Revoltella, 1775–1830). Likely due to the artificial mummification procedures, the inner part of the Baron's patella allowed the extraction of high-quality DNA yields, which were successfully used for PCR-CE and PCR-MPS typing of autosomal, Y-specific, and mitochondrial markers. The samples extracted from the trabecular inner part of the two femurs yielded no typing results by using the SNP identity panel, whereas the samples extracted from the compact cortical part of the same bone samples allowed genetic typing, even by the employment of PCR-CE technology. Altogether, 10/15 STR markers, 80/90 identity SNP markers, and HVR1, HVR2, and HVR3 regions of the mtDNA were successfully typed from the Baron's mother's remains by the combined use of PCR-CE and PCR-MPS technologies. The kinship analysis showed a likelihood ratio of at least 9.1 × 106 (corresponding to a probability of maternity of 99.9999999%), and thus confirmed the identity of the skeletal remains as those of the Baron's mother. This casework represented a challenging trial for testing forensic protocols on aged bones samples. It highlighted the importance of accurately sampling from the long bones, and that DNA degradation is not blocked by freezing at −80 °C. [ABSTRACT FROM AUTHOR]

Cancer is one of the main causes of human mortality worldwide. Despite the advances in the diagnostics, surgery, radiotherapy, and chemotherapy, the search for more effective treatment regimens and drug combinations are relevant. This work aimed to assess the radiomodifying effect and molecular mechanism of action of fucoidan from the brown alga Saccharina cichorioides (ScF) and product of its autohydrolysis (ScF_AH) in combination with pacificusoside D from the starfish Solaster pacificus (SpD) on the model of viability and invasion of three-dimension (3D) human melanoma cells SK-MEL-2. The cytotoxicity of ScF (IC50 JB6 Cl41 > 800 µg/mL; IC50 SK-MEL-2 = 685.7 µg/mL), ScF_AH (IC50 JB6 Cl41/SK-MEL-2 > 800 µg/mL), SpD (IC50 JB6 Cl41 = 22 µM; IC50 SK-MEL-2 = 5.5 µM), and X-ray (ID50 JB6 Cl41 = 11.7 Gy; ID50 SK-MEL-2 = 6.7 Gy) was determined using MTS assay. The efficiency of two-component treatment of 3D SK-MEL-2 cells was revealed for ScF in combination with SpD or X-ray but not for the combination of fucoidan derivative ScF_AH with SpD or X-ray. The pre-treatment of spheroids with ScF, followed by cell irradiation with X-ray and treatment with SpD (three-component treatment) at low non-toxic concentrations, led to significant inhibition of the spheroids' viability and invasion and appeared to be the most effective therapeutic scheme for SK-MEL-2 cells. The molecular mechanism of radiomodifying effect of ScF with SpD was associated with the activation of the initiator and effector caspases, which in turn caused the DNA degradation in SK-MEL-2 cells as determined by the Western blotting and DNA comet assays. Thus, the combination of fucoidan from brown algae and triterpene glycoside from starfish with radiotherapy might contribute to the development of highly effective method for melanoma therapy. [ABSTRACT FROM AUTHOR]

Accurate verification of the time passed since death is a challenging task in daily forensic investigations, chiefly in homicidal or unwitnessed death. The study evaluated the applicability of the time-dependent changes in histological structure, DNA content,and oxidant/antioxidant parameters in rat's skeletal muscles in an attempt to reach a reliable method for post-mortem interval (PMI) estimation. We used 56 adult male albino rats. These animals were sacrificed then and allocated randomly to seven groups (eight rats each). First group: rats were dissected to obtain samples from quadriceps femoris muscles promptly after death. While the rats in the second, third, fourth, fifth, sixth and seventh groups were dissected to get muscle samples from quadriceps femoris at 6, 24, 48, 72, 96 and 144 hours post-mortem (hpm). The results showed that a significant gradual histopathological alteration of muscle structure started at 48 hpm and progressed afterwards. Also, the DNA degradation percentage increased significantly parallel to the increased oxidant parameter levels from 6 to 144 hpm. Our results concluded that changes in oxidant/antioxidant parameters and DNA degradation, as well as the histological examination of muscle tissues, showed a strong correlation with post-mortem intervals. Consequently, the integration of all these parameters can provide a valuable method of post-mortem intervals estimation. [ABSTRACT FROM AUTHOR]

This is the first report on the usage of fruiting bodies of shiitake mushroom (Lentinula edodes) and silk sericin extracts as green reducer agents incorporated in the myco-synthesis of zinc oxide nanoparticles, coated with chitosan polymer that functions as a stabilizer was performed. The synthesized ZnO NCs were investigated by various techniques in order to confirm the production, particle size, and other properties. FTIR analysis confirmed the presence of functional groups in extracts and synthesized nanocomposites, which was further supported by X-ray photoelectron spectroscopy. The confirmation was verified by HRTEM analysis. XRD results revealed the crystalline structure with a size of 21 nm and 25 nm for silk sericin NCs and mushroom NCs, respectively, which was confirmed by HRTEM. Further, HRTEM proved the formation of cubic/hexagonal shapes with consistent sizes. The antibacterial effectiveness of nanocomposites was tested against two bacterial strains E. coli and Bacillus subtilis. The results showed that the E. coli strain was practically resistant to these NCs, whereas B. subtilis was quite sensitive. The findings point to a mechanism for ZnO NCs in cell death caused by complete DNA destruction. Under UV light, the photocatalytic effectiveness of biosynthesized ZnO NCs was evaluated for the methylene blue (MB) dye degradation process. 90% of the dye was degraded within 135 min. The prepared ZnO CH-SS NCs can therefore be employed as a photocatalyst for the breakdown of toxic dyes found in wastewater and it could be used as a promising antibacterial product. [Display omitted] • A first report on synergistic impact of ZnO-Chitosan- Lentinula edodes NCs. • Lentinula edodes and silk sericin have been used to synthesize ZnO NCs. • 90% of organic pollutant was degraded in 135 min. • Genomic DNA damage revealed activity against Bacillus subtilis. [ABSTRACT FROM AUTHOR]

Insect declines are a global issue with significant ecological and economic ramifications. Yet, we have a poor understanding of the genomic impact these losses can have. Genome‐wide data from historical specimens have the potential to provide baselines of population genetic measures to study population change, with natural history collections representing large repositories of such specimens. However, an initial challenge in conducting historical DNA data analyses is to understand how molecular preservation varies between specimens.Here, we highlight how Next‐Generation Sequencing methods developed for studying archaeological samples can be applied to determine DNA preservation from only a single leg taken from entomological museum specimens, some of which are more than a century old. An analysis of genome‐wide data from a set of 113 red‐tailed bumblebee Bombus lapidarius specimens, from five British museum collections, was used to quantify DNA preservation over time. Additionally, to improve our analysis and further enable future research, we generated a novel assembly of the red‐tailed bumblebee genome.Our approach shows that museum entomological specimens are comprised of short DNA fragments with mean lengths below 100 base pairs (BP), suggesting a rapid and large‐scale post‐mortem reduction in DNA fragment size. After this initial decline, however, we find a relatively consistent rate of DNA decay in our dataset, and estimate a mean reduction in fragment length of 1.9 bp per decade. The proportion of quality filtered reads mapping to our assembled reference genome was around 50%, and decreased by 1.1% per decade.We demonstrate that historical insects have significant potential to act as sources of DNA to create valuable genetic baselines. The relatively consistent rate of DNA degradation, both across collections and through time, mean that population‐level analyses—for example for conservation or evolutionary studies—are entirely feasible, as long as the degraded nature of DNA is accounted for. [ABSTRACT FROM AUTHOR]

Background: Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. Method: We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a − 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. Results: We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. Conclusions: These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify artificial single-nucleotide variants (SNVs) in degraded trace DNA samples. In a preliminary study, blood samples were stored for up to 120 days and whole-genome sequencing was performed using the Snakemake workflow dna-seq-gatk-variant-calling to identify positions that vary between the time point 0 sample and the aged samples. In a follow-up study on blood and saliva samples stored under humid and dry conditions, potential marker candidates for the estimation of the age of a blood stain (= time since deposition) were identified. Both studies show that a general decrease in the mean fragment size of the libraries over time was observed, presumably due to the formation of abasic sites during DNA degradation which are more susceptible to strand breaks by mechanical shearing of DNA. Unsurprisingly, an increase in the number of failed genotype calls (no coverage) was detected over time. Both studies indicated the presence of artificial SNVs with the majority of changes happening at guanine and cytosine positions. This confirms previous studies and can be explained by depurination through hydrolytic attacks which more likely deplete guanine while deamination leads to cytosine to thymine variants. Even complete genotype switches from homozygote 0/0 genotypes to the opposite 1/1 genotypes were observed. While positions with such drastic changes might provide suitable candidate markers for estimating short-term time since deposition (TsD), 11 markers were identified which show a slower gradual change of the relative abundance of the artificial variant in both blood and saliva samples, irrespective of storage conditions. [ABSTRACT FROM AUTHOR]

Food fraudulent activities have become a serious issue over the world recently. Seafood products have trading and profitable potential in Turkey owing to the abundance of fisheries and other species. While morphological features are commonly used for species identification in raw seafood products, this identification does not meet the correct classification in cryptic species and processed seafood products. Molecular techniques have been utilized for species authentication in processed seafood items successfully. In this study, the effect of different processing techniques on the DNA quality and DNA degradation isolated from raw and processed anchovy was investigated. Anchovy is one of the important species in both fisheries activities and processing and consumption in Turkey. For this aim, DNA was isolated from processed anchovy groups and un-processed anchovy groups as control by the same extraction methods and the quality of DNA was compared among the groups. The most common processing techniques, frying, baking, smoking, roasting, baking and grilling were applied to anchovy. The results revealed that not only different thermal processing but also treatment with acid and salt cause DNA degradation and quality loss of DNA parameters which are essential for authentication of species and traceability for public health.

Environmental DNA (eDNA) is a highly sensitive and cost‐effective tool that is increasingly being applied to studies of biodiversity and species detection. This non‐invasive method relies on the collection of environmental samples that contain genetic material being shed into surrounding environment by the target organism/s. While forensic science has a long history of using molecular tools for collecting DNA from the environment, the detection of human DNA from environmental water samples has been limited. This study investigated the detection and degradation rates of human eDNA in water samples under controlled laboratory conditions. Using a human‐specific qPCR assay targeting the ND1 region of human mitochondrial DNA, eDNA degradation over time in water spiked with human blood was assessed. Recovery of nuclear DNA was investigated by determining if routine DNA short tandem repeat (STR) profiles of the blood source could be generated. Results demonstrated that human eDNA remains detectable for up to 11 days under laboratory conditions in environmental water and up to 35 days in distilled water. Partial STR profiles could be recovered from environmental water only up to 24 h, while, in distilled water, partial profiles continued to be recovered up to 840 h. These findings demonstrate that sampling human eDNA from aquatic samples can provide reliable human DNA detection within relatively short time windows, assisting law enforcement agencies by providing information about the potential time an individual may have been present in an area or assisting in the detection and location of a body or remains in aquatic environments. [ABSTRACT FROM AUTHOR]

Objectives: The central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air., Methods: To test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish., Results: Considering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions., Conclusion: Our study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Modern forensic DNA quantitation assays provide information on the suitability of a DNA extract for a particular type of analysis, on the amount of sample to put into the analysis in order to yield an optimal (or best possible) result, and on the requirement for optional steps to improve the analysis. To achieve a high sensitivity and specificity, these assays are based on quantitative PCR (qPCR) and analyze target DNA loci that are present in multiple copies distributed across the genome. These target loci allow the determination of the amount of DNA, the degree of DNA degradation, and the proportion of DNA from male contributors. In addition, internal control DNA of a known amount is analyzed in order to inform about the presence of PCR inhibitors. These assays are nowadays provided as commercial kits that have been technically validated and are compatible with common qPCR instruments. In this review, the principles of forensic qPCR assays will be explained, followed by information on the nature of DNA loci targeted by modern forensic qPCR assays. Finally, we critically draw attention to the current trend of manufacturers not to disclose the exact nature of the target loci of their commercial kits.

In this work, we have presented a new method for species origin verification of collagen peptides based on DNA techniques. First, we investigate the changes in DNA during the preparation of collagen peptides including the total amount of collagen peptide DNA and the DNA degradation under different processing conditions. Secondly, we discussed the possibility of using polymerase chain reaction (PCR) for follow-up detection of collagen peptides. The results showed that the total amount of DNA decreased as the treatment intensity increased. The size of the cleaved fragments of DNA are mainly concentrated between 200 and 500 bp. On this basis, the combined PCR results finally determined that trace collagen peptide DNA can be effectively amplified with amplicons of about 300 bp to complete the verification of the species origin of collagen peptide. This study provides a new strategy for determining the authenticity of food labels for bovine collagen peptides., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Different types of disasters, whether natural or human in character, lead to the significant loss of human lives. In the latter case, the quick action of identification of corpses and human remains is mandatory. There are a variety of protocols to identify victims; however, genetics is one of the tools that allows an exact identification of the victim. However, several factors may interfere with this identification, from the biological samples’ degradation not allowing the analysis of nuclear information, to failure to dispose of biological samples from family members. Access to certain family members could be a determinant of the proper choice of genetic markers that allow the identification of the victim, or his/her inclusion in a given genetic maternal or paternal lineage. New advances in the field of genetics are soon expected to allow for the identification of victims from disasters with only their biological postmortem samples; it may also be possible to draw a robot portrait of a victim’s most likely physical characteristics. In all cases, genetics is the only modern tool with universal character and can be used in essentially all biological samples, giving and identification of more or less accurate statistical character, depending on whether nuclear or lineage markers are used.

To test the usefulness of the forensic PCR-MPS approach to eye and hair color prediction for aged skeletons, a customized version of the PCR-MPS HIrisPlex panel was used on two sets of samples. The first set contained 11 skeletons dated from the 3rd to the 18th centuries AD, and for each of them at least four bone types were analyzed (for a total of 47 samples). In the second set, 24 skeletons from the Second World War were analyzed, and only petrous bones from the skulls were tested. Good-quality libraries were achieved in 83.3% of the cases for the ancient skeletons and in all Second World War petrous bones, with 94.7% and 100% of the markers, respectively, suitable for SNP typing. Consensus typing was achieved for about 91.7% of the markers in 10 out of 11 ancient skeletons, and the HIrisPlex-S webtool was then used to generate phenotypic predictions. Full predictions were achieved for 3 (27.3%) ancient skeletons and 12 (50%) Second World War petrous bones. In the remaining cases, different levels of AUC (area under the receiver operating curve) loss were computed because of no available data (NA) for 8.3% of markers in ancient skeletons and 4.2% of markers in Second World War petrous bones. Although the PCR-based approach has been replaced with new techniques in ancient DNA studies, the results show that customized forensic technologies can be successfully applied to aged bone remains, highlighting the role of the template in the success of PCR-MPS analysis. However, because several typical errors of ancient DNA sequencing were scored, replicate tests and accurate evaluation by an expert remain indispensable tools. [ABSTRACT FROM AUTHOR]

DNA technology has entered our lives in every field as one of the most important scientific developments of this century. DNA analysis; It is widely used to identify suspicious persons in various forensic crimes, to identify unidentified corpses who died due to mass disasters and other reasons. In such cases, unsatisfactory results can be obtained from DNA analysis performed on rotten and decaying tissue samples taken from unidentified corpses coming from the crime scene to the forensic laboratories. This may be due to DNA contamination. The most common causes of DNA damage are enzymes secreted by microorganisms found in the corpse and environment. Microbial enzymes cause DNA damage, insufficient or no PCR amplification, thus irregularities in the peak levels in the DNA electropherogram and the appearance of meaningless and missing peaks. When DNA analysis methods are applied to a postmortem biological sample in which microorganisms multiply, it may be difficult to obtain accurate and precise results due to degradation. The aim of this study is to evaluate the importance of identification of microorganisms (bacteria) that reproduce in postmortem muscle tissues in terms of forensic sciences, to determine whether they prevent DNA analysis, and if so, to determine which bacteria may cause it. [ABSTRACT FROM AUTHOR]