DNA Preservation Research Articles

Comparison of DNA preservation and ATR-FTIR spectroscopy indices of cortical and trabecular bone of metacarpals and metatarsals

Shape, size, composition, and function of the bones in the human body vary on the macro, micro and nanoscale. This can influence changes caused by taphonomy and post-mortem preservation, including DNA. Highly mineralised compact bone is less susceptible to taphonomic factors than porous trabecular bone. Some studies imply that DNA can be better preserved in trabecular bone, due to remnants of the soft tissue or bacteria better digesting organic matter while not digesting DNA. The aim of this study was to understand the differences between compact (diaphyses) and trabecular (epiphyses) bone on a molecular level and thus the reasons for the better preservation of the DNA in the trabecular bone. The powder obtained from epiphyses and diaphyses of metacarpals and metatarsals was analysed using ATR-FTIR spectroscopy and compared. Samples with poorest DNA preservation originated from diaphyses, predominantly of metatarsals. They were characterised by higher concentrations of phosphates and crystallinity, while lower collagen quality in comparison to samples with the best DNA preservation. Epiphyses presented higher concentrations of better-preserved collagen while diaphyses had higher concentrations of carbonates and phosphates and higher crystallinity. Due to better-preserved collagen in the epiphyses, the soft tissue remnants hypothesis seems more likely than the bacteria hypothesis.

The Water‐Exclusion Trap (WET): A 3D printable window trap collector that prevents DNA degradation

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.

Performance of Different Cotton and Nylon Swabs on DNA Recovery and Storage

Touch DNA samples are routine yet challenging pieces of evidence that provide investigators with information that helps them solve crimes. However, this type of evidence can be easily lost if the correct collection method is not used. This problem could be overcome with an optimal method of collection that increases the amount of touch DNA collected from different types of surfaces. Better-quality touch DNA can increase the chances of getting a full genetic profile. This study was divided into two parts which aimed to assess whether the type of swab used on different surfaces will significantly increase DNA recovery, concentrations, and the DNA preservation during three different timeframes (24h, 1 month and 3 months). Two different cotton swabs and Nylon swabs were used to lift touch DNA on three different surfaces (glass, plastic and wood) to identify the most suitable method of collection across all three surfaces. A total of 72 samples were lifted (3 replicates from each swab on 3 different surfaces) from two different participants (Male and Female) which were left to dry for 14 days in room temperature prior to DNA extraction. DNA preservation of the swabs was observed while using three dilutions of blood sample which was prepared from one of the volunteers (1:1 – 1:10 – 1:20) where 10 uL of each dilution was pipetted onto the four types of swabs in three replicates (n=36) to observe the preservation over three different timeframes 24h storage, 1 Month and 3 Months with a total of 108 samples. The COPAN CLASSIQSwabsTM Dry swab showed an overall average result during the storage periods of 24h with (1:1) dilution by (2.694ng/μL), (1:10) dilution with (0.548ng/μL) and (1:20) dilution with (0.143ng/μL). Results for the period of 1 Month also showed an average of (1:1) dilution with (2.825ng/μL), (1:10) dilution with (0.361ng/μL) and (1:20) dilution with (0.156ng/μL). These findings can be helpful for laboratories and crime scene investigators to optimize DNA sample collection and preservation based on their workflow.

Detection of fish sedimentary DNA in aquatic systems: A review of methodological challenges and future opportunities

AbstractEnvironmental DNA studies have proliferated over the last decade, with promising data describing the diversity of organisms inhabiting aquatic and terrestrial ecosystems. The recovery of DNA present in the sediment of aquatic systems (sedDNA) has provided short‐ and long‐term data on a wide range of biological groups (e.g., photosynthetic organisms, zooplankton species) and has advanced our understanding of how environmental changes have affected aquatic communities. However, substantial challenges remain for recovering the genetic material of macro‐organisms (e.g., fish) from sediments, preventing complete reconstructions of past aquatic ecosystems, and limiting our understanding of historic, higher trophic level interactions. In this review, we outline the biotic and abiotic factors affecting the production, persistence, and transport of fish DNA from the water column to the sediments, and address questions regarding the preservation of fish DNA in sediment. We identify sources of uncertainties around the recovery of fish sedDNA arising during the sedDNA workflow. This includes methodological issues related to experimental design, DNA extraction procedures, and the selected molecular method (quantitative PCR, digital PCR, metabarcoding, metagenomics). By evaluating previous efforts (published and unpublished works) to recover fish sedDNA signals, we provide suggestions for future research and propose troubleshooting workflows for the effective detection and quantification of fish sedDNA. With further research, the use of sedDNA has the potential to be a powerful tool for inferring fish presence over time and reconstructing their population and community dynamics.

16S rRNA gene-based microbiota profiles from diverse avian faeces are largely independent of DNA preservation and extraction method.

The avian gut microbiota has been the subject of considerable recent attention, with potential implications for diverse fields such as the poultry industry, microbial ecology, and conservation. Faecal microbiotas are frequently used as a non-invasive proxy for the gut microbiota, however the extraction of high-quality microbial DNA from avian faeces has often proven challenging. Here we aimed to evaluate the performance of two DNA preservation methods (95% ethanol and RNAlater) and five extraction approaches (IndiSpin Pathogen Kit, QIAamp PowerFecal Pro DNA Kit, MicroGEM PrepGEM Bacteria Kit, ZymoBIOMICS DNA Miniprep Kit, and an in-house phase separation-based method) for studying the avian gut microbiota. Systematic testing of the efficacy of these approaches on faecal samples from an initial three avian species (chicken, ostrich, and the flightless parrot kākāpō) revealed substantial differences in the quality, quantity and integrity of extracted DNA, but negligible influence of applied method on 16S rRNA gene-based microbiota profiles. Subsequent testing with a selected combination of preservation and extraction method on 10 further phylogenetically and ecologically diverse avian species reiterated the efficacy of the chosen approach, with bacterial community structure clustering strongly by technical replicates for a given avian species. Our finding that marked differences in extraction efficacy do not appear to influence 16S rRNA gene-based bacterial community profiles provides an important foundation for ongoing research on the avian gut microbiota.

Magnetic Bead Spherical Nucleic Acid Microstructure for Reliable DNA Preservation and Repeated DNA Reading.

DNA is an attractive medium for long-term data storage because of its density, ease of copying, sustainability, and longevity. Recent advances have focused on the development of new encoding algorithms, automation, and sequencing technologies. Despite progress in these subareas, the most challenging hurdle in the deployment of DNA storage remains the reliability of preservation and the repeatability of reading. Herein, we report the construction of a magnetic bead spherical nucleic acid (MB-SNA) composite microstructure and its use as a cost-effective platform for reliable DNA preservation and repeated reading. MB-SNA has an inner core of silica@γ-Fe2O3@silica microbeads and an outer spherical shell of double-stranded DNA (dsDNA) with a density as high as 34 pmol/cm2. For MB-SNA, each strand of dsDNA stored a piece of data, and the high-density packing of dsDNA achieved high-capacity storage. MB-SNA was advantageous in terms of reliable preservation over free DNA. By accelerated aging tests, the data of MB-SNA is demonstrated to be readable after 0.23 million years of preservation at -18 °C and 50% relative humidity. Moreover, MB-SNA facilitated repeated reading by facile PCR-magnetic separation. After 10 cycles of PCR access, the retention rate of dsDNA for MB-SNA is demonstrated to be as high as 93%, and the accuracy of sequencing is more than 98%. In addition, MB-SNA makes cost-effective DNA storage feasible. By serial dilution, the physical limit for MB-SNA to achieve accurate reading is probed to be as low as two microstructures.

A comparison between petrous bone and tooth, femur and tibia DNA analysis from degraded skeletal remains.

Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15-30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.

Three dimensional structures of the inner and outer pig petrous bone using FIB-SEM: Implications for development and ancient DNA preservation

We report on the 3D ultrastructure of the mineralized petrous bone of mature pig using focused ion beam – scanning electron microscopy (FIB-SEM). We divide the petrous bone into two zones based on the degree of mineralization; one zone close to the otic chamber has higher mineral density than the second zone further away from the otic chamber. The hypermineralization of the petrous bone results in the collagen D-banding being poorly revealed in the lower mineral density zone (LMD), and absent in the high mineral density zone (HMD). We therefore could not use D-banding to decipher the 3D structure of the collagen assembly. Instead we exploited the anisotropy option in the Dragonfly image processing software to visualize the less mineralized collagen fibrils and/or nanopores that surround the more mineralized zones known as tesselles. This approach therefore indirectly tracks the orientations of the collagen fibrils in the matrix itself. We show that the HMD bone has a structure similar to that of woven bone, and the LMD is composed of lamellar bone with a plywood-like structural motif. This agrees with the fact that the bone close to the otic chamber is fetal bone and is not remodeled. The lamellar structure of the bone further away from the otic chamber is consistent with modeling/remodeling. The absence of the less mineralized collagen fibrils and nanopores resulting from the confluence of the mineral tesselles may contribute to shielding DNA during diagenesis. We show that anisotropy evaluation of the less mineralized collagen fibrils could be a useful tool to analyze bone ultrastructures and in particular the directionality of collagen fibril bundles that make up the bone matrix.

Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing.

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.

Diagnostic models to predict nuclear DNA and mitochondrial DNA recovery from incinerated teeth.

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).

Sustainable DNA Data Storage on Cellulose Paper.

DNA is a promising material for high density and long-term archival data storage. In addition to algorithms for encoding digital information into DNA sequences, the DNA writing (chemical synthesis) and reading (DNA sequencing), the preservation of DNA mixtures with high sequence diversity is another critical issue for sustainable, long-term, and large-scale DNA data storage. Here, this work demonstrates a method for low-cost, convenient and sustainable DNA data storage on cellulose paper. A DNA pool comprising thousands of sequences, in which archival data are encoded, is conveniently stored on a cellulose paper with a calculated density as high as 15TB per mm3 through electrostatic adsorption. This work demonstrates that these digitally encoded DNA pools can be stable for years on the cellulose paper after drying even when directly exposed to air. Furthermore, the reversible electrostatic adsorption enables repeated loading/retrieval of DNA on/off cellulose paper. Therefore, this sustainable DNA preservation on cellulose paper through the convenient electrostatic adsorption exhibits a great advantage in terms of storage capacity and cost that is crucial for practical systems to achieve large-scale and long-time data storage.

Evaluation of Storage Solutions for DNA Preservation in Various Conditions

DNA should be protected from factors such as temperature fluctuation, hydrolysis, and oxidative damage, to be preserved intact. Thus far, DNA has been conventionally stored in freezers to avoid the influence of these factors. However, as the required storage time and number of samples increase, so do the spatial and financial burden. DNAstable and DNAstable Plus are commercial solutions for long-term storage of DNA at room temperature. In this study, DNA extracted from human blood was stored at different temperatures (room temperature, 60 °C, −80 °C, −20 °C, and 4 °C), under hydration and illumination conditions, and with or without long-term storage solutions. After 1, 3, 6, and 12 months of storage, we analyzed the quantity and preservation of STR loci of the stored DNA. Storage of DNA for 1 year at 60 °C using DNAstable® Plus, simulating 16 years of storage at room temperature, had a preservative effect similar to that of freezing in dried state. On the other hand, DNAstable® was inefficient compared to conventional methods. Storage at ambient temperature using DNAstable® Plus thus presents a cost- and space-effective alternative for long-term storage, even for concentrations of DNA lower than 0.5 ng/μL.

Impact of storage conditions and time on DNA yield from ammunition cartridges.

Recovery of suitable amounts of DNA from ammunition cartridges for short tandem repeat (STR) or mitochondrial (mt) DNA analysis has been a challenge for crime laboratories. The metal composition of cartridge cases and projectiles exposes the DNA to harmful ions that damage and ultimately degrade the DNA such that it cannot be effectively amplified. The current study assessed the impact of time and storage conditions on touch DNA deposited on cartridge components of varying metal content: aluminum, nickel, brass, and copper. Elevated humidity levels facilitated greater DNA degradation and loss compared to low humidity (or "dry") conditions, indicating that recovered cartridge component evidence should be stored in a low-humidity environment immediately after collection, preferably with a desiccant. As expected, a relationship was observed between the amount of time elapsed since the cartridge components were handled and the associated DNA yield. Interestingly, while yields dropped considerably in the first 48-96h post-handling, regardless of the storage conditions, a layering effect was observed that helps maintain a relatively constant level of surface DNA over extended periods of time. An apparent layering effect was also observed on cartridge components following multiple surface depositions, where yields were two times higher than single deposition samples at similar timepoints. Overall, these findings suggest that storage conditions and a layering affect play an important role in the preservation of DNA on ammunition components.

Peptide analysis of tooth enamel - A sex estimation tool for archaeological, anthropological, or forensic research.

Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.

Lone star ticks (Acari: Ixodidae) infected with Bourbon virus in New Jersey, USA.

Lone star ticks (Amblyomma americanum L.) are expanding within the northeast United States, a region historically focused on Ixodes scapularis-transmitted diseases. In Monmouth County, NJ, the shift has been dramatic, and lone star ticks now vastly outnumber blacklegged ticks. As a result, there is an enhanced need to focus on the potential health risks of A. americanum-transmitted pathogens, such as the emerging Heartland (HRTV) and Bourbon (BRBV) viruses. We screened 1,205 nymphal lone star ticks for HRTV and BRBV using RT-qPCR assays and detected BRBV in 3 ticks collected in Monmouth County, NJ, in 2021. Additionally, we sequenced a complete BRBV genome from a single infected specimen, finding 99.4% identity with human pathogenic isolates from the eastern-central United States. Our results have important public health implications for a region only recently becoming aware of public health risks posed by lone star ticks. Of note, we report successful detection of viral RNA in samples that were stored and intended for DNA preservation, for example, kept in ethanol at room temperature, which may reduce barriers for public health agencies seeking to expand their tick testing to include viruses.

DNA Data Storage: The Fusion of Digital and Biological Information

With the development of Internet technology, human information data grows by a huge amount. Traditional data storage media are no longer suitable for large amounts of data storage due to their inherent shortcomings, such as high power consumption, large physical size, and short storage life. DNA information storage, on the other hand, can overcome these shortcomings to a certain extent. This paper introduces the process and mechanism of DNA storage, such as the DNA synthesis method, DNA coding, DNA preservation and sequencing, the history of DNA storage, its defects, and shortcomings. This article introduces the principles and mechanisms of DNA storage, including DNA synthesis method, data storage, and DNA preservation, as well as the history of DNA storage and its shortcomings and prospects for improvement, using the comparison of traditional storage methods and DNA storage methods as an import cut.

Conservation of plant biodiversity by biotechnology methods

Several methods of plant preservation are currently used in the Republic of Kazakhstan: 1) field gene banks in natural growing areas and pomological collections; 2) preservation of seeds at +4°С, –18°С, and –196°С; 3) сold storage of in vitro shoots at +4°С and +10 ± 2°С; 4) cryopreservation of plant tissues and organs at –196°C; 5) preservation of plant DNA at –80°C.Ex situ field collections of plants in Kazakhstan are maintained in botanical gardens of the Republic, arboretums, pomological gardens, nurseries, peasant and farm enterprises, and personal plots. The largest collection of fruit and berry plants is located in the Pomological Garden and includes about 4 thousand varieties, more than 40 thousand hybrids and wild forms of various crops.The Republic of Kazakhstan has about 42,000 accessions in the seed collection. The cereal crop collection is mainly concentrated in the Kazakh Research Institute of Agriculture and Plant Growing – 1/3 of the national genetic resources. The main seed collection of vegetable crops and potatoes (14,700 accessions) is held by the Kazakh Research Institute of Potato and Vegetable Growing. Collections are also available at the Institute of Botany and Phytointroduction and in the Institute of Plant Biology and Biotechnology (IPBB), with the storage mode of + 4°C and –18°C. IPBB also uses cryopreservation technology at –196°C for seed storage.The main in vitro collection of Kazakhstan is kept at the IPBB at + 4°С and +10 ± 2°С; it includes more than 140 accessions of various fruit, berry, nut, vegetable, ornamental, and woody crops, grapes, potatoes, etc. A cryogenic collection of seeds, shoot tips, dormant buds, and embryonic axes in liquid nitrogen (–196°C) is found only in IPBB. The collection includes more than 700 accessions.

Abstract 245: Development of a novel urine cell-free DNA preservation method for molecular profiling in genitourinary tumors

Abstract Introduction: Due to the benefits of its non-invasive testing, liquid biopsy technology has been applied therapeutically as an adjuvant to disease detection and monitoring in areas such as prenatal cell-free DNA (cfDNA) screening and cancer. Although the majority of current liquid biopsy techniques focus on DNA from blood, DNA from urine also conveys important genetic information from the human body. Studies have revealed that urine cfDNA can potentially be utilized for bladder cancer detection and monitoring. Urine samples can be quickly and painlessly collected; however, because urine DNA is readily damaged, maintaining high-quality cfDNA in urine is challenging. In this study, we developed a robust and user-friendly urine preservation method to maintain integrity of cfDNA in urine and to facilitate the transportation of urinary samples to clinical laboratories. Methods: Urine samples from 10 donors were collected with Predicine’s proprietary urine preservation method. Two urine samples were spiked with BL21(DE3) E.coli competent cells to simulate the urinary tract infection. All samples were incubated at 4°C, room temperature, and 37°C to mimic extreme storage or transporting environments. cfDNA was extracted after incubation of 0, 4, and 7 days and measured by Qubit and Fragment analyzer. Extracted DNA was also quantified by ddPCR with two markers: EGFR as an endogenous DNA marker and ARv7 (synthetic oligos containing intron-deleted sequence, post-incubation spike-in) as an inner control for the extraction and detection assays. Whole-genome sequencing was conducted to measure the quality of the cfDNA and bacterial contamination. Other commercial urine preserve buffers are used as control. Results: cfDNA derived from urine stored in Predicine’s proprietary urine preservation buffer remained stable till day 7 at different temperatures. There was no significant change in normalized copies of EGFR in the treated urine samples for up to 7 days. cfDNA yield was considerably higher in the novel preservation buffer than that using commercial buffers. Furthermore, the mapping rates in bacteria-contaminated samples were significantly improved. Conclusion: Predicine’s proprietary urine preservation method demonstrated excellent urine cfDNA preserving capacity at various temperatures for at least 7 days, outperforming commercially available preserve buffers in terms of DNA integrity and yield. The innovative technique offers an easy-to-use solution for obtaining high-quality urine cfDNA for clinical use. Citation Format: jie zhang, fang liu, min wang, cuishan deng, wei mo, seng tao, amy wang, shidong jia, zhixin zhao, pan du, binggang xiang, shujun luo. Development of a novel urine cell-free DNA preservation method for molecular profiling in genitourinary tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 245.

Oral metagenomes from Native American Ancestors reveal distinct microbial lineages in the pre-contact era.

Limited studies have focused on how European contact and colonialism impacted Native American oral microbiomes, specifically, the diversity of commensal or opportunistically pathogenic oral microbes, which may be associated with oral diseases. Here, we studied the oral microbiomes of pre-contact Wichita Ancestors, in partnership with the Descendant community, The Wichita and Affiliated Tribes, Oklahoma, USA. Skeletal remains of 28 Wichita Ancestors from 20 archeological sites (dating approximately to 1250-1450 CE) were paleopathologically assessed for presence of dental calculus and oral disease. DNA was extracted from calculus, and partial uracil deglycosylase-treated double-stranded DNA libraries were shotgun-sequenced using Illumina technology. DNA preservation was assessed, the microbial community was taxonomically profiled, and phylogenomic analyzes were conducted. Paleopathological analysis revealed signs of oral diseases such as caries and periodontitis. Calculus samples from 26 Ancestors yielded oral microbiomes with minimal extraneous contamination. Anaerolineaceae bacterium oral taxon 439 was found to be the most abundant bacterial species. Several Ancestors showed high abundance of bacteria typically associated with periodontitis such as Tannerella forsythia and Treponema denticola. Phylogenomic analyzes of Anaerolineaceae bacterium oral taxon 439 and T. forsythia revealed biogeographic structuring; strains present in the Wichita Ancestors clustered with strains from other pre-contact Native Americans and were distinct from European and/or post-contact American strains. We present the largest oral metagenome dataset from a pre-contact Native American population and demonstrate the presence of distinct lineages of oral microbes specific to the pre-contact Americas.

Role of L-Type Calcium Channels in the Increased Fatigue of Rat Soleus Muscle under Functional Unloading

Excessive long-term accumulation of calcium ions in the myoplasm of skeletal muscles can negatively affect mitochondria and lead to muscle dysfunction. The aim of our study was to identify the role of L-type calcium channels in the development of increased fatigue rat soleus muscle under functional unloading. Young male Wistar rats were divided into three groups of 8 animals each: the vivarium control group (C), the group subjected to hind limb unloading for 7 days (7HS) and the group with 7 days of hindlimb unloading with daily intraperitoneal injections of nifedipine (7 mg/kg body weight). The administration of nifidipine during hindlimb unloading prevented the upregulation of calcium-dependent phosphorylation of calcium-calmodulin kinase II (CaMK II), prevented the increase in fatigue and contributed to the preservation of mitochondrial proteins, DNA and mRNA expression of a number of genes that regulate mitochondrial biogenesis.