DNA Ratio Research Articles

The International Society for Biological and Environmental Repositories Presents Abstracts from Its Annual Meeting

The International Society for Biological and Environmental Repositories Presents Abstracts from Its Annual Meeting

Biologic profile to predict overall survival in non-metastatic breast cancer: A latent class analysis on aging biomarkers.

12037 Background: The aging process is heterogeneous and the result of different biological mechanisms. Blood biomarkers involved in the aging process, aside from comprehensive geriatric assessment, may help clinicians when facing equivocal therapeutic decisions, for example adjuvant treatment at high risk of toxicities. Methods: This study was conducted on a previously published cohort (PMID 25989735) including 162 patients aged ≥70 years (“Old”) and 82 patients aged ≤60 years (“Young”) with newly diagnosed non-metastatic breast cancer. Serum samples collected from all patients at breast cancer diagnosis, before any treatment were available from the extensive Leuven blood biobank. Overall survival was evaluated with October 7, 2022 as data cutoff. A panel of blood aging biomarkers attributed to inflammation: IL6, MCP1, RANTES, telomere shortening: telomeric DNA ratio T/S, telomeric dysfunction: CRAMP, chitinase activity, and anabolism : IGF1. Considering a high risk of collinearity, we performed a latent class analysis (LCA) on the whole cohort, to delineate the respective roles of inflammaging, replicative senescence and sarcopenia on patient clusters. Results: In univariate analysis were significantly associated with a poorer survival: high MCP1, hazard ratio (HR) = 1.57 (95%CI 1.32-1.87), high chitinase activity, HR = 1.33 (95%CI 1.12-1.57), and low IGF1, HR = 1.76 (95%CI 1.47-2.11). The biomarkers correlation matrix identified a significant correlation of Chitinase activity with MCP1, IGF1, CRAMP; of CRAMP with IL6; and IGF1 with T/S ratio. LCA identified 2 biologic profiles within the population, representing 137 (56.1%, class 1) and 107 (43,9%, class 2) patients. Class 1 was associated with a significantly poorer overall survival: HR = 3.52 (95%CI 2.28-5.45), and age-adjusted HR = 1.77 (95%CI 1.12-2.80). The best predictors for class 1 membership were low IGF1 (89%), high MCP1 (82%), high chitinase activity (79%), high IL6 (69%). Class 1 represented 77% of the “Old” and 15% of the “Young” populations. Conclusions: Our results delineate an unfavorable biologic profile that transcends chronological age and significantly predicts a poorer overall survival, underpinned by markers of both inflammaging, telomeric dysfunction and sarcopenia. The strong correlation between markers argues for a unique underlying biological pathway. Noteworthy, high IGF1, usually considered as a risk factor for breast cancer progression, was associated with a better prognosis. Low IGF1, high chitinase activity and high MCP1 appear as promising predictive biomarkers.

DNA Materials Assembled from One DNA Strand.

Due to the specific base-pairing recognition, clear nanostructure, programmable sequence and responsiveness of the DNA molecule, DNA materials have attracted extensive attention and been widely used in controlled release, drug delivery and tissue engineering. Generally, the strategies for preparing DNA materials are based on the assembly of multiple DNA strands. The construction of DNA materials using only one DNA strand can not only save time and cost, but also avoid defects in final assemblies generated by the inaccuracy of DNA ratios, which potentially promote the large-scale production and practical application of DNA materials. In order to use one DNA strand to form assemblies, the sequences have to be palindromes with lengths that need to be controlled carefully. In this review, we introduced the development of DNA assembly and mainly summarized current reported materials formed by one DNA strand. We also discussed the principle for the construction of DNA materials using one DNA strand.

Maternal Western-style diet in nonhuman primates leads to offspring islet adaptations including altered gene expression and insulin hypersecretion.

Maternal overnutrition is associated with increased susceptibility to type 2 diabetes in the offspring. Rodent models have shown that maternal overnutrition influences islet function in offspring. To determine whether maternal Western-style diet (WSD) alters prejuvenile islet function in a model that approximates that of human offspring, we utilized a well-characterized Japanese macaque model. We compared islet function from offspring exposed to WSD throughout pregnancy and lactation and weaned to WSD (WSD/WSD) compared with islets from offspring exposed only to postweaning WSD (CD/WSD) at 1 yr of age. WSD/WSD offspring islets showed increased basal insulin secretion and an exaggerated increase in glucose-stimulated insulin secretion, as assessed by dynamic ex vivo perifusion assays, relative to CD/WSD-exposed offspring. We probed potential mechanisms underlying insulin hypersecretion using transmission electron microscopy to evaluate β-cell ultrastructure, qRT-PCR to quantify candidate gene expression, and Seahorse assay to assess mitochondrial function. Insulin granule density, mitochondrial density, and mitochondrial DNA ratio were similar between groups. However, islets from WSD/WSD male and female offspring had increased expression of transcripts known to facilitate stimulus-secretion coupling and changes in the expression of cell stress genes. Seahorse assay revealed increased spare respiratory capacity in islets from WSD/WSD male offspring. Overall, these results show that maternal WSD feeding confers changes to genes governing insulin secretory coupling and results in insulin hypersecretion as early as the postweaning period. The results suggest a maternal diet leads to early adaptation and developmental programming in offspring islet genes that may underlie future β-cell dysfunction.NEW & NOTEWORTHY Programed adaptations in islets in response to maternal WSD exposure may alter β-cell response to metabolic stress in offspring. We show that islets from maternal WSD-exposed offspring hypersecrete insulin, possibly due to increased components of stimulus-secretion coupling. These findings suggest that islet hyperfunction is programed by maternal diet, and changes can be detected as early as the postweaning period in nonhuman primate offspring.

Identification and characterization of novel DIP-STRs from whole-genome sequencing data.

In an attempt to enhance forensic DNA mixture deconvolution several alternative DNA typing approaches have been developed. Among these, DIP-STR compound markers can resolve extremely unbalanced two-source DNA mixtures of same-or-opposite sex donors, up to a 1:1000 minor:major DNA ratio. A forensic set of 10 markers was validated for casework and a larger set of 23 DIP-STRs has proven suitable to biogeographic ancestry inference and for prenatal paternity testing. Yet, to promote the widespread use of this original approach, more markers and multiplex panels need to be developed. To this end, here we describe an extended set of forensic DIP-STRs identified using currently available whole-genome sequencing datasets. Complete lists of Indels and STRs were obtained from reported frequencies of genetic variants of 76,156 genomes. About 3000 identified DIP-STRs candidates were shorter than 200bp and 500 showed high haplotype variability estimated using the genotypes of individuals homozygous for the DIP or the STR. Here, we present 23 additional DIP-STRs validated for sensitivity, specificity and Swiss population variability. Finally, a set of 30 markers comprising seven previously validated ones is proposed for the prospective development of a forensic DIP-STR multiplex panel.

A duplex tetra-primer ARMS-PCR assay to discriminate three species of the Schistosoma haematobium group: Schistosoma curassoni, S. bovis, S. haematobium and their hybrids

BackgroundThe use of applications involving single nucleotide polymorphisms (SNPs) has greatly increased since the beginning of the 2000s, with the number of associated techniques expanding rapidly in the field of molecular research. Tetra-primer amplification refractory mutation system—PCR (T-ARMS-PCR) is one such technique involving SNP genotyping. It has the advantage of amplifying multiple alleles in a single reaction with the inclusion of an internal molecular control. We report here the development of a rapid, reliable and cost-effective duplex T-ARMS-PCR assay to distinguish between three Schistosoma species, namely Schistosoma haematobium (human parasite), Schistosoma bovis and Schistosoma curassoni (animal parasites), and their hybrids. This technique will facilitate studies of population genetics and the evolution of introgression events.MethodsDuring the development of the technique we focused on one of the five inter-species internal transcribed spacer (ITS) SNPs and one of the inter-species 18S SNPs which, when combined, discriminate between all three Schistosoma species and their hybrid forms. We designed T-ARMS-PCR primers to amplify amplicons of specific lengths for each species, which in turn can then be visualized on an electrophoresis gel. This was further tested using laboratory and field-collected adult worms and field-collected larval stages (miracidia) from Spain, Egypt, Mali, Senegal and Ivory Coast. The combined duplex T-ARMS-PCR and ITS + 18S primer set was then used to differentiate the three species in a single reaction.ResultsThe T-ARMS-PCR assay was able to detect DNA from both species being analysed at the maximum and minimum levels in the DNA ratios (95/5) tested. The duplex T-ARMS-PCR assay was also able to detect all hybrids tested and was validated by sequencing the ITS and the 18S amplicons of 148 of the field samples included in the study.ConclusionsThe duplex tetra-primer ARMS-PCR assay described here can be applied to differentiate between Schistosoma species and their hybrid forms that infect humans and animals, thereby providing a method to investigate the epidemiology of these species in endemic areas. The addition of several markers in a single reaction saves considerable time and is of long-standing interest for investigating genetic populations.Graphical

MP30-06 A UNIQUE EPIGENETIC-BASED TEST TO ACCURATELY PREDICT SUCCESSFUL TESE AND mTESE OUTCOMES

You have accessJournal of UrologyCME1 Apr 2023MP30-06 A UNIQUE EPIGENETIC-BASED TEST TO ACCURATELY PREDICT SUCCESSFUL TESE AND mTESE OUTCOMES Tim Jenkins, Ryan Barney, Kelaney Stalker, Ryan Miller, Kristin Brogaard, Jordan Kassab, and Larry Lipshultz Tim JenkinsTim Jenkins More articles by this author , Ryan BarneyRyan Barney More articles by this author , Kelaney StalkerKelaney Stalker More articles by this author , Ryan MillerRyan Miller More articles by this author , Kristin BrogaardKristin Brogaard More articles by this author , Jordan KassabJordan Kassab More articles by this author , and Larry LipshultzLarry Lipshultz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Affecting 1% of men, non-obstructive azoospermia (NOA) is the absence of sperm in the ejaculate, and is the most severe form of male infertility. Currently, men with NOA have decreased chances of conceiving a child of their own. Despite recent progress in reproductive medicine, men with NOA have essentially one invasive and expensive procedure to isolate sperm (microdissection testicular sperm extraction or mTESE). Unfortunately, mTESE has a ∼50% failure rate. Here we present the use of an innovative DNA methylation-based technology to non-invasively predict the presence of sperm in the testicle of men with NOA. METHODS: At a single academic center biopsies (N=18) and azoospermic semen samples (N=5) were procured from consented men who had undergone a testicular aspirate, biopsy, or mTESE procedure. Utilizing new epigenetic technology, the biopsies and semen samples were analyzed for sperm-specific DNA. Specifically, these samples were analyzed for a sequence of DNA that can distinguish sperm cells from all other somatic cells via a unique methylation pattern. The quantity of sperm-specific DNA can be used to determine the absence (men with <0.25 ratio of sperm to somatic DNA) or presence (>0.5 ratio of sperm to somatic DNA) of sperm in the testicles. These ratios were defined for each sample and compared to clinical outcomes of the TESA, TESE or mTESE procedures. RESULTS: Out of the 18 biopsies, 13 had >0.5 ratios of sperm DNA to somatic cell DNA in the testicular biopsies. All of these men were found to have sperm in their biopsies as defined from the clinical notes. 3 of the 18 men had <0.25 ratios of sperm DNA to somatic cell DNA. Again, consistent with previous analysis, none of these men had sperm in their biopsies. The remaining 2 men had ratios between 0.26 – 0.49, with one having sperm in his biopsy and one without sperm. Of the 5 azoospermic semen samples with associated TESA results, 3 has ratios of <0.25 and all 3 had no sperm in the corresponding TESA samples. 2 semen samples had ratios of >0.5 and were confirmed to have sperm in the TESA sample. CONCLUSIONS: In the analysis of testicular biopsies and semen samples, the sperm-specific DNA methylation ratios of <0.25% or >0.5% were 100% predictive of either the absence or presence of sperm in the testicle. With these data the next steps of our work will focus on continued analysis of sperm-specific cfDNA in seminal plasma to predict the presence or absence of sperm prior to a testicular extraction procedure. The ultimate goal will be to develop a non-invasive and cost-effective way to avoid unnecessary surgical procedures. Source of Funding: Venture Capital © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e393 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tim Jenkins More articles by this author Ryan Barney More articles by this author Kelaney Stalker More articles by this author Ryan Miller More articles by this author Kristin Brogaard More articles by this author Jordan Kassab More articles by this author Larry Lipshultz More articles by this author Expand All Advertisement PDF downloadLoading ...

WITHDRAWN: Mitochondrial replication, transcription, and function in obstructive sleep apnea

We assessed mitochondrial replication, transcription, and function in the upper airways of obstructive sleep apnea (OSA) patients and the effects of uvulopalatopharyngoplasty. Twenty subjects with mild and 40 with moderate to severe OSA requiring uvulopalatopharyngoplasty were included. Mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in uvula specimens were assessed by immunohistochemical staining, and their mRNA and protein expression was examined using reverse-transcription polymerase chain reaction and western blotting, respectively. The mitochondrial to nuclear DNA (Mt/N) ratio in the blood, exhaled breath condensate (EBC), and uvula was measured using quantitative reverse-transcription polymerase chain reaction. TFAM and PGC-1α protein concentrations in the plasma and EBC were determined using enzyme-linked immunosorbent assay. All tested parameters were higher in the OSA group than in the control. Three months later, 21 uvulopalatopharyngoplasty-responsive patients with OSA showed decreased TFAM and PGC-1α concentrations and EBC Mt/N ratio while these remained high in 19 uvulopalatopharyngoplasty-unresponsive patients. The OSA group showed severe inflammation, increased mitochondrial replication and transcription-related signaling, and mitochondrial dysfunction in the uvula. Successful OSA treatment using uvulopalatopharyngoplasty restored the TFAM and PGC-1α levels and EBC Mt/N ratio.

Response to Verticillium dahliae infection in a genetically diverse set of olive cultivars

Understanding the interaction between the soil-borne fungal pathogen Verticillium dahliae and olive is one of the major concerns for breeders and plant pathologists due to the wide variability in the disease response observed among cultivars. In this work, 40 representative olive cultivars of The World Olive Germplasm Bank of Cordoba (WOGBC, Spain) and six bred selections of IFAPA breeding program have been evaluated for the infection of the defoliating pathotype of V. dahliae under homogeneous conditions. The disease response has been characterized considering different traits of the disease development. Traits such as the relative area under disease progression curve (RAUDPC), the slope of the disease progression curve, the final dead plants and the ratio DNAV.dahliae/DNAolive showed a positive correlation with the susceptible genotypes, whereas the incubation period (IP), the final asymptomatic plants and the survival time were positively correlated to the resistant ones. Nonetheless, the DNA ratio detected in infected plants was highly variable. The clustering analysis considering all these parameters divided the genotypes in resistant and susceptible clusters. Cultivars like ‘Abbadi Abou Gabra’, ‘Abou Salt Mohazam’, ‘Chemlal del Kabylie’, ‘Jabali’, ‘Llumeta’, ‘Majhol-152′, ‘Mastoidis’, ‘Menya’, and ‘Temprano’ resulted extremely susceptible whereas ‘Frantoio’, ‘FrxAr_5′, ‘FrxAr_6′, and ‘KorOp_48′. resulted highly resistant. This study highlights the variability to the Verticillium wilt response of the most representative cultivars of the WOGBC and the relevance of breeding programs developing new cultivars with improved resistance characteristics.

RNA outperforms DNA-based metabarcoding in assessing the diversity and response of microeukaryotes to environmental variables in the Arctic Ocean

The Arctic Ocean (AO) has a harsh environment characterized by low temperatures, extensive ice coverage, and periodic freezing and melting of sea ice, which has provided diverse habitats for microorganisms. Prior studies primarily focused on microeukaryote communities in the upper water or sea ice based on environmental DNA, leaving the composition of active microeukaryotes in the diverse AO environments largely unknown. This study provided a vertical assessment of microeukaryote communities in the AO from snow and ice to sea water at a depth of 1670 m using high-throughput sequencing of co-extracted DNA and RNA. RNA extracts depicted microeukaryote community structure and intergroup correlations more accurately and responded more sensitively to environmental conditions than those derived from DNA. Using RNA:DNA ratios as a proxy for relative activity of major taxonomic groups, the metabolic activities of major microeukaryote groups were determined along depth. Analysis of co-occurrence networks showed that parasitism between Syndiniales and dinoflagellates/ciliates in the deep ocean may be significant. This study increased our knowledge of the diversity of active microeukaryote communities and highlighted the importance of using RNA-based sequencing over DNA-based sequencing to examine the relationship between microeukaryote assemblages and the responses of microeukaryotes to environmental variables in the AO.

Rapid Development of Cell State Identification Circuits with Poly-Transfection.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines. Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upontraditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

Environmental nucleic acids (eDNA and eRNA) metabarcoding analyses have attracted considerable research attention as they non-invasively and cost-effectively monitor ecosystems. For heretofore unknown reasons, however, they often detect false positives. Here, we focused on domestic wastewater as a possible source of false positives and characterized fish nucleic acids in the influent and effluent of a wastewater treatment plant (WWTP). The metabarcoding analysis revealed that DNA and RNA detected in the influent wastewater originated from over 120 fish species, and most of these were consumed fish. The nucleic acid content was significantly higher in the influent than in the effluent, and the copy numbers of DNA in the influent were significantly higher than those of RNA. These findings were corroborated by the current literature and indicated that eRNA could effectively mitigate false positives. The present study demonstrated that rivers may be extensively contaminated by fish nucleic acids, and WWTPs substantially alter the quantities and ratios of fish DNA and RNA. Therefore, analyses of the wastewater near sampling points and coverage of the sewage system enable us to select and use the appropriate nucleic acid type (DNA or RNA) for metabarcoding analysis and improve the accuracy of fish species detection and identification.

Relationship of Protein Synthesis to mRNA Levels in the Muscle of Chicks under Various Nutritional Conditions

The relationship between the rate of protein synthesis and the level of messenger RNA (mRNA) in the breast muscle (pectoralis major) was studied to examine the mechanisms regulating muscle protein synthesis in chicks under various nutritional conditions. The fractional synthesis rate (FSR) of protein was measured by a large-dose injection of L-[4-3H]phenylalanine. Poly(U)-Sepharose 4B affinity chromatography was used to extract Poly(A)+ RNA as mRNA. The first study examined the effect of dietary protein levels (0–60%) on protein synthesis rate and the contents of mRNA and total RNA. The fractional synthesis rate increased with increasing dietary protein levels from 0 to 20%. When chicks were fed high protein diets (40 and 60% of dietary protein), both the FSR and absolute synthesis rate (ASR) of protein were reduced compared with the 20% protein group. Multiple regression analysis indicated that for graded dietary protein levels below the dietary protein requirement, the response of muscle protein synthesis is largely regulated by variation in the ASR per unit RNA (the efficiency of RNA in synthesizing protein). At dietary protein levels above 20%, synthesis rates are also related to changes in the RNA:mRNA ratio (ribosome number relative to mRNA), the mRNA:DNA ratio (mRNA availability) and the DNA:protein ratio (DNA concentration). When chicks were food deprived for 2 d, FSR was reduced to about half that of well-fed controls. Upon refeeding of these chicks with the control diet, the FSR returned to the normal level within 0.5 h. When food-deprived chicks were refed with protein, carbohydrate or fat alone, the FSR returned to the control level within 0.5 h after consumption of each nutrient. The increase in muscle protein synthesis of unfed chicks following refeeding of various nutrients was explained by an increased ASR per unit RNA. Our results suggest that nutrient intake can change muscle protein synthesis rates in chicks largely by altering the ASR per unit RNA, in some cases associated also with changes in the levels of RNA, mRNA and DNA.

Are bipolar disorder, major depression, and suicidality linked with Toxoplasma gondii? A seromolecular case-control study

ABSTRACT Objective The existence of predisposing effects of latent Toxoplasma gondii (T. gondii) infection in bipolar disorder (BD), major depression (MD), and even suicide attempt (SA) has long been debatable. This conjecture remains unclear because there is a lack of evidence regarding how T. gondii manipulates the brain and behavior. Methods We investigated the influence of T. gondii infection on BD and MD patients with or without SA compared to age-, sex-, and province-matched healthy controls (HCs) concurrently with serology and molecular-based evaluations. We prospectively assessed 147 volunteers with BD, 161 with MD, and 310 HCs. Results T. gondii seropositivity rates were 57.1% for BD, 29.2% for MD, 64.8% for SA, and 21.3% for HC. Binary logistic regression analyses revealed that T. gondii positive Immunoglobulin G (IgG) status may be a prominent tendentious agent for BD (OR = 3.52; 95% CI [2.19–5.80]; p < 0.001) and SA (OR = 17.17; 95% CI [8.12–36.28]; p < 0.001), but not for MD (OR = 1.21; 95% CI [0.74–1.99]; p = 0.45). Nevertheless, the T. gondii DNA ratios determined by polymerase chain reaction (PCR) were linked to BD and MD. Conclusion Our findings strongly support the burgeoning interest in the possibility that latent T. gondii infection may be relevant to the etiology of BD and SA, although this connection remains ambiguous.

A glimpse into the trophic ecology of deep-water sharks in an important crustacean fishing ground.

Deep-water sharks are among the most vulnerable deep-water taxa because of their extremely conservative life-history strategies (i.e., late maturation, slow growth, and reproductive rates), yet little is known about their biology and ecology. Thus, this study aimed at investigating the trophic ecology of five deep-water shark species, the birdbeak dogfish (Deania calcea), the arrowhead (D. profundorum), the smooth lanternshark (Etmopterus pusillus), the blackmouth catshark (Galeus melastomus) and the knifetooth dogfish (Scymnodon ringens) sampled onboard a crustacean bottom-trawler off the south-west coast of Portugal. We combined carbon and nitrogen stable isotopes with RNA and DNA (RD) ratios to investigate the main groups of prey assimilated by these species and their nutritional condition, respectively. Stable isotopes revealed overall small interspecific variability in the contribution of different taxonomic groups to sharks' tissues, as well as in the origin of their prey. S. ringens presented higher δ15 N and δ13 C values than the other species, suggesting reliance on bathyal cephalopods, crustaceans and teleosts; the remaining species likely assimilated bathy-mesopelagic prey. The RD ratios indicated that most of the individuals had an overall adequate nutritional condition and had recently eaten. This information, combined with the fact that stable isotopes indicate that sharks assimilated prey from the local or nearby food webs (including commercially important shrimps), suggests a potential overlap between this fishing area and their foraging grounds, which requires further attention.

Developing a male-specific age predictive model based on Y-CpGs for forensic analysis

In forensic work, predicting the age of the criminal suspect or victim could provide beneficial clues for investigation. Epigenetic age estimation based on age-correlated DNA methylation has been one of the most widely studied methods of age estimation. However, almost all available epigenetic age prediction models are based on autosomal CpGs, which are only applicable to single-source DNA samples. In this study, we screened the available methylation data sets to identify loci with potential to meet the objectives of this study and then established a male-specific age prediction model based on 2 SNaPshot systems that contain 13 Y-CpGs and the mean absolute deviation (MAD) values were 4–6 years. The multiplex methylation SNaPshot systems and age-predictive model have been validated for sensitivity (the DNA input could be as low as 0.5 ng) and male specificity. They are supposed to have feasibility in forensic practice. In addition, it demonstrated that the method was also applicable to bloodstains, which were commonly found at crime scenes. The results showed good performance (the training set: R2 = 0.9341, MAD = 4.65 years; the test set: R2 = 0.8952, MAD = 5.73 years) in case investigation for predicting male age. For mixtures, when the male to female DNA ratio is 1:1, 1:10, the deviation between the actual age and the predicted age obtained by the model was less than 8 years, which offers great hope for future prediction of the age of males in mixtures and will be a powerful tool for special cases, such as sexual assault. Furthermore, the work provides a basis for the application of Y-CpGs in forensic science.

Examining the Cross-sectional Association Between Neighborhood Conditions, Discrimination, and Telomere Length in a Predominantly African American Sample.

Disproportionate exposure to adverse neighborhood conditions and greater discrimination may contribute to health disparities among African Americans (AAs). We examined whether adverse neighborhood conditions, alone or in conjunction with discrimination, associate with shorter leukocyte telomere length among a predominantly AA cohort. The sample included 200 residents from two low-income neighborhoods (96% AA; mean age = 67years). Perceived neighborhood conditions and discrimination were surveyed in 2018, and objective neighborhood conditions (total crime rate, neighborhood walkability, ambient air pollution (PM2.5, black carbon)) were collected in 2017/2018. Relative telomere length (T/S; ratio of telomeric DNA to a single-gene copy) was assessed from blood samples. Linear regression models estimated the main effects of each neighborhood condition and discrimination and their interactions on the T/S ratio. Less walkable neighborhoods were associated with shorter telomeres. Higher air pollution (PM2.5) was associated with shorter telomeres among those experiencing greater discrimination. Findings highlight the importance of understanding the intersecting influences of historic and contemporary sources of systemic racism and how they contribute to accelerated aging among adults.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.

Preterm infants at low risk for early-onset sepsis differ in early fecal microbiome assembly

ABSTRACT Antibiotics are administered near-universally to very low birth weight (VLBW) infants after birth for suspected early-onset sepsis (EOS). We previously identified a phenotypic group of VLBW infants, referred to as low-risk for EOS (LRE), whose risk of EOS is low enough to avoid routine antibiotic initiation. In this cohort study, we compared 18 such infants with 30 infants categorized as non-LRE to determine if the lower risk of pathogen transmission at birth is accompanied by differences in microbiome acquisition and development. We did shotgun metagenomic sequencing of 361 fecal samples obtained serially. LRE infants had a higher human-to-bacterial DNA ratio than non-LRE infants in fecal samples on days 1–3 after birth, confirming lower bacterial acquisition among LRE infants. The microbial diversity and composition in samples from days 4–7 differed between the groups with a predominance of Staphylococcus epidermidis in LRE infants and Enterobacteriaceae sp. in non-LRE infants. Compositional differences were congruent with the distribution of virulence factors and antibiotic resistant genes. After the first week, the overall composition was similar, but changes in relative abundance for several taxa with increasing age differed between groups. Of the nine late-onset bacteremia episodes, eight occurred in non-LRE infants. Species isolated from the blood culture was detected in the pre-antibiotic fecal samples of the infant for all episodes, though these species were also found in infants without bacteremia. In conclusion, LRE infants present a distinct pattern of microbiome development that is aligned with their low risk for EOS. Further investigation to determine the impact of these differences on later outcomes such as late-onset bacteremia is warranted.

Early warning of Diaporthe infection in kiwifruit soft rot by plasmonic dimer-enhanced Raman spectroscopy.

Diaporthe genus is the dominant pathogens of kiwifruit soft rot with long incubation period and rapid onset and very hard to detect in advance. It is of great significance to develop point-of-care tests for disease prevention and field management. Here we screen a pair of genus-level primers for constructing plasmonic dimer Rayleigh/Raman spectroscopy, which enables rapid, specific, and sensitive detection of Diaporthe genus in real kiwifruits. Dark-filed Rayleigh scattering clearly visualizes the target-induced dimer assembly of nanoprobes. Plasmonic dimer-enhanced Raman spectroscopy has high specificity for 12 common isolates of Diaporthe genus in one batch samples with highly accurate results and much higher sensitivity than polymerase chain reaction (PCR). It realizes the recognition of Diaporthe infection at an early stage of 24h when the kiwifruits do not have any noticeable symptoms. It demonstrates a bright prospect of point-of-care tests for early warning of kiwifruit soft rot infection and quality control.