Search Results (68)

Functional flours, defined as flours enriched with health-promoting compounds such as phenolics, fibers, or proteins, are gaining attention as wheat-free alternatives due to the nutritional limitations of wheat flour. This study introduces a novel liquid chromatographic time-of-flight tandem mass spectrometric method (LC-QTOF-MS/MS) to characterize the phenolic profiles of functional flours from different origins and evaluate their potential as flour substitutes in food products. The proposed method was validated and the limits of quantification (LOQs) were calculated over the ranges 0.1–1.0 mg/kg. Calculated recoveries were as low as 82.4%. Repeatability and reproducibility were expressed as intra-day (n = 6) and inter-day (n = 4 × 3) measurements and were lower than 8.1 and 10.9%, respectively. Target and suspect screening findings underscore the potential of pulse flours as nutritionally enriched ingredients for functional food development. Full article

White spot syndrome virus (WSSV) has emerged as a significant threat to global shrimp aquaculture, causing economic losses because of its rapid spread and high mortality rates. This study aims to elucidate the genetic and evolutionary dynamics of WSSV through a comprehensive genome analysis. Utilizing 27 complete genome sequences sourced from public databases, this study investigates the genetic variability, potential recombination events, and evolutionary patterns of WSSV. Our results identified multiple genomic deletions, 14 novel single-nucleotide polymorphism sites, and variable number tandem repeats across different strains, underscoring the virus’s genetic diversity. A recombination event between freshwater and marine strains highlights a complex transmission pathway, potentially facilitated by aquaculture practices. A phylogenetic tree constructed using ancestral genes suggests that WSSV originated in Southeast Asia and subsequently globally spread, influenced by both natural and anthropogenic factors. Genomic shrinkage of the virus occurred in time series, while the host’s viral infection induced transposon transposition and insertion into the earlier virus genome to provide a basis for genomic shrinkage. Our research emphasizes the importance of advanced molecular characterization and evolutionary models of the virus in understanding the spread of viral pathogens in aquaculture environments. Full article

Dimorphism of sex chromosomes often leads to a need for dosage compensation. In eutherian mammals, XIST, a long non-coding RNA, is expressed from the X chromosome that will be silenced, triggering X-chromosome inactivation (XCI). XIST originated from the ancestral protein-coding Lnx3 gene with contributions from various mobile elements that contributed to the striking domains of tandem repeats within the first and sixth exons. Modular domains of XIST are now involved in recruiting heterochromatic marks and proteins essential for XCI initiation and maintenance. This review presents a comparative analysis of human XIST with five other eutherian mammals—chimpanzees, cats, pigs, sheep, and mice—examining conservation across exons as well as the tandem repeats. Notably, repeats exhibited higher conservation than exons, underscoring their functional importance. Additionally, a species-specific G repeat, previously described in pigs, was also identified in sheep and cats. These findings provide insights into the domains of XIST, a cis-acting silencer that has been used to proposed to alleviate the impact of a supernumerary chromosome in Down syndrome. Full article

Backgrounds: Adamantinomatous craniopharyngiomas (ACPs) are benign intracranial tumors that behave aggressively due to their location, infiltration of the surrounding nervous tissue and high capacity for recurrence. In this study, we aimed to construct ACP primary cell models for further investigation of tumorigenic and recurrent mechanisms. Methods: Primary cells were isolated from primary (one case) and recurrent (one case) ACP. Short tandem repeat (STR) analysis was used to clarify the identity of the ACP primary cells we isolated. Whole exome sequencing (WES), immunofluorescence (IF) and immunohistochemistry (IHC) were performed on primary cells and corresponding ACP tissues, to determine the mutational profile and to clarify the tissue origin and phenotypic of primary cells. Transcriptome RNA-seq was performed to obtain the gene expression characteristics of ACP primary cells. Subsequently, a heterotopic ACP xenograft mouse model was established to confirm the tumorigenesis capacity of ACP primary cells. Results: ACP primary cells were successfully cultured. The genetic variants were similar to the original tumor tissue, and they owned expression of cancer-associated fibroblast (CAF) markers (FSP1/S100A4, Vimentin) and nuclear translocation β-catenin. Meanwhile, they had an high level expression of extracellular matrix components (Fibronectin). The tumor formation ability of ACP primary cells was verified. The transcriptional signatures of ACP primary cells were also explored. Conclusions: We successfully isolated and characterized ACP primary cells that acquired multiple CAF features and demonstrated stable propagation through dozens of passages. These PDC models laid the foundation for further research on ACP. Full article

(1) Background: Fetal chromosomal examination is a critical component of modern prenatal testing. Traditionally, maternal serum biomarkers such as free β-human chorionic gonadotropin (Free β-HCG) and pregnancy-associated plasma protein A (PAPPA) have been employed for screening, achieving a detection rate of approximately 90% for fetuses with Down syndrome, albeit with a false positive rate of 5%. While amniocentesis remains the gold standard for the prenatal diagnosis of chromosomal abnormalities, including Down syndrome and Edwards syndrome, its invasive nature carries a significant risk of complications, such as infection, preterm labor, or miscarriage, occurring at a rate of 7 per 1000 procedures. Beyond Down syndrome and Edwards syndrome, other chromosomal abnormalities, such as trisomy of chromosomes 9, 16, or Barr bodies, pose additional diagnostic challenges. Non-invasive prenatal testing (NIPT) has emerged as a powerful alternative for fetal genetic screening by leveraging maternal blood sampling. However, due to the extremely low abundance of fetal cells in maternal circulation, NIPT based on fetal cells faces substantial technical challenges. (2) Methods: Fetal nucleated red blood cells (FnRBCs) were first identified in maternal circulation in a landmark study published in The Lancet in 1959. Due to their fetal origin and presence in maternal peripheral blood, FnRBCs represent an ideal target for non-invasive prenatal testing (NIPT). In this study, we introduce a novel self-assembled cell array (SACA) chip system, a microfluidic-based platform designed to efficiently settle and align cells into a monolayer at the chip’s base within five minutes using lateral flow dynamics and gravity. This system is integrated with a fully automated, multi-channel fluorescence scanning module, enabling the real-time imaging and molecular profiling of fetal cells through fluorescence-tagged antibodies. By employing a combination of Hoechst+/CD71+/HbF+/CD45− markers, the platform achieves the precise enrichment and isolation of FnRBCs at the single-cell level from maternal peripheral blood. (3) Results: The SACA chip system effectively reduces the displacement of non-target cells by 31.2%, achieving a single-cell capture accuracy of 97.85%. This isolation and enrichment system for single cells is well suited for subsequent genetic analysis. Furthermore, the platform achieves a high purity of isolated cells, overcoming the concentration detection limit of short tandem repeat (STR) analysis, demonstrating its capability for reliable non-invasive prenatal testing. (4) Conclusions: This study demonstrates that the SACA chip, combined with an automated image positioning system, can efficiently isolate single fetal nucleated red blood cells (FnRBCs) from 50 million PBMCs in 2 mL of maternal blood, completing STR analysis within 120 min. With higher purification efficiency compared to existing NIPT methods, this platform shows great promise for prenatal diagnostics and potential applications in other clinical fields. Full article

The development of an effective and broadly protective influenza vaccine against circulating and emerging strains remains elusive. In this study, we evaluated a potentially universal influenza vaccine based on single-component self-assembling protein nanoparticles (1c-SApNPs) presenting the conserved matrix protein 2 ectodomain (M2e) from influenza A and B viruses (IAV and IBV, respectively). We previously designed a tandem antigen comprising three IAV M2e domains of human, avian/swine, and human/swine origins (termed M2ex3). The M2ex3-presenting 1c-SApNPs conferred complete protection in mice against sequential lethal challenges with H1N1 and H3N2. To broaden this protection to cover IBVs, we designed a series of antigens incorporating different arrangements of three IAV M2e domains and three copies of IBV M2e. Tandem repeats of IAV and IBV (termed influenza A-B) M2e arrayed on the I3-01v9a 60-mer 1c-SApNP, when formulated with an oil-in-water emulsion adjuvant, generated greater M2e-specific immunogenicity and protective efficacy than the soluble influenza A-B M2e trimer, indicated by higher survival rates and reduced weight loss post-challenge. Importantly, one of the influenza A-B M2e SApNP constructs elicited 100% protection against a lethal influenza A/Puerto Rico/8/1934 (H1N1) challenge in mice and 70% protection against a lethal influenza B/Florida/4/2006 (Yamagata lineage) challenge, the latter of which has not been reported in the literature to date. Our study thus provides a promising M2e-based single-component universal vaccine candidate against the two major types of influenza virus circulating in humans. Full article

The control region (CR) regulates the replication and transcription of the mitochondrial genome (mitogenome). Some avian mitogenomes possess two CRs, and the second control region (CR2) may enhance replication and transcription; however, the CR2 in lark mitogenome appears to be undergoing loss and is accompanied by tandem repeats. Here, we characterized six lark mitogenomes from Alaudala cheleensis, Eremophila alpestris, Alauda razae, and Calandrella cinerea and reconstructed the phylogeny of Passerida. Through further comparative analysis among larks, we traced the evolutionary process of CR2. The mitochondrial gene orders were conserved in all published lark mitogenomes, with Cytb-trnT-CR1-trnP-ND6-trnE-remnant CR2 with tandem repeat-trnF-rrnS. Phylogenetic analysis revealed Alaudidae and Panuridae are sister groups at the base of Sylvioidea, and sporadic losses of CR2 may occur in their common ancestor. CR sequence and phylogeny analysis indicated CR2 tandem repeats were generated within CR2, originating in the ancestor of all larks, rather than inherited from CR1. The secondary structure comparison of tandem repeat units within and between species suggested slipped-strand mispairing and DNA turnover as suitable models for explaining the origin and evolution of these repeats. This study reveals the evolutionary process of the CR2 containing tandem repeat in Alaudidae, providing reference for understanding the evolutionary characteristics and dynamics of tandem repeats. Full article

Ochrobactrum anthropi (O. anthropi) is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with low immunity. The identification of O. anthropi mainly uses biochemical methods, such as the API 20NE or Vitek-2. The typing studies of O. anthropi have mainly utilized PFGE, rep-PCR, AFLP, 16s rDNA sequencing, RecA-PCR RFLP, and MALDI-TOF MS. This study aims to evaluate the polymorphisms of variable-number tandem-repeats (VNTRs) within genomic DNA of O. anthropi strains. The tandem repeats (TRs) in genomic DNA are discovered using Tandem Repeat Finder software (version 4.09). Twelve different VNTRs are designated and assigned to the nomenclature. The primers for PCR of 12 loci are designed. The PCR product size is converted to the number of tandem repeats in every locus. The relatedness of 65 O. anthropi strains from geographically different countries are analyzed by means of 12-variable-number tandem-repeat analysis(MLVA-12). A total of 51 different genotypes are found in 65 O. anthropi strains. These strains, which were collected from the same environmental samples, hospitals, and countries, are clustered within the same or closely genotypes. The MLVA-12 assay has a good discriminatory power for species determination, typing of O. anthropi, and inferring the origin of bacteria. Full article

DNA replication is a fundamental process ensuring the maintenance of the genome each time cells divide. This is particularly relevant early in development when cells divide profusely, later giving rise to entire organs. Here, we analyze and compare the genome replication progression in human embryonic stem cells, induced pluripotent stem cells, and differentiated cells. Using single-cell microscopic approaches, we map the spatio-temporal genome replication as a function of chromatin marks/compaction level. Furthermore, we mapped the replication timing of subchromosomal tandem repeat regions and interspersed repeat sequence elements. Albeit the majority of these genomic repeats did not change their replication timing from pluripotent to differentiated cells, we found developmental changes in the replication timing of rDNA repeats. Comparing single-cell super-resolution microscopic data with data from genome-wide sequencing approaches showed comparable numbers of replicons and large overlap in origins numbers and genomic location among developmental states with a generally higher origin variability in pluripotent cells. Using ratiometric analysis of incorporated nucleotides normalized per replisome in single cells, we uncovered differences in fork speed throughout the S phase in pluripotent cells but not in somatic cells. Altogether, our data define similarities and differences on the replication program and characteristics in human cells at different developmental states. Full article

Pentatricopeptide repeat (PPR) proteins, with tandem 30–40 amino acids, were characterized as one kind of nucleus coding protein. They have been demonstrated to play important roles in RNA editing, plant growth and development, and plant immunity. Although the PPR gene family has been characterized in some plant species, less is known about this family in peanut, especially their functions in response to Ralstonia solanacearum. In this study, we performed a genome-wide analysis to identify PPR genes and their functions in resistance to R. solanacearum. Here, 389, 481, and 1079 PPR genes were identified from Arachis duranensis, Arachis ipaensis, and Arachis hypogaea, respectively. Allopolyploidization was the main reason for the increased number of the AhPPR members. Gene duplication brought about 367 pairs of homologous genes of PPRs in A. hypogaea. Whole-genome replication, tandem repeats, scattered repeats, and unconnected repeats constituted the replication types. The substitution rates of nonsynonymous (Ka) versus synonymous (Ks) of all homologous pairs were less than 1.0, suggesting that the homologous AhPPRs underwent intense purifying selection pressure and remained conserved in both structure and function. RNA-seq and RT-qPCR analyses showed that AhPPR598 gene was highly expressed in the aerial part of peanut and involved in response to R. solanacearum. The transient expression of AhPPR598 in Nicotiana benthamiana induced the HR-mediated cell death, up-regulated expression of resistant marker genes, and enhanced the resistance to R. solanacearum, suggesting AhPPR598 was a positive regulator of immunity by regulating the JA and SA pathways. These results provide a new understanding of the origin, distribution, and evolution of the AhPPR gene family and potential gene resources for peanut-resistant breeding. Full article

Yuca (Manihot esculenta esculenta; cassava, manioc) is a native Amazonian crop represented by myriad landraces. To investigate human influences on its diversification, we conducted field observations and analyzed 13 short tandem repeat (STR) loci in 43 landraces in the Peruvian Amazon. We found a different multilocus genotype (MLG) in every landrace. However, tests for Hardy–Weinberg equilibrium found a deficit of heterozygosity at every locus (p < 0.001 for 12 of 13 loci). Further, the fraction of genetic variance due to landrace differences was greater than expected (38.84%; p = 0.001). This suggested that landrace hybridization is restricted, a finding consistent with our field observations. However, we found an excess of within-landrace heterozygosity (p < 0.001) in 39 of 43 landraces, suggesting they originated through hybridization. Mantel tests identified associations between genetic and geographic distances (p < 0.001), but their correlation coefficients were low (Mantel’s r < 0.21). In addition, AMOVA analyses revealed that differences between landraces collected from five sampled rivers accounted for just 3.05% of observed genetic variance (p < 0.001). Neighbor joining and principal components analyses also revealed little evidence of differentiation between rivers. Finally, in a comparison with a secondary sample, we found that the closest relative of 27 of 28 specimens had a landrace name different from their own, suggesting that traditional nomenclature is a poor indicator of genetic relatedness. Full article

This study investigated the intra- and inter-herd diversity of Mycobacterium avium subsp. paratuberculosis (MAP) isolates from four goat herds in Thuringia (Germany) that were affected by paratuberculosis for several years. The main focus was on the characterization and distribution of genotypes among animals and the environment of goat herd 1. This study included 196 isolates from the feces of 121 infected goats, various tissues from 13 clinically diseased goats, 29 environmental samples from herd 1, and additionally, 22 isolates of different origin from herds 2 to 4. The isolates, sampled between 2018 and 2022, were genotyped using short-sequence-repeat (SSR) analysis, mycobacterial-interspersed repetitive units–variable-number tandem repeat (MIRU–VNTR) analysis, and a single nucleotide polymorphism (SNP)-based assay for phylogenetic grouping. All the isolates belonged to the MAP-C group. In herd 1, one predominant genotype was determined, while two other genotypes were identified very rarely and only in fecal and environmental samples. One of three further genotypes was found in each of herds 2 to 4. The assignment of genotypes to different phylogenetic clades suggested six different infection strains. The results indicated no epidemiological links between the examined herds. Based on the current MAP genotyping data from Germany, possible sources of infection are MAP-contaminated barns previously used by infected cattle and the purchase of sub-clinically infected goats. Full article

The increasing number of food frauds, mainly targeting high quality products, is a rising concern among producers and authorities appointed to food controls. Therefore, the development or implementation of methods to reveal frauds is desired. The genetic traceability of traditional or high-quality dairy products (i.e., products of protected designation of origin, PDO) represents a challenging issue due to the technical problems that arise. The aim of the study was to set up a genetic tool for the origin traceability of dairy products. We investigated the use of Short Tandem Repeats (STRs) to assign milk and cheese to the corresponding producer. Two farms were included in the study, and the blood of the cows, bulk milk, and derived cheese were sampled monthly for one year. Twenty STRs were selected and Polymerase Chain Reactions for each locus were carried out. The results showed that bulk milk and derived cheese express an STR profile composed of a subset of STRs of the lactating animals. A bioinformatics tool was used for the exclusion analysis. The study allowed the identification of a panel of 20 markers useful for the traceability of milk and cheeses, and its effectiveness in the traceability of dairy products obtained from small producers was demonstrated. Full article

Clear cell carcinomas of Müllerian origin have a strong female predominance and only extremely rarely will arise within the kidney, presumably due to ectopic Müllerian embryogenesis. Herein, we report a unique case of metastatic Müllerian type clear cell carcinoma in a 37-year-old patient who had previously received a transplanted kidney from his father at age 11 (due to severe bilateral vesicoureteral reflux) and remained on chronic immunosuppression. The tumor was highly aggressive and demonstrated somatic mutations in NF2 and SETD2. Imaging of the transplanted kidney did not reveal any clear evidence of malignancy. However, targeted multigene sequencing and short tandem repeat testing revealed that the cancer was of donor origin, presumably from ectopic Müllerian tissue transplanted to the patient along with the kidney graft. The tumor was resistant to first-line therapy with a triple combination of carboplatin plus paclitaxel plus bevacizumab, as well as to second-line immunotherapy with nivolumab plus ipilimumab after tapering down the patient’s immunosuppression. Despite the tumor being genetically distinct from the host, the use of immune checkpoint therapy with nivolumab plus ipilimumab did not yield a response. This unique case showcases the value of molecular testing in determining the tumor origin in patients with solid organ transplants who present with cancers of unknown primary. This can prompt the potential investigation of other recipients from the same donor. Full article

The enteropathogenic Yersinia genus is commonly detected in wildlife including wild boars. Difficulties in its cultivation may hamper subsequent epidemiological studies and outbreak investigations. Multiple-locus variable-number tandem repeat analysis (MLVA) of Yersinia (Y.) enterocolitica and Y. pseudotuberculosis has proven useful in source attribution and epidemiological studies but has hitherto relied on the analysis of isolates. In the present study, MLVA profiles generated from 254 isolates of Y. enterocolitica indicated similarities between human, pig and rodent isolates. Further, MLVA analyses of 13 Y. pseudotuberculosis pure-cultured isolates were compared to MLVA analyses performed directly on the 14 PCR-positive enrichment broths from which the isolates originated, which showed matching MLVA profiles. This indicates that MLVA analysis performed directly on enrichment broths could be a useful method for molecular epidemiological investigations. In addition, 10 out of 32 samples of wild boar minced meat obtained from private hunters and from approved wild-game-handling establishments were PCR-positive for the presence of Y. enterocolitica and may indicate a risk for public health. Full article