DNA Content Research Articles

Vgll2 as an integrative regulator of mitochondrial function and contractility specific to skeletal muscle.

During skeletal muscle adaptation to physiological or pathophysiological signals, contractile apparatus and mitochondrial function are coordinated to alter muscle fiber type. Although recent studies have identified various factors involved in modifying contractile proteins and mitochondrial function, the molecular mechanisms coordinating contractile and metabolic functions during muscle fiber transition are not fully understood. Using a gene-deficient mouse approach, our previous studies uncovered that vestigial-like family member 2 (Vgll2), a skeletal muscle-specific transcription cofactor activated by exercise, is essential for fast-to-slow adaptation of skeletal muscle. The current study provides evidence that Vgll2 plays a role in increasing muscle mitochondrial mass and oxidative capacity. Transgenic Vgll2 overexpression in mice altered muscle fiber composition toward the slow type and enhanced exercise endurance, which contradicted the outcomes observed with Vgll2 deficiency. Vgll2 expression was positively correlated with the expression of genes related to mitochondrial function in skeletal muscle, mitochondrial DNA content, and protein abundance of oxidative phosphorylation complexes. Additionally, Vgll2 overexpression significantly increased the maximal respiration of isolated muscle fibers and enhanced the suppressive effects of endurance training on weight gain. Notably, no additional alteration in expression of myosin heavy chain genes was observed after exercise, suggesting that Vgll2 plays a direct role in regulating mitochondrial function, independent of its effect on contractile components. The observed increase in exercise endurance and metabolic efficiency may be attributed to the acute upregulation of genes promoting fatty acid utilization as a direct consequence of Vgll2 activation facilitated by endurance exercise. Thus, the current study establishes that Vgll2 is an integrative regulator of mitochondrial function and contractility in skeletal muscle.

Dietary supplementation with N-acetylcysteine confers a protective effect on muscle and liver in lipopolysaccharide-challenged piglets.

N-acetylcysteine (NAC) is a well-established antioxidant that offers exciting opportunities for intestinal health in weaned piglets, while the effects of NAC on muscle and liver has not been fully characterized. Therefore, the present study was performed to investigate the effects of dietary supplementation with NAC on muscle and liver in weaned piglets challenged with lipopolysaccharide (LPS). Twenty-four piglets (24-day-old) were randomly assigned to three treatment groups, the piglets in the control (CTR) and LPS- challenged (LPS) groups were fed the basal diet and those in the LPS+ NAC group was fed the basal diet supplemented with 500 mg/kg NAC. The animal trial lasted for 21 days. At the end of the trial, piglets in the LPS and LPS+ NAC groups were injected intraperitoneally with LPS (100 μg/kg body weight) and piglets in the CTR group were administrated with an equal volume of normal saline. 3 h later, the blood was collected and tissue samples were obtained after 6 h of LPS or normal saline treatment. The results showed that the level of IL-1β, and the mRNA levels of C-X-C motif chemokine receptor 3 (CXCR3) and interferon-γ (IFN-γ) in the liver were up-regulated, and the mRNA levels of insulin-like growth factor 1 (IGF-1), total glutathione (T-GSH), and the ratio of total protein to DNA in the liver were decreased under LPS challenge (P < 0.05). At the same time, LPS increased the level of H2O2 and decreased the content of T-GSH and DNA in the longissimus dorsi and gastrocnemius muscles (P < 0.05). In addition, the percentage of monocytes and the level of epidermal growth factor (EGF) were down-regulated in the LPS treatment (P < 0.05). Interestingly, dietary NAC supplementation reversed the above changes induced by LPS (P < 0.05). Furthermore, NAC might alleviate the muscle and liver injury in LPS-challenged piglets by regulating the expression of genes related to the type I interferon signaling pathway, as well as hypoxia inducible factor 1 (HIF1) and nuclear factor erythroid-2 related factor 2 (Nrf-2). Our findings suggested that dietary supplementation with NAC could benefit the health of muscle and liver in LPS-challenged weaned piglets.

Preventive effects of dill oil on potassium bromated-induced oxidative DNA damage in garlic root tips

In this study, the potential antimutagenic/cytoprotective effect of the oil extract (2.5µg/ml) from Anethum graveolens L. (Dill) on oxidative DNA damage was tested. The experiments were carried out to inhibit mutagenicity induced by potassium bromated (KBrO3-9g/l) in Allium assay using mitotic activities and nuclear DNA contents. Result showed a clear reduction in radical length in all KBrO3-treated groups compared to control plants. Mitotic activity, measured by mitotic index (MI) and mitotic stages, were significantly decreased in meristematic cells after KBrO3 application, The results showed that mitotic index (MI) was decreased to reach about 0.8 at 24 hrs compared with 23.9% in untreated plants. The analysis of potential cyto and genoprotective of dill extract on nuclear DNA contents showed a statistically significant increase in the nuclear DNA content compared to KBrO3-treated roots was observed. Clearly, dill oil showed highly performance in protecting the mitotic cells against mutagenic KBrO3 effects by increasing the MI and decreasing the abnormal chromosomes. Also, the percentage of damage reduction was observed in the pre- (28.9, 57.9 and 46.4 %), simultaneous (53.0, 70.6 and 35.7 %) and post- (37.3, 61.8 and 50 %) treatment. The results of the qualitative study showed that this dill extracts were rich in chemical compounds belonging to several phytochemical classes. GC-MS analysis detected 29 chemical compounds with different concentrations. The predominant ones were Octadecanoic acid (31.75%) and 2-pentanone, 4-hydroxy-4-methyl (16.77). So that these experiments will help to understand and uncover the mechanism of action dill oil against the mutagenic effects of KBrO3 (i.e whether they have a prophylactic or treatment activity).

Molecular age prediction using skull bone samples from individuals with and without signs of decomposition: a multivariate approach combining analysis of posttranslational protein modifications and DNA methylation.

The prediction of the chronological age of a deceased individual at time of death can provide important information in case of unidentified bodies. The methodological possibilities in these cases depend on the availability of tissues, whereby bones are preserved for a long time due to their mineralization under normal environmental conditions. Age-dependent changes in DNA methylation (DNAm) as well as the accumulation of pentosidine (Pen) and D-aspartic acid (D-Asp) could be useful molecular markers for age prediction. A combination of such molecular clocks into one age prediction model seems favorable to minimize inter- and intra-individual variation. We therefore developed (I) age prediction models based on the three molecular clocks, (II) examined the improvement of age prediction by combination, and (III) investigated if samples with signs of decomposition can also be examined using these three molecular clocks. Skull bone from deceased individuals was collected to obtain a training dataset (n = 86), and two independent test sets (without signs of decomposition: n = 44, with signs of decomposition: n = 48). DNAm of 6 CpG sites in ELOVL2, KLF14, PDE4C, RPA2, TRIM59 and ZYG11A was analyzed using massive parallel sequencing (MPS). The D-Asp and Pen contents were analyzed by high performance liquid chromatography (HPLC). Age prediction models based on ridge regression were developed resulting in mean absolute errors (MAEs)/root mean square errors (RMSE) of 5.5years /6.6 years (DNAm), 7.7 years /9.3 years (Pen) and 11.7 years /14.6 years (D-Asp) in the test set. Unsurprisingly, a general lower accuracy for the DNAm, D-Asp, and Pen models was observed in samples from decomposed bodies (MAE: 7.4-11.8 years, RMSE: 10.4-15.4 years). This reduced accuracy could be caused by multiple factors with different impact on each molecular clock. To acknowledge general changes due to decomposition, a pilot model for a possible age prediction based on the decomposed samples as training set improved the accuracy evaluated by leave-one-out-cross validation (MAE: 6.6-12 years, RMSE: 8.1-15.9 years). The combination of all three molecular age clocks did reveal comparable MAE and RMSE results to the pure analysis of the DNA methylation for the test set without signs of decomposition. However, an improvement by the combination of all three clocks was possible for the decomposed samples, reducing especially the deviation in case of outliers in samples with very high decomposition and low DNA content. The results demonstrate the general potential in a combined analysis of different molecular clocks in specific cases.

Physioxia rewires mitochondrial complex composition to protect stem cell viability

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O2) or physioxic (5 % O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition.

Enhanced visualization of nuclear staining and cell cycle analysis for the human commensal Malassezia

Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.

MTOR potentiates senescent phenotypes and primary cilia formation after cisplatin-induced G2 arrest in retinal pigment epithelial cells

Cisplatin, a platinum-based anticancer drug, is used to treat several types of cancer. Despite its effectiveness, cisplatin-induced side effects have often been reported. Although cisplatin-induced toxicities, such as apoptosis and/or necrosis, have been well studied, the fate of cells after exposure to sublethal doses of cisplatin needs further elucidation. Treatment with a sublethal dose of cisplatin induced cell cycle arrest at the G2 phase in retinal pigment epithelial cells. Following cisplatin withdrawal, the cells irreversibly exited the cell cycle and became senescent. Notably, the progression from the G2 to the G1 phase occurred without mitotic entry, a phenomenon referred to as mitotic bypass, resulting in the accumulation of cells containing 4N DNA content. Cisplatin-exposed cells exhibited morphological changes associated with senescence, including an enlarged size of cell and nucleus and increased granularity. In addition, the senescent cells possessed primary cilia and persistent DNA lesions. Senescence induced by transient exposure to cisplatin involves mTOR activation. Although transient co-exposure with an mTORC1 inhibitor rapamycin did not prevent mitotic bypass and entry into senescence, it delayed the progression of senescence and attenuated senescent phenotypes, resulting in shorter primary cilia formation. Conclusively, cisplatin induces senescence in retinal pigment epithelial cells by promoting mTOR activation.

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

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

Knockdown of Fzr inhibited the growth of Nilaparvata lugens by blocking endocycle.

The endocycle can generate cells referred to as 'polyploid'. Fizzy-related protein (Fzr) plays an important role in driving the mitosis-to-endocycle transition. The brown planthopper (BPH), Nilaparvata lugens (Stål), a serious insect pest, feeds exclusively on rice. However, polyploidy and its regulatory mechanisms are poorly understood in BPH. Here, we found that the ploidy levels of follicles H (FH) and accessory gland (AG) significantly increased with BPH age when examining the polyploidy of FH and AG of salivary glands. Fzr was identified as an important regulator for polyploidy in BPH salivary gland. Knockdown of Fzr resulted in a decrease in cell size and DNA content in nymph salivary glands. Fzr knockdown transcriptionally upregulated cyclin-dependent kinase 1 (CDK1), CDK2, cyclin A (CycA) and CycB, and downregulated CycD, CycE, Myc and mini-chromosome maintenance protein 2-7 (MCM2-7). Phenotypically, Fzr knockdown significantly suppressed salivary protein production, feeding and survival in BPH nymphs. Our results show that BPH salivary glands exhibit obvious polyploidy, and Fzr positively regulates the endocycle in nymph salivary gland. These findings provide clues for the study of the regulatory mechanisms of insect polyploidy. © 2024 Society of Chemical Industry.

Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry

Abstract Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.

Satellite DNAs and the evolution of the multiple X1X2Y sex chromosomes in the wolf fish Hoplias malabaricus (Teleostei; Characiformes)

Multiple sex chromosomes usually arise from chromosomal rearrangements which involve ancestral sex chromosomes. There is a fundamental condition to be met for their long-term fixation: the meiosis must function, leading to the stability of the emerged system, mainly concerning the segregation of the sex multivalent. Here, we sought to analyze the degree of differentiation and meiotic pairing properties in the selected fish multiple sex chromosome system present in the wolf-fish Hoplias malabaricus (HMA). This species complex encompasses seven known karyotype forms (karyomorphs) where the karyomorph C (HMA-C) exhibits a nascent XY sex chromosomes from which the multiple X1X2Y system evolved in karyomorph HMA-D via a Y-autosome fusion. We combined genomic and cytogenetic approaches to analyze the satellite DNA (satDNA) content in the genome of HMA-D karyomorph and to investigate its potential contribution to X1X2Y sex chromosome differentiation. We revealed 56 satDNA monomers of which the majority was AT-rich and with repeat units longer than 100 bp. Seven out of 18 satDNA families chosen for chromosomal mapping by fluorescence in situ hybridization (FISH) formed detectable accumulation in at least one of the three sex chromosomes (X1, X2 and neo-Y). Nine satDNA monomers showed only two hybridization signals limited to HMA-D autosomes, and the two remaining ones provided no visible FISH signals. Out of seven satDNAs located on the HMA-D sex chromosomes, five mapped also to XY chromosomes of HMA-C. We showed that after the autosome-Y fusion event, the neo-Y chromosome has not substantially accumulated or eliminated satDNA sequences except for minor changes in the centromere-proximal region. Finally, based on the obtained FISHpatterns, we speculate on the possible contribution of satDNA to sex trivalent pairing and segregation.

Identification and culture of meniscons, meniscus cells with their pericellular matrix

Background aimsMeniscus injury is highly debilitating and often results in osteoarthritis. Treatment is generally symptomatic; no regenerative treatments are available. “Chondrons,” articular chondrocytes with preserved pericellular matrix, produce more hyaline cartilage extracellular matrix and improve cartilage repair. If meniscons exist in the meniscus and have similar therapeutic potential as chondrons, employing these cells has potential for meniscus cell therapy and tissue engineering. In this study, we isolated and cultured “meniscons,” meniscus cells surrounded by their native pericellular matrix, and investigated cell behavior in culture compared with chondrons. MethodsHuman meniscons were enzymatically isolated from osteoarthritic menisci and cultured up to 28 days in fibrin glue. Freshly isolated meniscons and chondrons were analyzed by histology and transmission electron microscopy. We used 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride labeling and type VI collagen immunohistochemistry to image pericellular matrix after 0 and 28 days of culture. Gene expression was quantified using real-time polymerase chain reaction and DNA content and proteoglycan production were analyzed using biochemical assays. ResultsMeniscons were successfully isolated from human meniscus tissue. The pericellular matrix of meniscons and chondrons was preserved during 28 days of culture. Meniscons and chondrons had similar cell proliferation and proteoglycan production. Meniscons and chondrons expressed similar levels of collagen type I alpha 1 chain, whereas collagen type II alpha 1 chain and aggrecan expression was lower in the meniscon population. ConclusionsFreshly isolated meniscons and meniscons cultured for 28 days share similarities with chondrons with regard to cell proliferation, morphology and biochemical activity. Rapid isolation of meniscons (45 min) demonstrates potential for one-stage meniscus regeneration and repair, which should be confirmed in vivo.

Proposal of Lactobacillus amylovorus subsp. animalis subsp. nov. and an emended description of Lactobacillus amylovorus.

Seven novel lactic acid bacterial strains (BF125T, BF186, TKL145, YK3, YK6, YK10 and NSK) were isolated from the fresh faeces of Japanese black beef cattle and weanling piglets, spent mushroom substrates, or steeping water of a corn starch production plant. These strains are rod-shaped, Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, cytochrome oxidase-negative, facultatively anaerobic, and homofermentative. Strain BF125T did not produce any gas from glucose; both d- and l-lactate were produced as end-products of glucose (D/L, 40 : 60). Growth occurred at 30-45 °C (optimum, 37 °C), pH 5.0-8.0 (optimum, pH 6.0), and with NaCl concentration of 1.0-3.0% (w/v). The G+C content of genomic DNA of strain BF125T was 37.8 mol% (whole-genome analysis). The major fatty acids were C16 : 0, C18 : 1 ω9c, C19 cyclopropane 9, 10, and summed feature 10. The 16S rRNA gene in strain BF125T showed high similarity to that of the type strain of Lactobacillus amylovorus (99.93%), and the other isolates were also identified as L. amylovorus based on these similarities. A phylogenetic tree based on the core genomes of L. amylovorus strains (n=54), including the seven isolates, showed that they could be divided into two clusters. Strains YK3, YK6, YK10, and NSK were in the first cluster, along with the type strain DSM 20531T, while the second cluster included isolates BF125T, BF186, TKL145, and other strains isolated from various animal origins. Phenotypic differences in fermentability were observed for lactose, salicin, and gentiobiose between these two groups. The intergroup digital DNA-DNA hybridization values (72.9-78.6%) and intergroup average nucleotide identity values (95.64-96.92%) were comparable to values calculated using datasets of other valid subspecies of the genus (ex-) Lactobacillus. In light of the physiological, genotypic, and phylogenetic evidence, we propose a novel subspecies of L. amylovorus, named Lactobacillus amylovorus subsp. animalis subsp. nov. (type strain BF125T=MAFF 212522T=DSM 115528T). Our findings also led to the automatic creation of Lactobacillus amylovorus subsp. amylovorus subsp. nov. and an emended description of the species L. amylovorus.

Characterization of pediatric porcine pulmonary valves as a model for tissue engineered heart valves

Heart valve tissue engineering holds the potential to transform the surgical management of congenital heart defects affecting the pediatric pulmonary valve (PV) by offering a viable valve replacement. While aiming to recapitulate the native valve, the minimum requirement for tissue engineered heart valves (TEHVs) has historically been adequate mechanical function at implantation. However, long-term in situ functionality of TEHVs remains elusive, suggesting that a closer approximation of the native valve is required. The realization of biomimetic engineered pediatric PV is impeded by insufficient characterization of healthy pediatric tissue. In this study, we comprehensively characterized the planar biaxial tensile behaviour, extracellular matrix (ECM) composition and organization, and valvular interstitial cell (VIC) phenotypes of PVs from piglets to provide benchmarks for TEHVs. The piglet PV possessed an anisotropic and non-linear tension-strain profile from which material constants for a predictive constitutive model were derived. The ECM of the piglet PV possessed a trilayer organization populated by collagen, glycosaminoglycans, and elastin. Biochemical quantification of ECM content normalized to wet weight and DNA content of PV tissue revealed homogeneous distribution across sampled regions of the leaflet. Finally, VICs in the piglet PV were primarily quiescent vimentin-expressing fibroblasts, with a small proportion of activated α-smooth muscle actin-expressing myofibroblasts. Overall, piglet PV properties were consistent with those reported anecdotally for pediatric human PVs and distinct from those of adult porcine and human PVs, supporting the utility of the properties determined here to inform the design of tissue engineered pediatric PVs. Statement of significanceHeart valve tissue engineering has the potential to transform treatment for children born with defective pulmonary valves by providing living replacement tissue that can grow with the child. The design of tissue engineered heart valves is best informed by native valve properties, but native pediatric pulmonary valves have not been fully described to date. Here, we provide comprehensive characterization of the planar biaxial tensile behaviour, extracellular matrix composition and organization, and valvular interstitial cell phenotypes of pulmonary valves from piglets as a model for the native human pediatric valve. Together, these findings provide standards that inform engineered heart valve design towards generation of biomimetic pediatric pulmonary valves.

Comparison of the trophic sources and pathways of mesozooplankton and ichthyoplankton in the Kuroshio current and its neighboring waters

Commercially important fish spend their vulnerable early life stages in the Kuroshio Current, resulting in high fishery production even in the vicinity of poor prey availability under oligotrophic conditions. Nevertheless, little information is available on how ichthyoplankton are supported by trophodynamics in complicated food webs. Here, we have explored trophic sources and pathways toward ichthyoplankton in the Kuroshio and its neighboring waters based on metabarcoding analysis of gut DNA content for major taxonomic groups of mesozooplankton and ichthyoplankton. Calanoids were found to be the most predominant and frequently appearing prey, whereas non-crustaceans were the secondary prey for most mesozooplankton and ichthyoplankton groups. Trophic networks based on gut DNA content demonstrated that calanoids were the most important sector with multiple linkages among their prey and predators, and gelatinous and non-crustacean mesozooplankton were the secondary sectors. These findings suggest that calanoids are important hubs of trophic pathways toward ichthyoplankton, and that gelatinous and non-crustacean mesozooplankton groups strengthen trophic relationships with multiple components. Contrary to general thought, our metabarcoding analysis has revealed that trophodynamics toward ichthyoplankton are not strongly dependent on the grazing food chain, but are supported by multiple trophic pathways in the Kuroshio and its neighboring waters.

FLOW CYTOMETRY ANALYSIS REVEALS NUCLEAR DNA CONTENT VARIATION IN PHALAENOPSIS YOUNG LEAF AND ROOT TIP CELLS

FLOW CYTOMETRY ANALYSIS REVEALS NUCLEAR DNA CONTENT VARIATION IN PHALAENOPSIS YOUNG LEAF AND ROOT TIP CELLS

Rapid detection of murine-derived ingredients in meat products using real-time enzymatic recombinase amplification.

Accurate identification of meat species is critical to prevent economic fraud and safeguard public health. The use of inappropriate meat sources, such as murine, poses significant health risks because of potential contamination with pathogens and allergens, leading to foodborne illnesses. The present study aimed to develop a novel real-time enzymatic recombinase amplification (ERA) method for the rapid and specific detection of murine DNA in meat products. A novel ERA primer and probe set was designed, targeting a murine-specific single-copy nuclear gene identified through bioinformatics analysis. The assay demonstrates high specificity, showing no amplification in commonly consumed meats, other animals or major crops. Additionally, it exhibits remarkable sensitivity, detecting as few as five copies of murine genomic DNA. For practical application, the ERA method could effectively identify mouse DNA in laboratory-prepared samples at concentrations as low as 0.5% and also quantify samples with mouse DNA content as low as 5%. It also accurately detects the presence of murine-derived ingredients in commercially available meat products. The detection process is straightforward, utilizing a simple isothermal device for incubation, blue light excitation and a smartphone camera for result interpretation. This rapid analysis can be completed within 20 min. The newly developed real-time ERA method provides a valuable tool for standardizing meat trade practices, promoting food safety and enhancing consumer confidence in the authenticity of meat products. © 2024 Society of Chemical Industry.

Genetic Characteristics of Echinococcus granulosus from Fixed Paraffin-Embedded Tissue Samples in Human Isolates Based on the High-Resolution Melting Point Analysis in Sabzevar, Northeast Iran.

There are ten genotypes of Echinococcus granulosus with different intermediate and final hosts affecting the parasite's life cycle and its transmission to humans. Therefore, this study was conducted to determine the genotype of isolated hydatid cysts using the simple and fast high-resolution melting point analysis (HRM) method. The paraffin tissue samples of patients who underwent surgery were obtained from the pathology sample bank of Vasei and Emdad Hospitals in Sabzevar, Iran during 2010-2020. The DNA content of the samples was extracted after collecting and determining the characteristics using the DNA extraction kit. PCR was performed on the samples and the presence of the hydatid cyst genome was confirmed using the special Master Kit. Mix PCR of Solis Biodyne Company and Real-Time device (Bio-Rad) were used, and the genetic identity of hydatid cysts were determined. Out of 33 paraffin samples, 21 samples contained hydatid cyst DNA, two of which were from the brain and 19 from the liver tissues; 12 samples did not contain hydatid cyst DNAs. All liver samples were from sheep species (G1), and the brain samples were from buffalo species (G3). Therefore, 9.53% of the Echinococcus species collected were buffalo (G3), and 90.47% were sheep (G1) strain. Based on previous patterns, HRM methods can be used for easy and quick identification of Echinococcus strains. The G1 strain was the dominant strain causing hydatid cyst in different human organs, including the liver and brain.

Single cell, Label free Characterisation of Human Mesenchymal Stromal cell Stemness and Future Growth Potential by Autofluorescence Multispectral Imaging.

To use autofluorescence multispectral imaging (AFMI) to develop a non-invasive assay for the in-depth characterisation of human bone marrow derived mesenchymal stromal cells (hBM-MSCs). hBM-MSCs were imaged by AFMI on gridded dishes, stained for endpoints of interest (STRO-1 positivity, alkaline phosphatase, beta galactosidase, DNA content) then relocated and results correlated. Intensity, texture and morphological features were used to characterise the colour distribution of regions of interest, and canonical discriminant analysis was used to separate groups. Additionally, hBM-MSC lines were cultured to arrest, with AFMI images taken after each passage to investigate whether an assay could be developed for growth potential. STRO-1 positivity could be predicted with a receiver operator characteristic area under the curve (AUC) of 0.67. For spontaneous differentiation this was 0.66, for entry to the cell-cycle it was 0.77 and for senescence it was 0.77. Growth potential (population doublings remaining) was estimated with an RMSPE = 2.296. The Mean Absolute Error of the final prediction model indicated that growth potential could be predicted with an error of ± 1.86 doublings remaining. This non-invasive methodology enabled the in-depth characterisation of hBM-MSCs from a single assay. This approach is advantageous for clinical applications as well as research and stands out for the characterisation of both present status as well as future behaviour. The use of data from five MSC lines with heterogenous AFMI profiles supports potential generalisability.

Whole-genome sequencing and identification of antimicrobial peptide coding genes in parsley (Petroselinum crispum), an important culinary and medicinal Apiaceae species.

Parsley is a commonly cultivated Apiaceae species of culinary and medicinal importance. Parsley has several recognized health benefits and the species has been utilized in traditional medicine since ancient times. Although parsley is among the most commonly cultivated members of Apiaceae, no systematic genomic research has been conducted on parsley. In the present work, parsley genome was sequenced using the long-read HiFi (high fidelity) sequencing technology and a draft contig assembly of 1.57 Gb that represents 80.9% of the estimated genome size was produced. The assembly was highly repeat-rich with a repetitive DNA content of 81%. The assembly was phased into a primary and alternate assembly in order to minimize redundant contigs. Scaffolds were constructed with the primary assembly contigs, which were used for the identification of AMP (antimicrobial peptide) genes. Characteristic AMP domains and 3D structures were used to detect and verify antimicrobial peptides. As a result, 23 genes (PcAMP1-23) representing defensin, snakin, thionin, lipid transfer protein and vicilin-like AMP classes were identified. Bioinformatic analyses for the characterization of peptide physicochemical properties indicated that parsley AMPs are extracellular peptides, therefore, plausibly exert their antimicrobial effects through the most commonly described AMP action mechanism of membrane attack. AMPs are attracting increasing attention since they display their fast antimicrobial effects in small doses on both plant and animal pathogens with a significantly reduced risk of resistance development. Therefore, identification and characterization of AMPs is important for their incorporation into plant disease management protocols as well as medicinal research for the treatment of multi-drug resistant infections.