DNA Content Research Articles

Sperm Mitochondrial Content and Mitochondrial DNA to Nuclear DNA Ratio Are Associated with Body Mass Index and Progressive Motility.

Mitochondrial dysfunction is a risk factor in the pathogenesis of metabolic disorders. According to the energy requirements, oxidative phosphorylation and the electron transport chain work together to produce ATP in sufficient quantities in the mitochondria of eukaryotic cells. Abnormal mitochondrial activity causes fat accumulation and insulin resistance as cells require a balance between the production of ATP by oxidative phosphorylation (OXPHOS) in the mitochondria and the dissipation of the proton gradient to reduce damage from reactive oxygen species (ROS). This study aims to explore the relationship between the mitochondrial content of sperm and the ratio of mitochondrial DNA to nuclear DNA in relation to body mass index (BMI) and how it may affect the progressive motility of sperm cell. Understanding the relationships between these important variables will help us better understand the possible mechanisms that could connect sperm motility and quality to BMI, as well as further our understanding of male fertility and reproductive health. Data were collected from 100 men who underwent IVF/ICSI at the University Hospital of Ioannina's IVF Unit in the Obstetrics and Gynecology Department. The body mass index (BMI) of the males tested was used to classify them as normal weight; overweight; and obese. Evaluations included sperm morphology; sperm count; sperm motility; and participant history. In the group of men with normal BMI, both BMI and progressive motility displayed a statistically significant association (p < 0.05) with mitochondrial DNA content, relative mitochondrial DNA copy number, and the mtDNA/nDNA ratio. Similar to this, there was a positive association between BMI and motility in the groups of men who were overweight and obese, as well as between the expression of mitochondrial DNA and the mtDNA/nDNA ratio, with statistically significant differences (p < 0.05). There was not a statistically significant difference observed in the association between the relative mtDNA copy number and BMI or motility for the overweight group. Finally, the relative mtDNA copy number in the obese group was only associated with motility (p = 0.034) and not with BMI (p = 0.24). We found that in all three groups, BMI and progressive motility exhibited comparable relationships with mitochondrial DNA expression and the mtDNA/nDNA ratio. However, only in the normal group and in the obese group, the relative mitochondrial DNA copy number showed a positive association with BMI and progressive motility.

The Decellularized Calf Testis: Introducing Suitable Scaffolds for Spermatogenesis Studies.

Men's infertility and lack of production of healthy and active sperm are concerns of recent years in most countries. Studies on the preparation of extracellular matrix (ECM) from decellularization of testis tissue and spermatogenesis could provide proper results to solve some of the men's infertility problems. This study aims to decellularize calf testis by different methods to reach a suitable scaffold and introduce it in spermatogenesis studies. In this experimental study, calf testis were decellularized by a freeze-de freeze, 1% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), 0.1% SDS-vacuum, 1% SDS, 1% SDS-vacuum, and Triton- X100 methods. The content of DNA, collagen, and glycosaminoglycan (GAG) was analyzed using the kit and staining with Hematoxylin-Eosin, Masson's trichrome, Alcian blue, and Orcein methods. The morphology of the scaffolds was analyzed with a scanning electron microscope (SEM). Methods of 1% SDS, 1% SDS-vacuum, and 1% SD completely removed the cells. The preservation of collagen and GAG was confirmed using the staining kit and methods. The use of a vacuum showed greater porosity in the SEM images. Toxicity and hemolysis were not observed in the scaffolds. Testis decellularization with 1% SDS and 1% SD, in addition to cell removal, could maintain the ECM structure to a large extent without having cytotoxic and hemolysis effects.

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

PSIII-7 Effect of Time of Estrus on Conception Rate for a Sexed Semen Artificial Insemination Protocol in Multiparous Beef Cows

Abstract Sexed semen artificial insemination is a breeding technique that allows producers to shorten breeding seasons, optimize genetics through use of superior semen, create calves based on sex preference, and increase profitability. Sexed semen is a technology that sorts semen by differences in DNA content between X (female) and Y (male) bearing sperm. However, sexed semen practices have yet to be widely used in beef cows due to reduced sperm numbers, low conception rates, and increased costs. The objective of this study was to determine the effect of time of estrus upon fixed-time artificial insemination (FTAI) of sex-sorted semen on conception rate using two different estrous synchronization protocols. Multiparous commercial beef cows (n = 73) were randomized by age, body condition score (BCS), and days post-partum at the University of Arkansas Livestock and Forestry Research Station in Batesville. Both synchronization treatments (TRT) used a controlled internal drug releasing device (Eazi-breed CIDR; 1.38g progesterone) inserted intravaginally on d -14 of the protocol simultaneous to administration of prostaglandin F2α (PGF; 500µg cloprostenol sodium; Estrumate). In the first treatment (TRT 1; 7&amp;7 Synch), gonadotropin-releasing hormone (GnRH; 86µg gonadorelin; Fertagyl) was administered on d -7. In the second treatment (TRT 2; 8&amp;6 Synch), GnRH was administered on d -6. Both treatments were then administered PGF simultaneous to CIDR removal on d 0, 66 ± 2h before FTAI and administration of GnRH. Standing heat (estrus) was visually detected every 3 h for 30 min intervals between CIDR removal and FTAI. Heat detection patches were used in conjunction with visual detection of estrus. Time of estrus was signified as time of standing immobile when mounted in combination with presenting a patch score of 4 (&amp;gt;75% color removed). Time of estrus (TD) was deviated from time of FTAI and categorized into three equal time groups (Grp 1 = 0 to 11.0 h; Grp 2 = 11.01 to 22.0 h; and Grp 3 = 22.01 to 33.0 h). Conception rate (CR) was determined via ultrasound 37 ± 2 d after FTAI. Data were analyzed using the GLIMMIX procedure of SAS 9.4. CR did not differ by age of dam (P = 0.58), BCS (P = 0.48), or TRT (P = 0.83). However, TD had an effect on CR (P = 0.002); and resulted in CR of 45% (Grp 1), 87% (Grp 2), and 42% (Grp 3). In conclusion, these results indicate artificially inseminating cows with sexed semen between 11.01 and 22.0 h after time of standing estrus will increase pregnancy rates for multiparous commercial beef cattle.

Seasonal dynamics of soil microbial growth, respiration, biomass, and carbon use efficiency in temperate soils

Soil microbial growth, respiration, and carbon (C) use efficiency (CUE) are essential parameters to understand, describe and model the soil carbon cycle. While seasonal dynamics of microbial respiration are well studied, little is known about how microbial growth and CUE change over the course of a year, especially outside the plant growing season. In this study, we measured soil microbial respiration, gross growth via 18O incorporation into DNA, and biomass in an agricultural field and a deciduous forest 16 times over the course of two years. We sampled soils to a depth of 5 cm from plots at which harvest residues or leaf litter remained on the plot or was removed. We observed strong seasonal variations of microbial respiration, growth, and biomass. All these microbial parameters were significantly higher at the forest site, which contained 4.3 % organic C compared to the agricultural site with 0.9 % organic C. CUE also varied strongly (0.1 to 0.7) but was overall significantly higher at the agricultural site compared to the forest site. We found that microbial respiration and to a lesser extent microbial growth followed the seasonal dynamics of soil temperature. Microbial growth was further affected by the presence of plants in the agricultural system or foliage in the forest. At low temperatures in winter, both microbial respiration and gross growth showed the lowest rates, whereas CUE (calculated from both respiration and growth) showed amongst the highest values determined during the two years, due to the higher temperature sensitivity of microbial respiration. Microbial biomass C strongly increased in winter. Surprisingly, this winter peak was not connected to high microbial growth or an increase in DNA content. This suggests that microorganisms accumulated C and N, potentially in the form of osmo- or cryoprotectants or increased in cell size but did not divide. This microbial winter bloom and following decline, where C is released from microbial biomass and freely available, might constitute a highly dynamic time in the annual C cycle in temperate soil systems. Highly variable CUE, which was observed in our study, and the fact that CUE is calculated from independently controlled microbial respiration and microbial growth, ask for great caution when CUE is used to describe soil microbial physiology, soil C dynamics or C sequestration. Instead, microbial respiration, microbial growth, and microbial biomass C should be investigated individually in combination to better understand the soil C cycle.

252 Use of Agomir and Antagomir Technologies to Alter Mirna Expression and Muscle Hypertrophy in Intrauterine Growth Restricted Lambs

Abstract microRNAs (miRNA) are present in skeletal muscle and play important roles in regulating gene expression. Previous research identified miR-22-3p and miR-127 as differentially regulated during muscle hypertrophy and in vitro experiments found that antagomir-22-3p and mimic-127 enhanced satellite cell proliferation. The objective of this study was to test in vivo intramuscular injection of agomiR-127 or antagomir-22-3p in the longissimus muscle (LM) on miRNA and target mRNA expression in intrauterine growth restricted lambs. Pregnant ewes (n = 18) with twins were nutrient restricted (60%) from gestational d 85 to parturition. On d 2 of age, lambs were randomly selected and assigned to one of two miRNA treatments, 1) agomiR-127 (AGO127, 100 nM, n = 9) to assess gain of function or 2) antagomiR-22-3p (ANT22, 500 nM, n = 9) to assess loss of function. AgomiR and antagomiR technologies are chemically modified miRNA mimics or inhibitors that do not require transfection for in vivo use. AgomiR-127 or antagomir-22-3p were reconstituted in phosphate buffered saline (PBS) and injected into the center of the LM on the left side every 3-d starting at the 10th rib and moving posterior by 1.27 cm at each injection for a total of 7 injections. A sham control treatment (SHAM127 or SHAM22) of PBS was administered following the same protocol in the right LM to serve as the in-animal control. Lambs were harvested 4 d after the last injection and samples were collected from the injection region of each LM for miRNA and mRNA expression, histology, and proteomic analyses. Data were analyzed using paired t-tests to compare each miRNA treatment to its SHAM within animal. AGO127 up-regulated (P = 0.0071) miR-127 expression compared with SHAM127. mRNA expression of PAX7 and MSTN were up-regulated (P &amp;lt; 0.05) and IGFBP2 expression was down-regulated (P &amp;lt; 0.05) for AGO127 compared with SHAM127. Both protein and DNA content of AGO127 were increased (P &amp;lt; 0.05) but the ratio of protein to DNA did not change (P &amp;gt; 0.05). Proteomic analyses found 11 proteins that were differentially expressed for AGO127 compared with SHAM127. ANT22 down-regulated (P = 0.0024) miR-22-3p expression from SHAM22. Expression of predicted mRNA targets of miR-22-3p (ACVR2a, ACVR2b, SIRT1), myosin heavy chain isoforms (MyHCI, MyHCIIa, MyHCIIx), and other myogenic genes (MAPK6, IGF1, MyoG) were also altered (P &amp;lt; 0.05) in the LM compared with SHAM22. DNA content and the protein to DNA ratio was greater (P &amp;lt; 0.05) in ANT22 than SHAM22. Proteomic analyses identified nine proteins that were differentially expressed (P &amp;lt; 0.05) for ANT22. The direct injection of agomiR and antagomir technologies in the LM up- and down-regulated expression, respectively, for the miRNA of interest, which altered mRNA expression of predicted targets, protein abundance and muscle fiber type.

Informed interpretation of metagenomic data by StrainPhlAn enables strain retention analyses of the upper airway microbiome.

The usage of 16S rRNA gene sequencing has become the state-of-the-art method for the characterization of the microbiota in health and respiratory disease. The method is reliable for low biomass samples due to prior amplification of the 16S rRNA gene but has limitations as species and certainly strain identification is not possible. However, the usage of metagenomic tools for the analyses of microbiome data from low biomass samples is not straight forward, and careful optimization is needed. In this work, we show that by validating StrainPhlAn 3 results with the data from bacterial cultures, the strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA being present when parameters are carefully optimized to fit low biomass microbiomes. This work further proposes that strain retention analyses are feasible, at least for more abundant species. This will help to better understand the longitudinal dynamics of the upper respiratory microbiome during health and disease.

Description of Fusibacillus kribbianus gen. nov., sp. nov., a fusiform anaerobe isolated from pig feces

ObjectiveThe family Lachnospiraceae is affiliated with the order Clostridiales and was originally contained within Clostridial cluster XIVa. The members of Lachnospiraceae inhabiting the gut comprise the chemoorganotrophic genera, generating sundry short-chain fatty acids to supply energy to the host, and are considered to be related to obesity and gut health. MethodsThe polyphasic taxonomic approach was used to characterize the isolate YH-rum2234T. A detailed metabolic analysis was conducted to compare the novel isolate with related strains within the family Lachnospiraceae. ResultsA fusiform, obligately anaerobic, Gram-stain-negative bacterium, YH-rum2234T, was isolated from pig feces. Analysis of the 16S rRNA gene sequence revealed that the similarities between the isolate and the familiarly interrelated strain Lientehia hominis KCTC 25345T was 94.3%. The average nucleotide identities and genome-to-genome distances of YH-rum2234T and its closely related strains were below 85.5% and 32.5%, respectively. The G + C content of the genomic DNA was 49.2 mol%. The main fatty acids were C16:0, C14:0, and C14:0 DMA. The major polar lipids were aminophospholipids. The cell wall did not contain the peptidoglycan meso-diaminopimelic acid. ConclusionGiven the chemotaxonomic, phenotypic, and phylogenetic properties, YH-rum2234T (=KCTC 25710T = DSMZ 116041T) represents a new genus and species in the family Lachnospiraceae. Fusibacillus kribbianus gen. nov., sp. nov. is the proposed name.

Jiella pelagia sp. nov., isolated from the phosphonate-amended seawater of the northwestern Pacific Ocean.

A novel Gram-stain-negative, aerobic, rod-shaped bacterium, designated as HL-NP1T, was isolated from the surface water of the northwestern Pacific Ocean after enrichment cultivation using the organic phosphorous compound of 2-aminoethylphosphonate. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Jiella, with the highest similarity to Jiella pacifica 40Bstr34T (98.7 %). The complete genome sequence of strain HL-NP1T comprised a circular chromosome of 5.58 Mbp and two circular plasmids of 0.15 and 0.22 Mbp. Comparison of the genome sequences between strains HL-NP1T and J. pacifica 40Bstr34T revealed that average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values (88.0, 86.4 and 33.9 %, respectively) were below the recommended cut-off levels for delineating bacterial species. Strain HL-NP1T showed optimal growth at 30 °C, pH 6.5-7.0, with 2.0-2.5 % (w/v) NaCl. The sole respiratory quinone was ubiquinone-10. The predominant fatty acid was summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c). The polar lipids comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, an unidentified aminolipid and four unidentified lipids. The G+C content of the genomic DNA was 65.1 %. Based on phylogenetic, genotypic, phenotypic and chemotaxonomic data, strain HL-NP1T is proposed to represent a novel species of the genus Jiella, for which the name Jiella pelagia sp. nov. is proposed. The type strain is HL-NP1T (= KCCM 90499T = JCM 35838T).

Evaluation of Vascular Endothelial Growth Factor Gene Expression in Recellularized Liver Tissue by Mouse Embryo Fibroblast.

The aim of the present study was to evaluate alterations in the vegf gene expression as an angiogenic factor in mouse embryo fibroblasts seeded on the decellularized liver fragments. Liver tissue samples (n = 10) collected from adult male mice were randomly divided into decellularized and native control groups. Tissues were decellularized by treating with 1% Triton X-100 and 0.1% SDS for 24 hours and assessed by H&E staining and SEM. Then DNA content analysis and toxicity tests were performed. By centrifugation, DiI-labeled mouse embryo fibroblasts were seeded on each scaffold and cultured for one week. The recellularized scaffolds were studied by H&E staining, SEM, and LSCM. After RNA extraction and cDNA synthesis, the expression of the vegf gene in these samples was investigated using real-time RT-PCR. Our observations showed that the decellularized tissues had morphology and porous structure similar to the control group, and their DNA content significantly reduced (p < 0.05) and reached to 4.12% of the control group. The MTT test indicated no significant cellular toxicity for the decellularized scaffolds. Light microscopy, SEM, and LSCM observations confirmed the attachment and penetration of embryonic fibroblast cells on the surface and into different depths of the scaffolds. There was no statistically significant difference in terms of vegf gene expression in the cultured cells in the presence and absence of a scaffold. The reconstructed scaffold had no effect on vegf gene expression. Decellularized mouse liver tissue recellularized by embryonic fibroblasts could have an application in regenerative medicine.

Combination of upstream primer-multuplex PCR (UP-mpcr) and capillary electrophoresis for equine genetic analysis

The purity and content of DNA extracted from the sample is important during PCR analysis. In the conditions of our country, there are many cases of working on samples that do not meet the requirements for some reason. In such cases, there is a need to further test and develop other sensitive methods. The upstream-primer multiplex PCR (UP-mPCR technique) is known for its high specificity and fidelity, and has been used for detecting multiple food borne pathogens, meat species testing and detecting different genetically modified organism (GMO) insertions in a plant genome. The purpose of this experiment is to apply the UP-mPCR method on DNA samples that do not meet the quality requirements, and to test it on domestically produced diagnostics.We combined UP-mPCR with fragment analysis on DNA capillary electrophoresis genetic analyzer by applying fluorescent labelled upstream primers which were tested by amplifying 8 STRs on 23 low-quality equine gDNA samples. These samples had formerly undergone unsuccessful testing by domestic equine genotyping 15-plex kit. Single trial of UP-mPCR on the same samples showed successful amplification and detection of amplicons from 4-6 STRs, and their alleles were genotyped. Combining UP-mPCR and DNA capillary electrophoresis can be helpful in extreme situations such as having limited amounts of sample, or a shortage of multiple fluorescent dye oligonucleotides. There is no former report about the same method as combining UP-mPCR with fragment analysis.

Enhancing Ezetimibe Anticancer Activity Through Development of Drug Nano-Micelles Formulations: A Promising Strategy Supported by Molecular Docking.

Ezetimibe, initially recognized as a cholesterol-lowering agent, has recently attracted attention due to its potential anticancer properties. We aimed to explore an innovative approach of enhancing the drug anticancer activity through the development of drug nano-formulations. Fifteen different nano-micelles formulations were prepared utilizing D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and pluronic F127. The prepared formulations were characterized for size, polydispersity index (PDI), zeta potential, and entrapment efficiency (EE). The formulations were morphologically characterized using light and transmission electron microscopies and the drug-binding mode with the active site was investigated using the molecular docking. Cell viability against MCF-7 and T47D was studied. Apoptosis and cell cycle were assessed. The prepared formulations were in the nano-size range (34.01 ± 2.00-278.34 ± 9.11 nm), zeta potential values were very close to zero, and the TPGS-based micelles formulations showed the highest ezetimibe EE (94.03 ± 1.71%). Morphological study illustrated a well-defined, spherical nanoparticles with a uniform size distribution. Molecular docking demonstrated good interaction of ezetimibe with Interleukin-1 Beta Convertase through multiple hydrogen bonding, covalent bond, and hydrophobic interaction. TPGS-based nano-micelle formulation (F5) demonstrated the lowest IC50 against MCF-7 (4.51 µg/mL) and T47D (8.22 µg/mL) cancer cells. When T47D cells were treated with IC50 concentrations of F5, it exhibited significant inhibition with late apoptosis (43.9%), a response comparable to T47D cells treated with an IC50 dose of ezetimibe. Cell cycle analysis revealed that both ezetimibe and F5-treated T47D cells exhibited an increase in the subG1 phase, indicating reduced DNA content and cell death. These findings suggest that F5 could serve as a proficient drug delivery system in augmenting the cytotoxic activity of ezetimibe against breast cancer.

Changes in the behavior of Staphylococcus aureus strains in the presence of oxacillin under the effect of gamma radiation

Staphylococcus aureus (S. aureus) as a major pathogen is implicated in a wide range of foodborne and hospital-acquired infections, its methicillin resistant variants contribute to the spread of β-lactam antibiotic resistance. It is essentially important to destroy these pathogens, their resistance genes and the antibiotics in wastewaters. For this purpose reactions of reactive radicals (advanced oxidation processes), first of all hydroxyl radicals (•OH), are suggested. Here the radiolysis of water supplied these radicals. In the experiments B.01755 oxacillin sensitive and B.02174 resistant S. aureus strains were used to study their behaviorr in suspensions under the effect of irradiation in presence and absence of oxacillin. Oxacillin inactivation depended on concentration of the antibiotic used (0.042 and 1 g dm−3), higher concentration required a higher dose. When 106–109 CFU cm−3 S. aureus suspensions were irradiated with γ-radiation the bacteria were inactivated at low absorbed doses: 4 orders of magnitude decrease ocurred in the number of culturable cells at ∼0.6 kGy dose. Both cell membrane and DNA suffered considerable damages during irradiation. Due to the membrane damage the cells could not be stained, and the DNA content of cells in several days period was released into the solution. In DNA damage the oxacillin resistance mecA gene was also modified, it did not multiply in PCR test. These findings are important from the point of view of applying irradiation technology to stop the spread of antibiotic resistance.

Polyploid cell dynamics and death before and after PEG-treatment of a NIH/3T3 derived culture: vinblastine effects on the regulation of cell subpopulations heterogeneity

BackgroundNeoplastic subpopulations can include polyploid cells that can be involved in tumor evolution and recurrence. Their origin can be traced back to the tumor microenvironment or chemotherapeutic treatment, which can alter cell division or favor cell fusion, generating multinucleated cells. Their progeny, frequently genetically unstable, can result in new aggressive and more resistant to chemotherapy subpopulations. In our work, we used NIHs cells, previously derived from the NIH/3T3 line after serum deprivation, that induced a polyploidization increase with the appearance of cells with DNA content ranging from 4 to 24c. This study aimed to analyze the cellular dynamics of NIHs culture subpopulations before and after treatment with the fusogenic agent polyethylene glycol (PEG), which allowed us to obtain new giant polyploid cells. Successively, PEG-untreated and PEG-treated cultures were incubated with the antimicrotubular poison vinblastine. The dynamics of appearance, decrease and loss of cell subpopulations were evaluated by correlating cell DNA content to mono-multinuclearity resulting from cell fusion and division process alteration and to the peculiarities of cell death events.ResultsDNA microfluorimetry and morphological techniques (phase contrast, fluorescence and TEM microscopies) indicated that PEG treatment induced a 4–24c cell increase and the appearance of new giant elements (64–140c DNA content). Ultrastructural analysis and autophagosomal–lysosomal compartment fluorochromization, which allowed us to correlate cytoplasmic changes to death events, indicated that cell depletion occurred through distinct mechanisms: apoptotic death involved 2c, 4c and 8c cells, while autophagic-like death involved intermediate 12–24c cells, showing nuclear (lobulation/micronucleation) and autophagic cytoplasm alterations. Death, spontaneously occurring, especially in intermediate-sized cells, was increased after vinblastine treatment. No evident cell loss by death events was detected in the 64–140c range.ConclusionsPEG-treated NIHs cultures can represent a model of heterogeneous subpopulations originating from cell fusion and division process anomalies. Altogether, our results suggest that the different cell dynamics of NIHs subpopulations can affect the variability of responses to stimuli able to induce cell degeneration and death. Apoptptic, autophagic or hybrid forms of cell death can also depend on the DNA content and ability to progress through the cell cycle, which may influence the persistence and fate of polyploid cell descendants, also concerning chemotherapeutic agent action.

Does Insulin Resistance Contribute to Neuroinflammation, Cognitive Decline and Brain Senescence in Obesity?

The burgeoning prevalence of obesity and overweight conditions from the age of adolescence and throughout the lifespan has reached epidemic proportions in industrialized nations throughout the globe. The development of insulin resistance is one of the most common observations in obesity and is associated with a systemic hypoxia in adipose tissue and a chronic inflammatory state. The insulin resistance affects multiple elements of substrate oxidation and oxidative free radical generation in both somatic and neural tissues and with cytokine-linked chronic inflammation likely originating in adipose tissue depots. In studies with aging congenic lean and obese rats chronic hyperinsulinemia and brain shrinkage has now been reported, albeit it in the absence of Type 2 diabetes (NIDDM), hypertension (HTN) or other comorbidities, thereby suggesting that aspects of disordered substrate metabolism including insulin resistance as commonly observed in obesity may be a key contributory factor in the development of a neuroinflammatory linked brain shrinkage with accompanying decreases in brain mass, protein and DNA content, and which were further compromised when fed an isocaloric high vs. a low glycemic insulinogenic diet containing sucrose vs. cornstarch respectively. These results suggest that chronic insulin resistance of obesity secondary at least in part to dietary factors represents a likely independent risk factor in the progression of neuroinflammation, DNA damage, brain shrinkage and neural senescence in this strain of obese rats.

Anticancer Studies of Newly Synthesized Thiazole Derivatives: Synthesis, Characterization, Biological Activity, and Molecular Docking

Thiazole and its derivatives have received a lot of attention from researchers due to its wide biological, pharmacological, and anticancer properties. A novel series of 2-[2-[4-Hydroxy-3-substituted benzylidene hydrazinyl]-thiazole-4[5H]-ones (4a–c) and acetoxy derivative (5) were synthesized via using thiosemicarbazones (2a–c). The structure of the thiazole derivatives (4a–c) and 5 in these compounds was confirmed by spectroscopic techniques (IR and NMR), as well as elemental investigations. The synthesized derivatives biological activity was assessed based on their capacity to suppress the growth of the cancer cell lines MCF-7 and HepG2, as well as to halt the cell cycle and trigger apoptosis. Among the synthesized thiazole derivatives, compound 4c was found the most active derivative, with inhibitory concentrations IC50 = 2.57 ± 0.16 and 7.26 ± 0.44 µM in MCF-7 and HepG2, respectively, compared to Staurosporine as the standard drug with IC50 6.77 ± 0.41 and 8.4 ± 0.51 µM, respectively. Additionally, compound 4c blocked vesicular endothelial growth factor receptor-2 (VEGFR-2), according to our results (IC50 = 0.15 µM), compared to Sorafenib (IC50 = 0.059 µM) as the standard drug. Moreover, compound 4c induced cell cycle arrest at the G1/S phase, increasing the percentage and accumulation of cancer cells (DNA content) in the pre-G1 phase by 37.36% in MCF-7 cancer cells compared to untreated MCF-7 cells at 2.02%. Also, compound 4c increased the percentage of early and late apoptosis from 0.51% and 0.29%, respectively, in the case of the MCF-7 untreated control sample to 22.39% and 9.51%, respectively, in the MCF-7 treated sample. Furthermore, molecular docking studies of compounds 4a–c and 5 were conducted with four key proteins (aromatase, epidermal growth factor receptor (EGFR), cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma 2 (Bcl-2)) that stimulate the growth, proliferation, and development of cancer cells. Compound 4c exhibited good docking scores with a promising and potential binding affinity toward the active site of selected docked proteins. According to these results, compound 4c showed efficient cytotoxic activity against the tested cancer cells.

Aurantibacillus circumpalustris gen. nov., sp. nov., the first characterized representative of the Bacteroidota candidate family env.OPS 17 and proposal of Aurantibacillaceae fam. nov.

16S rRNA sequence types associated with the candidate family env.OPS 17 have been reported from various environments, but no representatives have been characterized and validly named. Bacteria of env.OPS 17 are affiliated with the order Sphingobacteriales and were first detected more than two decades ago in the vicinity of a thermal spring in Yellowstone National Park. Strain Swamp196T, isolated from the soil surrounding a swamp in Northern Germany, is the first characterized representative of candidate family env.OPS 17. Cells of strain Swamp196T are rod-shaped, non-motile, non-spore-forming, non-capsulated and stain Gram-negative. Colonies are small and orange-coloured. The strain is mesophilic and grows under aerobic or microaerophilic conditions. It grows chemo-organotrophically over a narrow range of pH and exclusively on proteinaceous substrates. The major cellular fatty acids are iso-C15 : 0, iso-C15 : 1 ω10c, C18 : 1 ω9c and C16 : 1 ω7c and the major polar lipids are two unidentified aminophospholipids, one unidentified aminolipid and one unidentified lipid. The predominant respiratory quinone is MK-7. The DNA G+C content of genomic DNA is 35.5 mol%. Strain Swamp196T is related to Pedobacter cryophilus AR-3-17T, Arcticibacter pallidicorallinus Hh36T and Pedobacter daechungensis Dae 13T with 16S rRNA gene sequence similarity of 84.1, 83.8 and 83.5 %, respectively. Based on our phenotypic, genomic and phylogenetic analysis, we propose the novel species Aurantibacillus circumpalustris sp. nov (type strain Swamp196T=DSM 105849T=CECT 30420T) of the novel genus Aurantibacillus gen. nov. and the novel family Aurantibacillaceae fam. nov.

Characteristics of testis development in autotetraploid fish

Fertile autotetraploid fish are an excellent germplasm resource for producing improved triploids. However, males produce more diploid gametes than females during production, which means that males are more valuable in application. To better understand the testis development of autotetraploid fish (4nRR) derived from the distant hybridization of Carassius auratus red var. (RCC, ♀) × Megalobrama amblycephala (BSB, ♂), all-male autotetraploid fish (AM4nRR) were generated. In this study, the tissue structure and transcriptome changes in different periods of gonadal development of AM4nRR were analyzed. Histological sectioning revealed a small number of gonium at 45 days post-hatching (dph), the number of germ cells greatly increased during 65–95 dph, and primary spermatocytes and secondary spermatocytes were observed at 105 dph. Meanwhile, white semen could be extruded from most individuals at 120 dph, and the DNA content of sperm from AM4nRR was approximately twice that from RCC as determined by flow cytometry. These results indicated that AM4nRR reached sexual maturity at 120 dph and produced diploid sperm. In addition, a total of 149.55 Gb of clean data and 56,188 unigenes were obtained from the testicular tissue of AM4nRR at different developmental periods (25 dph, 35 dph, 40 dph, 45 dph, 65 dph, 95 dph and 120 dph). Differential expression analysis showed that the number of differentially expressed genes (DEGs) among the three periods of 40 dph, 45 dph, and 65 dph was the lowest (72–275). However, the expression levels of partial DEGs at 45 dph changed abruptly compared with those at 40 dph and 65 dph according to hierarchical clustering analysis, which was consistent with the expression characteristics of gonadal development-related genes at the early period of sex differentiation. These results suggested that 40–65 dph might be a critical period for the initiation of testis development in autotetraploid fish. These findings not only provide a new gamete resource for the large-scale preparation of improved triploids but also provide a basis for further study of sex differentiation in autotetraploid fish.

Intraoperative Flow Cytometry Upon and Beyond the Cell Cycle: A Case Study of the Characterization of a Bone Metastasis

The accurate assessment of malignancy and the precise characterization of cancer type are pivotal in guiding clinical decisions and ensuring optimal patient outcomes. The challenging task of identifying the primary site of carcinoma, especially during a surgical procedure, is not always possible and necessitates the exploration of novel, innovative diagnostic techniques. In this report, we detail a unique case of carcinoma of unknown primary origin encountered during an orthopedic surgical procedure. We introduce a novel intraoperative flow cytometry (iFC) methodology, distinctly different from traditional flow cytometry, which is tailored for real-time assessment during surgeries. This iFC technique, applied in a bone metastasis case of unknown primary origin, enabled DNA content analysis and the quantification of cytokeratin for rapid malignancy characterization, presenting an avenue for immediate surgical guidance and decision making. The case was distinctively characterized using iFC, shedding light on the potential of this emerging technique. iFC has been gaining traction because of its ability to facilitate cancer cell assessment and margin evaluation. It has been successfully applied across a wide spectrum of neoplastic conditions. Our innovative, two-step approach using iFC encompasses (1) DNA content analysis, which serves as a reliable indicator for the detection of cancer cells, and (2) the quantification of cytokeratin, a pivotal marker, which aids in the characterization and classification of a malignancy. In this case, the malignancy was classified as a carcinoma. The findings obtained from iFC were subsequently validated through pathology assessment, confirming the accuracy and reliability of our approach. This noteworthy case strengthens the potential of iFC as a novel tool in malignancy assessment, which is not just limited to cell cycle analysis but instead extends beyond this application. The promising results obtained from this case study serve as a testament to the need for more extensive research in clinical studies.

Significance of Crypt Atypia in Barrett’s Esophagus: A Clinical, Molecular, and Outcome Study

The aim of this study was to characterize baseline morphologic features of crypts in nondysplastic Barrett's esophagus and correlate them with DNA content abnormalities and risk of progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). The morphologic features of nondysplastic crypts in baseline biopsy specimens from 212 BE patients (2956 biopsy specimens) were graded histologically using a 4-point scale (crypt atypia levels, 0-3). DNA content abnormalities were detected using flow cytometry. In patients who had dysplasia in their baseline biopsy specimens, dysplasia was associated significantly with increasing grades of crypt atypia in the background nondysplastic Barrett's esophagus (P < .001). In a subset of patients without dysplasia at baseline (N= 149), a higher grade of crypt atypia was associated with longer Barrett's esophagus segment length (5.5 vs 3.3 cm; P= .0095), and a higher percentage of cells with 4N DNA content (3.67 ± 1.27 vs 2.93 ± 1.22; P= .018). Crypt atypia was associated with the development of any neoplasia (low-grade dysplasia and HGD/EAC). Although no significant association was noted between the grade of crypt atypia and increased 4N, aneuploidy, or progression to HGD/EAC, only patients with grade 2 or 3 crypt atypia showed increased 4N, aneuploidy, or progression to HGD/EAC. Patients with Barrett's esophagus likely develop dysplasia via a progressive increase in the level of crypt atypia before the onset of dysplasia, and these changes may reflect some alteration of DNA content.