DNA Content Research Articles

Using qPCR to Identify Potential Effects of Thermal Conditions during Embryogenesis on Mitochondrial DNA Copy Number in Juvenile Brown Trout Salmo trutta

Changes in the number, structure, and function of mitochondria during the early life stages of animals can play an important role for an organism’s metabolic rate, growth, and health. Previous studies have shown that juvenile brown trout (Salmo trutta) subjected to elevated temperatures during the embryonic stage respond phenotypically with a reduced metabolic rate. The aim of this study was to explore if embryonic temperature affects the mitochondria content of young brown trout and as such explains the previously found differences in metabolic rates. Here, we optimize a quantitative PCR (qPCR) method for the mitochondria cytochrome c oxidase subunit I gene, and then use the method as a proxy for mitochondrial DNA content. We hypothesize that young trout subjected to elevated temperatures during the embryonic stage respond phenotypically with a reduced mitochondrial DNA content. To test this hypothesis, we subjected brown trout to either control ambient (4.4 ± 1.5 °C) or elevated temperatures (7.1 ± 0.6 °C) during embryogenesis. Subsequently, we extracted DNA from liver and white muscle tissue of juvenile brown trout from the two different incubation temperature treatments and successively optimized qPCR for mitochondrial DNA. We found that the amount of mitochondria DNA in liver tissue was 18 times higher than in white muscle tissue, but there was no significant difference in mitochondria content in liver or muscle tissue between brown trout exposed to elevated and ambient control temperatures during embryogenesis. We conclude that reduced metabolic rate is not likely associated with mitochondria DNA content. We also suggest that qPCR is a simple and cost-effective method to quantify mitochondria DNA in frozen and partly degraded tissue from different treatment groups and a useful proxy for identification of differences in mitochondria number.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Comparative Analysis of Bisexual and Parthenogenetic Populations in Haemaphysalis Longicornis.

Haemaphysalis longicornis, a three-host tick with a wide host range, is widely distributed in different countries and regions. It stands out among ticks due to its unique feature of having both parthenogenetic and bisexual populations. Despite their morphological resemblance, the characteristics of the parthenogenetic population have been overlooked. In this comprehensive study, we systematically compared the similarities and differences between these two populations. Our investigation revealed that the parthenogenetic H. longicornis, widely distributed in China, was found in ten provinces, surpassing the previously reported distribution. Notably, individuals from the parthenogenetic population exhibited a prolonged blood-feeding duration during the larval and nymph stages compared to their bisexual counterparts. Additionally, the life cycle of the parthenogenetic population was observed to be longer. A flow cytometry analysis indicated a DNA content ratio of approximately 2:3 between the bisexual and parthenogenetic populations. A phylogenetic analysis using whole mitochondrial genome sequences resulted in the separation of the phylogenetic tree into two distinct branches. A molecular analysis unveiled a consistent single T-base deletion at nucleotide 8497 in the parthenogenetic population compared to the bisexual population. Both populations displayed high viral infection capability and significant resistance to ivermectin. Intriguingly, despite these differences, the parthenogenetic population exhibited a similar life cycle to the bisexual population, retaining the ability to transmit pathogens such as Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland Virus (HRTV). These findings contribute to a deeper understanding of the distinct characteristics and similarities between different populations of H. longicornis, laying the foundation for future research in this field.

Medaka Terb1 Mutant Displays Defects of Synaptonemal Complex Formation and Sexual Difference in Gametogenesis.

Formation of the synaptonemal complex (SC) is a prerequisite for proper recombination and chromosomal segregation during meiotic prophase I. One mechanism that ensures SC formation is chromosomal movement, which is driven by the force derived from cytoskeletal motors. Here, we report the phenotype of medaka mutants lacking the telomere repeat binding bouquet formation protein 1 (TERB1), which, in combination with the SUN/KASH protein, mediates chromosomal movement by connecting telomeres and cytoskeletal motors. Mutations in the terb1 gene exhibit defects in SC formation in medaka. Although SC formation was initiated, as seen by the punctate lateral elements and fragmented transverse filaments, it was not completed in the terb1 mutant meiocytes. The mutant phenotype further revealed that the introduction of double strand breaks was independent of synapsis completion. In association with these phenotypes, meiocytes in both the ovaries and testes exhibited an aberrant arrangement of homologous chromosomes. Interestingly, although oogenesis halted at the zygotene-like stage in terb1 mutant, testes continued to produce sperm-like cells with aberrant DNA content. This indicates that the mechanism of meiotic checkpoint is sexually different in medaka, similar to the mammalian checkpoint in which oogenesis proceeds while spermatogenesis is arrested. Moreover, our results suggest that spermatogenesis is mechanistically dissociable from meiosis.

Revealing the Satellite DNA Content in Ancistrus sp. (Siluriformes: Loricariidae) by Genomic and Bioinformatic Analysis

Introduction: Eukaryotic genomes are composed of simple, repetitive sequences, including satellite DNAs (satDNA), which are noncoding sequences arranged in tandem arrays. These sequences play a crucial role in genomic functions and innovations, influencing processes such as the maintenance of nuclear material, the formation of heterochromatin and the differentiation of sex chromosomes. In this genomic era, advances in next-generation sequencing and bioinformatics tools have facilitated the exhaustive cataloging of repetitive elements in genomes, particularly in non-model species. This study focuses on the satDNA content of Ancistrus sp., a diverse species of fish from the Loricariidae family. The genus Ancistrus shows significant karyotypic evolution, with extensive variability from the ancestral diploid number. Methods: By means of bioinformatic approaches, 40 satDNA families in Ancistrus sp., constituting 5.19% of the genome were identified. Analysis of the abundance and divergence landscape revealed diverse profiles, indicating recent amplification and homogenization of these satDNA sequences. Results: The most abundant satellite, AnSat1-142, constitutes 2.1% of the genome, while the least abundant, AnSat40-52, represents 0.0034%. The length of the monomer repeat varies from 16 to 142 base pairs, with an average length of 61 bp. These results contribute to understanding the genomic dynamics and evolution of satDNAs in Ancistrus sp. Conclusion: The study underscores the variability of satDNAs between fish species and provides valuable information on chromosome organization and the evolution of repetitive elements in non-model organisms.

ZLN005 alleviates PBDE-47 induced impairment of mitochondrial translation and neurotoxicity through PGC-1α/ERRα axis

Recent studies are identified the mitochondria as critical targets of 2, 2′, 4, 4′-tetrabromodiphenyl ether (PBDE-47) induced neurotoxicity. This study aimed at examining the impact of PBDE-47 exposure on mitochondrial translation, and its subsequent effect on PBDE-47 neurotoxicity. The Sprague-Dawley (SD) rat model and neuroendocrine pheochromocytoma (PC12) cells were adopted for the measurements of mitochondrial ATP levels, mitochondrial translation products, and expressions of important mitochondrial regulators, such as required meiotic nuclear division 1 (RMND1), estrogen-related receptor α (ERRα), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). To delve into the role of PGC-1α/ERRα axis in mitochondrial translation, 2-(4-tert-butylphenyl) benzimidazole (ZLN005) was employed. Both cellular and animal model results shown that PBDE-47 impeded PGC-1α/ERRα axis and mitochondrial translation. PBDE-47 suppressed mitochondrial function in rat hippocampus and PC12 cells by decreasing relative mitochondrial DNA (mtDNA) content, mitochondrial translation products, and mitochondrial ATP levels. Particularly, ZLN005 reversed PBDE-47 neurotoxicity by enhancing mitochondrial translation through activation of PGC-1α/ERRα axis, yet suppressing PGC-1α with siRNA attenuates its neuroprotective effect in vitro. In conclusion, this work highlights the importance of mitochondrial translation in PBDE-47 neurotoxicity by presenting results from cellular and animal models and suggests a potential therapeutic approach through activation of PGC-1α/ERRα axis. Environmental implicationPBDEs have attracted extensive attention because of their high lipophilicity, persistence, and detection levels in various environmental media. Increasing evidence has shown that neurodevelopmental disorders in children are associated with PBDE exposure. Several studies have also found that perinatal PBDE exposure can cause long-lasting neurobehavioral abnormalities in experimental animals. Our recent studies have also demonstrated the impact of PBDE-47 exposure on mitochondrial biogenesis and dynamics, leading to memory and neurobehavioral deficits. Therefore, we explore whether the pathological mechanism of PBDE-47-induced neurotoxicity involves the regulation of mitochondrial translation through the PGC-1α/ERRα axis.

Mcph1, mutated in primary microcephaly, is also crucial for erythropoiesis.

Microcephaly is a common feature in inherited bone marrow failure syndromes, prompting investigations into shared pathways between neurogenesis and hematopoiesis. To understand this association, we studied the role of the microcephaly gene Mcph1 in hematological development. Our research revealed that Mcph1-knockout mice exhibited congenital macrocytic anemia due to impaired terminal erythroid differentiation during fetal development. Anemia's cause is a failure to complete cell division, evident from tetraploid erythroid progenitors with DNA content exceeding 4n. Gene expression profiling demonstrated activation of the p53 pathway in Mcph1-deficient erythroid precursors, leading to overexpression of Cdkn1a/p21, a major mediator of p53-dependent cell cycle arrest. Surprisingly, fetal brain analysis revealed hypertrophied binucleated neuroprogenitors overexpressing p21 in Mcph1-knockout mice, indicating a shared pathophysiological mechanism underlying both erythroid and neurological defects. However, inactivating p53 in Mcph1-/- mice failed to reverse anemia and microcephaly, suggesting that p53 activation in Mcph1-deficient cells resulted from their proliferation defect rather than causing it. These findings shed new light on Mcph1's function in fetal hematopoietic development, emphasizing the impact of disrupted cell division on neurogenesis and erythropoiesis-a common limiting pathway.

Host-Pathogen Interactions and Correlated Factors That Are Affected in Replicative-Aged Cryptococcus neoformans.

Cryptococcus neoformans is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) C. neoformans cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN C. neoformans, such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed C. neoformans of different generational ages. Our results showed that old C. neoformans exhibited higher urease activity and enhanced Golgi activity. In addition, old C. neoformans were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old C. neoformans prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old C. neoformans to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host-pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design.

Development of decellularization protocols for female cat reproductive organs

Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2-cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns.

Development of a Novel Formulation of Bioactive Glass Based Calcium Phosphate Cement for Bone Grafting

AbstractParticulate bone grafting material can travel from the implantation site and diminish the magnitude of required bone formation response and hence its effectiveness. An easily prepared moldable graft that rapidly sets rigid in situ would alleviate this problem and in this study, such an injectable bone graft material is presented. By combining calcium phosphate (CP) salts with a bioactive glass (BG) mixed with a 2.5% Na2HPO4 solution a novel BG‐based calcium phosphate cement (CPC) is produced. Immersion in Tris buffer and simulated body fluid (SBF) solutions result in the rapid generation of octacalcium phosphate (OCP) and hydroxyapatite (HA) phases – a pre‐requisite for “bioactivity”. Cement‐conditioned media does not result in any cytotoxic potential with respect to MTT activity, DNA content, or alkaline phosphatase activity with an osteoblastic cell line. Application of the cement into standardized surgical defects in the minipig mandible results in short‐term, moderate irritation at 3 and 6 weeks and complete replacement of cement with new bone at 12 weeks.

Variability in genome size of Trigonella foenum-graecum, Trigonella corniculata and Trigonella caerulea as estimated by flow cytometry indicates complex evolutionary history of fenugreek.

The determination of genome size is a fundamental step which provides a basis to initiate studies aimed at deciphering the genetic similarity of a species and to carry out other genomics based investigations. Fenugreek (Trigonella spp.) is an important spice crop which has numerous health promoting phytochemicals. Many species within this genus are known for their various health benefits owing to the presence of a wide diversity of important phytochemicals like diosgenin, trigonelline, fenugreekine, galactomannan, 4-hydroxy isoleucine, etc. It is a multipurpose crop being cultivated for food, animal feed and industrial purposes. Despite its importance, research on the genomics aspect of fenugreek remains scant. In the absence of sufficient genomic information, crop improvement in fenugreek is severely lagging. Estimation of genome size of a species is the preliminary step for initiation of any genomic studies and therefore in the present study we have estimated the genome size for fenugreek. Here, we have determined the genome sizes of three different Trigonella spp. namely T. foenum-graecum, T. corniculata and T. caerulea through flow cytometry (FC). The 2C DNA content values were found to be 6.05 pg (T. foenum-graecum), 1.83 pg (T. corniculata) and 1.96 pg (T. caerulea). The genome size of T. foenum-graecum is approximately three times the genome size of T. corniculata and T. caerulea. This variation in genome size of more than three-fold indicates the level of genetic divergence among the three species, though within the same genus. The differences observed in the genome sizes of the three species provide conclusive evidence of their genetic divergence. Additionally, the information about the genome size would provide an impetus to the structural and functional genomics-based research in this crop.

Ruegeria marisflavi sp. nov. and Ruegeria aquimaris sp. nov., isolated from seawater of the Yellow Sea.

Two novel Gram-stain-negative, aerobic, non-motile and rod-shaped bacteria, designated as WL0004T and XHP0148T, were isolated from seawater samples collected from the coastal areas of Nantong and Lianyungang, PR China, respectively. Both strains were found to grow at 10-42 °C (optimum, 37 °C) and with 2.0-5.0 % (w/v) NaCl (optimum, 3.0 %). Strain WL0004T grew at pH 6.0-9.0 (optimum, pH 7.0-8.0), while XHP0148T grew at pH 6.0-10.0 (optimum, pH 7.0-8.0). The major cellular fatty acids (>10 %) of both strains included summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c). In addition, strain WL0004T contained 11-methyl C18 : 1 ω7c and strain XHP0148T contained C12 : 0 3-OH. The respiratory quinone of both strains was ubiquinone-10. The G+C content of genomic DNA of strains WL0004T and XHP0148T were 62.5 and 63.0 mol%, respectively. Strains WL0004T and XHP0148T showed the highest 16S rRNA gene sequence similarity to Ruegeria pomeroyi DSS-3T (99.4 and 99.0 %, respectively), and the 16S rRNA gene-based phylogenetic analysis indicated that the two strains were closely related to members of the genus Ruegeria. The average nucleotide identity and digital DNA-DNA hybridization values among the two strains and type strains of the genus Ruegeria were all below 95 and 70 %, respectively, and the phylogenetic tree reconstructed from the bac120 gene set indicated that the two strains are distinct from each other and the members of the genus Ruegeria. Based on this phenotypic and genotypic characterization, strains WL0004T (=MCCC 1K07523T=JCM 35565T=GDMCC 1.3083T) and XHP0148T (=MCCC 1K07543T=JCM 35569T=GDMCC 1.3089T) should be recognized as representing two novel species of the genus Ruegeria and the names Ruegeria marisflavi sp. nov. and Ruegeria aquimaris sp. nov. are proposed, respectively.

Nano zinc oxide mediated resuscitation of aged Cajanus cajan via modulating aquaporin, cell cycle regulatory genes and hormonal responses.

Nanoparticle pretreatment improved the health ofaged Cajanus cajan seeds viz., regulation ofredox status, gene expression, andrestoration ofhormonal homeostasis. Ageing deteriorates the quality of seeds by lowering their vigor and viability, and terminating with loss of germination. These days, nanotechnology has been seen to revolutionize the agricultural sectors, and particularly nano zinc oxide (nZnO) has gained considerable interests due to its distinctive properties. The aim of the present work was to decipher the possibilities of using nZnO to rejuvenate accelerated aged (AA) seeds of Cajanus cajan. Both chemically (CnZnO) and green (GnZnO; synthesized using Moringa oleifera) fabricated nZnOs were characterized via standard techniques to interpret their purity, size, and shape. Experimental results revealed erratic germination with a decline in viability and membrane stability as outcomes of reactive oxygen intermediate (ROI) buildup in AA seeds. Application of nZnO substantially rebated the accrual of ROI, along with enhanced production of antioxidants, α-amylase activity, total sugar, protein and DNA content. Higher level of zinc was assessed qualitatively/ histologically and quantitatively in nZnO pulsed AA seeds, supporting germination without inducing toxicity. Meantime, augmentation in the gibberellic acid with a simultaneous reduction in the abscisic acid level were noted in nZnO invigorated seeds than that determined in the AA seeds, suggesting possible involvement of ROI in hormonal signalling. Furthermore, nZnO-subjected AA seeds unveiled differential expression of aquaporins and cell cycle regulatory genes. Summarizing, among CnZnO and GnZnO, later one holds better potential for a revival of AA seeds of Cajanus cajan by providing considerable tolerance against ageing-associated deterioration via recouping the cellular redox homeostasis, hormonal signaling, and alteration in expression patterns of aquaporin and cell cycle regulatory genes.

Dynamic flow for efficient partial decellularization of tracheal grafts: A preliminary rabbit study.

Bioengineered tracheal grafts are a potential solution for the repair of long-segment tracheal defects. A recent advancement is partially decellularized tracheal grafts (PDTGs) which enable regeneration of host epithelium and retain viable donor chondrocytes for hypothesized benefits to mechanical properties. We propose a novel and tunable 3D-printed bioreactor for creating large animal PDTG that brings this technology closer to the bedside. Conventional agitated immersion with surfactant and enzymatic activity was used to partially decellularize New Zealand white rabbit (Oryctolagus cuniculus) tracheal segments (n = 3). In parallel, tracheal segments (n = 3) were decellularized in the bioreactor with continuous extraluminal flow of medium and alternating intraluminal flow of surfactant and medium. Unprocessed tracheal segments (n = 3) were also collected as a control. The grafts were assessed using the H&E stain, tissue DNA content, live/dead assay, Masson's trichrome stain, and mechanical testing. Conventional processing required 10 h to achieve decellularization of the epithelium and submucosa with poor chondrocyte viability and mechanical strength. Using the bioreactor reduced processing time by 6 h and resulted in chondrocyte viability and mechanical strength similar to that of native trachea. Large animal PDTG created using our novel 3D printed bioreactor is a promising approach to efficiently produce tracheal grafts. The bioreactor offers flexibility and adjustability favorable to creating PDTG for clinical research and use. Future research includes optimizing flow conditions and transplantation to assess post-implant regeneration and mechanical properties. NA.

The effect of fingerprint enhancement methods applied on adhesive surfaces on DNA recovery: a preliminary study

The presence of body fluids such as blood, saliva, semen or urine during fingerprint research on the evidence taken from the crime scene makes it necessary to protect biological materials to examine the evidence in multiple ways. Therefore, it is crucial that fingerprint development (FD) techniques do not disrupt the structure of biological materials during FD procedures. In this sense, it is essential to determine whether biological material or fingerprints should be the priority during the collection of evidentiary materials, to determine the systematic order and to determine whether the FD methods to be applied cause damage to the genetic material used in the identification of individuals and to evaluate them in terms of their evidentiary quality. This study investigated the effects of the application of trace detection methods on DNA profiling processes in evidence where fingerprints and biological samples are found at the same time. In this study, blood, saliva, semen and urine samples were taken from a male individual who signed an informed consent form at the laboratory stage. The samples were applied 50 µL on the adhesive tape surface. After application, the samples were treated with crystal violet (CV) and sticky side (SS) fingerprint development chemicals suitable for the surface type. The prepared samples were dried under room conditions. After 1 day and 45 days under normal room conditions, silica-based DNA extraction was performed. After extraction, DNA quantification was performed using the fluorimetry method. In the study, biological samples with known DNA content were used to focus on DNA quantification. Among the fresh samples prepared in the study, DNA recovery was higher in the SS-treated urine, blood and saliva samples and in the CV-treated semen sample group compared to the other groups. This shows that chemical treatment of some biological samples on adhesive tape increases the efficiency of DNA recovery. When the 45-day waiting samples were compared with the control group samples, DNA recovery decreased in CV-treated urine and blood samples, while DNA recovery increased in SS-treated urine and blood samples. In semen samples, both CV and SS treatment negatively affected DNA recovery. In saliva samples, DNA recovery increased ~2-fold in the CV-treated sample group, while SS treatment caused a ~75% decrease in DNA recovery. The results show that the non-porous adhesive tape does not adversely affect the amount of DNA in terms of STR profiling of latent FD chemicals used on the surfaces and that adhesive tape treated with fingerprint enhancement chemicals can actually be used for advanced forensic genetic analyses for DNA extraction on surfaces.

Elucidating the Anticancer Mechanisms of Cinnamoyl Sulfonamide Hydroxamate: Insights From DNA Content Analysis and Gene Expression Profiling in Squamous Cell Carcinoma.

Oral cancer is a major public health concern worldwide, with oral squamous cell carcinoma (OSCC) being one of its most common subtypes. Despite advances in diagnosis and management of this disease, there remains a need to develop new therapeutic approaches for better outcomes. This study aimed to investigate the molecular mechanisms through which cinnamoyl sulfonamide hydroxamate derivatives exert their anticancer effects on OSCC. The derivatives were synthesized via multi-step processes and then characterized at the molecular level. Flow cytometry assay for DNA content and cell cycle distribution, anisidine/toluidine double staining for apoptosis detection, as well as reverse transcription polymerase chain reaction (RT-PCR) gene expression analysis, were performed on OSCC cell lines exposed to cinnamoyl sulfonamide hydroxamate derivatives. Flow cytometry unveiled remarkable changes in the distribution of cells throughout the OSCC cell line upon treatment with cinnamoyl sulfonamide hydroxamate derivatives. Consequently, it led to a noticeable decrease in cells at the G0/G1 phase, together with an increase at the S phase, thereby indicating a retardation at various points of the cycle. In addition, apoptotic morphological alterations have been observed by anisidine/toluidine double staining after some treatments with the compounds. RT-PCR analysis showed a marked increase in p21 gene expression levels, further supporting the compounds' ability to induce cell cycle arrest and apoptosis. The research highlighted the potential of cinnamoyl sulfonamide hydroxamate derivatives as candidates for oral cancer, particularly OSCC treatment, shedding light on their operation at the molecular level and paving the way for the development of targeted therapies that could aid in the cure of oral cancer.

Efficacy of nano-curcumin on nicotine-induced genotoxicity and immunomodulatory disruption in protein- malnourished female rats

Background: Aggravated DNA damage and immunomodulatory disruptions by nicotine are more prominent in female populations under protein-restricted conditions. Females are more susceptible to nicotine-induced complications due to their low innate immunity. Anti-inflammatory and anti-genotoxic curcumin, though it effectively works against nicotine-induced complications, still its therapeutic use is hindered due to its poor aqueous solubility and low bioavailability. Nano-curcumin would be a better therapeutic agent because of its increased aqueous solubility and higher bioavailability than curcumin. Materials: This study investigates the effects of nicotine (2.5 mg/kg body weight injected subcutaneously for 21 days) on genotoxicity and immunomodulatory disruptions in female rats maintained under a protein-restricted diet (5% casein) and determines the ameliorative efficacy of nano-curcumin after one hour of nicotine dosage. Results: It is observed that nicotine decreases hemoglobin content in blood and DNA content in tissues. It causes severe DNA damage and disrupts the immune system and endocrine functions under protein-restricted conditions. Nano-curcumin ameliorates the nicotine-mediated genotoxic effects, maintains the female sex hormone levels, and restores the normalcy of immune responses in protein-restricted diet rats more efficiently than curcumin. Conclusion: Nano-curcumin looks like a potential blocker of nicotine, which may be used as a prospective therapeutic herbal agent to protect the health of the protein-malnourished female population.

The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models.

Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

DNA Content in Embryonic Extracellular Vesicles Is Independent of the Apoptotic Rate in Bovine Embryos Produced In Vitro.

Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis.

Development of robust in vitro propagation protocol and cyto-genetic fidelity assessment of Siraitia grosvenorii (monk fruit)

Siraitia grosvenorii (Swingle) C. Jeffrey of the family Cucurbitaceae is a unique and precious plant of medicinal and economic importance in the international market. Due to the huge market potential and to narrow down the gap between demand and supply, an efficient in vitro propagation system using nodal explants was established. Shoot nodal segments were cultured in medium supplemented with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kn 1.0–7.5 µM). A two-stage culture is recommended for micropropagation of S. grosvenorii. The highest shoot multiplication was observed in BAP-supplemented medium (5.0 µM), whereas rooting was observed in half-strength MS medium supplemented with 8.75 µM of indole-3-butyric acid (IBA). Fully developed plantlets were acclimatized with 100 % survival in sterilized sand. These hardened 15-day-old plants were successfully established under shade net conditions in fields. In vitro raised plants were found genetically stable using molecular markers, including RAPD and ISSR. The 2C nuclear DNA content using flow cytometry was observed to be 0.73–0.76 pg. Hence, the protocol developed for in vitro propagation of S. grosvenorii is cost-effective and can be efficiently used for the industrial production of this natural sweetener and pharmaceutically important plants.