DNA Fragmentation Analysis Research Articles

Protective Effects of Autologous ‎Platelet-Rich Plasma (PRP) on the Outcome of ‎Cryopreservation in Rabbit Sperm.

Sperm ‎cryopreservation is a cost-effective means of preserving gene resources and ‎transporting sperm across distant locations. However, due to the ‎general disadvantages of using freeze-thawed sperm, to prevent this irreversible damage, cryopreservation techniques must be ‎improved by the addition of additional cryoprotection agents. ‎This study aims to improve the freezability of buck semen using an ‎‎intratesticular injection of Autologous ‎Platelet-Rich Plasma (‎PRP) and ‎confirm this theory by Casa laboratory analysis and gene ‎expression detection of three genes (CATSPER1‎, SPAG5 and Hsp70). Twenty ‎rabbits a New ‎Zealand ‎healthy male were randomly ‎‎divided into two equal groups; ‎the control and PRP group ‎which enriched with PRP, ‎the semen ‎collection was ‎applied after 10 weeks, then each sample ‎was ‎divided into two ‎‎fractions; ‎First ‎‎fractions ‎‎ we apply the ‎ laboratory semen analysis ‎directly, and the second ‎‎fractions ‎‎were ‎ cryopreservation, then thawed after one ‎month to apply ‎‎the same ‎ laboratory analysis. The ‎results of CASA, DNA fragmentation, and‎ real ‎time-PCR analysis ‎had ‎ ‎‎statistically significant ‎differences (P<0.01) when compartment ‎of these values ‎after and ‎‎before freezing, yet we don't ‎record any statistical ‎differences between the C group ‎and CR ‎group.‎ This study's findings are extremely significant, indicating that intratesticular injection of PRP ‎ is a good method for using in enhancing the rabbit sperm procedure after ‎freeze-thawing.

Distribution and Identification of Pepper yellow leaf curl Indonesia virus Infecting Chili Pepper in Bali Island

Yellow leaf curl disease in chili pepper has been reported in Bali Island since the early 2012. Research was conducted to identify the virus causing this disease and disease distribution in Bali. Field survey was carried out to observe disease intensity and to collect field samples from several chili pepper growing areas in Bali (Karangasem, Bangli, Tabanan, and Gianyar). Begomovirus identification from field samples was then conducted by polymerase chain reaction method using universal primers SPG1/SPG2, followed by an analysis of the amplified target DNA sequences. The incidence of pepper yellow leaf curl disease reached 100% at all sites and disease severity reached 18%−87%. Begomovirus specific DNA fragment measuring 912 bp was successfully amplified from 12 field samples. Sequence analysis of DNA fragments showed the highest homology with Pepper yellow leaf curl Indonesia virus (PYLCIV). Further phylogenetic analysis confirmed the relationship between PYLCIV isolates from Bali and various PYLCIV isolates from Indonesia.

DNA Coil Dynamics and Hydrodynamic Gating of Pressure-Biased Nanopores.

Nanopores are ideally suited for the analysis of long DNA fragments including chromosomal DNA and synthetic DNA with applications in genome sequencing and DNA data storage, respectively. Hydrodynamic fluid flow has been shown to slow down DNA transit time within the pore, however other influences of hydrodynamic forces have yet to be explored. In this report, a broad analysis of pressure-biased nanopores and the impact of hydrodynamics on DNA transit time, capture rate, current blockade depth, and DNA folding are conducted. Using a 10nm pore, it is shown that hydrodynamic flow inhibits the early stages of linearization of DNA and produces predominately folded events which are initiated by folded DNA (2-strands) entering the pore. Furthermore, utilizing larger pores (30nm) leads to unique DNA gating behavior in which DNA events can be switched on and off with the application of pressure. A computational model, based on combining electrophoretic drift velocities with fluid velocities, accurately predicts the pore size required to observe DNA gating. Hydrodynamic fluid flow generated by a pressure bias, or potentially more generally by other mechanisms like electroosmotic flow, is shown to have significant effects on DNA sensing and can be useful for DNA sensing technologies.

MMR profile and microsatellite instability status in colorectal mucinous adenocarcinoma with synchronous metastasis: a new clue for the clinical practice

AimsMucinous adenocarcinoma (MA) is associated with a high frequency of microsatellite instability (MSI). In the metastatic setting, it is crucial to establish mismatch repair (MMR) and/or MSI status. However, genetic...

Comparative Analysis of the Complete Chloroplast Genomes of Nine Paphiopedilum Species.

Paphiopedilum is known as “lady’s or Venus” slipper orchids due to its prominent shoe-shaped labellum, with high ornamental value. Phylogenetic relationships among some species in Paphiopedilum genus cannot be effectively determined by morphological features alone or through the analysis of nuclear or chloroplast DNA fragments. In order to provide aid in understanding the evolutionary and phylogenetic relationship in Paphiopedilum at chloroplast (cp) genome-scale level, the complete cp genomes of six Paphiopedilum species were newly sequenced in this study, and three other published cp genome sequences of Paphiopedilum were included in the comparative analyses. The cp genomes of the six Paphiopedilum species ranged from 154,908 bp (P. hirsutissimum) to 161,300 bp (P. victoria-mariae) in size, all constituting four-part annular structures. Analyses of the nucleotide substitutions, insertions/deletions, and simple sequence repeats in the cp genomes were conducted. Ten highly variable regions that could serve as potential DNA barcodes or phylogenetic markers for this diverse genus were identified. Sequence variations in the non-coding regions were greater than that in the conserved protein-coding regions, as well as in the large single copy (LSC) and small single copy (SSC) regions than in the inverted repeat (IR) regions. Phylogenetic analysis revealed that all Paphiopedilum species clustered in one monophyletic clade in the Cypripedioideae subfamily and then subdivided into seven smaller branches corresponding to different subgenus or sections of the genus, with high bootstrap supports, indicate that cp genome sequencing can be an effective means in resolving the complex relationship in Paphiopedilum.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos.

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

A Capillary Electrophoresis Method for Analyzing the Diversity of Vaginal Microbiome

16S ribosomal RNA (rRNA) gene sequence analysis is the common method to identify the bacteria in human vaginal flora. While specific DNA primers were designed to target 16S rRNA gene sequences, DNA fragment analysis using capillary electrophoresis can obtain more accurate qualitative and quantitative information on the microbiome. This study aimed to assess the ability of capillary electrophoresis method to analyze the diversity of vaginal microbiome and provide a theoretical basis for the accurate gene detection of vaginal flora. We collected 75 vaginal secretion samples from female outpatients aged 25 - 50, who had undergone rou-tine gynecologic examinations in Fujian provincial hospital from March 2021 to April 2021. Clinical diagnosis was based on the results of microscopic examination of Gram-stained specimens and biochemical tests of bacteria (pH value, catalase, leukocyte esterase, sialidases, β-glucuronidase, and acetylglucosaminidase). Vaginal secretion samples were collected and then total bacterial DNA was extracted. We used six pairs of fluorescent dye tagged specific primers that were designed based on the 16S rRNA genes of four Lactobacillus species (L. iners, L. crispatus, L. jensenii, L. gasseri), Gardnerella vaginalis, and Atopobium vaginae. PCR products of six species of bacteria were detected and analyzed by a 3130 Genetic Analyzer. Seventy-five samples were divided into two groups according to the vaginal microbiome evaluation, including 50 cases which had vaginal bacteria balance and 25 cases which had vaginal bacteria disorder. PCR amplification of 16S ribosomal RNA genes of L. iners, L. crispatus, L. jensenii, L. gasseri, Gardnerella vaginalis, and Atopobium vaginae was successfully performed on the DNA extracted from vaginal secretion samples. Four Lac-tobacillus species were detected in 4 - 33 cases of "Balance" group, and Gardnerella vaginalis was detected in 23 cases of "Disorder" group and, simultaneously, Atopobium vaginae was detected in 20 cases. Based on the technique of DNA fragment analysis using capillary electrophoresis method, the most common vaginal bacteria in Chinese healthy women are L. iners and L. crispatus. Gardnerella vaginalis and Atopobium vaginae are the most common pathogenic bacteria detected in the patients who had vaginal bacteria disorder. Using capillary electrophoresis method to detect the vaginal bacteria will be useful for accurate identifica-tion of vaginal microbiome. There will be an application value to find out the composition of the vaginal microbiome rapidly and detect specific gene markers to identify potential pathogenic bacteria when women are at risk of serious illness before they develop obvious symptoms.

Two approaches for calculating female fetal DNA fraction in noninvasive prenatal testing based on size analysis of maternal DNA fragments

The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing (NIPT). The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation. It is important to measure fetal DNA fraction before NIPT. However, there is still little research on how to calculate the concentration of female fetuses. Two estimation approaches were proposed to calculate fetal DNA fraction, including the fragments size-based approach, aneuploid-based approach, which are all approaches based on chromosome segments. Based on high-throughput sequencing data, two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values, which were close to the actual values. The correlation coefficient of fragments size-based approach was 0.9243 (P < 0.0001) and the aneuploid-based approach reached 0.9339 (P < 0.0001). We calculated the concentration of female fetuses and obtained remarkable experimental results. We came up with two approaches for calculating the fetal DNA fraction of female fetuses. It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.

Two approaches for calculating female fetal DNA fraction in noninvasive prenatal testing based on size analysis of maternal DNA fragments

The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing (NIPT). The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation. It is important to measure fetal DNA fraction before NIPT. However, there is still little research on how to calculate the concentration of female fetuses. Two estimation approaches were proposed to calculate fetal DNA fraction, including the fragments size-based approach, aneuploid-based approach, which are all approaches based on chromosome segments. Based on high-throughput sequencing data, two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values, which were close to the actual values. The correlation coefficient of fragments size-based approach was 0.9243 (P < 0.0001) and the aneuploid-based approach reached 0.9339 (P < 0.0001). We calculated the concentration of female fetuses and obtained remarkable experimental results. We came up with two approaches for calculating the fetal DNA fraction of female fetuses. It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.

Mitochondrial Genome of Nonmodel Marine Metazoans by Next-Generation Sequencing (NGS).

Mitochondrial genomes (mtgenome) represent an important source of information for addressing fundamental evolutionary, phylogeographic, systematic, and ecological questions in marine organisms. In the last two decades the advent of high-throughput next-generation sequencing (NGS) has provided an unprecedented possibility to access large amount of genomic data and, as such, there has been a rapid growth in mtgenome resources and studies. In particular, NGS strategies represent a great advantage for investigating nonmodel marine organisms for which no or limited genomic resources are available. Here, we describe a routinely used standardized protocol to obtain mtgenome of nonmodel marine organisms by NGS. The protocol is composed of five main steps, including DNA extraction, DNA fragmentation, library preparation, high-throughput sequencing, and bioinformatic analyses. Each of the first three steps is followed by size/quality and concentration validations. The advantages of the described protocol rely on the assumption that no a priori information on mtgenome of the studied organism is needed and on its versatility as researchers may choose several kits for DNA extraction and library preparation and adopt different methods for DNA fragmentation depending on their needs, experience, and suppliers.

Evaluation of cytotoxic properties of two fluorescent fac-Re(CO)3 complexes bearing an N,N-bidentate benzimidazole coligand.

The reaction between 1H-benzimidazol-2-ylmethyl-(N-aryl)amine derivatives (LR) and [ReBr(CO)5] afforded octahedral Re(i) complexes of the general formula of [ReBr(CO)3LR] (R = 4-Cl and 4-COOCH3). The Re(i) complexes were screened for their potential cytotoxicity against three malignant cell lines and one normal cell line of different origins. The solvatochromic characteristics of the complexes were examined by UV/vis. spectroscopy with the aid of time-dependent density functional theory calculations. Strong autofluorescence emission can be seen in the two Re(i) complexes between 460 and 488 nm. They appeared to accumulate inside intercellular connections and surrounding cellular membranes. The substances gathered also, along the cell membrane, waiting for their entry. The mode of cell death staining and the DNA fragmentation analysis revealed that the 4-Cl complex showed increased apoptotic changes in the MCF-7, and the Caco-2 cell line, while the HepG2 cell line showed little apoptotic changes.

LIFGO: A modular laser-induced fluorescence detection system based on plug-in blocks

In this work, a laser-induced fluorescence (LIF) detection system built in a modular assembling mode was developed based on commercial LEGO blocks and 3D printed blocks. We designed and fabricated a variety of 3D printed building blocks fixed with optical components, including laser light source, filters, lens, dichroic mirror, photodiode detector, and control circuits. Utilizing the relatively high positioning precision of the plug-in blocks, a modular construction strategy was adopted using the flexible plug-in combination of the blocks to build a highly sensitive laser-induced fluorescence detection system, LIFGO. The LIFGO system has a simple structure which could be constructed by inexperienced users within 3 h. We optimized the structure and tested the performance of the LIFGO system, and its detection limits for sodium fluorescein solution in 100 μm i.d. and 250 μm i.d. capillaries were 7 nM and 0.9 nM, respectively. Based on the LIFGO system, we also built a simple capillary electrophoresis (CE) system and applied it to the analysis of DNA fragments to demonstrate its application possibility in biochemical analysis. The separation of 7 fragments in DL500 DNA markers were completed in 600 s. Because of the features of low cost (less than $100) and easy-to-build construction, we introduced the LIFGO system to the experimental teaching of instrumental analysis for undergraduate students. The modular construction form of the LIF detection system greatly reduces the threshold of instrument construction, which is conducive to the popularization of the LIF detection technique in routine laboratories as well as the reform of experimental teaching mode.

Genetic Diversity and Population Structure of Moroccan Isolates Belong to Alternaria spp. Causing Black Rot and Brown Spot in Citrus.

Alternaria alternata is one of the most important fungi causing various diseases on citrus worldwide. In Morocco, Alternaria black rot (ABR) and Alternaria brown spot (ABS) are two major diseases causing serious losses in commercial cultivars of citrus. The aim of the present work was to study the genetic diversity and the population structure of isolates belonging to sect. Alternaria obtained from infected citrus fruits, collected from seven provinces at different locations in Morocco (markets, packinghouses, and orchards). Forty-five isolates were analyzed by sequence-related amplified polymorphism (SRAP) markers, and cluster analysis of DNA fragments was performed using UPGMA method and Jaccard coefficient. Cluster analysis revealed that isolates were classified in four distinct groups. AMOVA revealed also a large extent of variation within sect. Alternaria isolates (99%). The results demonstrate that no correlation was found among SRAP pattern, host, and geographical origin of these isolates. Population structure analyses showed that the Alternaria isolates from the same collection origin had almost a similar level of admixture.

Preparation, Characterization, and Pharmacological Investigation of Withaferin-A Loaded Nanosponges for Cancer Therapy; In Vitro, In Vivo and Molecular Docking Studies.

The rapidly growing global burden of cancer poses a major challenge to public health and demands a robust approach to access promising anticancer therapeutics. In parallel, nanotechnology approaches with various pharmacological properties offer efficacious clinical outcomes. The use of new artificial variants of nanosponges (NS) as a transporter of chemotherapeutic drugs to target cells has emerged as a very promising tool. Therefore, in this research, ethylcellulose (EC) NS were prepared using the ultrasonication assisted-emulsion solvent evaporation technique. Withaferin-A (WFA), an active ingredient in Withania somnifera, has been implanted into the nanospongic framework with enhanced anticancer properties. Inside the polymeric structure, WFA was efficiently entrapped (85 ± 11%). The drug (WFA) was found to be stable within polymeric nanosponges, as demonstrated by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) studies. The WFA-NS had a diameter of 117 ± 4 nm and zeta potential of −39.02 ± 5.71 mV with a polydispersity index (PDI) of 0.419 ± 0.073. In addition, scanning electron microscopy (SEM) revealed the porous surface texture of WFA-NS. In vitro anticancer activity (SRB assay) results showed that WFA–NS exhibited almost twice the anticancer efficacy against MCF-7 cells (IC50 = 1.57 ± 0.091 µM), as quantified by flow cytometry and comet tests. Moreover, fluorescence microscopy with DAPI staining and analysis of DNA fragmentation revealed apoptosis as a mechanism of cancer cell death. The anticancer activity of WFA-NS was further determined in vivo and results were compared to cisplatin. The anticancer activity of WFA-NS was further investigated in vivo, and the data were consistent to those obtained with cisplatin. At Day 10, WFA-NS (10 mg/kg) significantly reduced tumour volume to 72 ± 6%, which was comparable to cisplatin (10 mg/kg), which reduced tumour volume to 78 ± 8%. Finally, the outcomes of molecular modeling (in silico) also suggested that WFA established a stable connection with nanosponges, generating persistent hydrophobic contacts (polar and nonpolar) and helping with the attractive delayed-release features of the formulation. Collectively, all the findings support the use of WFA in nanosponges as a prototype for cancer treatment, and opened up new avenues for increasing the efficacy of natural product-derived medications.

Paleo-diatom composition from Santa Barbara Basin deep-sea sediments: a comparison of 18S-V9 and diat-rbcL metabarcoding vs shotgun metagenomics

Sedimentary ancient DNA (sedaDNA) analyses are increasingly used to reconstruct marine ecosystems. The majority of marine sedaDNA studies use a metabarcoding approach (extraction and analysis of specific DNA fragments of a defined length), targeting short taxonomic marker genes. Promising examples are 18S-V9 rRNA (~121–130 base pairs, bp) and diat-rbcL (76 bp), targeting eukaryotes and diatoms, respectively. However, it remains unknown how 18S-V9 and diat-rbcL derived compositional profiles compare to metagenomic shotgun data, the preferred method for ancient DNA analyses as amplification biases are minimised. We extracted DNA from five Santa Barbara Basin sediment samples (up to ~11 000 years old) and applied both a metabarcoding (18S-V9 rRNA, diat-rbcL) and a metagenomic shotgun approach to (i) compare eukaryote, especially diatom, composition, and (ii) assess sequence length and database related biases. Eukaryote composition differed considerably between shotgun and metabarcoding data, which was related to differences in read lengths (~112 and ~161 bp, respectively), and overamplification of short reads in metabarcoding data. Diatom composition was influenced by reference bias that was exacerbated in metabarcoding data and characterised by increased representation of Chaetoceros, Thalassiosira and Pseudo-nitzschia. Our results are relevant to sedaDNA studies aiming to accurately characterise paleo-ecosystems from either metabarcoding or metagenomic data.

NFATC2 regulates Targets of MYC Signaling in MLL-AF9 AML

Background: Acute myeloid leukemia (AML) arises due to an accumulation of genetic lesions within myeloid progenitors and oncogenic transformation is often characterised by disordered transcription. Recently the histone lysine demethylase KDM4A was shown to be essential for AML blast survival and self-renewal. shRNA knockdown (KD) of KDM4A led to downregulated expression of the transcription factor NFATC2 an MLL-AF9 AML model, suggesting that it is a key target of KDM4A oncogenic function. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors control cell cycle genes and self-renewal pathways in hematopoietic tissues and are well-defined as oncogenic regulators in various malignancies. NFATs have recently been attributed roles in the development of FLT3 ITD AML and resistance to tyrosine kinase inhibitors (TKIs) in myeloid leukemias but there is little evidence detailing the role(s) of NFATC2 specifically in AML. We hypothesized that NFATc2 activity is essential for the survival of AML cells and the oncogenic transcriptional networks within these.Aims: To determine if AML cells are dependent on NFATC2 for survival and to elucidate the transcriptional and binding targets of NFATc2 in AML.Methods: NFATC2 was depleted using shRNA KD in numerous cell line models of AML and putative transcriptional targets were elucidated using RNA-seq following KD. Binding targets of NFATc2 were determined using ChIP-seq. Transcriptomic targets of NFATc2 were validated using the Fluidigm Biomark multiplex PCR system and real time quantitative PCR.Results: KD of NFATC2 significantly impaired the colony forming capacity and expansion in liquid cultures of AML cell lines from diverse (cyto)genetic backgrounds. MLL-AF9/TP53 mut THP-1 cells showed reduced entry to the S-phase of the cell cycle and downregulation of cyclin D1 following NFATC2 depletion, suggesting that NFATC2 is critical for cell cycle progression in these cells. Overexpression of human NFATC2 in THP-1 led to an increased rate of cell growth.RNA-seq analysis of THP-1 cells with NFATC2 KD revealed >20 genes with deregulated expression (FDR<0.1), which have been validated using PCR methods. Overexpression of human NFATC2 resulted in significant deregulation of 9 of these genes (FDR<0.1), defining a subset of genes which may regulate the observed phenotype. Additionally, these top genes were not all differentially regulated in other MLL-AF9 AML cell lines MOLM-13 and NOMO-1 following NFATC2 KD. Finally, in THP-1, gene set enrichment analysis (GSEA) of sequencing results revealed that targets of MYC and calmodulin kinase STK33 were enriched within the genes perturbed by NFATC2 depletion. Targets of MYC signaling were validated by PCR in THP-1 but were not found to be deregulated in MOLM-13 following NFATC2 KD.ChIP-seq analysis of NFATc2 binding in THP-1 cells showed that >30% of NFATc2 targets were at promoter regions within 5kb of the transcription start site. Motif analysis of precipitated DNA fragments discovered two novel motifs which were enriched at NFATc2 binding sites (p<0.0001).Discussion: NFATC2 was found to be essential for expansion of AML cells in various cell line models. In the MLL-AF9 driven THP-1 model a number of putative transcriptional and genomic targets were defined, which include novel targets not previously described in AML pathogenesis and targets of MYC, an established oncogenic protein in AML. The differing expression profiles observed across AML cell lines of diverse (cyto)genetic backgrounds with NFATC2 KD suggest that the regulatory targets of NFATc2 vary depending on the cellular signaling landscape. Together with the finding that NFATC2 is indispensable for AML cell survival this study has elucidated novel roles(s) for NFATC2 in AML oncogenesis. DisclosuresMassett: Kymab Ltd: Current Employment. Huang: Janssen Pharmaceutical Companies of Johnson & Johnson (China): Current Employment.

New mixed ligand copper(II) hydrazone‐based complexes: Synthesis, characterization, crystal structure, DNA/RNA/BSA binding, in vitro anticancer, apoptotic activity, and cell cycle analysis

AbstractFour new mixed ligand copper(II) complexes with the general formula [Cu(L)(B)]n (1)–(4) (H2L = dehydroacetic acid benzoyl hydrazone Schiff base, B = H2O (1), DMSO (2), imidazole (imz) (3), n = 1) or pyridine (py) (4), n = 2) were synthesized. Characterization was performed by elemental analyses, FTIR, 1H NMR, electron paramagnetic resonance (EPR), ESI‐mass, and UV–visible) spectroscopy, together with magnetic susceptibility and molar conductivity measurements. The molecular structure of (2) and (4) complexes was investigated by X‐ray crystallography. The ligand behaves as a di‐basic tridentate ONO donor, coordinating to copper(II) center via deprotonated hydroxyl group, azomethine nitrogen, and deprotonated amide oxygen in a square planar (1)–(3) /square pyramidal geometry (4). The intercalation binding mode of the compounds with calf thymus DNA (ct DNA) and yeast (tRNA) and strong static interaction with bovine serum albumin (BSA) protein have been evaluated by absorption and emission spectroscopy. Further, in vitro cytotoxic activity of the compounds was examined on three human cancer cell lines; breast cancer (MCF7), colon cancer (HCT116), liver cancer (HepG2), and a normal lung fibroblast cell line (WI38) using cisplatin as a standard. Based on IC50 and therapeutic indices (TI), complexes (3) and (4) showed better activity than that of cisplatin. So both complexes were subjected for further studies viz, DNA fragmentation, cell apoptosis (annexin), and cell cycle analysis. They induced DNA fragmentation, and the HCT116 and HePG2 cell death were induced using (3) and (4) complexes, respectively, by apoptosis and cell cycle arrest at the G2/M phase.

Homozygosity of short VNTR lengths in the CEL gene may confer susceptibility to idiopathic chronic pancreatitis

ObjectiveThe carboxyl-ester lipase (CEL) gene contains a variable number of tandem repeats (VNTR) region. It remains unclear whether the number of repeats in the CEL VNTR is related to the risk of pancreatic diseases. The aim of this study was to investigate whether CEL VNTR length is associated with idiopathic chronic pancreatitis (ICP), alcoholic chronic pancreatitis (ACP), or pancreatic cancer in a cohort of Chinese patients. MethodsCEL VNTRs were genotyped in patients diagnosed with ICP (n = 771), ACP (n = 222), or pancreatic cancer (n = 263), and in healthy controls (n = 927). CEL VNTR lengths were determined using a screening method combining PCR and DNA fragment analysis. ResultsOverall, the CEL VNTR lengths ranged from 5 to 22 repeats, with the 16-repeat allele (‘normal’ size, N) accounting for 73.82% of all observed alleles. The VNTR allele frequencies and genotype distributions were not significantly different between healthy controls and patients with ACP or pancreatic cancer. For the ICP group, allele frequencies did not differ significantly from the controls, while the frequency of the SS genotype (homozygosity for 5–15 repeats) was significantly higher in the patients (4.67%) than in the controls (1.94%) (p = 0.0014; OR = 2.47; 95% CI = 1.39–4.39). ConclusionsThere were no associations between the CEL VNTR length and ACP or pancreatic cancer. However, homozygosity for short VNTR lengths may confer susceptibility to ICP.

A systematic method for DNA fragment amplification and sequencing based on DNA indexing technology. Protocol and technical considerations.

DNA indexing is based on a presynthesized library of oligonucleotide adaptors (256 in total), named indexers, and type-IIS restriction endonucleases. It enables amplification and direct analysis of large DNA fragments with low overall redundancy and without subcloning. Here, we describe a detailed protocol for PCR-based amplification of DNA fragments followed by DNA sequencing by indexer walking and provide helpful hints on its practical use. The proposed protocol can be applied to the sequencing of plasmids, cDNA clones, and longer DNA fragments. It can also be used for gap filling at the final stage of genome sequencing projects.

Synthesis, characterization of ruthenium(II), nickel(II), palladium(II), and platinum(II) triphenylphosphine-based complexes bearing an ONS-donor chelating agent: Interaction with biomolecules, antioxidant, in vitro cytotoxic, apoptotic activity and cell cycle analysis

Four new transition metal complexes, [M(PPh3)(L)].CH3OH (M = Ni(II) (1), Pd(II) (2)) [Pt (PPh3)2(HL)]Cl (3) and [Ru(CO)(PPh3)2(L)] (4) (H2L = 2,4-dihydroxybenzaldehyde-S-methyldithiocarbazate, PPh3 = triphenylphosphine) have been synthesized and characterized by elemental analyses (C, H, N), FTIR, NMR (1H, 31P), ESI-MS and UV–visible spectroscopy. The molecular structure of (1) and (2) complexes was confirmed by single-crystal X-ray crystallography. It showed a distorted square planar geometry for both complexes around the metal center, and the H2L adopt a bi-negative tridentate chelating mode. The interaction with biomolecules viz., calf thymus DNA (ct DNA), yeast RNA (tRNA), and BSA (bovine serum albumin) was examined by both UV–visible and fluorescence spectroscopies. The antioxidant activity of all compounds is discussed on basis of DPPH• (2,2-diphenyl-1-picrylhydrazyl) scavenging activity and showed better antioxidant activity for complexes compared to the ligand. The in vitro cytotoxicity of the compounds was tested on human (breast cancer (MCF7), colon cancer (HCT116), liver cancer (HepG2), and normal lung fibroblast (WI38)) cell lines, showing that complex (1) the most potent against MCF7 and complex (4) against HCT116 cell lines based on IC50 and selective indices (SI) values. So, both complexes were chosen for further studies such as DNA fragmentation, cell apoptosis, and cell cycle analyses. Complex (1) induced MCF7 cell death by cellular apoptosis and arrest cells at S phase. Complex (4) induced HCT116 cell death predominantly by cellular necrosis and arrested cell division at G2/M phase due to DNA damage.