Cleavage Frequency Research Articles

P-150 Comparison of morphokinetic parameters of embryos generated from Capacitation In-vitro Maturation and Controlled ovarian stimulation-ICSI

Abstract Study question Is there any difference in the embryo morphokinetic parameters between embryos generated from Capacitation – In-vitro maturation (CAPA-IVM) and Controlled ovarian stimulation – ICSI (COS-ICSI)? Summary answer Embryos generated from CAPA-IVM cycles reached tPNa, t2, t4, t8, tSC, tM, tSB, and tB significantly later than COS-ICSI embryos. What is known already IVF with ovarian hyperstimulation has limitations in some subgroups of women at high risk of ovarian stimulation, such as those with polycystic ovary syndrome. IVM may be a feasible alternative to IVF in women with a high AFC. A recent randomized controlled trial produced statistically inconclusive findings regarding the noninferiority of the CAPA-IVM technique compared with COS-ICSI. Moreover, there is a lack of studies investigating the impact of CAPA-IVM on the morphokinetic parameters of embryos. Study design, size, duration This is a retrospective cohort study conducted from March 2021 to March 2022. The study included 115 couples (68 couples used CAPA-IVM, 47 couples used COS-ICSI) who had their embryos cultured to the blastocyst stage using a time-lapse system. Participants/materials, setting, methods The study was conducted at My Duc Phu Nhuan hospital, Ho Chi Minh City, Vietnam. Couples aged ≤ 40 years old, had blastocyst culture using time-lapse system were included in the study. Couples used oocyte or sperm donation, PGT treatment, had severe male factors, and fertilization failure in previous cycles were excluded. We analyzed 392 blastocysts from 115 couples for morphokinetic parameters including tPNa, tPNf, the onset of t2 to t8, tSC, tM, tSB, and tB. Main results and the role of chance There was no difference in tPNf, t3, and t5 between embryos from CAPA-IVM and COS-ICSI. However, embryos generated from CAPA-IVM reached tPNa, t2, t4, t8, tSC, tM, tSB, and tB significantly later than COS-ICSI embryos [tPNa (9.3 2.0 vs 8.0 1.9; P < 0.001), t2 (27.4 3.7 vs. 25.5 3.1; P < 0.001), t4 (38.7 4.5 vs. 37.3 4.3; P = 0.004), t8 (58.9 9.2 vs. 56.3 8.4; P = 0.011), tSC (76.2 11.1 vs. 71.4 10.4; P < 0.001), tM (90.4 8.6 vs. 85.5 10.0; P < 0.001); tSB (101 8.1 vs. 97.1 8.3; P < 0.001) and tB (108 7.6 vs 106 7.7; P < 0.05)]. The frequency of direct cleavage in the first and the second cell division cycle was similar between two groups (58.6% vs. 30.0%; 41.4% vs. 70%, respectively, P > 0.05). Limitations, reasons for caution The retrospective cohort study design could limit the ability to eliminate sources of bias. The characteristics of participants may influence the study findings, i.e. women in the CAPA-IVM group were PCOS while women in the COS-ICSI were not. In addition, the study population's single-ethnicity nature limits the results' external generalizability. Wider implications of the findings This study found that embryos from CAPA-IVM reached tPNa, tPNf, t2, t4, t8, tSC, tM, tSB, and tB significantly later than COS-ICSI. The findings of this study provide evidence for further studies to evaluate CAPA-IVM embryos and to improve the CAPA-IVM culture system. Trial registration number Not Applicable

Direct cleavage during the first mitosis is a sign of abnormal fertilization in cattle

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.

The Impact of Low-Temperature Inactivation of Protease AprX from Pseudomonas on Its Proteolytic Capacity and Specificity: A Peptidomic Study

The destabilization of UHT milk during its shelf life can be promoted by the residual proteolytic activity attributed to the protease AprX from Pseudomonas. To better understand the hydrolysis patterns of AprX, and to evaluate the feasibility of using low-temperature inactivation (LTI) for AprX, the release of peptides through AprX activity on milk proteins was examined using an LC-MS/MS-based peptidomic analysis. Milk samples were either directly incubated to be hydrolyzed by AprX, or preheated under LTI conditions (60 °C for 15 min) and then incubated. Peptides and parent proteins (the proteins from which the peptides originated) were identified and quantified. The peptides were mapped and the cleavage frequency of amino acids in the P1/P1′ positions was analyzed, after which the influence of LTI and the potential bitterness of the formed peptides were determined. Our results showed that a total of 2488 peptides were identified from 48 parent proteins, with the most abundant peptides originating from κ-casein and β-casein. AprX may also non-specifically hydrolyze other proteins in milk. Except for decreasing the bitterness potential in skim UHT milk, LTI did not significantly reduce the AprX-induced hydrolysis of milk proteins. Therefore, the inactivation of AprX by LTI may not be feasible in UHT milk production.

An Efficient Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein 9 Mutagenesis System for Oil Palm (Elaeis guineensis).

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5–83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.

Colorado potato beetle alpha-amylase: Purification, action pattern and subsite mapping for exploration of active centre

Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the active centre of Colorado potato beetle (Leptinotarsa decemlineata) α-amylase (LdAmy). Bond cleavage frequency values for LdAmy were determined by HPLC product analysis on a chromophore labelled maltooligomer substrate series. Binding energies between amino acid moieties of subsites and glucose residues of substrate were calculated. Active site contains six subsites in the binding region of LdAmy; four glycone- (−4, −3, −2, −1) and two aglycone-binding sites (+1, +2). Subsite map calculation resulted in apparent binding energies −11.8 and − 11.0 kJ/mol for subsites (+2) and (−3), respectively, which revealed very favorable interactions at these positions. Structures of binding sites of LdAmy and mammalian α-amylases show similarity, but there are variations in the binding energies at subsite (−2) and (−4). Differences were interpreted by comparison of amino acid sequences of human salivary α-amylase (HSA) and porcine pancreatic α-amylase (PPA) and two insect (Leptinotarsa decemlineata and Tenebrio molitor) enzymes. The observed substitution of positively charged His305 in HSA at subsite (−2) with an acidic Asp in LdAmy in the same position may explain the obtained energy reduction.

PhosphoShield: Improving Trypsin Digestion of Phosphoproteins by Shielding the Negatively Charged Phosphate Moiety.

Protein phosphorylation is a post-translational modification that is essential to cellular signaling, cellular function, and associated disease progression. Bottom-up proteomics based on enzymatic digestion is the most widely used approach for identifying and quantifying phosphoproteins in complex biological samples. Researchers have largely optimized the experimental conditions for trypsin digestion, and it is now a routine procedure. However, trypsin digestion is impaired by the presence of phosphorylated residues in the protein sequence. This impairment arises from the fact that there are commonly salt bridges between a negatively charged phosphate group and the side chain of protonated arginine or lysine. On average, 55% of all phosphopeptides have their phosphosites located less than three amino acid residues from a cleavage site. Salt bridges reduce the cleavage accessibility for trypsin by masking the basic site chain groups of arginine and lysine. Thus, there are frequent missed cleavages in the vicinity of phosphorylation sites, thereby lessening both the depth of proteome coverage and the quantification accuracy of phosphoproteomics. In this work, we propose a method termed PhosphoShield to mitigate salt bridge formation by adding a digallium complex that exhibits a high binding affinity to the phosphate group. We tested our method using quantitative mass spectrometry analysis of the phosphoproteome of human liver cancer cells (HepG2). PhosphoShield enhances the cleavage frequency of at least 17% of tryptic phosphopeptides having cleavage sites close to the phosphate group.

A Frequency-Based Approach to Predict the Low-Energy Collision-Induced Dissociation Fragmentation Spectra.

Peptide identification algorithms rely on the comparison between the experimental tandem mass spectrometry spectrum and the theoretical spectrum to identify a peptide from the tandem mass spectra. Hence, it is important to understand the fragmentation process and predict the tandem mass spectra for high-throughput proteomics research. In this study, a novel method was developed to predict the theoretical ion trap collision-induced dissociation (CID) tandem mass spectra of the singly, doubly, and triply charged tryptic peptides. The fragmentation statistics of the ion trap CID spectra were used to predict the theoretical tandem mass spectra of the peptide sequence. The study estimated the relative cleavage frequency for each pair of adjacent amino acids along the peptide length. The study showed that the cleavage frequency can be directly used to predict the tandem mass spectra. The predicted spectra show a high correlation with the experimental spectra used in this study; 99.73% of the high-quality reference spectra have correlation scores greater than 0.8. The new method predicts the theoretical spectrum and correlates significantly better with the experimental spectrum as compared to the existing spectrum prediction tools OpenMS_Simulator, MS2PIP, and MS2PBPI, where only 80, 85.76, and 85.80% of the spectral count, respectively, has a correlation score greater than 0.8.

Temperature–Salinity Dependence of the Mitotic Interval (0) for Chromosomal Manipulation in Marine Medaka, Oryzias dancena

Eggs of the marine medaka, Oryzias dancena were collected and fertilized to observe the temperature-salinity-related cleavage rates and mitotic intervals (0). We investigated the relationship between 0 and five different water temperatures (18, 22, 26, 30, and 34°C) and four different salinities (0, 10, 20, and 30 ppt NaCl). As the water temperature increased, the slope of the first cleavage frequency increased with elapsed time after fertilization, and approximately 30% of fertilized eggs reached the first cleavage frequency at every 15-minute intervals. However, the slope of the first cleavage frequency did not differ significantly between 0 ppt and the other salinities (10, 20, or 30 ppt). At higher water temperatures, the eggs developed more rapidly, but no other developmental process was affected. At higher salinity, the hatching rates of the eggs decreased, and the hatching times were delayed. There was a strong negative correlation between 0 and water temperature as shown in this equation (Y = –1.137X + 75.47, R2 = 0.977, where Y is 0 and X is water temperature). At a constant water temperature, 0 did not differ significantly in 0, 10, 20, and 30 ppt NaCl.

22 The zona pellucida is required for normal development of invitro-produced cat embryos

Limited information have been reported regarding to the role of the zona pellucida during embryo development in the domestic cat. In the present research we compared invitro and invivo development of cat embryos generated with versus without the zona pellucida. Embryos were produced by IVF and cultured up to blastocyst stage accordingly to experimental group: 1) with intact zona pellucida (ZI) and 2) without zona pellucida (ZF). Ovaries of domestic cats were collected by ovariohysterectomy and cumulus-oocyte complexes (COCs) were recovered by slicing. For IVM, COCs were cultured in supplemented medium-199 for 26-28h in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C. For IVF, 1.5-2.5×106 epididymal spermatozoa/mL were incubated with 20-30 COCs in supplemented Tyrode's albumin lactate pyruvate medium for 18h in a 5% CO2 atmosphere at 38.5°C. The zona pellucida of the presumed zygotes was removed by 2-4min of incubation in 2mgmL−1 pronase. In ZI and ZF groups, embryo culture was done in 4-well dishes; in the ZF group the well of the well system was used. The IVC was done in supplemented synthetic oviductal fluid medium, in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C for 8 days. The frequencies of cleavage and development to the morula and blastocyst stages were determined. The relative expression of the pluripotency markers OCT4, SOX2, and NANOG was evaluated by RT-qPCR in the blastocysts using the standard curve method. The SDHA gene was used as internal control. Additionally, the invivo development was evaluated. For this, cat recipients were synchronized with 200IU equine chorionic gonadotrophin followed by 100IU human chorionic gonadotrophin 4 days later, and embryo transfers (ET) were made by mid-ventral laparotomy. The Wilcoxon nonparametric test was used to evaluate the developmental competence and the gene expression analysis. Nine and six replicates were performed in the ZI and ZF groups, respectively. No differences were observed between the ZI and ZF groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morulae rate: 115/155 (74.2%) and 68/116 (58.6%), and blastocysts rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P>0.05). No differences were observed in the expression of OCT4 (P>0.05). However, the expression of SOX2 and NANOG was lower in blastocysts from the ZF group than in those from the ZI group (P<0.05). Finally, three ET were done at the blastocyst stage in both groups. In the ZI group, 8-16 Day 7 blastocysts were transferred per cat, two pregnancies were obtained with one gestational sac in each cat (2/3, 66.6%), and one live kitten was born at Day 64 of pregnancy (1/3, 33.3%). In the ZF group, 7-14 were transferred per cat, and no pregnancies were obtained after ET. In conclusion, in the domestic cat, zona pellucida removal at the presumed zygote stage did not affect invitro development to blastocyst but affect negatively the expression of SOX2 and NANOG. However, the low survival after ET of ZI embryos is a possible indicator that embryo quality might be affecting survival rates. Nonetheless, these results add credence to previous ET studies that provide indirect evidence of a crucial role of the zona pellucida for successful implantation of cat embryos.

КЛИНИЧЕСКАЯ ОЦЕНКА ФИКСИРОВАННЫХ ЗУБНЫХ ПРОТЕЗОВ НА КОБАЛЬТ-ХРОМОВОЙ ОСНОВЕ

Subject. Ceramic-metal prostheses are distinguished by high wear resistance, resistance to deformation, and tight fit to the gums. However, there are relatively large differences in determining the success / survival of cobalt chromium based restorations. The article presents the results of a study of cobalt-chromium fixed dentures.
 Objectives — evaluate the effectiveness of cobalt-chromium fixed dentures made using the direct metal laser sintering (DMLS) technique. 
 Material and methods. 32 patients (mean age 41.8 ± 2.46 years) were found to have 54 fixed cobalt-chromium fixed bridges made using direct laser sintering of metals. Dentures were evaluated between 6, 12, 24, 36 months according to G. Ryge criteria. The survival rate of restorations is calculated according to Kaplan-Meyer. 
 Results. Assessment of fixed prostheses revealed the absence of criteria C and D in all 3 categories. A comparative analysis of prostheses showed no change in the period 6, 12, 24 months. After 36 months, chips that did not violate the function of the prosthesis were revealed in 5.5 % of cases, visible signs of a cleft in the category of “marginal adaptation” in 1.8 % of cases, edge discoloration of the surface in 3.7 % of cases. Radiographically, 1 patient showed signs of incipient proximal caries and periapical changes, respectively. During the study period of 6-24 months. survival rate was 1.0. The survival of the restoration over a 3-year period was 88.9 %. 
 Conclusion. The frequency of cleavage was 5.5 %, which is comparable to the usual performance of ceramic-metal restorations. The visible signs of a cleft in the regional adaptation was 1.8 %, and the regional color mismatch was 3.7 %. 1.8 % of restoration was detected. Signs of incipient proximal caries were 1.8 %, periapical changes — 1.8 %. Survival was 88.9 %.

Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency.

Defining the variables that impact the specificity of CRISPR/Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targetedloci has also been hypothesized to be an important factor.In this study, functional data from two genome-wide assays,genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for invitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors thatmodulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency.

Evaluation and Reduction of CRISPR Off-Target Cleavage Events.

Introduction of CRISPR/Cas9 methods (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) have led to a huge surge in the use of precision genome editing for research applications. Translational medical efforts are likewise rapidly progressing, and Phase I clinical trials using these techniques have already started. As with any new technology that is applied to medical therapeutics, risks must be carefully defined and steps taken to mitigate side effects wherever possible. Effective methods are now available that permit identification of off-target cleavage events, a major class of potential side effects seen in mammalian genome editing. Off-target prediction algorithms are improving and have utility, but are insufficient to use alone. Empiric methods to define the off-target profile must also be used. Once defined, the frequency of off-target cleavage can be minimized using methods that limit the duration of exposure of the genome to the active genome editing complex, for example, using the ribonucleoprotein (RNP) approach. In addition, Cas9 mutants have been developed that markedly reduce the rate of off-target cleavage compared to the wild-type enzyme. Use of these new tools should become standard practice for medical applications.

Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells

BackgroundIt has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC.MethodsPotential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed.ResultsIn the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient.ConclusionsOur findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.

Development of feline embryos produced by Piezo-actuated intracytoplasmic sperm injection of elongated spermatids.

Piezo-actuated intracytoplasmic sperm injection (Piezo-ICSI) is used as an efficient in vitro fertilization method with various animals. With this method, elongated spermatids are collected from testicular tissues and are easier to obtain from animals that unexpectedly die than ejaculate sperm. Additionally, elongated spermatid injection often results in the development of embryos and offspring. To develop assisted reproductive techniques (ARTs) for domestic cats, we examined the effects of oocyte activation on cleavage and embryo development after Piezo-ICSI with motile sperm (experiment 1) and after Piezo-ICSI with either testicular sperm or elongated spermatids (experiment 2). In experiment 1, the proportions of cleaved embryos, morulas, and blastocysts following Piezo-ICSI with ethanol activation were significantly higher (P < 0.05) than in the non-activated groups. However, the proportion of blastocysts and the blastocyst quality did not differ significantly (P > 0.05) between the ethanol-activated and non-activated groups. In experiment 2, the cleavage frequencies of oocytes after Piezo-ICSI of testicular sperm or elongated spermatids and ethanol activation were higher (P < 0.05) than that of oocytes in the non-activated group, but the occurrence of blastocyst formation and quality of blastocysts did not differ between the activated and non-activated groups. In summary, cat embryos can be produced by Piezo-actuated microinjection of elongated spermatids. Ethanol activation increased the frequency of cleavage, but it affected neither the occurrence of blastocyst development nor the quality of blastocysts. These results represent an expansion in the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.

Matrix association region/scaffold attachment region (MAR/SAR) sequence: its vital role in mediating chromosome breakages in nasopharyngeal epithelial cells via oxidative stress-induced apoptosis

BackgroundOxidative stress is known to be involved in most of the aetiological factors of nasopharyngeal carcinoma (NPC). Cells that are under oxidative stress may undergo apoptosis. We have previously demonstrated that oxidative stress-induced apoptosis could be a potential mechanism mediating chromosome breakages in nasopharyngeal epithelial cells. Additionally, caspase-activated DNase (CAD) may be the vital player in mediating the chromosomal breakages during oxidative stress-induced apoptosis. Chromosomal breakage occurs during apoptosis and chromosome rearrangement. Chromosomal breakages tend to cluster in certain regions, such as matrix association region/scaffold attachment region (MAR/SAR). We hypothesised that oxidative stress-induced apoptosis may result in chromosome breaks preferentially at the MAR/SAR sites. The AF9 gene at 9p22 was targeted in this study because 9p22 is a deletion site commonly found in NPC.ResultsBy using MAR/SAR recognition signature (MRS), potential MAR/SAR sites were predicted in the AF9 gene. The predicted MAR/SAR sites precisely match to the experimentally determined MAR/SARs. Hydrogen peroxide (H2O2) was used to induce apoptosis in normal nasopharyngeal epithelial cells (NP69) and NPC cells (HK1). Nested inverse polymerase chain reaction was employed to identify the AF9 gene cleavages. In the SAR region, the gene cleavage frequency of H2O2-treated cells was significantly higher than that of the non-treated cells. A few chromosomal breakages were detected within the AF9 region which was previously found to be involved in the mixed lineage leukaemia (MLL)-AF9 translocation in an acute lymphoblastic leukaemia patient. As for the non-SAR region, no significant difference in the gene cleavage frequency was found between the untreated control and H2O2-treated cells. Furthermore, H2O2-induced cleavages within the SAR region were reduced by caspase-3 inhibitor, which indirectly inhibits CAD.ConclusionsThese results reaffirm our previous findings that oxidative stress-induced apoptosis could be one of the potential mechanisms underlying chromosome breakages in nasopharyngeal epithelial cells. MAR/SAR may play a vital role in defining the location of chromosomal breakages mediated by oxidative stress-induced apoptosis, where CAD is the major nuclease.

Structural interpretation of DNA-protein hydroxyl-radical footprinting experiments with high resolution using HYDROID.

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.

Characterization of Vv-miR156: Vv-SPL pairs involved in the modulation of grape berry development and ripening.

SPL is a plant-specific transcription factor family. Many researchers reported that SPL members targeted by miR156s could play crucial roles in the modulation of plant growth and development. Although there are similar reports on grapes, till now little is known about grape berry development and ripening. To gain more insight into how grape miR156s (Vv-miR156s) modulated the above given processes of grape berries by mediating their target gene Vv-SPLs, here we identified the precise sequences of Vv-miR156s in 'Giant Rose' grape berries, predicted their potential targets, and revealed that the matching degree of various Vv-miR156: Vv-SPL pairs exhibited some discrepancy, implying the divergence of their interaction. Subsequently, we also discovered similar motifs such as ABRE, CGTCA and ERE, which are more specific to berry development and ripening, within the promoters of both Vv-MIR156s and Vv-SPLs. With berry development and ripening, meanwhile, Vv-miR156a, b/c/d, e and f/g/i exhibited an overall increasing expression trend, while their targets showed opposite trends at the corresponding stages. Additionally, exogenous ABA and NAA application promoted or curbed the expression of Vv-miR156s to some extent, before grape berry ripening stage. The cleavage products, sites and frequencies of Vv-miR156a, b/c/d, e, f/g/i and their respective targets (Vv-SPL2, 9, 10, 16) during grape berry development and ripening process were validated by our developed PPM-RACE and modified RLM-RACE together with qRT-PCR, which demonstrated that Vv-miR156s can be involved in the modulation of grape berry development and ripening process by mediating the expression of Vv-SPL2, 9, 10, 16. Our findings lay an important foundation for further recognizing their functions in grape berries, and enrich the knowledge of the regulatory mechanism of miRNA-mediated grape berry development and ripening.

Randomized DNA libraries construction tool: a new 3-bp \u2018frequent cutter\u2019 TthHB27I/sinefungin endonuclease with chemically-induced specificity

BackgroundAcoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN.ResultsIn this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5’-CAARCA-3′ is converted into a combined 3.2–3.0-bp ‘site’ or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed.ConclusionsIn the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA.

Temperature Dependent of Mitotic Interval for Grass Puffer, Takifugu niphobles.

ABSTRACTThe objective of this study was to determine the mitotic intervals (τ0) of two consecutive cell divisions and synchronous embryonic cleavage in grass puffer, Takifugu niphobles at different water temperatures (18, 20, 22, and 24℃). The color of the fertilized egg was light yellowish. The egg type was demersal and unadhesive. Egg weight was 0.09±0.002 mg. The sizes of unfertilized eggs were smaller than fertilized eggs in major axis and minor axis at 20℃ (p<0.05). The size of the fertilized egg of 18℃ water temperature group at the blastodisc stage was the smallest (p<0.05), but no significant differences were observed in the other water temperatures group except 18℃ water temperature group (p>0.05). The first cleavage stages at 18, 20, 22, and 24℃ were at 75, 90, 105, and 120 mins, respectively. As water temperature was increased, embryonic development and formation time of the first cleavage furrow were accelerated. There were negative correlation between τ0 and water temperature for grass puffer (Y=–1.225X+70.05, R2=0.988, n=10, where Y was τ0 and X was temperature). This study confirmed that successful hatching of grass puffer was related to water temperature. Chromosome manipulation will be helpful for this species using cleavage frequency and τ0.

Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.