Successfully introduce maize DNA fragments into rice

The maize DNA fragments was successfully incorporated into rice by Associate Prof WAN Wenju’s research team at Hunan Agricultural College, Changsha, China. The new gene transferring rice is named Genetic Engineered Rice (GER) line. The team put dry embryo of rice cultivar Xiangzaoxian 8 in the solution containing maize DNA using a modified method of embryo soaking to introduce the exotic DNA into the rice embryo. By selections through successive generation, promising rice lines were finally developed from progenies having required variations. DNA molecular test showed that the lines have already got some maize DNA fragments. The line GER-1 is of the most productive among the new rice lines. It has strong tillering ability and large sized panicles with an average number of 200 grains per panicle and 80% seed-setting rate. It gives a

Analysis of parental strain DNA fragments existing in GEMs-Fhhh

There were 6 target DNA fragments of the three parental strains existing in the cell of GEMs(genetically engineered microorganism strain) Fhhh measured in this research by PCR(polymerase chain reaction). The determination showed that GEMs Fhhh contained all the 6 target DNA fragments, mnp 1, mnp 2、 lip 1、 lip 2, FLO 1 and 16S rDNA, and had the molecular genetic stability. Meanwhile the PCR production of each parental strain could only had its target DNA fragments and was different from each other. It may illustrate that the technique of the inter kingdom protoplast fusion for the construction of GEMs Fhhh through the process of intercellular gene recombination could be used as a reliable bioengineering technique to create the specific functional stain for the pollution control.

The relationship between DNA fragmentation and the intensity of morphologically abnormal human spermatozoa

Objective:To determine the relationship between teratozoospermia and sperm DNA fragmentation(SDF)in the human ejaculate.Methods:This retrospective study included 100 normozoospermic men as a control cohort(abnormal forms>14%),210 patients with a high level of abnormal forms(≤4%)and 65 patients presenting with a moderate level of abnormal forms(>4%to≤14%)based on the World Health Organization definitions.Sperm morphology was assessed using bright field microscopy.Sperm DNA fragmentation was assessed using the sperm chromatin dispersion assay.Non-parametric analyses were conducted to examine the relationship between abnormal sperm morphology and sperm DNA fragmentation;receiver operating characteristic(ROC)analyses were conducted to assess sensitivity and specificity of this relationship.Results:A correlation analysis revealed that the higher the proportion of abnormal spermatozoa in the ejaculate,the higher the level of SDF(Spearman's Rho=-0.230;P<0.001).Significant differences in the proportion of SDF were found when all cohorts were compared(P<0.001);these significant differences were also retained when the different cohorts were compared pairwise.ROC analysis showed a moderate but significant predictive value for SDF to differentiate patients with different levels of teratozoospemia.Conclusions:Although analysis of a more continuous range of values for teratozoospermia would help further clarify any causal relationship with SDF,there is clearly a synergistic or coincident affiliation between these variables that needs to be acknowledged by the clinician when interpreting the spermiogram.

Efficient de novo assembly and modification of large DNA fragments

Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes.The construction of the large,gigabase(Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly.But for the synthesis and assembly of such large-scale genomes,the development of new or expanded methods is required.In this study,we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases(kb)or above from scratches and describe an efficient method for“scar-free”engineering of the assembled sequences.Our method,therefore,should provide a standard framework for producing long DNA molecules,which are critical materials for synthetic genomics and metabolic engineering.

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments

Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.

Human circBOULE RNAs as potential biomarkers for sperm quality and male infertility

Reliable molecular biomarkers to predict fertility remain scarce.The current study investigated the potential of testis-specific circBOULE RNAs as biomarkers for male infertility and sperm quality.Using reverse transcription-PCR and real-time reverse transcription-PCR assays,we identified seven circular RNAs from the human BOULE gene in human sperm.We observed that the expression level of circEx3-6 was significantly reduced in asthenozoospermia,while the expression levels of both circEx2-6 and circEx2-7 were decreased in terato-zoospermia,compared with the controls.Furthermore,we demonstrated that the expression level of circEx2-6 was negatively correlated with the sperm DNA fragmentation index,and the expression level of circEx2-7 was correlated with both fertilization and cleavage rates in those treated with the assisted reproductive technologies.Further functional analyses in a transgenic fly model supported the roles of circBOULE RNAs in sperm development and human male fertility.Collectively,our findings support that sperm circBOULE RNAs may serve as diagnostic biomarkers for assessing sperm motility and DNA quality.Therefore,clinical application and significance of sperm circBOULE RNAs in the assisted reproductive technologies warrant further investigation.

Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

Aim： To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality. Methods： Two early apoptotic changes in the semen of 56 men were assessed using Annexin V （AN）/propidium iodide （PI） staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential （MMP）. The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results： The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN^-/PI^- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL （＋） spermatozoa. As for the AN^-/PI^＋ fraction, we found an opposite result in comparison to AN^-/PI^- spermatozoa. The level of early apoptotic AN^＋/PI^- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN^＋/PI^＋ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL （＋） spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL （＋） spermatozoa. Conclusion： Although early apoptotic AN^＋/PI^- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa （especially, the percentage of AN^-/PI^- spermatozoa）, and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization. （Asian J Androl 2008 Mar; 10： 227-235）

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.

Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment

Over the last years, major improvements in the field of male infertility diagnosis have been achieved. The aim of this study was to determine the diagnostic usefulness of sperm DNA integrity and sperm vacuolisation for predicting outcome in infertile couples undergoing in vitroferti lisation （IVF） and intracytoplasmic sperm injection （ICSI） treatments. A cohort study from 152 infertile couples undergoing sperm DNA fragmentation and high-magnification tests prior to an assisted reproduction treatment was designed. We found that the most predictive cutoff for pregnancy was 25.5% of DNA fragmentation with a negative predictive value of 72.7% （P=0.02）. For the degree of vacuolisation, the best predictor of pregnancy was 73.5% of vacuolated sperm grades Ⅲ ＋ Ⅳ with a negative predictive value of 39.4% （P=0.09）, which was not statistically significant. In conclusion, sperm DNA fragmentation greater than 25.5% could be associated with higher probability of failure IVF treatment. Regarding the results of the sperm analysis at high magnification, they do not allow us to predict whether or not oatients will become pregnant.

DNA fragmentation in apoptosis

Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

Lethal effect and apoptotic DNA fragmentation in response of D-GalN-treated mice to bacterial LPS can be suppressed by pre-exposure to minute amount of bacterial LPS: Dual role of TNF receptor 1

AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.

Sperm nuclear histone H2B： correlation with sperm DNA denaturation and DNA stainability

Aim： To examine the relationship between sperm DNA damage and sperm nuclear histone （H2B） staining. Methods： We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone （H2B） staining and sperm chromatin integrity （assessed by sperm chromatin structure assay and expressed using the percentage of （i） DNA fragmentation index [%DFI] and （ii） high DNA stainability [%HDS）]） were evaluated. Results： Histone H2B immunocytochemistry demonstrated two nuclear staining patterns： （i） focal punctate staining; and （ii） diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men （7.7%± 4.6% vs. 1.6% ±1.2%, respectively, P 〈 0.01）. We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm %DFI （r = 0.63, P 〈 0.01） and sperm %HDS （r = 0.63, P 〈 0.01）. Conclusion： The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.

Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors？

This study compared the potential of assessing sperm DNA fragmentation （SDF） from neat semen and the subsequent swim-up （SU） procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females （n=81） were transferred embryos resulting from intracytoplasmic sperm injection （ICSI） of their partner＇s spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation （NS） and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol （P=0.000）. Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden＇s indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology （ART） procedures. Consequently, we propose a modification of the so called ＇iceberg model＇ as a possible rationale for understanding the role of SDF in reproductive outcome.

Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments.METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD)with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated,purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data.RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size,histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene.CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcinogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis.

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination （MSOME） criteria, and hyaluronic acid （HA） binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method （control）, high magnification at x6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate （2.6% vs. 1.7%; P=0.032）, with no significant difference in aneuploidy rate （0.8% vs0.7%; P=0.583）, than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls （7% aneuploidy and 26.8% DNA fragmentation rates, respectively）. In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference （0.9%） might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

The ability of sperm selection techniques to remove single- or double-strand DNA damage

A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are densitygradient centrifugation （DGC） and swim-up （SUP）. To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques （ART）, but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged （degraded） DNA, as characterized by the presence of both single- and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART.

LDFF,the large molecular weight DNA fragmentation factor,is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

Sperm DNA fragmentation does not affect the clinical outcomes in the cumulative transfers of an ICSI cycle along with blastocyst transfers in couples with normozoospermic male patients

Objective:To know whether sperm DNA fragmentation(SDF)affects the clinical outcomes in the cumulative transfers of an intracytoplasmic sperm injection(ICSI)cycle along with blastocyst transfers in couples with normozoospermic males.Methods:The study included 252 couples who underwent their first ICSI cycles along with blastocyst transfer and whose male partner semen samples were normozoospermic according to the World Health Organization 2010 criteria.All the couples were classified into two groups based on the SDF:the low SDF group(SDF≤30%,n=162)and the high SDF group(SDF>30%,n=90).Clinical as well as laboratory outcomes were correlated between the two groups.Sperm DNA fragmentation was assessed on the post-wash semen samples by acridine orange test.The main outcome measures were the live birth rate and miscarriage rate.Results:A significant decrease in the live birth rates was observed in the high SDF group compared to the low SDF group in fresh embryo transfer cycles(P<0.05).However,no significant difference was observed in the clinical outcomes either in the frozen embryo transfer cycles or in the overall cumulative transfer cycles(P>0.05).No significant difference was observed in the laboratory outcomes between the two SDF groups.A remarkable decrease in sperm motility was observed in the high SDF group compared to the low SDF group(P<0.05).Conclusions:Sperm DNA fragmentation does not affect the clinical outcomes in the cumulative transfers of an ICSI cycle along with blastocyst transfers in couples with normozoospermic males.

New approach to assess sperm DNA fragmentation dynamics： Fine-tuning mathematical models

Background： Sperm DNA fragmentation（sDF） has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if s DF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model（linear regression, exponential or polynomial） for DNA fragmentation over time in frozen-thawed donkey semen.Results： After submitting post-thaw semen samples to no centrifugation（UDC）, sperm washing（SW） or single layer centrifugation（SLC） protocols, sD F values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC.Coefficient of determination（R-2） values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation（aSDF） were obtained for SW, fol owed by SLC and UDC.Conclusion： SLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover,the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

Acceleration of DNA Replication of Klenow Fragment by Small Resisting Force

DNA polymerases are an essential class of enzymes or molecular motors that catalyze processive DNA syntheses during DNA replications. A critical issue for DNA polymerases is their molecular mechanism of processive DNA replication. We have proposed a model for chemomechanical coupling of DNA polymerases before, based on which the predicted results have been provided about the dependence of DNA replication velocity upon the external force on Klenow fragment of DNA polymerase I. Here, we performed single molecule measurements of the replication velocity of Klenow fragment under the external force by using magnetic tweezers. The single molecule data verified quantitatively the previous theoretical predictions, which is critical to the chemomechanical coupling mechanism of DNA polymerases. A prominent characteristic for the Klenow fragment is that the replication velocity is independent of the assisting force whereas the velocity increases largely with the increase of the resisting force,attains the maximum velocity at about 3.8 pN and then decreases with the further increase of the resisting force.