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.

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））.

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.

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.

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.

Nonextensivity and Tsallis Entropy in DNA Fragmentation Patterns by Ionizing Radiation

Nonextensive statistical mechanics as in Tsallis formalism was used in this study, along with the dynamical Hamiltonian rod-like DNA model and the maximum entropy criteria for Tsallis’ entropy, so as to obtain length distribution of plasmid fragments, after irradiation with very high doses, assuming that the system reaches metaequilibrium. By intensively working out the Grand Canonical Ensemble (used to take into account the variation of the number of base pairs) a simplified expression for Fragment Size Distribution Function (FSDF) was obtained. This expression is dependent on two parameters only, the Tsallis q value and the minimal length of the fragments. Results obtained from fittings to available experimental data were adequate and the characteristic behavior of the shortest fragments was clearly documented and reproduced by the model, a circumstance never verified from theoretical distributions. The results point to the existence of an entropy which characterizes fragmentation processes and depending only on the q entropic index.

Inter-center variation in the efficiency of sperm DNA damage reduction following density gradient centrifugation

This was a prospective multicenter study aiming at comparing the efficiency of sperm selection by density gradient centrifugation (DGC) in reducing sperm DNA fragmentation (SDF) in different ART centers. The study was designed using 290 semen samples collected from 10 different ART centers performing artificial insemination, in vitro fertilization and blind assessment of SDF at the University facilities. The results showed that while there was a significant reduction in the SDF levels in sperm isolated from the gradient centrifuged pellet (DGC) compared to neat semen samples (NSS), there was also significant inter-center variability in the efficiency to reduce SDF values by DGC (78.5% to 29.2%). Surprisingly, for some patients, the level of SDF actually increased following sperm selection. The main conclusions derived from this study were that 1) isolation of sperm from the gradient pellet by DGC must be performed using validated, optimized protocols;2) routine comparison of SDF values in NSS semen and in processed sperm after DGC or swim-up must be recommended as part of the internal quality control (QC) of ART laboratories to test the efficacy of sperm processing;and 3) SDF values in processed spermatozoa should be obtained to compare with the pregnancy rate when insemination or fertilization is about to be performed, otherwise, attempts to predict pregnancy outcome from SDF could be biased or are essentially meaningless.

The effect of natural products combination on MCF-7 cells exceeds tamoxifen therapeutic dose effects in vitro

Cancer remains to be one of the most severe sicknesses globally.Cases have kept rising over the years.Breast cancer(BC),which is among the leading types of cancers and predominantly affects women,is the second leading cause of cancer mortality.Researchers have developed interventions over the years;however,the BC survival rate has not improved since the 1980s.This has created the need for novel drug interventions that would manage and treat BC more effectively.This study focused on using a combination of natural product extracts such as phytoestrogen(Ziziphus jujube)and Tannin nanoparticles(NP99)together,which we have referred to as(Z.NP99)and tamoxifen(Tam)as one of the leading BC drugs since the 70s,in treating BC.The effectiveness of Tam if used alone in the treatment and if combined with Z.NP99 was evaluated using MCF-7 cells in vitro.The findings showed that the combination treatment of Z.NP99 affected the proliferation and viability of MCF-7 cells more than Tam at a 10μg/mL dose.Moreover,Z.NP99 with Tam stimulated the maximum reduction of MCF-7 proliferation and viability in a time-dependent manner.Furthermore,Tam and Z.NP99 augmented the DNA fragmentation percentage combined with the upregulation of the apoptotic genes.Additionally,the results showed that the apoptotic impact of Z.NP99 and Tam on MCF-7 cells may be intermediated by down-regulating some genes such as Claudin-1 followed by down-regulating mRNA expression of MMP-9,VEGF,and BCL-2 genes of treated cells.Combining Tam with Z.NP99 considerably enhanced the effectiveness of conventional therapy.As a result,this study suggested that the Z.NP99 was ideal for developing effective natural treatments that would improve BC outcomes.

Can the Prediction of Intrauterine Insemination Results by Used Aniline Blue Stain (ABS) and Sperm Chromatin Dispersion (SCD) Levels?

Introduction: This study aimed to perform routine seminal fluid analysis, sperm DNA fragmentation, and sperm function tests at the chromatin maturation level and evaluate pregnancy in the patients passing intrauterine insemination before starting Intrauterine Insemination (IUI) method. Materials and Methods: In this prospective study, 111 couples who underwent Intrauterine Insemination (IUI) in unexplained infertility patients were admitted to Al-Farah IVF and assisted reproductive center in Baghdad, Iraq between November 2020 and February 2021 were evaluated. Semen fluid analysis was performed based on (WHO 4th) guiding rules. In addition, Sperm Chromatin Dispersion (halo test) and sperm maturation were performed with Aniline Blue Stain (ABS). Results: Sperm Chromatin Dispersion (SCD) groups were compared in terms of pregnancy outcome;the positive pregnancy rate was found to be above in the normal SCD groups (p = 0.0005). In addition, Aniline Blue Stain (ABS) groups were compared in the terms of pregnancy outcome;the positive pregnancy rate was found to be higher in the normal ABS group (p = 0.017). Conclusion: Our study showed that the use of DNA fragmentation (SCD) and sperm maturation tests (ABS) together with routine semen analysis in intrauterine insemination cases will make a significant contribution to the prediction of Intrauterine Insemination (IUI) increased results. So, these results indicate a defect in the effect of DNA fragmentation on the outcome of intrauterine insemination.

Microsurgical varicocelectomy： a review

Varicocelectomy is the most commonly performed surgical procedure for the treatment of male infertility. Although several different techniques for varicocele repair have been described in the literature, microsurgical varicocelectomy performed through a subinguinal or inguinal incision is recognized as the gold-standard approach for varicocelectomy, due to high success rates with minimal complications. Standard indications for varicocelectomy include palpable varicocele（s）, with one or more abnormal semen parameters, and, for the couple trying to conceive, in the setting of normal or correctable female infertility. However, varicocele repair is often recommended and undertaken for reasons other than infertility, including low serum testosterone, testicular pain, testicular hypotrophy and poor sperm DNA quality. This article reviews the technical aspects of microsurgical varicocelectomy, and its indications in adults and adolescents.

Local anesthetic lidocaine induces apoptosis in human corneal stromal cells in vitro

AIM:To demonstrate the apoptosis-inducing effect of iidocalne on human corneal stromal(HCS)cells fn vitm,and provide experimental basis for safety anesthetic usage In clinic of ophthalmology.METHODS:In vitro cultured HCS cells were treated with lidocaine at different doses and times,and their morphology was monitored successively with inverted phase contrast microscopy.The membrane permeability of them was detected by acridine orange/ethidium bromide(AO/EB)double staining.The DNA fragmentation of them was examined by agarose gel electrophoresis,and their ultrastructure was observed by transmission electron microscopy(TEM),respectively.RESULTS:Exposure to lidocaine at doses from0.3125g/L to 20g/L induced morphological changes of HCS cells such as cytoplasmic vacuolation,cellular shrinkage,and turning round,and elevated membrane permeability of these cells in AO/EB staining.The change of morphology and membrane permeability was doseand time-dependent,while lidocaine at dose below0.15625g/L could not induce these changes.Furthermore,lidocaine induced DNA fragmentation and ultrastructural changes such as cytoplasmic vacuolation,structural disorganization,chromatin condensation,and apoptotic body appearance of the cells.CONCLUSION:Lidocaine has significant cytotoxicity on human corneal stromal cells in vitro in a dose-and time-dependent manner by inducing apoptosis of these cells.The established experimental model and findingsbased on this model here help provide new insight into the apoptosis-inducing effect of local anesthetics in eye clinic.

Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing

In this study, we compared the effects of repeated freezing/thawing of human sperm by our in-house method of rapid freezing with slow programmable freezing. Sperm samples from 11 normozoospermic subjects were processed through density gradients and divided into three aliquots： non-frozen, rapid freezing and slow programmable freezing. Sperm in the rapid freezing group had better motility and viability than those in the slow freezing group （P〈O.01） after the first, second and third cycles of freezing/thawing, but there was no difference in morphology. In the second experiment, rapid freezing was repeated three times in 20 subjects. The samples from each thawing cycle were evaluated for DNA fragmentation using the alkaline comet assay. DNA fragmentation began to increase considerably after the second cycle of freezing/thawing, but to a level that was not clinically important. In the third experiment, rapid freezing was done repeatedly in 10 subjects, until no motile sperm were observed after thawing. The median number of repeated freezing/thawing that yielded no motile sperm was seven （range： 5-8, mean： 6.8）. In conclusion, we demonstrated that repeated freezing/thawing of processed semen using our rapid freezing method gave better results than standard slow programmable freezing. This method can help maximize the usage of precious cryopreserved sperm samples in assisted reproduction technology.

Chlorogenic acid complex(CGA7),standardized extract from green coffee beans exerts anticancer effects against cultured human colon cancer HCT-116 cells

Coffee is commonly consumed beverage in the world and it has been suggested to have beneficial effect.Chlorogenic acids(CGAs)are main ingredient of coffee beans which has been extensively used in nutraceuticals and medicine.Recently,various therapeutic effects of chlorogenic acids have been investigated.However,there are limited studies to investigate its anticancer properties.In the present study,we have used chlorogenic acid complex(CGA7)a decaffeinated water soluble green coffee bean extract to evaluate its cytotoxic effect on human and mouse cancer cell lines by using different approaches.From our results we found CGA7 treatment induces cell death in a dose and time dependent manner in different cancer cell lines.Further,CGA7 induced apoptosis was characterized by DNA fragmentation,PARP-1 cleavage,caspase-9 activation,and down regulation of Bcl-2,an anti-apoptotic protein and up regulation of pro-apoptotic protein BAX.Overall findings indicated that CGA7 complex a potent anticancer molecule found in green coffee beans could be a safe bioactive ingredient for prevention of cancer.