Vitrified before and after genome editing via electroporation.

BACKGROUND: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation. OBJECTIVE: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes. MATERIALS AND METHODS: In vitro-derived bovine zygotes were electroporated with the CRISPR/Cas9 system immediately (Vitrified-EP) or 2 h after incubation (Vitrified-2h-EP) following vitrification and warming, or electroporated before vitrification (EP-vitrified). RESULTS: The development rates of vitrified-warmed zygotes were significantly lower (p < 0.05) than those of control zygotes that were not vitrified. Moreover, no differences were observed in the mutation rates and mutation efficiency of the blastocysts resulting from electroporated zygotes, irrespective of the timing of electroporation treatment. CONCLUSION: Our results suggest that vitrification before and after electroporation treatments does not affect the genome editing of zygotes.

[Analysis of occupational health status of radiation workers in Guangdong Province].

Objective: To discuss the effect of low-dose ionizing radiation on the health of radiation workers, and provide a basis for occupational health risk assessment of radiation workers. Methods: In January 2020, 3165 radiation workers who performed radiation occupational health examinations in Guangzhou Prevention and Treatment Hospital for Occupational Disease from January 2017 to December 2019 were selected as the research objects, and compared and analyzed the health status of radiation workers with different examination types (pre-job, in-job and off-job) , types of work, gender, and length of service. Results: The off-job occupational radiological health examination was rare at 2.3% (74/3165) . The abnormal detection rate of chest radiographs, renal function, thyroid function, and blood routine of the radiation workers in-job group was higher than that of the pre-job group (P<0.05) . No statistical difference was found in the abnormal detection rate of the examination items during the in-job group and the off-job group (P>0.05) . The blood routine abnormality detection rate of medical application group and industrial application group were higher than those of nuclear fuel group (P<0.05) . The abnormal detection rate of blood pressure and renal function of male radiation workers was higher than that of females, while the abnormal detection rate of blood routine of females was higher than that of males (P<0.05) . The abnormal detection rate of electrocardiogram, chest radiograph, blood pressure, renal function, thyroid function, and blood routine of radiation workers increased with increasing working age (P<0.05) . Conclusion: Occupational health status of radiation workers is not optimistic. Radiation occupational health monitoring should be strengthened, special attention should be paid to off-job radiation occupational health examination, focusing on the sensitive indicators of sensitive personnel, improving radiation protection conditions, and effectively protecting the occupational health of radiation workers.

[Related factors for microalbuminuria in adult type 1 diabetes patients of short disease duration].

Objective: To investigate related factors for microalbuminuria in adult type 1 diabetes (T1D) patients of short disease duration (less than 5 years), and provide evidence for prevention of early diabetic kidney disease in this population. Methods: All adult patients enrolled in the Guangdong T1D translational medicine study between 2011 and 2017 with a disease duration of less than 5 years were included in this analysis. At enrollment, patients' demographic and clinical data were documented, and blood and urine samples were collected for the measurements of blood lipids, glycated hemoglobin A1c and urine albuminuria. Insulin resistance was evaluated by estimated glucose disposal rate (eGDR). Patients were categorized into groups based on urine albumin creatitine ratio (UACR): normoalbuminuric group (UACR<30 mg/g) and microalbuminuric group (UACR≥30 mg/g). Stepwise multivariate linear regression analysis was used to analyze risk factors for microalbuminuria in adult T1D patients of short disease duration. Results: A total of 384 patients were included in this analysis, and 51.3% (197/384) of which was female. The onset age of patients was (24.6±12.5) years, with a disease duration of 2.1(0.6, 3.5) years, body mass index of (19.8±3.2) kg/m(2), waist hip ratio of 0.85±0.21, and glycated hemoglobin A1c of (9.8±3.3)% at enrollment. Microalbuminuria occurred in 62 patients (16.1%). Multivariate linear analysis showed that higher glycated hemoglobin A1c, higher systolic blood pressure and more severe insulin resistance were related factors for microalbuminuria (t=2.322, 2.868 and -2.373, respectively, all P<0.05). Conclusions: Microalbuminuria was not rare in adult T1D patients of short disease duration. Inadequate glycemic control and insulin resistance were independent related factors for microalbuminuria in this population.

MicroRNA-140-5p suppresses invasion and proliferation of glioma cells by targeting glutamate-ammonia ligase (GLUL).

Glutamine addiction is a major feature of glioma cells and plays an important role in its growth and proliferation. GLUL (glutamate-ammonia ligase), which catalyzes glutamate and ammonia to synthesize glutamine, plays a crucial role in tumor growth and proliferation. We attempt to determine a pathway that limits the growth of glioma by targeting GLUL and explore effective strategies blocking glutamine metabolism. We note that miRNAs mediate regulation of genes participating directly or indirectly in cancer cell metabolism. The regulatory roles of miRNAs on metabolic enzymes are widely discussed, however miRNAs regulation of glutamine metabolism by targeting GLUL in glioma has not yet been reported. Here, we examined both the expression and functions of GLUL in glioma cells. Findings indicated that the expression of GLUL was upregulated in high-grade compared to low-grade glioma cells. Knockdown of GLUL effectively inhibited proliferation, migration and invasion of glioma cells in vitro. Bioinformatics analyses, as well as dual-luciferase reporter assays, revealed that miR-140-5p bound to GLUL mRNA at the 3'-UTR location. Furthermore, the proliferation, migration and invasion of glioma cells were also repressed by miR-140-5p. Overall, these results showed that miR-140-5p exerted its inhibitory effects on proliferation, migration and invasion in glioma cells through downregulating GLUL. Thus, the miR-140-5p/GLUL axis may function as a potential target for glioma treatment.

[Analysis of common gynecological diseases in 1142 married female workers].

Objective: To understand the prevalence of gynecologic diseases among married female workers. Methods: The data of married female workers who underwent occupational health examination in a physical examination center from January to December 2017 were collected. The relationship between the detection of common gynecological diseases, age and occupational types examined by gynecological routine, TCT, breast B-mode ultrasound, uterus and appendix B-mode ultrasound were analyzed. Results: Among the 1142 female workers, the total detection rate of reproductive tract infections was 67.25% (768/1142), the total detection rate of breast-related diseases was 75.22% (859/1142) ; the total detection rate of gynecological tumors and benign lesions was 14.71% (168/1142). The detection rate of breast hyperplasia was the highest 67.08% (766/1142), followed by vaginitis 51.66% (590/1142). Among the abnormalities detected in breast-related diseases, gynecological tumors and benign lesions, the highest detection rate was found in public institutions (85.66% and 27.13%), and the lowest was found in factory workers (70.24% and 7.89%). With the increase of age, the detection rate of breastrelated diseases (breast hyperplasia, breast cyst), gynecological tumors, benign lesions (uterine myoma), and Nessler's cyst abnormalities in married female workers increased (χ(2)(trend)=7.647、21.653、107.411、53.802, P<0.05), while the detection rate of columnar epithelium of cervix decreased (χ(2)(trend)=7.404, P<0.05). There was no significant difference in the total detection rate of reproductive tract infectious diseases (vaginitis, cervical polyps, cervical hypertrophy) among married famale workers of different ages (P<0.05) . Conclusion: The common gynecological diseases of married female workers are affected by many factors such as age and occupation. Health examination and health education should be carried out regularly to reduce the incidence of gynecological diseases among female workers according to different ages and occupations.

[Awareness of preconceptional care and its related factors in women of child-bearing age with type 1 diabetes].

Objective: To investigate the awareness of preconception care among women of child-bearing age with type 1 diabetes (T1DM) and their self-management status, in order to provide evidence for establishment of management pathway for women with T1DM in pregnancy in China. Methods: This cross-sectional survey recruited female participants of child-bearing age from the cohort of Guangdong Type 1 Diabetes Translational Medicine Study conducted between June 2011 and December 2017. The participants were asked to fill out a questionnaire on the awareness of preconception care, their frequency of self-monitoring of blood glucose (SMBG) and other related variables. Chi-squared test or chi-squared test for trend was used in comparisons of categorical variables, and logistic regression analysis was performed to assess associated factors. Results: Totally, 441 women of child-bearing age with T1DM were investigated. The results show that their awareness of preconception care was poor (15.42%, 68/441). Higher educational level (χ(2trend)=3.990, P=0.046), experience of post-diabetes education evaluation (P<0.001), and better coverage of different modules in diabetes education (survival skills: χ(2)=7.525, P=0.004; basic knowledge: χ(2)=8.598, P=0.002; advanced knowledge: P<0.001) were associated with better awareness of preconception care. The average frequency of SMBG in these participants was 0.29 (0.14, 2.00) times per day, and only 8.5% (37/435) of them reached the frequency (≥4 times per day) recommended by guidelines. Moreover, 21.1% (92/435) of them hardly ever performed SMBG. Conclusion: Child-bearing age women with T1DM in Gunangdong had poor awareness of preconception care, with a much lower SMBG frequency than recommendation.

[Association of insulin resistance with dyslipidemia in adults with type 1 diabetes].

Objective: To investigate the relationship between insulin resistance (IR) and dyslipidemia in adults with type 1 diabetes (T1DM) and provide more insights on diabetes-related cardiovascular disease management. Methods: A cross-sectional study recruiting patients from Guangdong T1DM Translational Study cohort was conducted between 2011 and 2017. The patients aged ≥18 years, with a diabetes duration of ≥1 year were enrolled in the study. Plasma lipid profile data of eligible patients, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were collected and their relationships with insulin resistance were analyzed. IR in these adults with T1DM was estimated by glucose disposal rate (eGDR) calculated by a model published previously. Patients with eGDR lower than 25 percentiles were grouped as severe IR, otherwise non-severe IR. Results: In total, 499 eligible patients were studied, among which 274 were women (54.9%). The level of eGDR was 8.43 (6.11, 10.63) mg kg(-1) min(-1) and the overall incidence of lipid disorders was 65.3% (326/499) in the study population. The result showed that eGDR was correlated with TC, TG, HDL-C and LDL-C (r=-0.163, -0.303, 0.170 and -0.150, respectively, all P<0.05). After adjusting for gender, age and diabetes duration, eGDR was still associated with TG, TC and LDL-C (all P<0.05). Stepwise multiple linear regression analysis showed that gender (female), elevated TC and declined HDL-C were independent factors associated with the severity of IR (t=5.651, 5.823 and 2.908, respectively, all P<0.05). Conclusions: IR is associated with dyslipidemiain in adults with T1DM. Elevated TC and decreased HDL-C are independent associated factors for insulin resistance.

[Effect of long-term low-concentration mixed benzene exposure on male peripheral blood in an automobile manufacturing enterprise].

Objective: To investigated the effect of long-term low-concentration mixed benzene exposure on peripheral blood of male workers. Methods: A case-control study was conducted to select 452 male workers exposed to mixed benzene (benzene, toluene, xylene) for five consecutive years from January 2012 to December 2016 in an automobile manufacturer as case group, and 438 male administrative and logistic managers who underwent physical examination during the same period as control group. The peripheral blood of the two groups was tested and compared, and the occupational hazards in the workplace were detected. Results: There were low dose exposure to mixed benzene in the enterprise, but the test results met the occupational exposure limit requirements. During the five years from 2010 to 2016, between the two groups of workers, the mean values of WBC, NEUT, RBC and Hb were statistically different (P<0.05). There was no statistical difference (P>0.05) in the mean value of PLT. The abnormal rate of main peripheral blood indexes in the control group was higher than that in the exposed group. There were significant differences in NEUT, RBC and Hb (P< 0.05), but no significant differences in WBC and PLT (P>0.05). Conclusion: It can not be concluded that long-term low-concentration mixed benzene exposure can cause the change of peripheral blood index.

Neuroprotection effect of interleukin (IL)-17 secreted by reactive astrocytes is emerged from a high-level IL-17-containing environment during acute neuroinflammation.

An increase in interleukin (IL)-17A-producing cells, particularly at sites of tissue inflammation, is observed frequently, yet the mechanism is not fully understood. This study aims to dissect the role of IL-17 in autoimmunity-mediated neuroinflammation. The cytokine milieu containing elevated IL-17, which often appears in active states of autoimmunity, was mimicked in vitro by a supernatant obtained from rat peripheral blood monocytes stimulated with phorbol mystistate acetate (PMA)/ionomycin. The application of such inflammatory media on only primary cultured cerebellar granule neurones resulted in significant apoptosis, but the presence of astrocytes largely prevented the effect. The supernatants of the stimulated astrocytes, especially those that contained the highest level of IL-17, achieved the best protection, and this effect could be blocked by anti-IL-17 antibodies. Protein IL-17 inhibited intracellular calcium increase and protected the neurones under inflammatory attack from apoptosis. IL-17, but not interferon (IFN)-γ, in the inflammatory media contributed to astrocyte secretion of IL-17, which depended on the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway activation. The astrocytes that were treated with IL-17 alone or with prolonged treatment of the inflammatory media failed to produce sufficient levels of IL-17. Moreover, confirmatory data were obtained in vivo in a monophasic experimental autoimmune uveitis (EAU) in Lewis rats; in this preparation, the high-level IL-17-containing the cytokine milieu was demonstrated, along with IL-17 secretion by the resident neural cells. The antagonism of IL-17 at a late stage disturbed the disease resolution and resulted in significant neural apoptosis. Our data show a dynamic role of IL-17 in the maintenance of homeostasis and neuroprotection in active neuroinflammation.

Optimization of selected cultivation parameters for Cordyceps guangdongensis.

AIMS: To increase the fruit body production of Cordyceps guangdongensis, selected cultivation conditions, especially nutritional parameters were optimized. METHODS AND RESULTS: Cordyceps guangdongensis was inoculated on potato dextrose agar slants with pH values from 4.5 to 9.0 and cultivated in artificial media with different carbon and nitrogen supplements. Primordium formation in C. guangdongensis was favoured by slightly acidic conditions. Fruit body yields and biological efficiency (BE) recorded were all highest in cultures of C. guangdongensis supplemented with sucrose and KNO3 as carbon and nitrogen supplements, respectively. Highest fruit body yields and BE values were recorded with C : N ratio of 12 : 1. The optimal medium consisted of (g l(-1)) 20.0 sucrose, 4.0 soya bean powder, 5.0 beef extract and 10.0 KNO3. Cultivation experiments using this medium confirmed its reliability; 18.35% of BE was obtained, compared with a calculated maximum BE of 18.65% based on orthogonal test data. CONCLUSIONS: Cordyceps guangdongensis preferred sucrose and potassium nitrate as best carbon and nitrogen supplements. It produced satisfying yield of fruit body with optimized medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimized artificial cultivation conditions could promote the yield of C. guangdongensis and decreased the cost of production.

Detecting early breast tumour by finite element thermal analysis.

Thermography has been proved to be an effective technique for indicating breast disease abnormalities or risks. However, the abnormalities might not express clearly due to various factors, such as when a small tumour is located in a deep region, or environmental influences that make breast disease difficult to find. This study aims to solve these problems for early detection of breast tumour. A three-dimensional breast model is presented to investigate the relationship between an embedded tumour and the surface temperature distribution. Then a subtraction technique is used to enhance the thermal signature of breast tumour. It was showed that the surface thermal characteristics of a small tumour even in a deep region could be found easily by this method. Furthermore, it was also found that the surface thermal characteristics of tumour obscured due to environmental cooling effect can be clearly displayed. The results are very useful for analysing breast thermograms.

First Report of Ageratum yellow vein virus Causing Tobacco Leaf Curl Disease in Fujian Province, China.

A leaf curling disease was observed on 7% of tobacco plants during December 2005 in research plots in the Cangshan District of Fuzhou, Fujian, China. Tobacco plants were infested with Bemisia tabaci, suggesting begomovirus etiology. To identify possible begomoviruses, total DNA was extracted from four symptomatic leaf samples (F1, F2, F3, and F4). The degenerate primers PA and PB were used to amplify part of the intergenic region and AV2 gene of DNA-A-like molecules (3). A 500-bp DNA fragment was amplified by PCR from all four samples. The PCR products were cloned and sequenced (GenBank Accession Nos. EF531601-EF531603 and EF527823). Alignment of the 500-bp sequences for the four isolates indicated that they shared 98.5 to 99.6% nt identity, suggesting that the plants were all infected by the same virus. Overlapping primers TV-Full-F (5'-GGATCCTCTTTTGAACGAGTTTCC-3') and TV-Full-R (5'-GGATCCCACATGTTTAAAATAATAC-3') were then designed to amplify the full-length DNA-A from sample F2. The sequence was 2,754 nucleotides long (GenBank Accession No. EF527823). A comparison with other begomoviruses indicated the F2 DNA-A had the highest nucleotide sequence identity (95.7%) with Ageratum yellow vein virus (AYVV; GenBank Accession No. X74516) from Singapore. To further test whether DNAß was associated with the four viral isolates, a universal DNAß primer pair (beta 01 and beta 02) was used (4). An amplicon of approximately 1.3 kb was obtained from all samples. The DNAß molecule from F2 was then cloned and sequenced. F2 DNAß was 1,345 nucleotides long (GenBank Accession No. EF527824), sharing the highest nucleotide sequence identity with the DNAß of Tomato leaf curl virus (97.2%) from Taiwan (GenBank Accession No. AJ542495) and AYVV (88.8%) from Singapore (GenBank Accession No. AJ252072). The disease agent was transmitted to Nicotiana tabacum, N. glutinosa, Ageratum conyzoides, Oxalis corymbosa, and Phyllanthus urinaria plants by whiteflies (B. tabaci) when field infected virus isolate F2 was used as inoculum. In N. tabacum and N. glutinosa plants, yellow vein symptoms were initially observed in young leaves. However, these symptoms disappeared later during infection and vein swelling and downward leaf curling symptoms in N. tabacum and vein swelling and upward leaf curling in N. glutinosa were observed. In A. conyzoides, O. corymbosa, and P. urinaria plants, typical yellow vein symptoms were observed. The presence of the virus and DNAß in symptomatic plants was verified by PCR with primer pairs TV-Full-F/TV-Full-R and beta 01/beta 02, respectively. The above sequence and whitefly transmission results confirmed that the tobacco samples were infected by AYVV. In China, Tobacco leaf curl Yunnan virus, Tobacco curly shoot virus, and Tomato yellow leaf curl China virus were reported to be associated with tobacco leaf curl disease (1,3). To our knowledge, this is the first report of AYVV infecting tobacco in China. A. conyzoides is a widely distributed weed in south China and AYVV was reported in A. conyzoides in Hainan Island, China (2). Therefore, this virus may pose a serious threat to tobacco production in south China. References: (1) Z. Li et al. Phytopathology 95:902, 2005. (2) Q. Xiong et al. Phytopathology 97:405, 2007. (3) X. Zhou et al. Arch. Virol. 146:1599, 2001. (4) X. Zhou et al. J. Gen. Virol. 84:237, 2003.

Four beams evanescent waves interference lithography for patterning of two dimensional features.

This work presents a theoretical study of using the interference of multiple counter-propagating evanescent waves as a lithography technique to print periodic two dimensional features. The formulation of the three dimensional Cartesian space expression of an evanescent wave is presented. In this work, the evanescent wave is generated by the total internal reflection of a plane wave at the interface between a incident dielectric material and a weakly absorbing transmission medium. The influences of polarization, incident angle and the phase shifting of the incident plane waves on the evanescent wave interference are studied. Numerical simulation results suggest that this technique enables fabrication of periodic two dimensional features with resolution less than one third the wavelength of the irradiation source.

Rapid donor liver procurement with only aortic perfusion.

AIM: To describe a rapid technique for procurement of donor liver with aortic perfusion only (APO). METHODS: Only the aorta is cannulated and perfused with chilled preservation solution. RESULTS: The quality of donor liver can ensure the grafted liver functions. CONCLUSION: The method of APO can simplify the operative procedure, compared with the dual cannulation. It also can minimize the danger of injuring vascular structures and involve less dissection.

Multiple-image shearography: a direct method to determine curvatures.

We present a modified method of shearography, known herein as multiple-image shearography, whereby the curvatures of an object can be measured directly from the resulting fringes. It employs an image-shearing camera that produces three sheared images simultaneously to interfere with each other in the image plane. When film is doubly exposed before and after an object is deformed, three sets of fringes are observed of which one set would depict the second-order derivatives of surface displacement.The theory of the multiple-image shearography technique and its application to curvature measurements in plate bending are presented.

Analysis of shearogram reconstruction.

The high-pass Fourier filtering technique with an opaque stopper is commonly used for the reconstruction of shearograms. We believe that no analysis of this technique as applied to the shearogram reconstruction has been described. We present here a theoretical analysis of this reconstruction system taking into account the stopper that blocks the low-frequency region in the Fourier transform plane. The results show that the light-intensity distribution after reconstruction is different from that of the original shearogram. High fringe visibility is obtained with high-pass filtering.