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The estimation of unsaturated permeability coefficients (UPC) is of great importance on the properties of the matrix. Aim to study the relationship between the UPC and oxidation - reduction potential (ORP) in different matrix depths in the subsurface wastewater infiltration system (SWIS) and provide scientific basis for regulating SWIS and increasing pollutant removal, a test experiment of simulating the SWIS which included an inflow period (12 h) and a drying period (12 h) in one cycle was designed with a hydraulic load of 0.10 m3·(m2·d)-1. Results investigated that ORP could increase with UPC increasing in 70 and 115 cm and decrease with the UPC increasing in 100 and 130 cm matrix depths. Phenomena indicated that capillary action could affect UPC and ORP obviously. Moreover, the existence of oxygen and low volumetric water contents could impose UPC and ORP. UPC in the 100 cm matrix depth below proved that anaerobic area could be found in aerobic environment under alternation conditions. UPC in different matrix depths of a satisfactory SWIS could change from 2.49 × 10-7 to 1.16 × 10-3 cm·s-1. Treated water met reused requirements and no clogging was found. [ABSTRACT FROM AUTHOR]

A correction to the article "Reduced semen Quality in Chronic Prostatitis Patients That Induce the Release of Apoptotic Protein Omi/HtrA2 From Spermatozoa," that was published in the 2007 issue of this journal.

Objectives: To investigate the factors influencing the occurrence of small for gestational age (SGA) at different degrees and provide a basis for early identification of severe SGA cases., Methods: Neonatal and maternal prenatal information were retrospectively collected from January 2018 to December 2022 at Peking University People's Hospital. The neonates were divided into three groups: severe SGA group (birth weight below the 3rd percentile for gestational age and sex), mild SGA group (birth weight ≥3rd percentile and <10th percentile), and non-SGA group (birth weight ≥10th percentile). An ordered multinomial logistic regression model was used to analyze the factors influencing the occurrence of SGA at different degrees., Results: A total of 14 821 neonates were included, including 258 cases (1.74%) in the severe SGA group, 902 cases (6.09%) in the mild SGA group, and 13 661 cases (92.17%) in the non-SGA group. The proportions of preterm births and stillbirths were higher in the severe SGA group compared to the mild SGA and non-SGA groups ( P <0.0125). The proportion of neonatal asphyxia was higher in both the severe SGA and mild SGA groups compared to the non-SGA group ( P <0.0125). Ordered multinomial logistic regression analysis showed that maternal pre-pregnancy underweight ( OR =1.838), maternal pre-pregnancy obesity ( OR =3.024), in vitro fertilization-embryo transfer ( OR =2.649), preeclampsia ( OR =1.743), connective tissue disease during pregnancy ( OR =1.795), nuchal cord ( OR =1.213), oligohydramnios ( OR =1.848), and intrauterine growth restriction ( OR =27.691) were all associated with a higher risk of severe SGA ( P <0.05). Maternal parity as a multipara ( OR =0.457) was associated with a lower likelihood of severe SGA ( P <0.05)., Conclusions: Maternal pre-pregnancy underweight, maternal pre-pregnancy obesity, in vitro fertilization-embryo transfer, preeclampsia, connective tissue disease during pregnancy, oligohydramnios, nuchal cord, and intrauterine growth restriction are closely related to the occurrence of more severe SGA. Maternal parity as a multipara acts as a protective factor against the occurrence of severe SGA.

Neonatal lupus erythematosus (NLE) is caused by the transmission of maternal anti-Ro/SSA antibodies, anti-La/SSB antibodies, and other autoantibodies to the fetus through the placenta. Usually, with the disappearance of autoantibodies in the children's body, abnormal changes in the mucocutaneous, blood system, and digestive system can spontaneously subside, but the damage to various systems caused by autoantibodies may persist for a long time. This article provides a comprehensive review of the manifestations and prognosis of NLE in various systems, including mucocutaneous, blood system, circulatory system, nervous system, digestive system, respiratory system, aiming to provide reference for clinical work.

Objective: To explore the clinical risk factors and susceptibility genes of diabetes after kidney transplantation (PTDM) and construct a risk prediction model for PTDM. Methods: The data of kidney transplant recipients who underwent follow-up in the Affiliated Lihuili Hospital, Ningbo University and Sir Run Run Shaw Hospital, Zhejiang University School of Medicine from January 2001 to December 2022 were retrospectively analyzed. The recipients were divided into PTDM group and Non-PTDM group according to whether they were complicated with PTDM. The differences in clinical indicators between the two groups were compared, the risk factors affecting the incidence of PTDM were determined, and susceptibility genes of PTDM were screened by genome-wide association study (GWAS). PTDM risk prediction models based only on clinical indicators (Model 1) and clinical indicators combined with susceptibility genes (Model 2) were established respectively, and the predictive performance of the two prediction models was compared. Finally, the Nomogram of the optimal model was drawn, and the discrimination, calibration and clinical applicability of the model were evaluated. Results: A total of 113 kidney transplant recipients (70 males and 43 females) were included, with an average age of (46.2±10.8) years. There were 51 cases in PTDM group and 62 cases in Non-PTDM group. The related factors screened by GWAS and logistic regression analysis included family history of diabetes ( OR =88.912, 95% CI : 5.827-1 356.601, P =0.001), preoperative triglyceride (TG) ( OR =1.888, 95 % CI : 1.150-3.098, P =0.012), uric acid (UA) ( OR =1.011, 95% CI : 1.000-1.022, P =0.045) and rs802707 ( OR =10.046, 95% CI : 1.462-69.042, P =0.019). The area under the curve (AUC) of the receiver operating characteristics analysis (ROC) predicted by Model 1 for PTDM was 0.891 (95% CI : 0.811-0.972), with the sensitivity of 0.889 and the specificity of 0.742. The AUC of ROC curve predicted by Model 2 for PTDM was 0.930 (95% CI : 0.864-0.995), with the sensitivity of 0.885 and the specificity of 0.900. Conclusions: Family history of diabetes, preoperative TG and UA, and rs802707 are significantly associated with the occurrence of PTDM. In addition, the combination of susceptibility genes could improve the predictive ability of clinical indicators for the risk of PTDM.

To explore the efficacy of commonly used forensic identification panels in complex paternity testing of trios that involved close relatives, we wrote a code by R to generate 10,000 pedigrees at 20 CODIS STR, 21 non-CODIS STR and 30 InDel loci in Chinese five ethnic groups based on their allele frequencies. Parentage identification index--cumulative paternity index (CPI) value was set as output and was further analyzed to evaluate the performance of the aforementioned panels in complex paternity testing when the alleged parent is a random individual, biological parent, grandparent, sibling of biological parent, half-sibling of biological parent, etc. The results showed that the false inclusion of parent sibling posed as parent demonstrated no statistically significant difference from that of grandparent posed as parent. The scenarios where both biological parent and alleged parent were consanguineous to the other parent were also simulated. The results revealed that the complexity of paternity testing would raise when biological parents were consanguineous and the alleged parent was a close relative of theirs. Despite the values of non-conformity number could vary in different genetic relationships, populations and panels, 20 CODIS STRs and 21 non-CODIS STRs performed satisfactorily in most simulated scenarios. However, the joint use of 20 CODIS STRs and 21 non-CODIS STRs is more recommendable when resolving the paternity testing of the incest mating case. Overall, the current study could be regarded as a worthwhile reference in complex paternity testing of trios that involved close relatives., Competing Interests: Declaration of interest This study was funded by National Natural Science Foundation of China (No. 81901926) and Natural Science Basic Research Program of Shaanxi Province (No. 2022JQ-926). All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: To construct zebrafish models for the screening of intracranial hemorrhage (ICH) associated genes. Methods: ICH zebrafish models were constructed through morpholino oligonucleotides (MOs) technique and microinjection technique, and multiple verification was performed from macro and micro perspectives. First, the normal wild-type AB strain zebrafish injected with control MO was used as the control group, and AB zebrafish embryos microinjected with MOs of genes related to development of neural crest-derived cells (NCDCs) were used as the study group, such as col8a1 MO, tfap2α MO, msx1a MO, msx2 MO, and dkk1a MO. Preliminary verification of the model was conducted under a white-light optical microscope. Then, the model was verified by Tg (flk1: gfp; gata1: dsRed) double transgenic zebrafish, with vascular endothelial cells labeled by green fluorescent protein (GFP) and red blood cell labeled by fluorescent protein (dsRed), and thus the location of cerebral hemorrhage can be observed more clearly. Specifically, zebrafish embryos were microinjected with Control MO as the control group and those microinjected with col8a1 MO as the study group. Then the embryos were cultured until 48 hours post-fertilization to observe the leakage of red blood cells under the confocal laser scanning microscope. Finally, Tg (flk1: gfp) transgenic zebrafish was used to verify the model based on the blood-brain barrier (BBB). Through the leakage of dextran-rhodamine and DAPI dyes, the destruction of BBB and the occurrence of cerebral hemorrhage in zebrafish were further clarified, and quantitative statistics were carried out to verify the relationship between NCDCs development related genes and cerebral hemorrhage phenotype, which proved that the modeling was effective. Results: The zebrafish with col8a1, tfap2α, and msx1 mutations in the study group had apparent ICH compared with wildtype zebrafish, and the prevalence of ICH was 18.18% (52/286), 23.04% (62/251), and 35.94% (23/64), respectively. While, the zebrafish with msx2 and dkk1a mutations rarely had ICH, with the ICH prevalence of 1.03% (1/97) and 1.15% (1/87), respectively. The prevalence of red blood cells leakage in Tg (flk1:gfp; gata1:dsred) double transgenic zebrafish injected with Control Mo and col8a1 Mo was 0.37% (1/273) and 18.18% (52/286) ( P <0.001). The number of DAPI positive nuclei of Tg (flk1: gfp) transgenic zebrafish injected with Control Mo and col8a1 Mo was 10.05±5.27 and 60.35±3.96 ( P <0.001), and the fluorescent intensity of midbrain parenchymal induced by dextran-rhodamin leakage was 2.54±4.70 and 5.13±3.52 ( P <0.001). Conclusion: This study successfully constructs the ICH zebrafish models, and ICH-related genes are screened out, such as col8a1, tfap2α, msx1, and so on.

Objective: To investigate the association between sleep duration and activity of daily living (ADL) in the elderly aged 65 years and older in China. Methods: A total of 11 247 subjects aged 65 and above were included in the Chinese Elderly Health Factors Tracking Survey from March 29, 2005 to April 8, 2019. Self-made questionnaire was used to collect the data of population sociological characteristics, health status and disease status. ADL status was assessed by basic activities of daily living. The association between sleep duration and ADL impairment was assessed by Cox proportional risk regression model. The dose-response relationship between sleep duration and ADL impairment was analyzed using restricted cubic spline function. Results: The age of the subjects was (79±10) years, including 5 793(51.5%) females. The incidence of ADL impairment was 33.3% (3 747/11 247). Subjects were divided into short, medium, and long sleep groups according to sleep duration of fewer than seven hours, seven to eight hours, or more than eight hours. The number of short, medium and long sleepers was 2 974 (26.4%), 4 922 (43.8%) and 3 351(29.8%), respectively. The intermediate sleep group had the lowest incidence of impaired ADL (4.98/100 person-years). Cox proportional risk regression model analysis showed that: taking the intermediate sleep group as reference, after adjustment of gender, age, marital status, educational level, place of residence, living with family, smoking, drinking, exercise, frequency of fruit consumption, vegetable intake frequency, sleep quality, factors such as hypertension, diabetes, heart disease and cerebrovascular disease, the long sleep time increased the risk of impaired ADL [ HR (95% CI ): 1.148 (1.062-1.241)]. Subgroup analysis showed a weak positive multiplicative interaction between sleep duration and age [ HR (95% CI ): 1.004 (1.000-1.009)], but no multiplicative interaction between sleep duration and sex [ HR (95% CI ): 0.948 (0.870-1.034)]. Longer sleep duration increased the risk of ADL impairment in women [ HR (95% CI ): 1.195 (1.074-1.329)], but not in men [ HR (95% CI ): 1.084 (0.966-1.217)]. Longer sleep duration increased the risk of ADL impairment in people aged 80 years and older [ HR (95% CI ): 1.185 (1.076-1.305)], but not in people younger than 80 years [ HR (95% CI ): 1.020 (0.890-1.169)]. There was a non-linear dose-response relationship between sleep duration and ADL damage ( P =0.007), and the risk of ADL damage was lowest when sleep duration was 7.5 h. Conclusion: Sleep duration was positively correlated with the risk of ADL impairment in the elderly in a nonlinear dose-response relationship.

With the increase of the incidence of pre-pregnancy diabetes mellitus and gestational diabetes mellitus, the number of infants of diabetic mothers (IDMs) are increasing year by year. IDMs may be associated with poor perinatal outcomes and may have a negative impact on neurodevelopment, but there are relatively few studies on the neurodevelopmental outcomes of IDMs. This article reviews the relevant literature and summarizes the neurodevelopmental outcomes of IDMs from the aspects of sensory and perception, motor, language, intellectual development, neuropsychiatric disorders, neurological examination and drug effect, so as to provide reference for clinical work. Citation .

This study focused on the distribution, combined pollution, potential source and risk assessment of 17 antibiotics in an aquaculture ecosystem surrounding the Yellow sea, North China. Antibiotics were detected in various matrices (seawater, sediment/biofilm, organism and feed) in different aquaculture modes (greenhouse and outdoor aquaculture) during the wet and dry seasons in coastal areas of Shandong province. The innovation points of the study were as follows: (1) To the best of our knowledge, this study was one of the few to investigate the occurrence and distribution of antibiotics in mariculture environments along the Yellow Sea coast; (2) Biofilms, a focus of the study, might act as a sink for antibiotics in the aquaculture ecosystem; and (3) The correlation of heavy metals and antibiotic concentrations was proved, which could correspondingly be used as an indicator for antibiotic concentrations in the studied area. The levels of antibiotics in water were observed to be relatively low, at the ng/L level. Trimethoprim was the most prevalent antibiotic, and was detected in all water samples. Oxytetracycline was detected at high concentrations in biofilms (up to 1478.29 ng/g). Moreover, biofilms exhibited a higher antibiotic accumulation capacity compared to sediments. Concentrations of oxytetracycline and doxycycline were high in feed, while other antibiotics were almost undetected. Tetracycline was widely detected and the concentration of enrofloxacin was highest in organisms. Correlation analysis demonstrated that environmental parameters and other coexisting contaminants (e.g. heavy metals) significantly affected antibiotic concentrations. In addition, the concentration of Zn was significantly correlated with the total antibiotic concentration and was proportional to several antibiotics in water and sediment (biofilm) samples (p < 0.01). High Mn concentrations were closely related to total and individual (e.g. sulfadiazine, sulfamethazine and enrofloxacin) antibiotic levels, which may result in the combined contamination of the environment. Antibiotics in estuaries and groundwater generally originated from aquaculture wastewater and untreated/treated domestic sewage. Most of the detected antibiotics posed no risk to the environment. Ciprofloxacin and enrofloxacin found in water may present high ecological and resistance risks, while the two antibiotics observed to accumulate in fish may pose a considerable risk to human health through diet consumption. All antibiotics detected in seafood were lower than the respective maximum residue limits. This study can act as a reference for the government for the determination of antibiotic discharge standards in aquaculture wastewater and the establishment of a standardized antibiotic monitoring and management system., Competing Interests: Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To explore the application of adaptive statistical iterative reconstruction Veo (ASIR-V) and 80 kV in renal computed tomography angiography(CTA). Methods: Eighty patients with renal computed tomography angiography were prospectively collected from April 2018 to July 2018 in the Affiliated Hospital of Shaanxi University of Chinese Medicine and randomly divided into group A and group B. The patients in group A adopted tube voltage 120 kV and contrast agent concentration 600 mgI/kg and reconstructed with filtered back projection (FBP), while the patients in group B were scanned with tube voltage 80 kV and contrast agent concentration 350 mgI/kg and reconstructed with FBP and ASIR-V from 10% to 100% with 10% interval. The CT values and standard deviation (SD) of the right renal artery, left renal artery were measured respectively to calculate the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR).The image quality of renal CTA was subjectively scored by two experienced radiologists blindly using a 5-point criteria.The contrast agent,CT volume dose index(CTDI(vol)) and dose length product(DLP) in both groups were recorded and the effective radiation dose(ED) was calculated. Results: The ED ((2.11±0.19)mSv) and contrast agent ((21.9±3.0)g) in group B were 65.1% ( P< 0.05) and 42.2% ( P< 0.05) lower than those in group A ((6.04±1.89)mSv and (38.0±3.8)g).With the increase of ASIR-V percentage in group B, CT values showed no significant difference, SD values gradually decreased, SNR values and CNR values gradually increased.The CT values with different reconstruction algorithm showed no statistically significant difference (all P> 0.05) between group A and group B. The SD values with 40%ASIR-V to 100%ASIR-V reconstruction in group B were significantly lower than those of group A (all P< 0.05).The SNR values with 50% ASIR-V to 100% ASIR-V reconstruction and CNR values with 70%ASIR-V to 100%ASIR-V were significantly higher than those of group A(all P< 0.5).Two radiologists had excellent consistency in subjective scores of image quality for renal CTA(all kappa >0.75, P< 0.05). The subjective scores with 60% ASIR-V to 90% ASIR-V in group B were significantly higher than those in group A ( P< 0.05), of which 70%ASIR-V reconstruction achieved the highest subjective score for renal CTA. Conclusion: ASIR-V and 80 kV can significantly reduce radiation dose (about 65.1%) and contrast agent (about 42.2%) in renal CTA, ASIR-V reconstruction can significantly improve the image quality of renal CTA, of which 70% ASIR-V reconstruction achieved the best image quality in 80 kV renal CTA.

Although RNA-Seq is an effective method for identifying and exploring novel functional genes in mammals, it has rarely been applied to study fertility-related genes in the goat. In this study, RNA-Seq was used to screen the estrus ovaries of uniparous and multiparous Anhui white goats (AWGs). In total, 15,890 genes were identified and 2201 of these were found to be differentially expressed between the genetic libraries from uniparous and multiparous goats. Compared to the uniparous library, 1583 genes were up-regulated and 618 genes were down-regulated in the multiparous library. The FER1L4 gene showed the level of highest up-regulation in the multiparous library, while SRD5A2 expression showed the greatest down-regulation. In order to determine the functions of FER1L4 and SRD5A2 in goats, the expression profiles of the two genes in different tissues from AWGs and Boer goats at diestrus were analyzed by quantitative PCR. FER1L4 and SRD5A2 showed tissue specific expression patterns and were highly expressed in ovaries from both AWGs and Boer goats. FER1L4 was more highly expressed in ovaries from multiparous than uniparous AWGs. In contrast, SRD5A2 was expressed at a lower level in multiparous AWGs. These results indicated that FER1L4 and SRD5A2 may be associated with the high fecundity of AWGs.

The desmoglein 4 (DSG4) gene is a potential candidate in the search for genes that may affect wool traits, because of its function. This study aimed to screen for polymorphisms in partial exon 16 and 3ꞌUTR of the sheep desmoglein 4 DSG4 gene, and to test its possible association with wool length and crimp associated with fur. Overall, 326 sheep were scanned via single-strand conformational polymorphism assay, through three pairs of primers. The breeds included Tan, Han, and TanxHan from China, Polled Dorset from Australia, and Suffolk from Britain genotypes AA, BB, and AB for primer2 and genotypes DD, EE, and DE for primer3 were detected in native breeds. Six SNPs and 3-bp insertion/deletions were found in exon 16, of which 4 lead to amino acid substitutions. In addition, 1 SNP was found in 3ꞌUTR. The DSG4 genotype was found to be strongly associated with all wool traits that were considered in this study (P < 0.01). Sheep with the genotype MM had a higher least square mean compared to sheep with the genotype WW or WM with respect to birth scapular wool length (P < 0.01), crimp number of birth scapular wool crimp (P < 0.01), crimp number of weaning scapular wool crimp (P < 0.01), and crimp number of weaning rump wool crimp (P < 0.01, P < 0.05). In conclusion, our study is the first to demonstrate that the DSG4 gene may be a candidate, or major gene, which influences important wool traits.

MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 species-conserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of real-time quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

Mutations in the myostatin (MSTN) gene can inactivate its expression and result in a non-functional protein, which leads to dramatic muscularity and a "double-muscling" phenomenon in many species. Using gene sequencing and polymerase chain reaction-single-strand conformation polymorphism methods, polymorphisms of the MSTN gene were investigated as a candidate marker for growth in 288 goats. The results showed 2 novel single nucleotide polymorphisms: DQ167575 g.197G>A and 345A>T. Three potential genotypes (AA, AB, and BB) of substitution 197G>A in the 5'-untranslated region were detected in the 2 breeds. The polymorphism (CC and CD) of substitution 345A>T in exon I was segregated. The genetic diversity analysis revealed that Boer goat and Anhui white goat possessed intermediate genetic diversity in the P1 and P3 loci. Significant associations between the genotypes of the P3 locus and body weight, body length, and body height were observed in Boer goat and Anhui white goat (P < 0.05). It could be inferred that the MSTN gene may be a major gene or linked to the major gene affecting the goat growth traits. The polymorphic site could be a molecular marker-assisted selection program for body weight.

To investigate the genetic diversity of seven Chinese indigenous meat goat breeds (Tibet goat, Guizhou white goat, Shannan white goat, Yichang white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat), explain their genetic relationship and assess their integrity and degree of admixture, 302 individuals from these breeds and 42 Boer goats introduced from Africa as reference samples were genotyped for 11 microsatellite markers. Results indicated that the genetic diversity of Chinese indigenous meat goats was rich. The mean heterozygosity and the mean allelic richness (AR) for the 8 goat breeds varied from 0.697 to 0.738 and 6.21 to 7.35, respectively. Structure analysis showed that Tibet goat breed was genetically distinct and was the first to separate and the other Chinese goats were then divided into two sub-clusters: Shannan white goat and Yichang white goat in one cluster; and Guizhou white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat in the other cluster. This grouping pattern was further supported by clustering analysis and Principal component analysis. These results may provide a scientific basis for the characteristization, conservation and utilization of Chinese meat goats.

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

Previously, we showed that NO induces thymocyte apoptosis via a caspase-1-dependent mechanism [(1) ]. In the present study, we investigated the role of heme oxygenase, catalase, bax, and p53 in this process. The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), induced DNA fragmentation in thymocytes in a time- and concentration-dependent way. SNAP (100 microM) induced 50--60% apoptosis; higher doses did not increase the rate of apoptosis significantly. SNAP decreased catalase and heme iron (Fe) levels without affecting superoxide dismutase, glutathione, or total Fe stores in thymocytes. SNAP significantly increased the expression of heme oxygenase 1 (HSP-32), p53, and bax but not bcl-2. Treatment with the heme oxygenase inhibitor, tin protoporphyrin IX inhibited SNAP-induced thymocyte apoptosis. Furthermore, thymocytes from p53 null mice were resistant to NO-induced apoptosis. Our data suggest that NO may induce its cytotoxic effects on thymocytes by modulating heme oxygenase and catalase activity as well as up-regulating pro-apoptotic proteins p53 and bax.

Nuclear factor-kappaB (N-kappaB) plays a key role in gut inflammation. NF-kappaB up-regulates proinflammatory genes encoding cytokines, adhesion molecules, and inducible nitric oxide synthase (iNOS). However, NF-kappaB has also been shown to up-regulate protective or anti-apoptotic factors. We utilized an adenoviral vector carrying a super-repressor form of the inhibitor of NF-kappaB, IkappaB, to examine the effects of NF-kappaB inhibition on cytokine-induced nitric oxide production and apoptosis in rat small intestinal epithelial cells (IEC-6). Chemical inhibitors of NF-kappaB, including pyrrolidine dithiocarbamate (PDTC), tosyl-lysine-chloromethylketone (TLCK), genistein, and N-acetyl-leu-leu-norleucinal (n-LLnL) were also utilized. Treatment of AdIkappaB-transfected cells with cytomix [1000 U/mL IFN-gamma, 1 nM IL-1beta, and 10 ng/mL tumor necrosis factor alpha (TNFalpha)] or TNFalpha-containing cytokine combinations resulted in inhibition of cytokine-induced nitrite production and a marked increase in apoptosis compared to control cells. Apoptosis occurred independently of nitric oxide (NO) production since exogenous sources of NO did not inhibit apoptosis. Inducible NOS and clAP were down-regulated in AdIkappaB-transfected cells treated with cytomix. TLCK and LLnL treatment also induced apoptosis in cytomix-treated cells, while PDTC and genistein did not. Thus, although NF-kappaB up-regulates various pro-inflammatory genes, it may also have protective or anti-apoptotic effects in enterocytes. NF-kappaB appears necessary for upregulating cIAP in IEC-6 cells upon cytokine exposure.

Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post-infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non-invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S. typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.

The biological effects of static magnetic fields, and their combined effects with ionizing radiation, were studied using a cultured mammalian cell line (FM3A). The three different modes of magnetic fields evaluated in this report were the 0.3 Tesla (T) field with a gradient of 0.3T/m, the 0.7T field with a gradient of 0.7T/m and the 6.34T field with no gradient. Exposure to the 0.3T and 0.7T fields had no effect on cell survival. Exposure to the 6.34T field decreased cell survival. Survival curves showing the combined effect of the 0.3T and 0.7T fields with radiation had a smaller mean lethal dose (D37) value. The survival curve of the 6.34T field was influenced by the interval between magnetic exposure and ionizing irradiation. When the interval was 6 or 12 h, the survival curve showing the combined effect of the 6.34T field had smaller D37 and quasithreshold dose (Dq) values, indicating the potentiation of the radiation effect. Flow cytometric analysis indicated that exposure to the 0.3T and 0.7T fields showed no change and that exposure to the 6.34T field showed an increase in the percentage of G1 phase cells. Our conclusions were as follows: 1) magnetic fields decreased the colony-forming abilities of cultured mammalian cells; 2) magnetic fields can affect the cell cycle; 3) a stronger magnetic field strength does not always have stronger biological effects and 4) the gradient of a magnetic field may be an important factor when combined with ionizing radiation. Despite the foregoing analysis, the biological effects of magnetic fields on mammalian cells remains a complex phenomena.