[Attention should be paid to the importance of genetic testing in clinical practice of amyotrophic lateral sclerosis].

Amyotrophic lateral sclerosis(ALS) is a progressive and fatal neurodegenerative disease that mainly involves upper and lower motor neurons. It lacks clear biomarkers and can be clearly diagnosed only one and a half years after the onset. Gene test is of great significance for diagnosis, prognosis and genetic counseling. In recent years, several gene therapy studies have entered the clinical trial stage of ALS, among which the antisense oligonucleotide therapy targeting the pathogenic variation of the superoxide dismutase 1 (SOD1) gene has been launched, and it is urgent to carry out routine gene test in clinical practice. On the basis of progress of ALS gene research in recent years, family history, age of onset and typical clinical manifestations of patients are no longer considered as the basis for genetic testing. However, the target genes of clinical gene testing needs to be further clarified according to the diagnostic purpose, the testing method and scheme need to be standardized, and the genetic consultation before testing should be paid attention to, and the informed consent should be fully achieved.

Evaluation of exposure risk for healthcare personnel performing the open technique HIPEC procedure using cisplatin.

OBJECTIVE: To perform an evaluation of the risk to healthcare personnel of exposure to cisplatin during a Hyperthermic Intraperitoneal Chemotherapy (HIPEC) procedure in an operating room environment. METHODS: Breathing zone air samples were taken from the operating room (OR) before, during and after the procedure of HIPEC filter membrane adsorption and the liquid impact method was applied to collect air samples. The samples of surface wipe from the floor of the OR were taken after the procedure. Inductively coupled plasma mass spectrometry(ICP-MS) was used to detect the content of cisplatin in all the samples. RESULTS: Thirty-six air samples and three surface wipes were collected from three different locations of healthcare personnel breathing zones. All the breathing zone air samples were negative for cisplatin; however, cisplatin contamination was detected on three surface wipes from the floor, but in a lowconcentration(≤ 2.25 ng). CONCLUSION: The results suggest that the risk of inhalation of cisplatin was extremely low for the healthcare personnel during the procedure of HIPEC, but the contamination of the OR floor should be taken into consideration.

[The value of serum dehydroepiandrosterone sulfate in differential diagnosis of Cushing's syndrome].

Objective: To evaluate the changes and diagnostic value of serum dehydroepiandrosterone sulfate (DHEAS) in Cushing's syndrome (CS) with different etiologies. Methods: The study retrospectively recruited patients diagnosed as CS in Drum Tower Hospital affiliated to Nanjing University Medical School between January 2012 and June 2019, including 36 patients (8 males, 28 females, with an average age of 44 years) with Cushing disease (CD) and 64 patients (6 males, 58 females, with an average age of 39 years) with adrenal CS (ACS). Meanwhile, 97 patients diagnosed as nonfunctional adrenal adenoma (NFA) were also included as controls. Clinical characteristics, laboratory data, adrenocorticotropic hormone (ACTH), serum DHEAS level and sex-and age-adjusted DHEAS ratio of the three groups were collected. The sensitivity and specificity of DHEAS and its ratio in differential etiology diagnosis of CS were compared using receiver operating characteristic (ROC) curve analysis. Results: Compared to NFA group, ACS patients had lower DHEAS levels [0.39 (0.39, 0.63) µmol/L vs 2.96 (1.92, 4.60) µmol/L, P<0.01] and lower DHEAS ratio [0.58 (0.27, 0.98) vs 3.95 (3.08, 6.83), P<0.01]. DHEAS [6.49 (4.32, 11.63) µmol/L] and DHEAS ratio [9.17 (4.49, 15.41)] in CD patients were significantly higher compared to those in NFA and ACS patients (all P<0.01). There were 53 ACS patients (82.8%) with suppressed ACTH level (<2.2 pmol/L) and 11 patients (17.2%) with normal/high ACTH level (≥2.2 pmol/L). The level of 24 hour urine free cortisol in normal/high ACTH level group was lower than the suppressed ACTH group [(1 299±511) nmol/24 h vs (1 972±876) nmol/24 h, P=0.04]. No significant differences were found in the DHEAS and DHEAS ratio between the two groups. ROC analysis showed that the area under the curve of serum DHEAS and DHEAS ratio in diagnosing ACS from CD was 0.997 and 0.990, respectively. The optimal cut-off values for DHEAS and its ratio were 2.06 µmol/L and 2.10, respectively. The diagnostic sensitivity and specificity of DHEAS were 97.5% and 100%, and those of DHEAS ratio were 95.0% and 100%, respectively. Conclusion: There are significant differences in serum DHEAS level and DHEAS ratio between ACS and CD patients, which might be used as indicators for the identification of the two main CS etiologies, especially in the identification of ACS patients without plasma ACTH suppression from CD patients.

[Clinical features and outcomes of surgical versus conservative management in patients with subclinical Cushing's syndrome].

Objective: To examine the clinical characteristics and metabolic features of subclinical Cushing's syndrome (SCS), and determine the effects of surgical or conservative approaches on the hormone levels and metabolic comorbidities in patients with SCS, thereby providing the evidence for decision-making in SCS management. Methods: A total of 56 consecutive SCS patients were selected in Drum Tower Hospital Affiliated to Nanjing University Medical School between 2010 and 2018, with 41 patients undergoing surgical treatment and 15 patients receiving conservative therapy. Meanwhile, 56 and 68 cases of sex-and age-matched patients diagnosed as nonfunctional adrenal adenoma (NFA) and adrenal Cushing's syndrome (CS) were included respectively. Clinical characteristics of patients in different groups were compared. Hormone levels and metabolic comorbidities were also observed during follow-up. Results: There were 56 SCS patients, including 15 males and 41 females, with an age of (52.0±12.6) years. The circadian rhythms of adrenocorticotropic hormone (ACTH) and cortisol disappeared in CS and SCS groups. Compared to NFA group, patients with SCS were characterized by suppressed plasma ACTH level [2.40 (1.11, 4.33) pmol/L vs 4.23 (2.74, 6.26) pmol/L], elevated midnight cortisol level [(240±121) nmol/L vs (59±8) nmol/L] and increased cortisol level after 1 mg overnight dexamethasone suppression test [(241±130) nmol/L vs (34±12) nmol/L] (all P<0.01). The derangement of ACTH-cortisol axis was more obvious in CS patients compared to SCS patients. The prevalence of hypertension, glucose intolerance, dyslipidemia and osteopenia/osteoporosis were higher in SCS patients compared to NFA patients (75.0% vs 41.1%, 33.9% vs 12.5%, 62.5% vs 28.6%, 35.7% vs 8.9%, all P<0.05). The 24-hour urine free cortisol correlated positively with systolic blood pressure, glycated hemoglobin A1c (HbA1c) and fasting blood glucose in SCS patients (r=0.335, 0.562 and 0.463, respectively, all P<0.05). In the surgical group, body weight, body mass index (BMI) and blood pressure decreased significantly after surgery (all P<0.05). Glucose intolerance/diabetes mellitus improved in 6 of 9 patients, BMI of 4 of 11 overweight/obesity patients normalized, and hypertension in 54.5% of patients (12/22) showed improvement after surgery. However, no alterations of hormone levels and metabolic parameters were observed in conservatively-managed patients. Conclusions: Patients with SCS are characterized by mild autonomous cortisol secretion and increased risk of metabolic comorbidities. Compared with conservative management, hormone abnormalities were corrected and metabolic abnormalities were improved in some SCS patients after surgery.

[Resting-state functional MRI studies of amyotrophic lateral sclerosis patients with various levels of cognitive impairment].

Objective: To characterize the brain functional changes of amyotrophic lateral sclerosis (ALS) patients with various levels of cognitive impairment as measured by resting-state functional MRI (RS-fMRI). Methods: From September 2013 to March 2017, a total of 55 patients diagnosed with ALS in Peking Union Medical College Hospital and 20 healthy controls (HCs) were included in this study, and all participants underwent neuropsychological assessments and diffusion tensor imaging scans. According to their cognitive performance, ALS patients were further subclassified into ALS with normal cognition (ALS-Cn, n=27), those with cognitive impairment (ALS-Ci, n=17) and ALS-FTD (n=11). Comparisons of fractional amplitude of low frequency fluctuation (fALFF) value and regional homogeneity (ReHo) value were conducted among the 4 subgroups. Results: The fALFF showed significant differences in bilateral frontal lobe, left temporal lobe and cingulate gyrus, (P<0.001, uncorrected) and the ReHo showed significant differences in left frontal lobe, right temporal lobe and left cingulate gyrus (P<0.001, FDR corrected). The differences mainly stemmed from that patients with ALS-FTD showed decreased fALFF and ReHo in these areas when compared to the other three groups, especially in relation to HCs, mainly locating in left prefrontal lobe and anterior cingulate cortex. The whole-brain comparisons of fALFF and ReHo between ALS-Ci, ALS-Cn and HCs revealed no significant difference (P<0.001, uncorrected). Conclusion: Hypoactivities are detected in extramotor areas in patients with ALS-FTD. RS-fMRI is helpful in investigating the pathophysiologic mechanism of cognitive impairment in ALS.

[Sample size for the estimation of F-wave parameters in healthy volunteers and amyotrophic lateral sclerosis patients].

Objective: The study aimed to investigate whether sample sizes of F-wave study differed according to different nerves, different F-wave parameters, and amyotrophic lateral sclerosis(ALS) patients or healthy subjects. Methods: The F-waves in the median, ulnar, tibial, and deep peroneal nerves of 55 amyotrophic lateral sclerosis (ALS) patients and 52 healthy subjects were studied to assess the effect of sample size on the accuracy of measurements of the following F-wave parameters: F-wave minimum latency, maximum latency, mean latency, F-wave persistence, F-wave chronodispersion, mean and maximum F-wave amplitude. A hundred stimuli were used in F-wave study. The values obtained from 100 stimuli were considered "true" values and were compared with the corresponding values from smaller samples of 20, 40, 60 and 80 stimuli. F-wave parameters obtained from different sample sizes were compared between the ALS patients and the normal controls. Results: Significant differences were not detected with samples above 60 stimuli for chronodispersion in all four nerves in normal participants. Significant differences were not detected with samples above 40 stimuli for maximum F-wave amplitude in median, ulnar and tibial nerves in normal participants. When comparing ALS patients and normal controls, significant differences were detected in the maximum (median nerve, Z=-3.560, P<0.01; ulnar nerve, t=5.019, P<0.01; tibial nerve, Z=-2.475, P<0.05; peroneal nerve, Z=-2.088, P<0.05)and mean F-wave latency (median nerve, Z=-3.243, P<0.01; ulnar nerve, t=3.876, P<0.01; tibial nerve, Z=-2.206, P<0.05; peroneal nerve, Z=-2.205, P<0.05)in all four nerves, F-wave chronodispersion (Z=-3.152, P<0.01)in the ulnar nerve, F-wave persistence in the median (Z=6.139, P<0.01)and ulnar nerves(Z=5.350, P<0.01), mean F-wave amplitude in the tibial nerve(t=2.981, P<0.01), maximum F-wave amplitude in the ulnar (Z=-2.134, P<0.05)and tibial nerves (t=2.746, P<0.01)with 20 stimuli; for chronodispersion in tibial nerve (t=2.551, P<0.05)100 stimuli, for chronodispersion in peroneal nerve (Z=-2.086, P<0.05)80 stimuli, for F-wave persistence in tibial nerve (Z=2.119, P<0.05) 60 stimuli, for mean F-wave amplitude in ulnar (Z=-2.552, P<0.05)and peroneal nerve (Z=-2.228, P<0.05)40 stimuli, for maximum F-wave amplitude in peroneal nerve (t=2.693, P<0.01)60 stimuli were necessary to detect differences. Conclusions: Sample sizes of F-wave study differed according to different nerves, different F-wave parameters , and ALS patients or healthy subjects.

[Clinical value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle and follow-up after birth].

Objective: To explore the value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle (ventriculomegaly) , and follow up the nervous system development status after birth. Methods: Simple expansion of the lateral ventricle fetus by prenatal MRI examination were collected in Huzhou Maternal and Child Care Hospital from May 2013 to June 2015, 126 cases of live births in expansion group, 50 normal cases were recruited in the same period as the control group. In expansion group, fetal subgroup analysis was done: (1) unilateral or bilateral lateral ventricle expasion: one group was 98 cases was lateral ventricle expansion (77.8%, 98/126), expansion of bilateral ventricle group was 28 cases (22.2%, 28/126). (2) Prenatal MRI in the diagnosis of the lateral ventricle of expansion: expansion of the lateral ventricle width was greater than 10.0 mm, if both sides were expanding, the expand width was the heavier one side, divided into 3 subgroups: ①Expansion in group A (lateral ventricle width 10.0-12.0 mm) were 88 cases (69.8%, 88/126). ②Expansion in group B (lateral ventricle width 12.1-15.0 mm) were 29 cases (23.0%, 29/126). ③Expansion of group C (lateral ventricle width> 15.0 mm) were 9 cases (7.12%, 9/126). All 176 cases were followed up after birth at the 3rd, 6th, 12th, 18th month (corrected age was used for premature babies), and Gesell developmental schedules (GDS) were used to evaluate the neurobehavioral development. Results: (1) The MRI results after birth: 21 cases were followed up by MRI after birth. In group A, 11 cases had MRI and 9 were normal (the ventricular width <10.0 mm after birth), the other 2 cases were stable (the ventricular width measured first time after birth was ≥10.0 mm, but the difference was within 2.0 mm from the MRI before birth). In group B, 4 cases had MRI, 1 was normal, 1 was stable, and 2 cases were getting better (the ventricular width measured first time after birth was ≥10.0 mm, but the width decreased more than 2.0 mm from the MRI before birth). In group C, 6 cases had MRI. 3 cases were getting better and 3 cases were stable. (2) Overall GDS results: expansion group after the birth of the 3rd, 6th, 12th, 18th month GDS evaluation results compared with control group, respectively, the differences were not statistically significant (all P>0.05). (3) The GDS results among the subgroups: in each evaluation after birth, there were no statistically significant differences between group A and the control group (all P>0.05). The GDS results of group B at the 3rd and 6th month were lower than those of the control group (P<0.05); while there were no statistically significant differences between the 2 goups at the 12th and 18th month (P>0.05). And for group C, statistically significant differences were found compared to the control group at each follow-up time (all P<0.05). (4) GDS results at different times after birth in the expansion group: there was no statistically significant difference between the results at the 3rd and 6th month (P>0.05). But when the result at the 3rd month was compared to the results of the 12th or 18th month, the differences were statistically significant (P<0.05). GDS result of 6th months after birth compared with 12th and 18th months, respectively, there were no statistically significant differences (P>0.05). There was no statistically significant difference between the results at the 12th and 18th month (P>0.05). (5) The GDS results in unilateral and bilateral ventricle expansion: at the 18th month, among the 98 unilateral cases, 86 (87.8%, 86/98) had normal GDS results(>85 scores); 8 (8.2%, 8/98) had borderline results (75-85 scores); 4 (4.1%, 4/98) had delayed results (<75 scores). Among the 28 bilateral cases, 23 (82.1%, 23/28) had normal GDS results; 3 (10.7%, 3/28) had borderline results; 2 (7.1%, 2/28) had delayed results. There was no statistically significant difference (P>0.05). Conclusions: Among the simple expansion of lateral ventricle, those whose ventricular width are ≤12.0 mm may not need clinical treatment. If the width is between 12.1 to 15.0 mm, closely follow-up and targeted rehabilitation training after birth are recommended. When the width is more than 15.0 mm, the risk of the central nervous system function delay is significantly increased, and early intervention might improve the prognosis.

Age at onset of amyotrophic lateral sclerosis in China.

BACKGROUND: For patients with amyotrophic lateral sclerosis (ALS), age at onset is not only a key factor for diagnosis and prognosis, but also a clue for exploring pathogenesis. Reports based on results from a single medical center suggested that the mean age at onset of ALS in China was earlier than in other developed countries. A larger, multicenter-based study is needed to confirm this finding. METHODS: A registry-based study of ALS was conducted at 10 ALS centers of the Chinese ALS Association from March 1, 2009 to August 31, 2009. The demographical and clinical features of patients with ALS were collected. RESULTS: Data from a total of 455 patients with ALS were available for analysis. The mean age at onset for the entire cohort was 52.4 ± 12.1 years. The peak age at onset was in the 45- to 49-year-old age group for women and the 55- to 59-year-old age group for men. The age at onset for patients from Guangzhou (a southern region) was significantly earlier than it was for patients from Shanghai (an eastern region) (t = 2.270, P = 0.025). CONCLUSIONS: This investigation confirmed the earlier age at onset of ALS in China as compared with other countries. Further population-based case-control investigations of genetic and environmental factors are needed to identify the potential risk factors for Chinese ALS patients.

Screening of the FUS gene in familial and sporadic amyotrophic lateral sclerosis patients of Chinese origin.

BACKGROUND: According to studies in European, North American, Australian, and Asian populations, FUS gene mutations occur in 0.6-20.2% of the patients with familial amyotrophic lateral sclerosis (ALS) and 0.4-2.0% of sporadic ALS cases. In China, FUS mutations have been reported in several familial ALS pedigrees but not in sporadic ALS cases. Here, we screened for FUS mutations in Chinese patients with ALS. METHODS: We sequenced all of the 15 exons of FUS in 10 familial ALS pedigrees, exons 5, 6, 14, and 15 in 210 patients with sporadic ALS and 151 healthy controls. All patients were negative for SOD1, TARDBP, and ANG mutations. RESULTS: A c.1562G>T (p.R521L) missense mutation was identified in one familial ALS proband and her asymptomatic daughter. A c.1562G>A (p.R521H) missense mutation was identified in two patients with sporadic ALS. Three synonymous mutations (c.453C>T, c.648C>T, and c.1464C>T) were detected among four patients with sporadic ALS, and a untranslated region variant (*14C>T) was identified in one familial ALS proband and one patient with sporadic ALS. CONCLUSIONS: The frequency of FUS mutations is approximately 1.0% in our SOD1-, ANG-, TARDBP-mutation-negative sporadic ALS cohort and similar to that reported in previous studies from Asia in our familial ALS cohort. [Correction added on 31 May 2012, after first online publication: the gene FUS- was changed to ANG-]. Our findings provide an overview of the occurrence of FUS mutations in Chinese sporadic and familial ALS cases and highlight the importance of screening for FUS mutations in ALS patients of Chinese origin.

Tangential hard x-ray diagnostic array on the EXL-50 spherical tokamak.

A tangential hard x-ray (HXR) diagnostic on the newly constructed ENN XuanLong-50 (EXL-50) spherical tokamak for fast electron emission studies is presented. The HXR detection system consists of a symmetrical CdZnTe semiconductor detector array with a spectral sensitivity range of 20-300 keV. 25 channels have been designed on the 270° horizontal vacuum port with 12 sight lines to observe the forward emission, 12 sight lines to observe the backward emission of fast electrons, and 1 for viewing the central. Currently, ten channels have been in operation in the EXL-50 experiments. The systems are designed to measure the x-ray spectra for the estimation of fast electron temperature and electron velocity distribution in the EXL-50 experiment, which will be useful for understanding the dynamics of fast electrons generated by electron cyclotron resonance heating, for plasma instability and transport studies and for the analysis of plasma heating efficiency.

Time resolved absolute extreme ultraviolet radiation measurement on the ENN XuanLong-50 spherical tokamak.

A plasma radiation measurement system for a wide spectral range, based on compact Absolute eXtreme UltraViolet (AXUV) silicon photodiodes, has been implemented on the newly constructed ENN XuanLong-50 (EXL-50) spherical tokamak. The system consists of two 16-channel AXUV16ELG arrays and one AXUV63HS1 single-cell detector mounted on ceramic sockets. The two arrays, facing toward the EXL-50 slim central post from two locations inside a top and a side ConFlat 400 port, have 32 view chords covering the interested plasma region in a poloidal cross section at toroidal 330°. The single-cell detector, seated on a retractable feedthrough, could be arranged flexibly with the help of an ultra-high vacuum compatible gate valve. The design details together with considerations on the EXL-50 specific engineering realities and physics requirements are described. Preliminary results from the EXL-50 2020 experimental campaign are presented.

Toroidal soft x-ray array on the EXL-50 spherical tokamak.

A toroidal soft x-ray array system for spectrum and intensity measurements on the EXL-50 spherical tokamak is described. Silicon drift detectors and digital multichannel analyzers are adopted for all 21 channels of the array, and an average energy resolution of 147 eV at 5.89 keV has been achieved at count rates over 500 kcps. In total, 20 channels of the array are symmetrically observed in both co- and counter-current directions on the EXL-50 mid-plane with a spatial resolution of around 10 cm, and the remaining one serves as a background reference channel. Tungsten emissions from tungsten coating of the limiters on the central post are observed. The influence of hard x rays on measured soft x-ray spectra and system operation is discussed.

Potential correlation between EDN1 gene polymorphisms with preeclampsia.

OBJECTIVE: To explore the potential correlation between endothelin 1 (EDN1) gene polymorphisms with preeclampsia (PE). PATIENTS AND METHODS: The single nucleotide polymorphisms (SNPs) of 248 PE patients and 232 healthy controls were genotyped by Polymerase Chain Reaction (PCR). The possible association between EDN1 polymorphisms and PE was revealed through the t-test and the Chi-square test. RESULTS: PE risk was significantly correlated with the C allele of polymorphism rs5370 in EDN1. The polymorphism rs5370 in EDN1 was remarkably associated with the onset of severe PE, rather than mild PE. The markedly increased risk of early-onset PE was related to the C allele of polymorphism rs5370 in EDN1, while no significant difference in the allele frequency of polymorphism rs1800541 was detected between the PE group and the control group. In the co-dominant model, the CC genotype of polymorphism rs5370 in EDN1 was associated with the increased PE risk. PE risk in the population carrying TC genotype was 1.59 times higher than those with TT/CC genotype, while polymorphism rs1800541 had no apparent association with PE risk. In the severe PE group, there was an evident difference in the genotype frequency between the dominant and over-dominant models of polymorphism rs5370. In the recessive model, the raised risk of early-onset PE was notably correlated with the TT/CC genotype compared with that of TT genotype. However, no evident association with the genotype frequency of polymorphism rs1800541 was observed between PE patients and controls. CONCLUSIONS: EDN1 gene polymorphism rs5370 is correlated with the increased risk of PE.

High-Pressure Polymorph of Thulium: An X-ray Diffraction Study.

An x-ray diffractiotn study of thulium at room temperature and high pressure by means of a diamond-anvil press has shown that thulium transforms from a hexagonal close-packed structure to the samarium type, as other rareearth elements (gadolinium, terbium, dysprosium, and holmium) do. Unlike the other rare-earth elements, thulium (hexagonal close-packed) has an axial ratio (c/a) that is independent of pressure within experimental error and the transition is reversible. The transition occurs with increasing pressure in the range of 60 to 116 kilobars. The lattice paralieters of the samarium-type phase of thulium at about 116 kilobars are a = 3.327 +/- 0.005 angstroms and c = 23.48 +/- 0.04 angstroms, and the volume change at the transition is estimated to be - 0.5 percent of the volume of the hexagonal close-packed phase at the transition.

Improving strategies for syphilis control in China: selective testing of sexually transmitted disease patients--too little, too late?

SUMMARY: Syphilis testing guidelines in China are usually based on symptomatic criteria, overlooking risk assessment and ultimately opportunities for disease detection and control. We used data from 10,695 sexually transmitted disease (STD) clinic patients in Guangxi, China, to assess the efficacy of a potential screening tool inquiring about behavioural and health risk factors in identifying the STD patients who should not be triaged for syphilis testing under current guidelines, but on the contrary receive such testing. Validity testing of the screening tool was performed and receiver-operating characteristic curves were plotted to determine an optimal total risk score cut-off for testing. About 40.9% of patients with positive toluidine red unheated serum test and Treponema pallidum particle agglutination test did not show hallmark signs of syphilis. The screening tool was more sensitive in detecting infection in non-triaged male versus female patients (highest sensitivity = 90% vs. 55%) and the cut-off score to warrant testing was lower in non-triaged female patients than in non-triaged male patients (cut-off = 1 vs. 2). Most of the cases were missed among female STD patients. In spite of selective testing based on behavioural and health indicators that improve case detection, cases were still missed. Our study supports universal testing for syphilis in the STD population.

Transgenic cloned mice expressing enhanced green fluorescent protein generated by activation stimuli combined with 6-dimethylaminopurine.

Most studies of mouse cloning successfully achieved activation of the reconstructed oocytes by strontium (Sr) combined with cytochalasin B (CB) treatment. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was used to inhibit the activity of maturation promoting factor for activation of oocytes, but it has never been successfully applied in mouse cloning. This study investigates the activation efficiency of 6-DMAP in mouse somatic cell nuclear transfer (SCNT). Higher parthenogenetic blastocyst rates (71-72%, p < 0.05) were achieved in the oocytes treated with Sr6D (10 mM Sr combined with 2 mM 6-DMAP for 4 h) and Sr6D + SrCB (Sr6D for 2 h then Sr combined with 5 mug/ml CB for another 2 h), and a higher rate of hatching and hatched blastocyst was observed in the Sr6D + SrCB group (31%, p < 0.01) compared with other treatment groups (1-8%). For mouse cloning, cumulus cells of enhanced green fluorescent protein (EGFP)-expressed ESC chimera F1 were used as donor nuclei. Following activation, better development of the cloned embryos was observed in Sr6D + SrCB treatment. Moreover, different media, i.e. KSOM-AA, MEM-alpha and MK, for culturing cloned embryos were also compared in this study. Better morula/blastocyst (40%) and blastocyst (29%) rates were achieved in the embryos cultured in MEM-alpha medium (p < 0.05). Consequently, four EGFP cloned mice were generated in the activation treatment containing 6-DMAP following embryo transfer. In conclusion, treatment with 6-DMAP in combination with other activation stimuli successfully activates mouse reconstructed oocytes and support full-term development of the transgenic SCNT cloned embryos.

Gene-environment interaction in the conversion of a mild-to-severe phenotype in a patient homozygous for a Ser172-->Cys mutation in the lipoprotein lipase gene.

Normal pregnancy is associated with a two- to threefold increase in plasma triglyceride levels, particularly in the third trimester, due both to the overproduction of VLDLs and to the possible suppression of lipoprotein lipase (LPL) activity. Numerous mutations in the human LPL gene causing complete LPL deficiency have been described, but naturally occurring mutations that result in defective LPL with partial activity have not yet been reported. Here we describe a 30-yr-old woman who was first diagnosed with LPL deficiency during pregnancy after she developed pancreatitis. Her plasma triglyceride levels remained mildly elevated at approximately 300 mg/dl (3.4 mmol/liter) after the first pregnancy but rose significantly after she became pregnant again (1800 to 2000 mg/dl) (20.2 to 22.5 mmol/liter). DNA sequence analysis of the LPL gene showed that the patient is homozygous for a Ser172-->Cys missense mutation in exon 5. In vitro mutagenesis revealed that the Ser172-->Cys mutation caused a mutant LPL protein that had residual activity higher than that seen in all eight other missense mutations in patients with LPL deficiency identified in our laboratory. We propose that some mutations in the LPL gene produce a defective LPL with partial activity, which usually leads to mild hypertriglyceridemia.

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin.

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca2+ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl2 (0.1--2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.

Changes in phase transition temperature of phospholipids induced by endotoxin.

The effects of endotoxin (lipopolysaccharide from Salmonella minnesota Re 595) on the phase transition temperature (Tm) of various phospholipids were studied. Endotoxin had no effect on the Tm and the width of the phase transition of dipalmitoyl-sn-3-phosphatidylcholine. Endotoxin at 100 micrograms/ml increased the Tm of dipalmitoyl-sn-3-phosphatidylethanolamine by 1.1 degrees C (P less than 0.01) and narrowed the range of transition from 4.5 to 2.6 degrees C; the endotoxin-induced changes in the Tm and the transition range were abolished by the presence of 0.25 mM CaCl2. Endotoxin increased the Tm of dipalmitoyl-sn-3-phosphatidic acid by 1.1 (P less than 0.01), 1.2 (P less than 0.01), and 3.1 (P less than 0.01) degrees C at 25, 50 and 100 micrograms/ml, respectively. Furthermore, the width of phase transition of phosphatidic acid was narrowed from 6.5 to 4.0 degrees C by endotoxin at 100 micrograms/ml. The endotoxin-induced changes in the Tm and the transition range of phosphatidic acid were not affected by the presence of EDTA (1 mM) or CaCl2 (0.05-0.1 mM). These results suggest that endotoxin decreases the fluidity of negatively charged phospholipids such as phosphatidic acid and phosphatidylethanolamine. A change in the physical properties of membrane lipid bilayers induced by endotoxin may have an adverse effect on the function of biological membranes.