The relationship between famine exposure in early life and left atrial enlargement in adulthood.

BACKGROUND: Increased left atrium diameter (LAD) is associated with an elevated risk of cardiovascular diseases. The relationship between nutrition status and left atrial enlargement (LAE) is still unclear. The present study aimed to investigate the association of famine exposure in early life with LAE in adulthood. METHODS: Participants were divided into non-exposed, fetal, early, middle and late childhood exposed groups according to birth data. LAE was defined when LAD was ≥3.9 cm in women and ≥4.1 cm in men, or ≥2.3 cm m-2 by a sex-independent cut-off normalised for body surface area. Multivariate logistic regression was performed to calculate the odds ratio (OR) and confidence interval (CI) between famine exposure and LAE. RESULTS: In total, 2522 [905 male, mean (SD) age 59.1 (3.65) years] subjects were enrolled, including 392 (15.5%) LAE subjects. The prevalence of LAE in non-exposed, fetal, early, middle and late childhood exposed groups was 55 (10.8%), 38 (11.2%), 88 (18.1%), 102 (16.7%) and 109 (19.0%), respectively. Compared to the non-exposed group, the ORs for LAE were in fetal (OR = 0.956, 95% CI = 0.605-1.500, P = 0.847), late (OR = 1.748, 95% CI = 1.208-2.555, P = 0.003), middle (OR = 1.647, 95% CI = 1.140-2.403, P = 0.008) and early (OR = 1.630, 95% CI = 1.116-2.399, P = 0.012) childhood exposed groups after adjusting potential cofounders. When stratified by gender, smoking, body mass index, hypertension and diabetes, we found that the effect of famine exposure on LAE was only modified by diabetes (Pinteraction  = 0.007). CONCLUSIONS: Famine exposure during childhood stage might increase the risk of LAE in adults, and this effect interacts with diabetes.

[Prevalence trends and related factors of hypertension patients complicating with dyslipidemia in community of Guandong province between 2013 and 2018].

Objective: To analyze the prevalence trends and related factors of hypertension patients complicating with dyslipidemia in community. Methods: This was a cross-sectional survey, patients with hypertension were selected from the different communities of Guangdong province in 2013 and 2018 respectively. General clinical characteristics, including demographic information, past history, family history, and medication history, were collected. Dyslipidemia was defined as follows: at least 1 item elevation of total cholesterol (TC)≥5.2 mmol/L, triglyceride (TG) ≥1.7 mmol/L, low-density lipoprotein cholesterol (LDL-C)≥3.4 mmol/L, or reduced high-density lipoprotein cholesterol (HDL-C)<1.0 mmol/L. The incidence of dyslipidemia was standardized based on the 2010 China Census data, and further subgroup analysis was performed according to age (<50, 50-60, ≥60 years old) and sex (male, female). Multivariate logistic regression was used to analyze the related factors of dyslipidemia. Results: In 2013 and 2018, 7 866 (4 148 (52.7%) females, with the age of (62.4±13.6) years) and 11 611 (6 692 (57.6%) females, with the age of (58.2±9.3)years) patients with hypertension were enrolled for data analysis, respectively. In 2013, the total prevalence rate of dyslipidemia in patients with hypertension in the community of Guangdong province was 56.3%, among which the prevalence rates of hypercholesterolemia, hypertriglyceridemia, high LDL-Cemia, and low HDL-Cemia were 17.1. %, 21.3%, 2.3% and 24.4%, respectively. The total prevalence of dyslipidemia in patients with hypertension in the community of Guangdong in 2018 was 47.3%, prevalence of hypercholesterolemia, hypertriglyceridemia, high LDL-Cemia and low HDL-Cemia was 14.1%, 20.3%, 12.0% and 19.4%, respectively. Subgroup analysis showed that the total prevalence of dyslipidemia in male patients with hypertension in the community of Guangdong in 2013 and 2018 was 59.0% and 50.7%, respectively, among which hypercholesterolemia was 13.8% and 8.0%, and hypertriglyceridemia was 22.3%, 20.9%, high LDL-Cemia was 1.7%, 8.1%, low HDL-Cemia was 32.9%, 30.3%, respectively. In 2013 and 2018, the total prevalence of dyslipidemia in female patients with hypertension in the community of Guangdong province was 53.9% and 44.8%, among which prevalence of hypercholesterolemia was 20.5% and 18.5%, hypertriglyceridemia was 20.4% and 19.8%, and high LDL-Cemia was 2.7% and 14.9%, and hypo-HDL-Cemia was 16.8% and 11.3%, respectively. Age subgroup analysis showed that the prevalence of dyslipidemia among hypertensive patients aged<50, 50-60, and ≥60 years in Guangdong community in 2013 were 60.1%, 60.6%, and 53.7%, respectively; and 46.2%, 49.3% and 46.5% in 2018, respectively. Multivariate logistic regression analysis showed that women (OR=0.860,95%CI 0.761-0.973,P=0.017), obese (OR=2.295,95%CI 2.007-2.624,P<0.001), diabetes (OR=1.314,95%CI 1.090-1.583,P=0.004), stroke (OR=1.894,95%CI 1.227-2.924,P=0.004) and the level of fasting blood glucose (OR=1.105,95%CI 1.066-1.146,P<0.001) were independently related with the occurrence of dyslipidemia. Conclusions: The prevalence of dyslipidemia in patients with hypertension in the communities of Guangdong province is relatively high, and the prevalence differs in sex and age. Between 2013 and 2018, the total prevalence of dyslipidemia, hyper-TCemia, and hypo-HDL-Cemia in hypertensive patients shows a downward trend. The prevalence of hyper-TGemia remains unchanged, but the prevalence of high LDL-C shows an upward trend. Several factors are related to the prevalence of dislipidemia in hypertension patients in Guandong community.

[Expression of GTF2IP23 in breast cancer and it mediated regulation of GTF2I].

Objective: To investigate the expression level of antisense transcript of pseudogene, general transcription factor Ⅱi psedugen23 (GTF2IP23), in breast cancer and its effect on the host gene general transcription factor Ⅱi (GTF2I). Methods: The expressions of GTF2IP23 and GTF2I were detected in 40 cases of invasive breast cancer tumors and their counterparts by using quantitative real-time polymerase chain reaction (qRT-PCR). The effects of GTF2IP23 on the expression of GTF2I gene and cell proliferation and migration were analyzed by overexpression of GTF2IP23 in breast cancer cells. Results: The expression of GTF2IP23 mRNA in breast cancer tissues was significantly higher than that in adjacent tissues (P<0.001), while the expression of GTF2I mRNA was significantly lower than that in adjacent tissues (P=0.007). The expression of GTF2IP23 was negatively correlated with GTF2I (r=-0.335, P=0.025). The expression of GTF2IP23 in breast cancer cells was significantly higher than in normal breast cells (P<0.01), while GTF2I expression in breast cancer cells was significantly lower than that in normal breast cells (P<0.01). Overexpression of GTF2IP23 in ZR-75-30 cells significantly reduced the expression of GTF2I (P=0.034) and enhanced cell proliferation (P=0.017) and migration (P=0.026) capacity. Conclusions: GTF2IP23 is distinctly upregulated in breast cancer, it inhibits the expression of real gene GTF2I and promotes the proliferation of breast cancer cells.

Significant improvement of intestinal microbiota of gibel carp (Carassius auratus gibelio) after traditional Chinese medicine feeding.

AIMS: Increasing attention has been attracted to intestinal microbiota, due to interactions with nutrition, metabolism and immune defence of the host. Traditional Chinese medicine (TCM) feed additives have been applied in aquaculture to improve fish health, but the interaction with fish gut microbiota is still poorly understood. This study aimed to explore the effect of adding TCM in feed on the intestinal microbiota of gibel carp (Carassius auratus gibelio). METHODS AND RESULTS: Bacterial communities of 16 fish intestinal contents and one water sample were characterized by high-throughput sequencing and analysis of the V4-V5 region of the 16S rRNA gene. The results showed that the composition and structure of the bacterial community were significantly altered by the TCM feeding. Some phyla increased markedly (Proteobacteria, Actinobacteria, Acidobacteria, etc.), while Fusobacteria were significantly reduced. Concurrently, the richness and diversity of the taxonomic units increased, and the microbiota composition of TCM-treated fish was more homogeneous among individuals. At the genus level, the addition of TCM tended to reduce the incidence of potential pathogens (Aeromonas, Acinetobacter and Shewanella), while stimulating the emergence of some potential probiotics (Lactobacillus, Lactococcus, Bacillus and Pseudomonas). CONCLUSIONS: These data suggested that the feed additive could regulate the fish intestinal microbiota by reinforcing the microbial balance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may provide useful information for further application of TCM for diseases prevention and stress management in aquaculture.

Association between interleukin-17 gene polymorphisms and the risk of laryngeal cancer in a Chinese population.

IL-17 is associated with the occurrence and development of laryngeal cancer. However, no study has reported the association between IL-17 polymorphisms and laryngeal cancer susceptibility. Therefore, we analyzed the association of three polymorphism loci (rs2275913, 197 G/A; rs3748067, 383 A/G; and rs763780, 7488 T/C) of IL-17A and IL-17F with laryngeal cancer in the Chinese population. A case-control study was performed with 325 patients and 325 controls. Polymorphisms were detected by polymerase chain reaction and sequencing methods. SPSS17.0 software was used for statistical analysis. Allele and genotype frequencies of IL-17A rs2275913 were significantly different between patients and controls (P < 0.05). Frequencies of rs2275913 (197 G/A) AA and GA+AA genotypes compared to the GG genotype were significantly higher in patients than in controls, indicating the association of these genes with laryngeal cancer susceptibility; adjusted OR values were 2.54 (1.50-4.23) and 1.62 (1.19-2.17), respectively. Furthermore, individuals with the GA+AA genotype, compared to the GG genotype, aged ≤60 years, with smoking and alcohol consumption habits, and without a family history of cancer showed a higher cancer risk (OR = 2.74, 95%CI = 1.41-5.23; OR = 2.11, 95%CI = 1.21-3.55; OR = 1.91, 95%CI = 1.02-3.70; OR = 1.99, 95%CI = 1.08-3.39, respectively). In conclusion, the rs2275913 IL-17A (197 G/A) is associated with the incidence and development of laryngeal cancer in the Chinese population, and the AA and GA+AA genotypes harbor a high laryngeal cancer risk.

Anti-tumor role of Bacillus subtilis fmbJ-derived fengycin on human colon cancer HT29 cell line.

To explore the potential clinical anti-tumor roles of Bacillus subtilis fmbJ-derived fengycin on cell growth and apoptosis in colon cancer HT29 cell line.Fengycin was extracted from Bacillus subtilis fmbJ and detected using HPLC. The effects of different concentration of fengycin on colon cell HT29 cell activity at different time points were analyzed using MTT assay. ROS level in colon HT29 cells affected by fengycin was detected using DCFH-DA method, followed by measuring the effects of fengycin on HT29 cell apoptosis and cell cycle by flow cytometry. The effects of fengycin on Bax/Bcl-2, CDK4/cyclin D1, Caspase-6 and Caspase-3 expressions in HT29 cells were analyzed using western blot. Also, mRNA levels of Bax/Bcl-2 and CDK4/cyclin D1 in HT29 cells affected by fengycin were analyzed using qRT-PCR.Compared with controlss, 20 µg/mL of fengycin performed an inhibit role on HT29 cell growth of at 3 day (P<0.05), and high dose of fengycin showed more excellent effect on inhibiting HT29 cell growth with time increasing. Besides, fengycin could induce HT29 cell apoptosis and affect the cell cycle arrest at G1. ROS level in HT29 cells treated by fengycin was significantly increased compared with that in control group (P<0.05). Western blot analysis showed that after being treated with fengycin, Bax, Caspase-3, and Caspase-6 expressions were increased, however, Bcl-2, and CDK4/cyclin D1 expressions were decreased (P<0.05).Our study suggested that fengycin may play certain inhibit roles in the development and progression of colon cancer through involving in the cell apoptosis and cell cycle processes by targeting the Bax/Bcl-2 pathway.

[Study on culture, identification and differentiation of primary rat skeletal muscle satellite cells].

OBJECTIVE: To establish the isolation, culture and identification methods of primary rat skeletal muscle satellite cells (SMSC) and observe its characterization of differentiation in vitro. METHODS: Skeletal muscle satellite cells were obtained by tissue block culture method in combination with pre-plating techniques, and the purity of these cells was detected by both immunocytochemistry and fluorescence activated cell sorter (FACS) with Pax7 as marker of SMSC. Myogenesis of these cells was induced in differentiation medium and the mRNA expressions of myogenic differentiation gene (MyoD) and Myogenin were determined by Real-time polymerase chain reaction (PCR). RESULTS: Cells crawled out from the edge of tissue blocks after 1 week of culture. After purification by pre-plating techniques, more than 97.6% of the cells expressed Pax7, a unique marker of satellite cells. The mRNA of MyoD and Myogenin showed time-specific expression in the myogenesis induction process in vitro. CONCLUSION: Skeletal muscle satellite cells with high purity and strong differentiation ability can be obtained by means of tissue block culture method.

Lack of association between the cyclooxygenase 2 -765G>C polymorphism and prostate cancer risk: a meta-analysis.

The aim of this study was to investigate the association between the cyclooxygenase 2 (COX2) -765G>C (rs20417) polymorphism and prostate cancer (PC) risk using meta-analysis. A systematic literature search was performed using the PubMed, Embase, Cochrane Library, and Google Scholar databases by using the terms "cyclooxygenase-2/COX-2/PTGs2", "polymorphism" or "variation", and "prostate" and "cancer" or "carcinoma" to identify relevant articles up to June 14, 2014. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were assessed for PC risk associated with COX2 -765G>C polymorphism using fixed- and random-effect models. We identified a total of nine publications, including 5952 cases and 5078 controls, to investigate the effect of COX2 -765G>C on PC risk, and found no significant association in any genetic model tested (CC vs GG: OR = 0.993, 95%CI = 0.923-1.068; GC+CC vs GG: OR = 1.041, 95%CI = 0.931-1.103; CC vs GC+GG: OR = 0.858, 95%CI = 0.689-1.067; CC vs GG: OR = 0.871, 95%CI = 0.689-1.086; GC vs GG: OR = 1.032, 95%CI = 0.945-1.127). Power analysis and tests for publication bias ensured the reliability of our results. This meta-analysis suggested that the functional COX2 -765G>C polymorphism, located in the COX2 gene promoter, is unlikely to be associated with PC risk. However, additional larger, well-designed studies are still required to reach a conclusive result on this issue.

[Analysis of the feasibility and prognostic value of circulating tumor DNA monitoring in detecting gene mutations in patients with diffuse large B-cell lymphoma receiving chimeric antigen receptor T-cell therapy].

Objective: To explore the prognostic value of circulating tumor DNA (ctDNA) testing in patients with refractory/relapsed diffuse large B-cell lymphoma (R/R DLBCL) undergoing chimeric antigen receptor T-cell (CAR-T) therapy, and to guide the prevention and subsequent treatment of CAR-T-cell therapy failure. Methods: In this study, 48 patients with R/R DLBCL who received CAR-T-cell therapy at the First Affiliated Hospital of Zhejiang University School of Medicine between December 2017 and March 2022 were included. Furthermore, ctDNA testing of 187 lymphoma-related gene sets was performed on peripheral blood samples obtained before treatment. The patients were divided into complete remission and noncomplete remission groups. The chi-square test and t-test were used to compare group differences, and the Log-rank test was used to compare the differences in survival. Results: Among the patients who did not achieve complete remission after CAR-T-cell therapy for R/R DLBCL, the top ten genes with the highest mutation frequencies were TP53 (41%), TTN (36%), BCR (27%), KMT2D (27%), IGLL5 (23%), KMT2C (23%), MYD88 (23%), BTG2 (18%), MUC16 (18%), and SGK1 (18%). Kaplan-Meier survival analysis revealed that patients with ctDNA mutation genes >10 had poorer overall survival (OS) rate (1-year OS rate: 0 vs 73.8%, P<0.001) and progression-free survival (PFS) rate (1-year PFS rate: 0 vs 51.8%, P=0.011) compared with patients with ctDNA mutation genes ≤10. Moreover, patients with MUC16 mutation positivity before treatment had better OS (2-year OS rate: 56.8% vs 26.7%, P=0.046), whereas patients with BTG2 mutation positivity had poorer OS (1-year OS rate: 0 vs 72.5%, P=0.005) . Conclusion: ctDNA detection can serve as a tool for evaluating the efficacy of CAR-T-cell therapy in patients with R/R DLBCL. The pretreatment gene mutation burden, mutations in MUC16 and BTG2 have potential prognostic value.

Systolic Blood Pressure and Mortality in Community-Dwelling Older Adults: The Role of Frailty.

OBJECTIVES: To investigate whether frailty modifies the association of systolic blood pressure (SBP) with cardiovascular mortality and all-cause mortality in community-dwelling older adults. DESIGN: A prospective cohort study. SETTING: A population-based study of nationally representative older Chinese adults in a community setting. PARTICIPANTS: This study included participants aged 65 years or older from the Chinese Longitudinal Healthy Longevity Survey 2002-2014 and followed up to 2018. MEASUREMENTS: Participants were divided into two groups according to a frailty index based on the accumulation of a 44-items deficits model. The association between SBP and mortality was analyzed using multivariable-adjusted Cox proportional hazards models. RESULTS: Among 18,503 participants included, the mean age was 87.2 years and the overall median follow-up time was 42.7 months. We identified 7808 (42.2%) frail participants (mean frailty index=0.33), in which 7533 (96.5%) died during the follow-up. Effect modification by frailty was detected (P for interaction=0.032). Among frail participants, a U-shaped association was found with hazard ratios of 1.16 (95% CI, 1.02-1.32) for SBP < 100 mmHg, and 1.11 (95% CI, 1.00-1.24) for SBP ≥ 150 mmHg compared with SBP 120-130 mmHg. For non-frail older adults, a tendency toward higher risk among those with SBP ≥ 130 mmHg was observed. The analyses towards cardiovascular mortality showed similar results. CONCLUSION: Our results suggest the presence of effect modification by frailty indicating a possible negative effect for elevated SBP in non-frail older adults and a U-shaped relationship of SBP in frail older adults with respect to mortality even after adjusting for diastolic blood pressure.

The Relationship between Telomere Length and Cancer Mortality: Data from the 1999-2002 National Healthy and Nutrition Examination Survey (NHANES).

OBJECTIVES: The association between telomeres length (TL) and cancer mortality is uncertain. We tested the hypotheses that long TL are associated with reduced cancer mortality. DESIGN: Prospective cohort study. SETTING: the National Health and Nutrition Survey (NHANES, 1999-2002). PARTICIPANTS: The analytic sample included adults (n = 7183) who had TL measurements. MEASUREMENTS: DNA was obtained via blood samples. Telomere length was assessed using the quantitative polymerase chain reaction method. RESULTS: During follow-up (0.08-12.7 person-years, median = 9.5 years), we observed 195 participants had cancer as causes of death. TL was negatively corelated with age, body mass index (BMI), systolic blood pressure (SBP), C-reactive protein (CRP), race, diabetes, hypertension, cardiovascular diseases (CVD) and cancer mortality, conversely, positively corelated with alcohol use, but not related to diastolic blood pressure (DBP) and smoking. Kaplan-Meier analysis revealed that TL was significantly associated with cancer mortality (log-rank, P <0.001). CONCLUSIONS: Our study expands upon previous evidence of a relationship between TL and cancer mortality. TL may be a useful tool for evaluating risk of cancer mortality in American adults.

Enhancer-dependent transcriptional oscillations in mouse erythroleukemia cells.

By using recombinase-mediated cassette exchange, a method that allows integration of single copies of different constructs at the same predetermined chromosomal location, several expression cassettes have been integrated at a randomly chosen locus in the genome of mouse erythroleukemia cells. The cassettes studied contain the human beta-globin promoter fused to lacZ coding sequences either alone or linked to DNase I-hypersensitive site HS2, HS3, or HS234 (a large locus control region fragment containing HS2, HS3, and HS4) of the human beta-globin locus control region. Analysis of expression of these cassettes revealed mosaic expression patterns reminiscent of, but clearly different from, position effect variegation. Further investigations demonstrated that these mosaic expression patterns are caused by dynamic activation and inactivation of the transcription unit, resulting in oscillations of expression. These oscillations occur once in every few cell cycles at a rate specific for the enhancer present at the locus. DNase I sensitivity studies revealed that the chromatin is accessible and that DNase-hypersensitive sites were present whether or not the transcription unit is active, suggesting that the oscillations occur between transcriptionally competent and transcriptionally active chromatin conformations, rather than between open and closed chromatin conformations. Treatment of oscillating cells with trichostatin A eliminates the oscillations only after the cells have passed through late G1 or early S, suggesting that these oscillations might be caused by changes in histone acetylation patterns.

Position effects are influenced by the orientation of a transgene with respect to flanking chromatin.

We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement.

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.