To explore racial-ethnic disparities in adverse pregnancy outcomes among Medicaid recipients, and to estimate excess Medicaid costs associated with the disparities. Cross-sectional study of adverse pregnancy outcomes and Medicaid payments using data from Medicaid Analytic eXtract files on all Medicaid enrollees in fourteen southern states. Compared to other racial and ethnic groups, African American women tended to be younger, more likely to have a Cesarean section, to stay longer in the hospital and to incur higher Medicaid costs. African-American women were also more likely to experience preeclampsia, placental abruption, preterm birth, small birth size for gestational age, and fetal death/stillbirth. Eliminating racial disparities in adverse pregnancy outcomes (not counting infant costs), could generate Medicaid cost savings of $114 to $214 million per year in these 14 states. Despite having the same insurance coverage and meeting the same poverty guidelines for Medicaid eligibility, African American women have a higher rate of adverse pregnancy outcomes than White or Hispanic women. Racial disparities in adverse pregnancy outcomes not only represent potentially preventable human suffering, but also avoidable economic costs. There is a significant financial return-on-investment opportunity tied to eliminating racial disparities in birth outcomes. With the Affordable Care Act expansion of Medicaid coverage for the year 2014, Medicaid could be powerful public health tool for improving pregnancy outcomes.
Disparities; Medicaid; Economic burden; Adverse maternal-child health outcomes and eliminating disparities
Health promotion for prevention and control of Tuberculosis (TB) is implemented worldwide because of its importance, but few reports have evaluated its impact on behavior due to a lack of standard outcome indicators. The objective of this study was to establish a framework of behavioral indicators for outcome evaluation of TB health promotion among TB suspects and patients.
A two-round modified Delphi method involving sixteen TB control experts was used to establish a framework of behavioral indicators for outcome evaluation of TB health promotion targeted at TB suspects and patients.
Sixteen of seventeen invited experts in TB control (authority score of 0.91 on a 1.0 scale) participated in round 1 survey. All sixteen experts also participated in a second round survey. After two rounds of surveys and several iterations among the experts, there was consensus on a framework of indicators for measuring outcomes of TB health promotion for TB suspects and patients. For TB suspects, the experts reached consensus on 2 domains (“Healthcare seeking behavior” and “Transmission prevention”), 3 subdomains (“Seeking care after onset of TB symptoms”, “Pathways of seeking care” and “Interpersonal contact etiquette”), and 8 indicators (including among others, “Length of patient delay”). For TB patients, consensus was reached on 3 domains (“Adherence to treatment”, “Healthy lifestyle” and “Transmission prevention”), 8 subdomains (including among others, “Adherence to their medication”), and 14 indicators (including “Percentage of patients who adhered to their medication”). Operational definitions and data sources were provided for each indicator.
The findings of this study provide the basis for debate among international experts on a framework for achieving global consensus on outcome indicators for TB health promotion interventions targeted at TB patients and suspects. Such consensus will help to increase effectiveness of TB health promotion, while ensuring international comparability of outcome data.
Health promotion; Outcome evaluation; TB suspect; TB patient; Indicator
The purpose of this study was to explore the racial and ethnic disparities in initiation of antiretroviral treatment (ARV treatment or ART) among HIV-infected Medicaid enrollees 18–64 years of age in 14 southern states which have high prevalence of HIV/AIDS and high racial disparities in HIV treatment access and mortality.
We used Medicaid claims data from 2005 to 2007 for a retrospective cohort study. We compared frequency variances of HIV treatment uptake among persons of different racial- ethnic groups using univariate and multivariate methods. The unadjusted odds ratio was estimated through multinomial logistic regression. The multinomial logistic regression model was repeated with adjustment for multiple covariates.
Of the 23,801 Medicaid enrollees who met criteria for initiation of ARV treatment, only one third (34.6%) received ART consistent with national guideline treatment protocols, and 21.5% received some ARV medication, but with sub-optimal treatment profiles. There was no significant difference in the proportion of people who received ARV treatment between black (35.8%) and non-Hispanic whites (35.7%), but Hispanic/Latino persons (26%) were significantly less likely to receive ARV treatment.
Overall ARV treatment levels for all segments of the population are less than optimal. Among the Medicaid population there are no racial HIV treatment disparities between Black and White persons living with HIV, which suggests the potential relevance of Medicaid to currently uninsured populations, and the potential to achieve similar levels of equality within Medicaid for Hispanic/Latino enrollees and other segments of the Medicaid population.
Several lines of evidence suggest that genes involved in dopamine (DA) transmission may contribute to creativity. Among these genes, the catechol-O-methyltransferase gene (COMT) and the dopamine D2 receptor gene (DRD2) are the most promising candidates. Our previous study has revealed evidence for the involvement of DRD2 in creative potential. The present study extended our previous study by systematically exploring the association of COMT with creative potential as well as the interaction between COMT and DRD2. Twelve single nucleotide polymorphisms (SNPs) covering COMT were genotyped in 543 healthy Chinese college students whose creative potentials were assessed by divergent thinking tests. Single SNP analysis showed that rs174697 was nominally associated with verbal originality, two SNPs (rs737865 and rs5993883) were nominally associated with figural fluency, and two SNPs (rs737865 and rs4680) were nominally associated with figural originality. Haplotype analysis showed that, the TCT and CCT haplotype (rs737865-rs174675-rs5993882) were nominally associated with figural originality, and the TATGCAG and CGCGGGA haplotype (rs4646312-rs6269-rs4633-rs6267-rs4818-rs4680-rs769224) were nominally associated with figural originality and verbal flexibility, respectively. However, none of these nominal findings survived correction for multiple testing. Gene–gene interaction analysis identified one significant four-way interaction of rs174675 (COMT), rs174697 (COMT), rs1076560 (DRD2), and rs4436578 (DRD2) on verbal fluency, one significant four-way interaction of rs174675 (COMT), rs4818 (COMT), rs1076560 (DRD2), and rs4648317 (DRD2) on verbal flexibility, and one significant three-way interaction of rs5993883 (COMT), rs4648319 (DRD2), and rs4648317 (DRD2) on figural flexibility. In conclusion, the present study provides nominal evidence for the involvement of COMT in creative potential and suggests that DA related genes may act in coordination to contribute to creativity.
creativity; creative potential; divergent thinking; dopamine; COMT; DRD2; gene–gene interaction
Objective: The efflux pump (EP) is one of the major mechanisms of antibiotic resistance in Klebsiella pneumoniae. However, there are few reports on the effect of the abuse of antibiotic use on the activity of EPs. To determine whether the use of low efficacy antibiotics has any effect on the activity of EPs and induces drug resistance in K. pneumoniae, we investigated the effect of ciprofloxacin on the activity of EPs in K. pneumoniae strains. Methods: Sixteen susceptible K. pneumoniae strains were isolated from patients and their minimum inhibitory concentrations (MICs) of ciprofloxacin were measured in the absence and presence of the pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The strains were then induced with a gradient of ciprofloxacin until the MICs of the strains showed no further increase, to obtain induced resistant strains. The EP activities of the strains before and after induction were compared using EP inhibition and ethidium bromide (EtBr) accumulation assays. Results: The MIC values of the strains were 16‒256 times higher after induction than before induction. In the presence of CCCP, the MIC values of 50% of the induced strains were 2‒4-fold lower than that in the absence of this inhibitor. The EtBr accumulation assay showed that the fluorescence of EtBr in the induced cells was lower than that in the cells before induction. Conclusions: EPs are widespread in susceptible and drug-resistant K. pneumoniae strains. Induction with ciprofloxacin may increase the activity of EPs in K. pneumoniae. The EtBr accumulation assay is more sensitive than the EP inhibition assay in evaluating the activity of EPs in K. pneumoniae.
Klebsiella pneumoniae; Efflux pump; Ciprofloxacin; Antibiotic resistance
Leukocyte cell–derived chemotaxin 2 enhances phagocytosis and bacterial killing of macrophages to improve the outcome of bacterial-induced sepsis.
Leukocyte cell–derived chemotaxin 2 (LECT2) is a multifunctional cytokine and reduced plasma levels were found in patients with sepsis. However, precise functions and mechanisms of LECT2 remain unclear. The aim of the present study was to determine the role of LECT2 in modulating immune responses using mouse sepsis models. We found that LECT2 treatment improved outcome in mice with bacterial sepsis. Macrophages (MΦ), but not polymorphonuclear neutrophils, mediated the beneficial effect of LECT2 on bacterial sepsis. LECT2 treatment could alter gene expression and enhance phagocytosis and bacterial killing of MΦ in vitro. CD209a was identified to specifically interact with LECT2 and mediate LECT2-induced MΦ activation. CD209a-expressing MΦ was further confirmed to mediate the effect of LECT2 on sepsis in vivo. Our data demonstrate that LECT2 improves protective immunity in bacterial sepsis, possibly as a result of enhanced MΦ functions via the CD209a receptor. The modulation of MΦ functions by LECT2 may serve as a novel potential treatment for sepsis.
Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70) concentrations and PCOS in a non-obese Chinese population.
Methods and Results
Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI) levels, as well as Insulin resistance index (HOMA-IR) (P < 0.05). Identically, markers of oxidative stress (malondialdehyde (MDA), 8-Hydroxy-desoxyguanosine (8-OHdG), Nitric oxide (NO)) and inflammation (tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP)) were markedly increased when compared to controls (P < 0.05). Elevated serum Hsp70 was positively correlated with IR, oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI). The receiver-operating characteristic curve (ROC) analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946) vs. 0.822 (95% CI 0.744-0.900); P for difference = 0.015).
Conclusions and Significance
Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.
Insulin resistance triggers the developments of diabetes mellitus and atherosclerosis. Tribbles homolog 3 (TRIB3) is involved in insulin resistance. We aimed to investigate whether TRIB3 is implicated in diabetic atherosclerosis. Sixty 3-week-old apolipoprotein E (ApoE−/−)/LDR receptor (LDLR−/−) mice were randomly divided into chow and diabetes groups. Diabetes was induced by a high-fat and high-sugar diet combined with low-dose streptozotocin. Mice in both groups were randomly divided into vehicle and TRIB3-silencing groups. After transfection, all mice were killed to evaluate the effects of TRIB3 on atherosclerosis. Silence of TRIB3 markedly decreased insulin resistance (P = 0.039) and glucose (P = 0.019), regardless of diabetes. Ultrasonography-measured parameters were similar in both groups, with and without silence of TRIB3. However, silence of TRIB3 decreased the aortic atherosclerotic burden (P = 1 × 10−13). Further study showed that in brachiocephalic lesions, fibrous cap thickness, cap-to-core ratio, collagen content, and the number of smooth muscle cells were significantly increased (P < 0.01 for all) by silence of TRIB3, whereas lipid and macrophage contents remained unaltered, with the vulnerability index significantly reduced. Moreover, the numbers of apoptotic cells and macrophages in brachiocephalic lesions were both significantly decreased (P < 0.01 for both). Macrophage migration was decreased (P = 4 × 10−4) by knocking down TRIB3, whereas adhesion and phagocytosis were increased (P < 0.05 for both). Silence of TRIB3 would diminish atherosclerotic burden and increase the plaque stability in diabetic mice.
At present there is no effective and accepted therapy for hepatic fibrosis. Transforming growth factor (TGF)-β1 signaling pathway contributes greatly to hepatic fibrosis. Reducing TGF-β synthesis or inhibiting components of its complex signaling pathway represent important therapeutic targets. The aim of the study was to investigate the effect of curcumin on liver fibrosis and whether curcumin attenuates the TGF-β1 signaling pathway.
Sprague–Dawley rat was induced liver fibrosis by carbon tetrachloride (CCl4) for six weeks together with or without curcumin, and hepatic histopathology and collagen content were employed to quantify liver necro-inflammation and fibrosis. Moreover, the mRNA and protein expression levels of TGF-β1, Smad2, phosphorylated Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) were determined by quantitative real time-PCR, Western blot, or immunohistochemistry.
Rats treated with curcumin improved liver necro-inflammation, and reduced liver fibrosis in association with decreased α-smooth muscle actin expression, and decreased collagen deposition. Furthermore, curcumin significantly attenuated expressions of TGFβ1, Smad2, phosphorylated Smad2, Smad3, and CTGF and induced expression of the Smad7.
Curcumin significantly attenuated the severity of CCl4-induced liver inflammation and fibrosis through inhibition of TGF-β1/Smad signalling pathway and CTGF expression. These data suggest that curcumin might be an effective antifibrotic drug in the prevention of liver disease progression.
Curcumin; Hepatic stellate cells; Liver fibrosis; Transforming growth factor-beta; Smads; Connective tissue growth factor
AIM: To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following lamivudine treatment of HepG2.2.15 cells.
METHODS: HepG2.2.15 cells were treated with 2 μmol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (cytokine group), or treated with 2 μmol/L lamivudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 × 105 HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circular DNA that may have remained. Both HBV covalently closed circular DNA (cccDNA) and HBV DNA were examined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantified with enzyme-linked immunosorbent assay. Cell viability was measured with the cell counting kit-8 assay.
RESULTS: Compared to lamivudine alone (22.63% ± 0.12%), both sequential (51.50% ± 0.17%, P = 0.034) and cytokine treatment (49.66% ± 0.06%, P = 0.041) showed a stronger inhibition of HBV cccDNA; the difference between the sequential and cytokine groups was not statistically significant (51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88). The sequential group showed less inhibition of HBV DNA replication than the lamivudine group (67.47% ± 0.02% vs 82.48% ± 0.05%, P = 0.014); the difference between the sequential and cytokine groups was not statistically significant (67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071). The levels of HBsAg and HBeAg were significantly decreased in the sequential treatment group compared to the other groups [HBsAg: 3.48 ± 0.04 (control), 3.09 ± 0.08 (lamivudine), 2.55 ± 0.13 (cytokine), 2.32 ± 0.08 (sequential), P = 0.042 for each between-group comparison; HBeAg: 3.48 ± 0.01 (control), 3.08 ± 0.08 (lamivudine), 2.57 ± 0.15 (cytokine), 2.34 ± 0.12 (sequential), P = 0.048 for each between-group comparison]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells (58.03% ± 8.03% vs 100%, P = 0.000). Lamivudine pretreatment significantly reduced IFN-γ + TNF-α-mediated toxicity of HepG2.2.15 cells [85.82% ± 5.43% (sequential) vs 58.03% ± 8.03% (cytokine), P = 0.002].
CONCLUSION: Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-γ and TNF-α by decreasing the viral load.
Hepatitis B virus; Covalently closed circular DNA; Interferon-γ; Tumor necrosis factor-α; Lamivudine
Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.
DNA methylation; physical activity; peripheral blood
Although the protective effect of transient ureteral obstruction (UO) prior to ischemia on subsequent renal ischemia/reperfusion (I/R) injury has been documented, the underlying molecular mechanism remains to be understood. We showed in the current study that 24 h of UO led to renal tubular hypoxia in the ipsilateral kidney in mice, with the accumulation of hypoxia-inducible factor (HIF)-2α, which lasted for a week after the release of UO. To address the functions of HIF-2α in UO-mediated protection of renal IRI, we utilized the Mx-Cre/loxP recombination system to knock out target genes. Inactivation of HIF-2α, but not HIF-1α blunted the renal protective effects of UO, as demonstrated by much higher serum creatinine level and severer histological damage. UO failed to prevent postischemic neutrophil infiltration and apoptosis induction in HIF-2α knockout mice, which also diminished the postobstructive up-regulation of the protective molecule, heat shock protein (HSP)-27. The renal protective effects of UO were associated with the improvement of the postischemic recovery of intra-renal microvascular blood flow, which was also dependent on the activation of HIF-2α. Our results demonstrated that UO protected the kidney via activation of HIF-2α, which reduced tubular damages via preservation of adequate renal microvascular perfusion after ischemia. Thus, preconditional HIF-2α activation might serve as a novel therapeutic strategy for the treatment of ischemic acute renal failure.
AIM: To evaluate the impacts of Schistosoma japonicum (S. japonicum) ova on the tight junction barriers in a trinitrobenzenesulfonic acid (TNBS)-induced colitis model.
METHODS: Balb/c mice were randomly divided into three groups: control group; TNBS+ova- group and TNBS+ova+ group. TNBS was used intracolonic to induce colitis and mice of the TNBS+ova+ group were pre-exposed to S. japonicum ova as a prophylactic intervention. Colon inflammation was quantified using following variables: mouse mortality, weight loss, colon extent and microscopic inflammation score. Serum expression of tumor necrosis factor-α and interferon-γ were assessed to evaluate the systemic inflammatory response. NOD2 and its mRNA were also tested. Bacterial translocations were tested by culturing blood and several tissues. ZO-1 and occludin were chosen as the representations of tight junction proteins. Both the proteins and mRNA were assessed.
RESULTS: Ova pre-treatment contributed to the relief of colitis and decreased the mortality of the models. NOD2 expression was significantly downregulated when pretreated with the ova. The TNBS injection caused a significant downregulation of ZO-1 and occludin mRNA together with their proteins in the colon; ova pre-exposure reversed these alterations. Treatment with S. japonicum ova in the colitis model caused lower intestinal bacterial translocation frequency.
CONCLUSION: S. japonicum ova can maintain epithelial barrier function through increasing tight junction proteins, thus causing less exposure of NOD2 to the luminal antigens which may activate a series of inflammatory factors and induce colitis.
Crohn’s disease; Schistosoma japonicum ova; Tight junction protein; ZO-1; Occludin
The congenital long QT syndrome is a heterogeneous genetic disease associated with delayed cardiac repolarization, prolonged QT intervals, the development of ventricular arrhythmias and sudden death. Type 2 congenital long QT syndrome (LQT2) results from KCNH2 or hERG gene mutations. hERG encodes the Kv11.1 alpha subunit of the rapidly activating delayed rectifier K+ current in the heart. Studies of mutant hERG channels indicate that most LQT2 missense mutations generate trafficking-deficient Kv11.1 channels.
To identify the mechanism underlying G572R-hERG by using molecular and electrophysiological analyses.
METHODS AND RESULTS:
To elucidate the electrophysiological properties of the G572R-hERG mutant channels, mutant hERG subunits were heterologously expressed in HEK293 cells alone or in combination with wild-type (WT)-hERG subunits. Patch-clamp techniques were used to record currents, and double immunofluorescence protein tagging and Western blotting were performed to examine the cellular trafficking of mutant subunits. When expressed alone, G572R-hERG subunits were not present in the cell membrane and did not produce detectable currents. When coexpressed with WT-hERG subunits, G572R-hERG decreased current density and altered gating properties of the WT-hERG channel.
The hERG-associated missense mutation G572R, like most LQT2 missense mutations, generates a trafficking-deficient phenotype. Furthermore, G572R-hERG causes a loss of function in hERG by a strong dominant negative effect on the WT-hERG channel.
hERG; Long QT syndrome; Mutation; Potassium channel; Protein trafficking
AIM: To evaluate the clinical value of serum CA19-9 levels in predicting the respectability of pancreatic carcinoma according to receiver operating characteristic (ROC) curve analysis.
METHODS: Serum CA19-9 levels were measured in 104 patients with pancreatic cancer which were possible to be resected according to the imaging. ROC curve was plotted for the CA19-9 levels. The point closest to the upper left-hand corner of the graph were chosen as the cut-off point. The sensitivity, specificity, positive and negative predictive values of CA19-9 at this cut-off point were calculated.
RESULTS: Resectable pancreatic cancer was detected in 58 (55.77%) patients and unresectable pancreatic cancer was detected in 46 (44.23%) patients. The area under the ROC curve was 0.918 and 95% CI was 0.843-0.992. The CA19-9 level was 353.15 U/mL, and the sensitivity and specificity of CA19-9 at this cut-off point were 93.1% and 78.3%, respectively. The positive and negative predictive value was 84.38% and 90%, respectively.
CONCLUSION: Preoperative serum CA19-9 level is a useful marker for further evaluating the resectability of pancreatic cancer. Obviously increased serum levels of CA19-9 (> 353.15 U/mL) can be regarded as an ancillary parameter for unresectable pancreatic cancer.
Pancreatic carcinoma; Resection; Tumor markers; CA19-9; Receiver operating characteristic curve
AIM: To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations.
METHODS: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods.
RESULTS: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non-cancerous liver tissues (11/19 vs 18/19, P = 0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P < 0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45) - 56.8% (25/45) tumors and 40.9% (18/45) - 52.3% (23/45) non-tumor tissues.
CONCLUSION: HBx deletion, especially the COOH-terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.
Hepatitis B virus; X gene; Hepatocellular carcinoma; COOH-terminal deletion mutation; Integration