Deficiency of ASGR1 in pigs recapitulates reduced risk factor for cardiovascular disease in humans.

Genetic variants in the asialoglycoprotein receptor 1 (ASGR1) are associated with a reduced risk of cardiovascular disease (CVD) in humans. However, the underlying molecular mechanism remains elusive. Given the cardiovascular similarities between pigs and humans, we generated ASGR1-deficient pigs using the CRISPR/Cas9 system. These pigs show age-dependent low levels of non-HDL-C under standard diet. When received an atherogenic diet for 6 months, ASGR1-deficient pigs show lower levels of non-HDL-C and less atherosclerotic lesions than that of controls. Furthermore, by analysis of hepatic transcriptome and in vivo cholesterol metabolism, we show that ASGR1 deficiency reduces hepatic de novo cholesterol synthesis by downregulating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and increases cholesterol clearance by upregulating the hepatic low-density lipoprotein receptor (LDLR), which together contribute to the low levels of non-HDL-C. Despite the cardioprotective effect, we unexpectedly observed mild to moderate hepatic injury in ASGR1-deficient pigs, which has not been documented in humans with ASGR1 variants. Thus, targeting ASGR1 might be an effective strategy to reduce hypercholesterolemia and atherosclerosis, whereas further clinical evidence is required to assess its hepatic impact.

FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs.

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.

Calcineurin modulates growth, stress tolerance, and virulence in Metarhizium acridum and its regulatory network.

Calcineurin is highly conserved and regulates growth, conidiation, stress response, and pathogenicity in fungi. However, the functions of calcineurin and its regulatory network in entomopathogenic fungi are not clear. In this study, calcineurin was functionally analyzed by deleting the catalytic subunit MaCnA from the entomopathogenic fungus Metarhizium acridum. The ΔMaCnA mutant had aberrant, compact colonies and blunt, shortened hyphae. Conidia production was reduced, and phialide differentiation into conidiogenous cells was impaired in the ΔMaCnA mutant. ΔMaCnA had thinner cell walls and greatly reduced chitin and ß-1,3-glucan content compared to the wild type. The ΔMaCnA mutant was more tolerant to cell wall-perturbing agents and elevated or decreased exogenous calcium but less tolerant to heat, ultraviolet irradiation, and caspofungin than the wild type. Bioassays showed that ΔMaCnA had decreased virulence. Digital gene expression profiling revealed that genes involved in cell wall construction, conidiation, stress tolerance, cell cycle control, and calcium transport were downregulated in ΔMaCnA. Calcineurin affected some components of small G proteins, mitogen-activated protein kinase, and cyclic AMP (cAMP)-protein kinase A signaling pathways in M. acridum. In conclusion, our results gave a global survey of the genes downstream of calcineurin in M. acridum, providing molecular explanations for the changes in phenotypes observed when calcineurin was deleted.

The CTCF insulator protein is posttranslationally modified by SUMO.

The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.

[Effects of ventilation in prone position combined with recruitment maneuver on lung injury in dogs with acute respiratory distress syndrome].

OBJECTIVE: To evaluate protective effect and its mechanism of prone position ventilation (PPV) combined with recruitment maneuver (RM) as a lung protective ventilation strategy on oleic acid-induced acute respiratory distress syndrome (ARDS) in dogs. METHODS: Twenty-four oleic acid-induced ARDS dogs were ventilated with volume controlled ventilation (VCV): 16 cm H2O (1 cm H2O=0.098 kPa) of positive end-expiratory pressure (PEEP) and 10 ml/kg of tidal volume (VT). All dogs were randomly divided by random digit table into four groups: supine position (SP group), prone position (PP group), supine position+RM (SPRM group), and prone position+RM (PPRM group, 6 in each group), and ventilated by VCV for 4 hours and then sacrificed by exsanguination. The serum levels of inflammatory mediators were measured respectively at 0.5, 2 and 4 hours. After they were sacrificed, the levels of cytokines in left lung tissue homogenate were measured. The wet/dry weight ratio of right lung was determined and histological sections of the lungs were prepared and examined. RESULTS: (1) At 4 hours, interleukin-8 (IL-8) in serum in the SPRM group was significantly higher than in other three groups (all P<0.05), tumor necrosis factor-alpha (TNF-alpha) in serum in the SPRM group was significantly higher than in the PP group and the PPRM group (all P<0.05). (2) IL-8 in lung tissue homogenate of the dorsal aspect of the lung in the SP group was higher than in the PP group and the PPRM group (both P<0.05). TNF-alpha in lung tissue homogenate at the dorsal aspect of the lung in the SPRM group was higher than in the PP group and the PPRM group (both P<0.05). (3) Wet/dry weight ratio of right lung in the PP group and the PPRM group were significantly lower than that in the SP group and the SPRM group (all P<0.05). (4)Pathology score of lung tissue at the dorsal aspect of the lung in the PP group and PPRM group was significantly lower than in the SP group and the SPRM group (all P<0.05). CONCLUSION: Protective ventilation strategy combined with RM is safer in prone position than supine position, and it alleviates lung injury in dog with ARDS.

[Nitric oxide inhalation improves alveolar liquid clearance in rabbit with endotoxin induced acute lung injury].

OBJECTIVE: To investigate the effects of nitric oxide (NO) inhalation (iNO) on alveolar liquid clearance (ALC), alveolar permeability, and lung edema, and its possible mechanism. METHODS: Eighteen male rabbits were challenged with endotoxin, and they were randomly assigned into three groups: mechanical ventilator (MV) group [volume control ventilation: tidal volume (V(T)) 15 ml/kg, respiration rate (RR) 40 beats/minute, positive end-expiratory pressure (PEEP): 5 cm H(2)O (1 cm H(2)O=0.098 kPa)], high; NO ( 40 x 10(-6) NO, HNO) group and low concentration; NO (10 x 10(-6) NO, LNO) group. Indexes including haemodynamics, blood gas analysis, and mechanics of breathing were determined after MV at different time points in each group. The rabbits were sacrificed after MV lasting for 4 hours. Wet/dry weight (W/D) ratio of the lung was calculated, and the condition of alveolar exudation was observed. RESULTS: (1) Oxygenation index (PaO(2)/FiO(2)) in HNO group and LNO group were higher than those in MV group at 0.5 hour after treatment. (2) The peak pressure of airway (Ppeak) and plateau pressure of airway (Pplat) in LNO group were lower than those in MV group at 0.5, 2 and 4 hours after treatment (P<0.05 or P<0.01). Ppeak at 4 hours, Pplat at 2 hours and 4 hours were lower in HNO group as compared with those in MV group (P<0.05 or P<0.01). Pplat in LNO group was lower than that in HNO group at 2 hours and 4 hours (both P<0.01). (3) Alveolar exudation in HNO group and LNO group was milder than that in MV group (F=22.756, P<0.01). ALC in HNO group and LNO group were higher than that in MV group (F=3.965, P<0.05). The W/D ratio of lung in MV group was higher than that in HNO group, and the W/D rate in HNO group was higher than that in LNO group (F=11.740, P<0.01). (4) Lung injury score in HNO group and LNO group was higher than that in MV group, but without significant difference. CONCLUSION: iNO can reduce lung edema by increasing ALC and improving alveolar permeability, and the LNO may be more effective than HNO in treatment of early acute lung injury induced by endotoxin.

[The role of polymorphonuclear cells in lung ischemia-reperfusion injury in a canine model of pulmonary thromboembolism].

OBJECTIVE: To explore the effects of polymorphonuclear cells (PMN) on lung ischemia-reperfusion (I/R) injury in a canine model of pulmonary thromboembolism. METHODS: Fifteen dogs were divided into three groups: a sham group (n = 5), an ischemia group (n = 5) and a reperfusion group (n = 5). PMN in the whole blood were isolated with density gradient centrifugation. Apoptosis rates of the PMN was measured through flow cytometer after the cells were labeled by Annexin V-FITC before embolectomy and at 2, 4, 6 h after the operation. The myeloperoxidase (MPO) concentrations in lung homogenates were measured by ELISA. Alveolar PMN in the reperfusion lobar were observed by optical microscopy. The lung ultrastructure was studied by transmission electron microscopy. RESULTS: Alveolar PMN infiltration and the concentrations of MPO in the reperfusion group were significantly higher than those in the ischemia group (PMN (31 +/- 11) vs (8 +/- 4)/ten high power fields, MPO (11.7 +/- 1.6) vs (9.1 +/- 0.5) microg/L, P < 0.05). In the reperfusion group, abundant inflammatory cell infiltration was observed, predominantly with PMN in the alveoli. Apoptosis rates of the blood PMN at 6 h after reperfusion were much lower than before reperfusion (3.0 +/- 2.5)% vs (7.4 +/- 4.5)%, P < 0.05). At 4 and 6 h after operation, the PMN apoptosis rates in the reperfusion group were significantly lower than the ischemia group (4 h: (4.8 +/- 2.6)% vs (9.3 +/- 2.0)%, 6 h: (3.0 +/- 2.5)% vs (8.0 +/- 1.6)%, P < 0.05). PMN attaching firmly to the alveolar septum were observed by electron microscope. CONCLUSION: PMN with enhanced activities and decreased apoptosis rate, are involved in the cellular mechanisms of the lung I/R injury in this model of pulmonary thromboembolism.

[Effects of different durations of thromboembolism on blood gases, hemodynamics, pulmonary arteriography and thrombo-pathology in a canine model with selective embolization of pulmonary lobar arteries].

OBJECTIVE: To investigate the effects of different durations of thromboembolism on blood gases, hemodynamic parameters, pulmonary arteriography and thrombo-pathology in an animal model mimicking chronic pulmonary thromboembolism (PTE). METHODS: Sixteen dogs were embolized with five thrombi developed by autologous blood into the left lower pulmonary artery (n = 15) and the right lower pulmonary artery (n = 1, used to confirm the available method of selective embolization). The 15 dogs were divided into three groups: sham group (n = 5), one-week group (n = 5) and two-week group (n = 5) according to the different durations of embolization. Swan-Ganz catheter was used to guide a plastic duct, through which the thrombi were injected selectively into the left or right lower pulmonary artery by X-ray fluoroscopy. Local pulmonary arteriography of lower pulmonary arteries was taken. Blood pressure (BP), and blood gases were measured. Central venous pressure (CVP), mean pulmonary arterial pressure (MPAP), pulmonary arteriole wedge pressure (PAWP), and cardiac output (CO) were recorded, and pulmonary vascular resistance (PVR) was calculated. Each dog underwent muscular injection with tranexamic acid for one or two weeks to prevent thrombolysis. The lower lung lobe was dissected to confirm the thromboembolism after one or two weeks. The lung sections were stained with phosphotungstic acid hematoxylin (PTAH) to observe thromboemboli with optical microscopy. RESULTS: In the PTE group, PaO(2)/FiO(2), MPAP and PVR changed significantly as compared to baseline values (P < 0.05) after one hour of embolization, with MPAP increasing from (15 +/- 3) mm Hg to (21 +/- 4) mm Hg, PVR increasing from (178 +/- 114) mm Hg.s/L to (404 +/- 260) mm Hg.s/L, and PaO(2)/FiO(2) decreasing from (508 +/- 58) mm Hg to (395 +/- 100) mm Hg; these parameters returned to the baseline values one or two weeks later. After embolization, pulmonary arteriography demonstrated lower lobar artery cut-off perfusion defects. One week later, pulmonary arteriography demonstrated irregularities and stiffness of the arterial wall, enlarged proximal part of lower pulmonary artery and cut-off perfusion defects. Poor filling at embolus site was evident after embolization for two weeks. In the 1-week PTE group, organized tissue covered with the blue-purple fibrin nest was observed in the thrombus with PTAH stain. In the two-week group, the well organized thrombi were partially recanalized and surrounded and invaded by hyperplastic tissues from pulmonary artery wall. CONCLUSIONS: A canine model mimicking chronic PTE can be established by the use of fibrinolytic inhibitor tranexamic acid. Different manifestations on pulmonary arteriography and varied degree of organization of thrombi are evident at different times after embolization.

Association of cottontail rabbit papillomavirus E6 oncoproteins with the hDlg/SAP97 tumor suppressor.

Papillomaviruses are small DNA viruses that infect epithelial tissues and cause warts. Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The E6 and E7 oncogenes are the only genes consistently expressed in HPV-positive cervical cancer cells. Cottontail rabbit papillomavirus (CRPV) induces papillomas and carcinomas on cottontail and domestic rabbits and provides an excellent animal model of HPV infection and vaccine development. CRPV encodes three transforming proteins; LE6, SE6, and E7. Each of these proteins is required for papilloma formation. Like HPV E7, the CRPV E7 protein binds to the tumor suppressor pRB. In contrast, unlike HPV E6, the CRPV E6 proteins do not bind the tumor suppressor p53. Although more than a dozen cellular proteins have been identified as HPV E6 interacting proteins, nothing is known about the cellular interacting proteins of CRPV E6s. Here we describe the association of CRPV E6s with hDlg/SAP97, the mammalian homolog of the Drosophila discs large tumor suppressor protein. HPV E6 has previously shown to bind and target hDlg/SAP97 for degradation. Our results demonstrate that both LE6 and SE6 interact with hDlg/SAP97, although their association does not lead to the degradation of hDlg/SAP97. The PDZ domains of hDlg were shown to be sufficient for interaction with CRPV E6 proteins while the C-terminus of CRPV E6 is essential for the interaction with hDlg. The association of hDlg with SE6 may be important but not sufficient for the transformation of NIH 3T3 cells by SE6. Importantly, a CRPV SE6 mutant defective for papilloma formation did not interact with hDlg. These results suggest that interaction with hDlg/SAP97 plays a role in the biological function of CRPV E6s.

The KCNQ1OT1 promoter, a key regulator of genomic imprinting in human chromosome 11p15.5.

The human 11p15.5 region contains several maternally and paternally imprinted genes. Dysregulation of imprinting of some of these genes occurs in the Beckwith-Wiedemann syndrome and several tumors. Imprinting in this region is controlled by two imprinting control regions (ICR). ICR1 acts as an insulator that regulates the reciprocal imprinting of the IGF2 and H19 genes. A differentially methylated region in ICR2 regulates the expression of a long transcript called KCNQ1OT1. This paternally expressed transcript negatively regulates several paternally imprinted genes around ICR2. Biallelic expression of the KCNQ1OT1 transcript is the primary molecular defect in over 50% of cases of Beckwith-Wiedemann syndrome. To understand the role of KCNQ1OT1 in regulating ICR2 we characterized its promoter. The critical promoter is approximately 300 bp and it is surrounded by inhibitory elements within the CpG island. The promoter activity is strongly inhibited by cytosine methylation in keeping with the finding that the inactive maternal promoter is methylated in vivo. We have identified the transcription start sites and four CCAAT boxes upstream of the 5'-most start site. Mutation of the CCAAT boxes produced impairment of promoter activity. Transfection and gel mobility shift experiments suggest that binding of the factor NF-Y to the CCAAT boxes is important for promoter activity.

Relationship between polymorphisms of genes encoding microsomal epoxide hydrolase and glutathione S-transferase P1 and chronic obstructive pulmonary disease.

BACKGROUND: Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). However, only 10% - 20% of chronic heavy cigarette smokers develop symptomatic disease. COPD is most likely the result of complex interactions between environmental and genetic factors. Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST). In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113-->His), exon 4 (His139-->Arg) and GSTP1 polymorphism in exon 5 (Ile105-->Val) in 100 COPD patients and 100 age- and sex-matched healthy controls. RESULTS: The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarette years was 2.96 (95% CI 1.24 - 7.09). There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls. When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1.00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1.00. There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls. CONCLUSIONS: mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China. The interaction might exist between mEH genotype and smoke. The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population.

[Association between polymorphisms in the microsomal epoxide hydrolase (mEH) gene and chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the association between polymorphisms in the microsomal epoxide hydrolase (mEH) gene and susceptibility to chronic obstructive pulmonary disease (COPD) in a Chinese population. METHODS: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to genotype mEH polymorphisms in exon3 (Tyr113-->His) and exon4 (His139-->Arg) in 100 COPD patients and 100 age and sex matched healthy controls. RESULTS: (1) The proportion of mEH heterozygotes in exon3 was significantly higher in the patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarettes years was 2.96 (95% CI 1.24 - 7.09). (2) There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and controls. (3) When COPD patients were nonsmokers, the OR of very slow activity genotype versus other genotypes was more than 1.00, and when COPD patients were smokers (Current smokers and ex-smokers), the OR was less than 1.00. CONCLUSIONS: (1) mEH heterozygotes in exon3 might be associated with the susceptibility to COPD in China. (2) The interaction might be existed between mEH genotype and smoke.

[Association between polymorphisms in the gene coding for glutathione S-transferase P1 and chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the association between polymorphisms in the gene coding for glutathione S-transferase P1 (GSTP1) and susceptibility to chronic obstructive pulmonary disease (COPD) in a Chinese population. METHODS: This was a pilot study of the molecular epidemiology in patients with COPD. The research design was a case-control study. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to genotype GSTP1 polymorphisms in exon 5 in 100 COPD patients and 100 age and sex matched healthy controls. Logistic regression analysis method was used to calculate the odds ratio (OR) and the 95% confidence interval (CI). RESULT: There was no significant difference in GSTP1 polymorphisms adjusted by age, sex, body mass index and smoking between COPD patients and controls. CONCLUSION: The gene polymorphism for GSTP1 was not associated with susceptibility to COPD in the Chinese population.

Insulator and silencer sequences in the imprinted region of human chromosome 11p15.5.

The imprinting of the genes on human chromosome 11p15.5 is thought to be controlled by two imprinting control regions located in two differentially methylated CpG islands upstream of the H19 gene (H19 DMR) and in intron 10 of the KCNQ1 gene (KvDMR). We have examined sequences in the human 11p15.5 genomic imprinted region for the presence of insulators and silencers using a position- and enhancer-dependent stable transfection assay. We have confirmed the existence of insulators in H19 DMR and discovered two novel insulators in the IGF2 gene. We have also found two novel silencer sequences; one is located in KvDMR, a region that is thought to contain the promoter for the KCNQ1OT1 transcript, and another is in the CDKN1C gene. We have demonstrated binding of CTCF protein in vitro to all the insulator and silencer sequences that we have detected. We discuss the differences in the regulation of imprinting controlled by the two imprinting control regions in chromosome 11p.

Interaction of oncogenic papillomavirus E6 proteins with fibulin-1.

Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calcium-binding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6.