[Gene transcriptome analysis of nasal epithelial cells in chronic rhinosinusitis with nasal polyps].

Objective: To identify the differentially expressed genes in nasal epithelial cells from chronic rhinosinusitis with nasal polyps (CRSwNP), and to analyze related genes which are involved in deficiency of nasal epithelial barrier in CRSwNP patients by analyzing the datasets download from the gene expression omnibus(GEO) database. Methods: The mRNA expression microarray data numbered GSE107624 (7 CRSwNP and 7 controls) and GSE69093 (13 CRSwNP and 11 controls) were downloaded from the publicly available GEO database. These two datasets were jointly analyzed to screen the differentially expressed genes in nasal epithelial cells of controls and CRSwNP patients. In the meanwhile, we further evaluated the function annotation and regulatory pathways of the differentially expressed genes. To further confirmed what we have observed, sinus tissues were collected from patients with CRSwNP (14 cases, 46.8±17.9 years) and uncinate process tissues were collected from patients with nasal septum deviation (7 cases, 23.4±2.3 years) as control group. The primary epithelial cells of nasal mucosa were cultured and the mRNA level of screened genes were measured by Q-PCR. SPSS 22.0 software was used to for statistical analysis. Results: GSE107624 dataset showed that there were 3 856 differentially genes in nasal epithelial cells between CRSwNP and control group, while there were 771 differentially expressed genes in GSE69093 dataset. Finally, 55 up-regulated genes and 3 down-regulated genes were noticed in nasal epithelial cells of CRSwNP patients in the two datasets. GO gene functional annotation analysis showed that SPTBN1, FNBP1L, VAPB and SNX1 were involved in cell adhesion function, MAP1B was participated in the formation of microtubule related complex. KEGG pathway enrichment analysis indicated that BAMBI and SIAH1 were involved in regulation of Wnt pathway, COL6A1 and EIF4E were involved in the regulation of PI3K-AKT pathway. String protein interaction network analysis assumed that MAP1B and VAPB were the core functional proteins. Among top 3 differentially expressed genes COL6A1, MAP1B and BAMBI, only MAP1B gene was increased in nasal epithelial cells of CRSwNP patients in comparison to controls. Conclusion: The increased MAP1B gene in epithelial cells of CRSwNP, as well as abnormal regulation of Wnt and PI3K-AKT signal pathways may mediate the barrier dysfunction in CRSwNP.

Effects of universal masking on Massachusetts healthcare workers' COVID-19 incidence.

BACKGROUND: Healthcare workers (HCWs) and other essential workers are at risk of occupational infection during the COVID-19 pandemic. Several infection control strategies have been implemented. Evidence shows that universal masking can mitigate COVID-19 infection, though existing research is limited by secular trend bias. AIMS: To investigate the effect of hospital universal masking on COVID-19 incidence among HCWs compared to the general population. METHODS: We compared the 7-day average incidence rates between a Massachusetts (USA) healthcare system and Massachusetts residents statewide. The study period was from 17 March (the date of first incident case in the healthcare system) to 6 May (the date Massachusetts implemented public masking). The healthcare system implemented universal masking on 26 March, we allotted a 5-day lag for effect onset and peak COVID-19 incidence in Massachusetts was 20 April. Thus, we categorized 17-31 March as the pre-intervention phase, 1-20 April the intervention phase and 21 April to 6 May the epidemic decline phase. Temporal incidence trends (i.e. 7-day average slopes) were compared using standardized coefficients from linear regression models. RESULTS: The standardized coefficients were similar between the healthcare system and the state in both the pre-intervention and epidemic decline phases. During the intervention phase, the healthcare system's epidemic slope became negative (standardized ß: -0.68, 95% CI: -1.06 to -0.31), while Massachusetts' slope remained positive (standardized ß: 0.99, 95% CI: 0.94 to 1.05). CONCLUSIONS: Universal masking was associated with a decreasing COVID-19 incidence trend among HCWs, while the infection rate continued to rise in the surrounding community.

The relationship between red cell distribution width and the risk of Henoch-Schönlein purpura nephritis.

INTRODUCTION: Red blood cell distribution width (RDW) is elevated in various inflammatory diseases, but its clinical significance in Henoch-Schönlein purpura nephritis (HSPN) in unknown. The aim of this study was to determine the value of RDW as a risk factor or marker for HSPN in children. METHODS: This was a case-control study of 105 Henoch-Schönlein purpura (HSP) patients, 120 HSPN patients and 192 healthy controls. The relationship between RDW-coefficient of variation (RDW-CV) and the clinical characteristics of HSPN patients was determined by a multiple logistic regression analysis (MVLRA). Receiver operating characteristic (ROC) curves were applied to compare the diagnostic potential of the RDW-CV, a panel of routine markers and combinations of these indices. RESULTS: The RDW-CV values were significantly higher in the HSPN group than the HSP group and controls (P < 0.001). Significant correlations were found between RDW-CV and ESR (P = 0.001). A combination of RDW-CV and ESR in a ROC curve showed 80% sensitivity and 84.9% specificity in the HSP patients, and 85.8% sensitivity and 93.8% specificity in the HSPN patients. The MVLRA revealed that RDW-CV (OR 1.69, 95% CI 1.16-2.48, P = 0.007) was an independent predictor of HSPN. CONCLUSIONS: The RDW levels were highest in the HPSN group, suggesting that RDW, especially the combination of RDW and ESR, may have value when assessing the risk of HSPN.

[Associations between airflow obstruction and the risks of morbidity on major chronic diseases in Chinese adults: a prospective cohort study].

Objective: To examine the prospective associations between airflow obstruction (AFO) and risks of major chronic diseases morbidity in Chinese adults. Methods: Samples of this study were from the China Kadoorie Biobank. A total of 486 996 participants aged 30 to 79 years (mean 51.5 years) at the baseline study, were included after excluding those who self-reported of having heart disease, stroke and cancer at baseline. AFO was defined under the Global Initiative on Obstructive Lung Disease (GOLD) criteria and forced expiratory volume per one second in percentage of the expected one (FEV(1)% P). Cox regression models were used to investigate the associations of AFO with incidence rates of ischemic heart disease, cerebrovascular disease and lung cancer after adjusted for potential confounders. Results: Over a period of 7 years through the follow-up program, the incident cases of ischemic heart disease, cerebrovascular disease and lung cancer appeared as 24 644, 36 336 and 3 218, respectively. Compared with people without AFO, the HR (95% CI) of GOLD-1 to GOLD-4 were 0.89 (0.78-1.01), 1.05 (0.98-1.12), 1.29 (1.18-1.40) and 1.65 (1.42-1.91) respectively for ischemic heart disease. The HR (95%CI) of GOLD-1 to GOLD-4 were 0.96 (0.70-1.26), 1.12 (0.96-1.31), 1.38 (1.14-1.65) and 1.48 (1.05-2.02) respectively for lung cancer. No statistically significant differences in the associations between GOLD level and cerebrovascular disease morbidity were found. However, each 10% decrease in FEV(1)% P was associated with 7.2% (95%CI: 6.4%-8.0%), 3.6% (95%CI: 3.0%-4.3%) and 10.5% (95%CI: 8.4%-12.6%) increased the risks of ischemic heart disease, cerebrovascular disease and lung cancer respectively. The results were persistant when stratified by smoking status. Conclusion: Higher degree of AFO seemed to be associated with the risks of ischemic heart disease, cerebrovascular disease and lung cancer morbidity among the Chinese adults.

[Associations between airflow obstruction and total and cause-specific mortality in adults in China].

Objective: To examine the prospective associations between airflow obstruction and total and cause-specific mortality. Methods: The study was based on China Kadoorie Biobank, in which 199 099 men and 287 895 women aged 30-79 years at baseline survey were included after excluding those with heart disease, stroke and cancer. The Global Initiative on Obstructive Lung Disease (GOLD) guideline was used to classify airflow obstruction. Cox regression models were used to estimate adjusted HR and 95%CI. Results: During 3 494 079 person-years of follow-up between 2004 and 2013 (median 7.2 years), a total of 21 649 people died. Absolute mortality rates were 5.5, 9.9, 13.1, 32.4 and 63.3 deaths per 1 000 person-years for participants who had normal airflow, GOLD-1 to GOLD-4 airflow obstruction, respectively. After adjusting potential confounders, compared with participants with normal lung function, the HRs for death were 0.98 (95%CI: 0.88-1.09), 1.03 (95%CI: 0.97-1.09), 1.62 (95% CI: 1.53-1.73) and 2.83 (95% CI: 2.59-3.10) for those whose airflow obstruction were classified as GOLD-1 to GOLD-4, respectively. The airflow obstruction was also associated with increased risk for deaths due to ischemic heart disease, cerebrovascular disease and chronic obstructive pulmonary disease. Conclusion: Airflow obstruction is associated with total and certain cause-specific mortality, the higher the airflow obstruction degree is, the higher the death risk is.

Programmed cell death-1 expression correlates with disease severity and IL-5 in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Programmed cell death-1 (PD-1) is a negative regulator of T-cell responses. Expression of PD-1 and its ligands PD-L1 and PD-L2 in chronic rhinosinusitis with nasal polyps (CRSwNP) is poorly studied. METHODS: Expression of PD-1, PD-L1, PD-L2, TGF-ß, IL-5, and IL-10 mRNA was measured by real-time quantitative PCR on tissue homogenates of patients with CRSwNP (n = 21) and healthy controls (n = 21) and on primary epithelial cells. Disease severity was scored using the Lund-Mackay scores of maxillofacial computed tomography (CT) scans. Expression of PD-1 and PD-L1/L2 was evaluated at the cellular and tissue levels (n = 6) by flow cytometry and immunohistochemistry. RESULTS: Programmed cell death-1 mRNA expression was increased in tissue homogenates from patients with CRSwNP compared with controls, irrespective of the atopy status. Importantly, expression of PD-1 correlated with the total CT scan scores (r = 0.5, P = 0.02). Additionally, a significant association was found between PD-1 mRNA and expression of IL-5 mRNA in control nasal tissue (r = 0.95, P < 0.0001) and in CRSwNP (r = 0.63, P = 0.002). PD-1 was expressed on different subsets of T cells and CD11b- dendritic cells. Both PD-1 and its ligands were expressed on primary epithelial cells from control nasal tissue and nasal polyp tissue. CONCLUSIONS: Higher PD-1 expression was found in CRSwNP than in nasal tissue from controls. This was associated with disease severity and tissue IL-5 expression but unrelated to the patients' atopy status.

Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor.

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

Viruses and bacteria in Th2-biased allergic airway disease.

Allergic airway diseases are typically characterized by a type 2-biased inflammation. Multiple distinct viruses and bacteria have been detected in the airways. Recently, it has been confirmed that the microbiome of allergic individuals differs from that of healthy subjects, showing a close relationship with the type 2 response in allergic airway disease. In this review, we summarize the recent findings on the prevalence of viruses and bacteria in type 2-biased airway diseases and on the mechanisms employed by viruses and bacteria in propagating type 2 responses. The understanding of the microbial composition and postinfectious immune programming is critical for the reconstruction of the normal microflora and immune status in allergic airway diseases.

Eosinophil differentiation in the bone marrow is promoted by protein tyrosine phosphatase SHP2.

SHP2 participates in multiple signaling events by mediating T-cell development and function, and regulates cytokine-dependent granulopoiesis. To explore whether and how SHP2 can regulate bone-marrow eosinophil differentiation, we investigate the contribution of SHP2 in the bone-marrow eosinophil development in allergic mice. Blockade of SHP2 function by SHP2 inhibitor PHPS-1 or conditional shp2 knockdown by adenovirus-inhibited bone-marrow-derived eosinophil differentiation in vitro, with no detectable effects on the apoptosis of eosinophils. Furthermore, SHP2 induced eosinophil differentiation via regulation of the extracellular signal-regulated kinase pathway. Myeloid shp2 conditional knockout mice (LysM(cre)shp2(flox/flox)) failed to induce eosinophilia as well as airway hyper-responsiveness. The SHP2 inhibitor PHPS-1 also alleviated eosinophilic airway inflammation and airway hyper-responsiveness, accompanied by significantly reduced levels of systemic eosinophils and eosinophil lineage-committed progenitors in allergic mice. We demonstrate that inhibition of eosinophil development is SHP2-dependent and SHP2 is sufficient to promote eosinophil formation in vivo. Our data reveal SHP2 as a critical regulator of eosinophil differentiation, and inhibition of SHP2 specifically in myeloid cells alleviates allergic airway inflammation.

Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

The spread of adenoviral vectors to central nervous system through pathway of cochlea in mimetic aging and young rats.

There is no definitive conclusion concerning the spread of viral vectors to the brain after a cochlear inoculation. In addition, some studies have reported different distribution profiles of viral vectors in the central auditory system after a cochlear inoculation. Thus, rats were grouped into either a mimetic aging group or a young group and transfected with adenoviral vectors (AdVs) by round window membrane injection. The distribution of AdV in central nervous system (CNS) was demonstrated in the two groups with transmission electron microscopy and immunofluorescence. We found that the AdV could disseminate into the CNS and that the neuronal damage and stress-induced GRP78 expression were reduced after transfection with PGC-1α, as compared with the control vectors, especially in the mimetic aging group. We also found that the host immune response was degraded in CNS in the mimetic aging group after transduction through the cochlea, as compared with the young group. These results demonstrate that viral vectors can disseminate into the CNS through the cochlea. Moreover, mimetic aging induced by D-galactose could facilitate the spread of viral vectors into the CNS from the cochlea. These findings may indicate a new potential approach for gene therapy against age-related diseases in the CNS.

14-3-3 tau (YWHAQ) gene promoter hypermethylation in human placenta of preeclampsia.

INTRODUCTION: Disruption of the 14-3-3 tau (YWHAQ) gene has been shown to be involved in preeclampsia (PE). The YWHAQ promoter could be differentially regulated by methylation in severe PE patients. METHODS: Placental genomic DNA from patients with severe PE (n = 21) and controls who experienced a normal pregnancy (n = 16) was analyzed using dot-blot and immunohistochemistry. The placental methylation patterns of YWHAQ, expression of 14-3-3 tau and ten-eleven translocation (TET), were confirmed by bisulfite sequencing, immunohistochemistry, western blot and real-time PCR, respectively. RESULTS: Genomic 5 hmC (P < 0.001), expression of 14-3-3 tau (P < 0.01) and TET (P < 0.05) were down-regulated, whereas 5 mC was up-regulated (P < 0.001) in preeclamptic placentas. Significant hypermethylation of the YWHAQ promoter was detected in PE placentas compared with control samples (19.1% vs. 9.4%, P = 0.0095). PE-specific hypermethylation of CpG2 - 4, CpG9, CpG17, CpG19 was identified in PE patients compared with controls (CpG2: 13.3% vs. 2.5%, P < 0.0001; CpG3: 14.8% vs. 3.1%, P < 0.0001; CpG4: 19.5% vs. 5.0%, P < 0.0001; CpG9: 15.7% vs. 5.0%, P = 0.0018; CpG17: 16.2% vs. 6.3%, P = 0.0003; and CpG19: 78.1% vs. 59.4%, P < 0.0001). DISCUSSION: The observed participation of 14-3-3 tau in the regulation of the placental epigenome may participate in the molecular mechanisms that govern the pathological process of PE, although this requires further evaluation.

Transforming growth factor-beta 1 pathways in inflammatory airway diseases.

Transforming growth factor-beta 1 (TGF-ß1) has been reported being involved in the remodeling and immunosuppression processes of inflammatory airway diseases; understanding the regulation of TGF-ß1 is therefore a key to unravel the pathomechanisms of these diseases. This review briefly summarizes the current knowledge on the influencing factors for driving TGF-ß1 and its regulatory pathways in inflammatory airway diseases and discusses possible therapeutic approaches to TGF-ß1 control. The factors include smoking and oxidative stress, prostaglandins (PGs), leukotrienes (LTs), bradykinin (BK), and microRNAs (miRs). Based on the summary, new innovative treatment strategies may be developed for inflammatory airway diseases with an impaired expression of TGF-ß1.

Transcutaneous electrical nerve stimulation on acupoints reduces fentanyl requirement for postoperative pain relief after total hip arthroplasty in elderly patients.

BACKGROUND: Transcutaneous electrical nerve stimulation (TENS) is regarded as an effective treatment for various types of pain. However, no randomized controlled trial has investigated TENS on acupoints for postoperative analgesia in elderly patients. This study aim to investigate whether TENS on acupoints has any favorable effect on complementary analgesia after total hip arthroplasty (THA) for elderly patients compared with a sham control treatment. METHODS: Sixty-eight elderly patients requiring THA surgery were enrolled and randomly allocated to one of two groups. Group Acu received true TENS on acupoints (bilateral P6, L14; ST36, GB31 ipsilateral to the surgery site) and Group Sham received sham treatment. All patients received patient-controlled analgesia for two days postoperatively. Analgesia was assessed by postoperative fentanyl requirement and pain intensity using a visual analogue scale (VAS-10 cm). The incidence of analgesia-related side effects, optional medication use and effects of patients' blinding were recorded. RESULTS: Fentanyl consumption in Group Acu was lower than that in Group Sham at 24 h (mean ± SD; 360±117 vs. 572±132 µg; P<0.001) and 48 h (712±184 vs. 1022±197 µg; P<0.001) after surgery. Postoperative pain intensity measured by VAS was similar in both groups. The incidence of opioid-related side effects and rescue medication for postoperative analgesia was significantly higher in Group Sham than in Group Acu. Differences between the groups regarding the effects of patients' blinding were not significant. CONCLUSION: TENS on specific acupoints is an effective and complementary approach to reduce postoperative analgesic requirement in elderly patients after THA.

Mortalin-p53 interaction in cancer cells is stress dependent and constitutes a selective target for cancer therapy.

Stress protein mortalin is a multifunctional protein and is highly expressed in cancers. It has been shown to interact with tumor suppressor protein-p53 (both wild and mutant types) and inactivates its transcriptional activation and apoptotic functions in cancer cells. In the present study, we found that, unlike most of the cancer cells, HepG2 hepatoma lacked mortalin-p53 interaction. We demonstrate that the mortalin-p53 interaction exists in cancer cells that are either physiologically stressed (frequently associated with p53 mutations) or treated with stress-inducing chemicals. Targeting mortalin-p53 interaction with either mortalin small hairpin RNA or a chemical or peptide inhibitor could induce p53-mediated tumor cell-specific apoptosis in hepatocellular carcinoma; p53-null hepatoma or normal hepatocytes remain unaffected.

Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa.

AIMS: To investigate the bactericidal activity of lactoferrin-derived peptides and a new LF-derived peptides chimera (LFchimera) against P. aeruginosa and the influence on virulence factors of P. aeruginosa. METHODS AND RESULTS: Lactoferricin (LFcin) and lactoferrampin (LFampin) are highly bioactive peptides isolated from the N-terminal region of lactoferrin (LF) by pepsin digestion. In this study, we designed LFchimera containing LFcin amino acids 17-30 and LFampin amino acids 268-284. Pseudomonas aeruginosa cells were incubated in medium with peptides at different concentrations, and then the assays of viability, pyocyanin, elastase activity and biofilm formation of P. aeruginosa were performed. We found that the concentration-dependent antibactericidal activity and down-regulating pyocyanin, elastase and biofilm formation of LFchimera were significantly stronger than those of LF, LFcin, LFampin or LFcin plus LFampin. CONCLUSIONS: Our results indicated that LF, LFcin, LFampin and LFchimera were potential candidates to combat P. aeruginosa, and LFchimera was the most effective in them. SIGNIFICANCE AND IMPACT OF THE STUDY: The new LFchimera has better activity against P. aeruginosa than LF, LFcin and LFampin and may be a promising new compound for treatment of P. aeruginosa infection.

Genetic polymorphisms and haplotype structures of HSPA5 gene in the Han population of Southern China.

The heat shock 70 kDa protein 5 (HSPA5) gene is known to be involved in stress-associated diseases. In this study, the promoter, exons, 3' untranslated region (3'UTR), and subtotal introns of the HSPA5 gene were sequenced in a sub-population of 161 healthy Han Chinese. Nine single-nucleotide polymorphisms (SNPs) including a new one (-86bp T > A from the estimated translation start site) were found and 15 haplotypes (frequencies > 1%) were inferred. Polymorphisms rs391957 and rs11355458 were completely linked in our population (r (2) = 1.00). Using this information, fellow scientists may be able to decrease the number of SNPs to be genotyped in future disease case-control studies.

Adenovirus-mediated CTLA4Ig gene transfer improves the survival of grafted human hepatic progenitors in mouse liver.

The invasive nature of surgery and limited numbers of donor livers for end-stage patients has prompted the search for alternative cell therapies for intractable hepatic disease. Hepatocyte transplantations have been performed for a variety of indications, but sustained benefits have not been observed in most cases. Rat fetal liver epithelial cells (liver stem cells) have demonstrated self-renewal in vivo and functional repopulation of the liver. We have previously isolated and expanded epithelial progenitor cells (EPC) from the human fetal liver to investigate their differentiation potential. In this study, we applied suppression of immunorejection by adenoviral CTLA4Ig gene delivery mediated to examine the survival and differentiation of human fetal EPC transplanted into normal mouse liver. The grafted EPC showed extensive proliferation at both 1 and 2 months after transplantation compared with controls. Moreover, most EPC differentiated into hepatocytes, while a small fraction became bile ductular cells. This finding suggested that human fetal EPC may be a ideal source of cell-based therapy for various liver diseases.

Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells.

AMP-activated protein kinase (AMPK) serves as a fuel-sensing enzyme that is activated by binding of AMP and subsequent phophorylation by upstream kinases such as the tumor suppressor LKB1, when cells sense an increase in the ratio of AMP to ATP. Acute activation of AMPK stimulates fatty acid oxidation to generate more ATP and simultaneously inhibits ATP-consuming processes including fatty acid and protein syntheses, thereby preserving energy for acute cell-surviving program, whereas chronic activation leads to inhibition of cell growth. The goal of the present study is to explore the mechanisms by which AMPK regulates cell growth. Toward this end, we established stable cell lines by introducing a dominant-negative mutant of AMPK alpha1 subunit or its shRNA into the prostate cancer C4-2 cells and other cells, or wild type LKB1 into the lung adenocarcinoma A549 and breast MB-MDA-231 cancer cells, both of which lack functional LKB1. Our results showed that the inhibition of AMPK accelerated cell proliferation and promoted malignant behavior such as increased cell migration and anchorage-independent growth. This was associated with decreased G1 population, downregulation of p53 and p21, and upregulation of S6K, IGF-1 and IGF1R. Conversely, treatment of the C4-2 cells with 5-aminoimidazole-4-carboxamide 1-D-ribonucleoside (AICAR), a prototypical AMPK activator, caused opposite changes. In addition, our study using microarray and RT-PCR revealed that AMPK regulated gene expression involved in tumor cell growth and survival. Thus, our study provides novel insights into the mechanisms of AMPK action in cancer cells and presents AMPK as an ideal drug target for cancer therapy.

Compound heterozygosity for two novel mutations (1203insG/Y1456X) in the von Willebrand factor gene causing type 3 von Willebrand disease.

A 23-year-old Chinese woman with severe von Willebrand factor (VWF) deficiency and her parents were investigated by PCR/direct sequencing of the VWF gene. The patient was found to be compound heterozygous for two novel null mutations. The first was a microinsertion in exon 8 (1203insG) that introduced a frameshift at codon 298 leading to premature translational termination at codon 302. The second was a C to A transversion in exon 28 which resulted in the replacement of tyrosine 562 by a stop codon (Y1456X). The failure to amplify VWF cDNA from the patient by semi-nested PCR is consistent with the induction of nonsense-mediated mRNA decay.