Association of sepsis with risk for osteoporosis: a population-based cohort study.

We conducted a large, retrospective cohort study using data from Taiwan's National Health Insurance Research Database to evaluate whether the risk of developing osteoporosis is associated with sepsis. Our study found that adults younger than 65 years with sepsis had a significantly increased risk of developing osteoporosis. INTRODUCTION: There have been limited studies regarding the osteoporosis risk associated with sepsis. Our purpose is to evaluate whether the risk of developing osteoporosis is associated with sepsis. METHODS: We conducted a large, retrospective cohort study using data from Taiwan's National Health Insurance Research Database. From the insurance claims data, a total of 13,178 patients diagnosed with sepsis from 2000 to 2012 were included in the sepsis cohort, and a propensity score-matched cohort included 13,178 individuals without sepsis. To calculate the incidence of osteoporosis, both groups were followed until 2013. Cox regression analysis was performed to obtain the hazard ratios (HRs) to assess the risk of developing osteoporosis. The Kaplan-Meier method was used to estimate the cumulative incidence of osteoporosis. RESULTS: The overall incidences of osteoporosis (per 1,000 person-years) in the sepsis and non-sepsis groups were 10.2 and 10.7, respectively. The risk of osteoporosis significantly increased in the presence of sepsis (adjusted HR = 1.17, 95% confidence interval (CI) = 1.04-1.31). The risk of osteoporosis in the sepsis group was significantly higher than that in the non-sepsis group for young patients aged 20-49 years and patients aged 50-64 years (adjusted HR = 1.93, 95% CI = 1.08-3.44; adjusted HR = 2.01, 95% CI = 1.52-2.65, respectively). The Kaplan-Meier curves of cumulative probability also showed a significantly increased risk of osteoporosis in patients aged 20-49 years and aged 50-64 years with sepsis compared with non-sepsis (P = 0.025; P < 0.001, respectively). CONCLUSION: Adults younger than 65 years with sepsis had a significantly increased risk of developing osteoporosis.

Deficiency in VHR/DUSP3, a suppressor of focal adhesion kinase, reveals its role in regulating cell adhesion and migration.

Vaccinia H1-related phosphatase (VHR/DUSP3) is a member of the dual-specificity phosphatase family. Deregulation of VHR is observed in various malignant diseases. We identified focal adhesion kinase (FAK) as a VHR-interacting molecule. Over-expression of VHR decreased tyrosine phosphorylation of FAK and decreasing VHR promoted FAK tyrosine phosphorylation. In vitro assays proved that recombinant VHR directly dephosphorylated FAK and paxillin. VHR-knockout mice did not have obvious abnormality; however, VHR-knockout cells showed decreased expression of integrins and FAK but stronger FAK and paxillin phosphorylation upon attachment to fibronectin. Additionally, VHR-knockout fibroblast and lung epithelial cells had elevated ligand-induced epidermal growth factor receptor (EGFR) phosphorylation. Inducible expression of VHR suppressed directional cell migration, and VHR deficiency resulted in a higher cell migratory ability. VHR-knockout cells have stronger FAK phosphorylation in cell adhesions, long-lasting trailing ends and slower turnover of focal adhesions. These collective data indicate that VHR is a FAK phosphatase and participates in regulating the formation and disassembly of focal adhesions.

A KRAS mutation status-stratified randomized phase II trial of gemcitabine and oxaliplatin alone or in combination with cetuximab in advanced biliary tract cancer.

BACKGROUND: Previous clinical trials have not proved that adding epidermal growth factor receptor inhibitors to chemotherapy confers a survival benefit for patients with advanced biliary tract cancer (ABTC). Whether the KRAS mutation status of tumor cells confounded the results of past studies is unknown. PATIENTS AND METHODS: ABTC patients stratified by KRAS status, Eastern Cooperative Oncology Group performance status, and primary tumor location were randomized 1 : 1 to receive GEMOX (800 mg/m(2) gemcitabine and 85 mg/m(2) oxaliplatin) or C-GEMOX (500 mg/m(2) cetuximab plus GEMOX) every 2 weeks. The primary end point was objective response rate (ORR). RESULTS: The study enrolled 122 patients between December 2010 and May 2012 (62 treated with C-GEMOX and 60 with GEMOX). Compared with GEMOX alone, C-GEMOX was associated with trend to better ORR (27% versus 15%; P = 0.12) and progression-free survival (PFS, 6.7 versus 4.1 months; P = 0.05), but not overall survival (OS, 10.6 versus 9.8 months; P = 0.91). KRAS mutations, which were detected in 36% of tumor samples, did not affect the trends of difference in ORR and PFS between C-GEMOX and GEMOX. The two treatment arms had similar adverse events, except that more patients had skin rashes, allergic reactions, and neutropenia in the C-GEMOX arm. Of patients with C-GEMOX, the presence of a grade 2 or 3 skin rash was associated with significantly better ORR, PFS, and OS. CONCLUSIONS: Addition of cetuximab did not significantly improve the ORR of GEMOX chemotherapy in ABTC, although a trend of PFS improvement was observed. The trend of improvement did not correlate with KRAS mutation status. CLINICAL TRIALS NUMBER: This study is registered at ClinicalTrials.gov (NCT01267344). All patients gave written informed consent.

Cluster headache in the Taiwanese -- a clinic-based study.

Cluster headache has not been fully investigated in Asians. One hundred and four patients (90M/14F; mean age 39.2 +/- 12.2 years) with cluster headache were recruited from two major headache clinics in Taiwan. They filled out a structured cluster headache questionnaire. All participants were diagnosed to have episodic cluster headache. Mean age of onset was 26.9 years; mean latency of diagnosis was 8.1 years. A trend of decrease in male/female ratio with time was noted. Seventy-three percent were ex- or current smokers (M: 79%, F: 36%). Restlessness was reported by 51% patients. Only 1 patient (1%) reported visual aura. Patients responded well to standard acute and prophylactic treatment. The monthly incidence of cluster period was inversely related to sunshine duration. Compared to Western series, our patients were different in several aspects including the absence of chronic cluster headaches and a low prevalence of restlessness and aura. Racial and geographical factors might contribute to these discrepancies.

Effects of single nucleotide polymorphisms in the 5'-flanking region of heat shock protein 70.2 gene on semen quality in boars.

This study aims to elucidate the effects of single nucleotide polymorphisms (SNPs) in the 5'-flanking region of porcine heat shock protein 70.2 gene (HSP70.2) on semen quality in boars. Genomic DNA isolated from 55 boars (41 Duroc, nine Landrace, and five Yorkshire) was subjected to PCR amplification of the 5'-flanking region of HSP70.2. The nucleotide sequences were determined by automated sequencing. Five SNPs (sites 44, 232, 250, 345, and 393) were detected in this region. Semen quality was evaluated in terms of sperm motility, percentage of normal sperm, percentage of sperm with proximal plasma droplet, percentage of abnormal sperm, sperm concentration, semen volume per ejaculate and total sperm number per ejaculate. The effect of the SNPs on semen quality was evaluated based on breed-corrected data within a season. During the cool season, the sperm motility of boars with AA genotype at the 232 site was significantly higher than that of boars with CC genotype (P<0.05). Meanwhile, boars with AC genotype at the 232 site had higher total sperm number per ejaculate than did those with CC genotype. In the hot season, heterozygotes at both the 232 and 250 sites had significantly higher total sperm number of per ejaculate than AA homozygotes (P<0.05). Semen volume of boars with TT and TC genotypes at the 345 site was significantly larger than that of those with CC genotype (P<0.05). Meanwhile, semen quality for boars with TT genotype at the 345 site was significantly higher than that of boars with TC or CC genotype (P<0.05), that is the semen contained higher percentages of normal sperm and lower percentages of abnormal sperm or sperm with proximal plasma droplets. Results herein suggest that the SNPs in the 5'-flanking region of porcine HSP70.2 are associated with semen quality traits in the hot season.

6-Substituted-4-(3-bromophenylamino)quinazolines as putative irreversible inhibitors of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity.

A series of new 6-substituted-4-(3-bromophenylamino)quinazoline derivatives that may function as irreversible inhibitors of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases have been prepared. These inhibitors have, at the C-6 position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solublilizing substituents. These compounds were prepared by acylation of 6-amino-4-(3-bromophenylamino)quinazoline with unsaturated acid chlorides or mixed anhydrides. We show that attaching a basic functional group onto the Michael acceptor results in greater reactivity, due to intramolecular catalysis of the Michael addition and/or an inductive effect of the protonated basic group. This, along with improved water solubility, results in compounds with enhanced biological properties. We present molecular modeling and experimental evidence that these inhibitors interact covalently with the target enzymes. One compound, 16a, was shown to have excellent oral activity in a human epidermoid carcinoma (A431) xenograft model in nude mice.

PTCH mutations in squamous cell carcinoma of the skin.

Ultraviolet light exposure is the major risk factor for the development of squamous cell carcinoma in Caucasians. Mutations in the tumor suppressor gene p53 have been identified in both squamous cell carcinomas and basal cell carcinomas. The human homolog of the Drosophila patched gene, has been shown to be mutated in sporadic basal cell carcinomas; however, mutations in the patched gene have not been found in squamous cell carcinoma. In this study, we screened a total of 20 squamous cell carcinoma samples for mutations in the patched gene. Using polymerase chain reaction-single strand conformation polymorphism as an initial screening method, we identified one non-sense mutation, two mis-sense mutations and three silent mutations in five squamous cell carcinoma samples. In one squamous cell carcinoma sample, we identified a tandem GG-->AA transitional change at nucleotide 3152 in exon 18 of the patched gene that resulted in a premature stop codon at codon 1051. The three squamous cell carcinoma samples containing non-sense and mis-sense mutations were isolated from individuals with histories of multiple basal cell carcinoma. Sequence analysis of the p53 gene in these five squamous cell carcinoma samples identified one CC-->TT and three C-->T ultraviolet-specific nucleotide changes. Our study provides evidence that the patched gene is mutated in squamous cell carcinoma from individuals with a history of multiple basal cell carcinoma. The identification of ultraviolet-specific nucleotide changes in both tumor suppressor genes supports the notion that ultraviolet exposure plays an important part in the development of squamous cell carcinoma.

UV-specific p53 and PTCH mutations in sporadic basal cell carcinoma of sun-exposed skin.

UVB irradiation is known to produce DNA damage at mutation hotspots in the p53 tumor suppressor gene, leading to the development of skin cancers. Mutations in the PTCH tumor suppressor gene, which is known to be responsible for the development of nevoid basal cell carcinoma syndrome, have also been identified in sporadic basal cell carcinomas (BCCs). We describe the case of an 80-year-old welder in whom 3 novel p53 mutations, as well as UV-specific PTCH mutations, were detected in two BCC samples from sun-exposed skin. The simultaneous presence of UV-specific p53 and PTCH mutations in the same BCC sample has not previously been reported.

Role of PTCH and p53 genes in early-onset basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common skin cancer in the Western world. Ultraviolet (UV) exposure, race, age, gender, and decreased DNA repair capacity are known risk factors for the development of BCC. Of these, UVB irradiation from sunlight is the most significant risk factor. The incidence of sporadic BCC increases in individuals older than age 55, with the greatest incidence reported in individuals who are older than 70, and is rare in individuals who are younger than 30. In this study, we analyzed 24 BCC samples from individuals who had BCC diagnosed by the age of 30. Fifteen single-stranded conformation polymorphism variants in the PTCH gene were identified in 13 BCC samples. Sequence analysis of these single-stranded conformation polymorphism variants revealed 13 single nucleotide changes, one AT insertion, and one 15-bp deletion. Most of these nucleotide changes (nine of 15) were predicted to result in truncated PTCH proteins. Fifteen p53 mutations were also found in 11 of the 24 BCC samples. Thirty-three percent (five of 15) and 60% (nine of 15) of the nucleotide changes in the PTCH and p53 genes, respectively, were UV-specific C-->T and CC-->TT nucleotide changes. Our data demonstrate that the p53 and PTCH genes are both implicated in the development of early-onset BCC. The identification of UV-specific nucleotide changes in both tumor suppressor genes suggests that UV exposure is an important risk factor in early onset of BCC.

Effects of birth season, breed, sex, and sire family on cardiac morphology determined in pigs (Sus scrofa domestica) by use of echocardiography.

PURPOSE: Echocardiography played an important role in the screening and diagnosis of hypertrophic cardiomyopathy. In the study reported here, we attempted to evaluate the effects of birth season, breed, sex, and sire family on cardiac morphology determined in pigs by use of echocardiography. METHODS: A total of 411 pigs (mean body weight and age of 105.7 +/- 10.6 kg and 214.4 +/- 25.5 days, respectively) with different genetic backgrounds (Landrace, Yorkshire, and their two-way crossbred) were studied. Cardiac morphologic measurements included thickness of left ventricle and interventricular septum at end-systolic and end-diastolic phases. Meanwhile, the statistical model included the following effects: birth season, breed, sex, interaction between breed and sex, sire family, body weight, and age. RESULTS: Mean cardiac morphologic measurements were as follows: thickness of the interventricular septum at end-systolic and end-diastolic phases was 1.74 and 1.14 cm, respectively; and thickness of the left ventricular free wall at end-systolic and end-diastolic phases was 1.81 and 0.98 cm, respectively. Medium positive correlations existed among the cardiac morphologic measurements r = 0.31 to 0.53; P < 0.001). Pigs born in spring had significantly (P < 0.05) lower cardiac thickness at the end systolic phase than did pigs born in other seasons, and Landrace pigs had higher cardiac morphologic measurements than did Yorkshire and two-way crossbred pigs. Additionally, thickness of interventricular septum at the end-diastolic phase in male pigs was significantly higher than that in female pigs (P < 0.05). Cardiac morphologic measurements for the sire family were significantly (P < 0.05) different, and contributed 77.2 to 87.9% of the total variation, suggesting that genetic variation in cardiac morphology might exist in pigs. CONCLUSIONS: Cardiac morphology of pigs might be influenced by genetic background. The effects of birth season, breed, sire family, and sex should be adjusted when using pigs as an animal model for comparative cardiovascular studies.

Association of heat shock protein 70 with semen quality in boars.

This study attempted to clarify the relationship between the levels of 70kDa heat shock protein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) boars (mean age=25.2+/-2.2 months) were examined. Three to four ejaculates per boar, collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proximal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality.

4-Anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors.

The synthesis and SAR of a series of 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor (EGF-R) kinase are described. Condensation of 3, 4-dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave, regiospecifically, 6,7-dialkoxy-4-oxo-1, 4-dihydroquinoline-3-carbonitriles. Chlorination (POCl(3)) followed by the reaction with substituted anilines furnished the 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts with a methyl 3, 4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH(4)Cl, MeOH-H(2)O) gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed by cyclization using LiCH(2)CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitrile, which was transformed as before. Compounds containing acid, ester, amide, carbinol, and aldehyde groups at the 3-position of the quinoline ring were also prepared for comparison, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the aniline substituents, and the substituent at the 3-position was studied. The compounds were further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is displaced by the cyano group which interacts with the same Thr residue.

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90).

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.

Germline PTEN mutations in three families with Cowden syndrome.

Cowden syndrome (CS) is an autosomal dominant inherited disorder characterized by hamartomas in a variety of tissues including the skin, thyroid, breast, endometrium, and the brain. Individuals with CS are predisposed to development of malignancy in these organs, especially the breast and the thyroid. We describe 3 unrelated individuals with CS associated with germline PTEN mutations. While the frameshift (375insTTTA) and the missense (Gly69Arg) mutations reported herein are novel in CS, the nonsense (Arg130stop) mutation has been described in 2 families with CS and in a single family exhibiting both CS and Bannayan Zonana phenotype.

Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer.

Epidemiological studies have demonstrated that men with a family history of prostate cancer are at an increased risk for this disease. This important observation has led a number of research teams, including our own, to collect DNA samples and clinical data from prostate cancer families, with the goal of localizing and characterizing prostate cancer susceptibility genes. The candidate tumor suppressor gene PTEN (also called MMAC1) has recently been shown to be somatically altered in several common malignancies, including cancers of the brain, kidney, skin, thyroid, endometrium, breast, and prostate. Germ-line mutations in this gene, which maps to chromosome 10q23, have been associated with Cowden disease, an autosomal dominant cancer predisposition syndrome that is characterized by multiple hamartomas. Although prostate cancer is not typically associated with Cowden disease, previous studies of sporadic prostate cancers demonstrate loss of heterozygosity at 10q23 loci in approximately 25% of cases. We, therefore, hypothesized that germ-line mutations in the PTEN gene may predispose to prostate cancer in a subset of families, particularly those in which cancers of the breast, kidney, and/or thyroid also segregate. To test this hypothesis, DNA was isolated from whole blood of 11 prostate cancer patients from 10 unrelated families. Four of the 10 families met the previously established clinical criteria for hereditary prostate cancer. Eight of the II men had at least one second primary malignancy, including cases of neuroendocrine cancer, glioblastoma multiforme, melanoma, kidney, and thyroid cancer. Although we identified some common as well as some unique polymorphisms, no nonsense or missense mutations were identified in any of the 11 samples. To further examine the possibility that PTEN mutations contribute to prostate cancer predisposition, we also studied the probands from each of 10 families with early-onset and/or multiple individuals with prostate cancer. Sequence analysis of the PTEN gene in these 10 men also revealed no mutations or novel polymorphisms. We conclude that germ-line mutations in the PTEN are unlikely to contribute in a significant way to the inherited predisposition to prostate cancer.

Statistical method for characterization of hypertrophic cardiomyopathy by use of morphologic and pathologic measurements in pigs (Sus scrofa domestica).

BACKGROUND AND PURPOSE: Hypertrophic cardiomyopathy (HCM) is characterized by symmetric or asymmetric hypertrophy of the left and/or right ventricle. Morphologic and pathologic indices (MI and PI) of hearts were established for classification of HCM in pigs. METHODS: Fifty on-farm-performance-tested pigs (average body weight, 104.3 kg; age, 224.5 days) were randomly selected. Heart weight, length, width, heart-to-body weight ratio, and thickness of the cranial and middle portions of ventricular septum and left ventricular free wall were measured. Myocyte disorganization and necrosis, myocardial and endocardial fibrosis, and intramural coronary arterial occlusion were scored. Principal component analysis and stepwise regression analysis were used to establish MI and PI. RESULTS: MI was established by using the first principal component as the dependent variable and applying stepwise regression analysis. Hearts were classified as morphologically normal, suspicious, and hypertrophic according to the range of MI. The same statistical method was used to find PI. Hearts were classified as pathologically normal, moderately affected, or seriously affected according to the range of PI. Combining MI and PI, hearts could be classified into five groups: no hypertrophy with minor lesion (normal); hypertrophy but with rare lesion; no hypertrophy but seriously affected; suspicious; and hypertrophy and seriously affected (heart with HCM). Another 119 hearts were collected and classified. The variation of heart measurements was consistent with the original purpose of classification. CONCLUSIONS: Using fewer measurements for identification of HCM objectively in pigs seems to have practical application.

Phenotypic findings of Cowden syndrome and Bannayan-Zonana syndrome in a family associated with a single germline mutation in PTEN.

Cowden syndrome (CS) and Bannayan-Zonana syndrome (BZS) are two hamartoma syndromes with distinct phenotypic features. Although partial clinical overlap exists between CS and BZS, they are considered to be separate entities. PTEN has been identified as the susceptibility gene for both disorders, suggesting allelism. We have identified a germline mutation, R335X, in PTEN in a family consisting of two female members with the phenotypic findings of CS and two male members with the phenotypic findings of BZS. To our knowledge, this is the first report that shows the presence of separate subjects with CS and with BZS in a single family associated with a single germline PTEN mutation.