Three new isocoumarin analogues from an endolichenic fungus Aspergillus flavus CPCC 400810.

Five isocoumarin derivatives including three new compounds, aspermarolides A-C (1-3), and two known analogues, 8-methoxyldiaporthin (4) and diaporthin (5) were obtained from the culture extract of Aspergillus flavus CPCC 400810. The structures of these compounds were elucidated by spectroscopic methods. The double bond geometry of 1 and 2 were assigned by the coupling constants. The absolute configuration of 3 was determined by electronic circular dichroism experiment. All compounds showed no cytotoxic activities against the two human cancer cells HepG2 and Hela.

N-myristoyltransferase-1 is necessary for lysosomal degradation and mTORC1 activation in cancer cells.

N-myristoyltransferase-1 (NMT1) catalyzes protein myristoylation, a lipid modification that is elevated in cancer cells. NMT1 sustains proliferation and/or survival of cancer cells through mechanisms that are not completely understood. We used genetic and pharmacological inhibition of NMT1 to further dissect the role of this enzyme in cancer, and found an unexpected essential role for NMT1 at promoting lysosomal metabolic functions. Lysosomes mediate enzymatic degradation of vesicle cargo, and also serve as functional platforms for mTORC1 activation. We show that NMT1 is required for both lysosomal functions in cancer cells. Inhibition of NMT1 impaired lysosomal degradation leading to autophagy flux blockade, and simultaneously caused the dissociation of mTOR from the surface of lysosomes leading to decreased mTORC1 activation. The regulation of lysosomal metabolic functions by NMT1 was largely mediated through the lysosomal adaptor LAMTOR1. Accordingly, genetic targeting of LAMTOR1 recapitulated most of the lysosomal defects of targeting NMT1, including defective lysosomal degradation. Pharmacological inhibition of NMT1 reduced tumor growth, and tumors from treated animals had increased apoptosis and displayed markers of lysosomal dysfunction. Our findings suggest that compounds targeting NMT1 may have therapeutic benefit in cancer by preventing mTORC1 activation and simultaneously blocking lysosomal degradation, leading to cancer cell death.

Identification of invadopodia by TKS5 staining in human cancer lines and patient tumor samples.

Invadopodia, cancer cell protrusive structures with associated proteolytic activity, provide cancer cells with the ability to remodel the extracellular matrix. Invadopodia facilitate invasive migration and their formation correlates with cancer cell invasiveness and metastatic potential. The unambiguous identification of invadopodia is an important step to undergo studies on invadopodia regulatory inputs, functional outputs, as well as the prevalence and significance of invadopodia for cancer cells and human tumors. The adaptor protein TKS5 is a known invadopodia regulatory protein, which is necessary for invadopodia formation and activity. TKS5 is highly enriched at invadopodia and, unlike other commonly used invadopodia markers, it does not accumulate significantly in other types of cellular protrusions. However, the use of TKS5 as a marker of invadopodia has not been generalized, in part due to the availability of suitable antibodies against the human protein. We have evaluated two commercial antibodies raised against human TKS5. Here, we detail protocols for the detection of invadopodia-associated TKS5 in human cells in culture and in paraffin-embedded archived tumor surgical specimens using commercial antibodies. These methods should facilitate the identification and study of human invadopodia. •TKS5 staining identifies invadopodia in human cancer cell lines and archived surgical tumor specimens.

TKS5-positive invadopodia-like structures in human tumor surgical specimens.

Invadopodia, cancer cell protrusions with proteolytic activity, are functionally associated with active remodeling of the extracellular matrix. Here, we show that the invadopodia-related protein TKS5 is expressed in human pancreatic adenocarcinoma lines, and demonstrate that pancreatic cancer cells depend on TKS5 for invadopodia formation and function. Immunofluorescence staining of human pancreatic cancer cells reveals that TKS5 is a marker of mature and immature invadopodia. We also analyze the co-staining patterns of TKS5 and the commonly used invadopodia marker Cortactin, and find only partial co-localization of these two proteins at invadopodia, with a large fraction of TKS5-positive invadopodia lacking detectable levels of Cortactin. Whereas compelling evidence exist on the role of invadopodia as mediators of invasive migration in cultured cells and in animal models of cancer, these structures have never been detected inside human tumors. Here, using antibodies against TKS5 and Cortactin, we describe for the first time structures strongly resembling invadopodia in various paraffin-embedded human tumor surgical specimens from pancreas and other organs. Our results strongly suggest that invadopodia are present inside human tumors, and warrants further investigation on their regulation and occurrence in surgical specimens, and on the value of TKS5 antibodies as pathological research and diagnostic tools.

Experimental supporting data on TKS5 and Cortactin expression and localization in human pancreatic cancer cells and tumors.

In this article, using human pancreatic cancer cell lines and tumor specimens, we analyze the expression and localization of the invadopodia-related proteins TKS5 and Cortactin. Specifically, we present data on: a) TKS5 expression and localization by immunofluorescence in human pancreatic tumors, b) Cortactin expression by western blotting in various human pancreatic adenocarcinoma cell lines, c) TKS5 and Cortactin localization at invadopodia in BxPC-3 pancreatic adenocarcinoma cells, and d) TKS5 and Cortactin localization by co-immunofluorescence in human pancreatic cancer specimens. Data presented here is related to and supportive of the research article by Chen et al., "TKS5-positive invadopodia-like structures in human tumor surgical specimens" (Chen et al., 2019), where interpretation of the research data presented here is available.

Development of predictive signatures for treatment selection in precision medicine with survival outcomes.

For survival endpoints in subgroup selection, a score conversion model is often used to convert the set of biomarkers for each patient into a univariate score and using the median of the univariate scores to divide the patients into biomarker-positive and biomarker-negative subgroups. However, this may lead to bias in patient subgroup identification regarding the 2 issues: (1) treatment is equally effective for all patients and/or there is no subgroup difference; (2) the median value of the univariate scores as a cutoff may be inappropriate if the sizes of the 2 subgroups are differ substantially. We utilize a univariate composite score method to convert the set of patient's candidate biomarkers to a univariate response score. We propose applying the likelihood ratio test (LRT) to assess homogeneity of the sampled patients to address the first issue. In the context of identification of the subgroup of responders in adaptive design to demonstrate improvement of treatment efficacy (adaptive power), we suggest that subgroup selection is carried out if the LRT is significant. For the second issue, we utilize a likelihood-based change-point algorithm to find an optimal cutoff. Our simulation study shows that type I error generally is controlled, while the overall adaptive power to detect treatment effects sacrifices approximately 4.5% for the simulation designs considered by performing the LRT; furthermore, the change-point algorithm outperforms the median cutoff considerably when the subgroup sizes differ substantially.

Ensemble survival trees for identifying subpopulations in personalized medicine.

Recently, personalized medicine has received great attention to improve safety and effectiveness in drug development. Personalized medicine aims to provide medical treatment that is tailored to the patient's characteristics such as genomic biomarkers, disease history, etc., so that the benefit of treatment can be optimized. Subpopulations identification is to divide patients into several different subgroups where each subgroup corresponds to an optimal treatment. For two subgroups, traditionally the multivariate Cox proportional hazards model is fitted and used to calculate the risk score when outcome is survival time endpoint. Median is commonly chosen as the cutoff value to separate patients. However, using median as the cutoff value is quite subjective and sometimes may be inappropriate in situations where data are imbalanced. Here, we propose a novel tree-based method that adopts the algorithm of relative risk trees to identify subgroup patients. After growing a relative risk tree, we apply k-means clustering to group the terminal nodes based on the averaged covariates. We adopt an ensemble Bagging method to improve the performance of a single tree since it is well known that the performance of a single tree is quite unstable. A simulation study is conducted to compare the performance between our proposed method and the multivariate Cox model. The applications of our proposed method to two public cancer data sets are also conducted for illustration.

Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 µM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent inhibition of bile salt transport.

Epidural abscess caused by Veillonella parvula: Case report and review of the literature.

Veillonella parvula, an anaerobic, Gram-negative coccus is part of the normal flora of the oral, gastrointestinal, respiratory, and genitourinary tracts in humans and animals. We herein present a case of epidural abscess caused by V. parvula in a 68-year-old man with sinus squamous cell carcinoma who presented with a 3-week history of low back pain. Blood and pus cultures were positive for Veillonella spp. After sequencing of the 16S ribosomal DNA, the pathogen was identified as V. parvula. Surgical debridement was performed following which the patient received intravenous administration of amoxicillin/clavulanate. To our knowledge, there are only seven reported cases of spinal infection caused by Veillonella spp. and these are reviewed here.

Association between measurements of thyroid function and the acrylamide metabolite N-Acetyl-S-(propionamide)-cysteine in adolescents and young adults.

Acrylamide is present in mainstream cigarette smoke and in some foods prepared at high temperatures. Animal studies have shown that acrylamide exposure alters thyroid function; however, it is not known if this also occurs in humans. The study examined the association between the urinary levels of the acrylamide metabolite and serum thyroid measures in adolescents and young adults. We recruited 793 subjects (mean age, 21.3 years; range, 12-30 years) from a population-based sample of Taiwanese adolescents and young adults to determine if the urinary levels of the acrylamide metabolite N-acetyl-S-(propionamide)-cysteine (AAMA) and the 6 serum thyroid measures are associated. The mean (SD) AAMA were 76.54 (76.42) µg/L. Linear regression analyzes showed a 1-unit increase in natural log AAMA was significantly associated with a decrease in serum free thyroxine (T4) (ng/dL) (ß=-0.041, SE=0.013, p=0.001) after controlling for covariates. Subpopulation analyzes showed AAMA and free T4 were significantly associated with females, age 20-30 years, non-current smokers, and non-alcohol consumers. In conclusion, higher urinary AAMA concentrations were associated with decreased levels of free T4 in this cohort. Further studies are warranted to determine if there is a causal relationship between acrylamide exposure and thyroid function.

Positive association between urinary levels of 8-hydroxydeoxyguanosine and the acrylamide metabolite N-acetyl-S-(propionamide)-cysteine in adolescents and young adults.

Acrylamide is present in mainstream cigarette smoke and in some foods prepared at high temperatures. Animal studies have shown that acrylamide exposure increases oxidative stress; however, it is not known if this also occurs in humans. We recruited 800 subjects (mean age, 21.3 years, range, 12-30 years) from a population-based sample of Taiwanese adolescents and young adults to determine if urinary levels of the acrylamide metabolite N-acetyl-S-(propionamide)-cysteine (AAMA) and the oxidative stress product 8-hydroxydeoxyguanosine (8-OHdG) are associated. The mean (SD) AAMA and 8-OHdG were 76.54 (76.42)µg/L and 3.48 (2.37)µg/L, respectively. In linear regression analyses, a 1-unit increase in natural log AAMA was significantly associated with an increase in natural log 8-OHdG (µg/g creatinine) (ß=0.044, SE=0.019, P=0.020) after controlling for covariates. Subpopulation analyses showed AAMA and 8-OHdG were significantly associated with males, adolescents, non-current smokers, without alcohol consumption, subjects, body mass index ≥ 24, and homeostasis model assessment of insulin resistance score ≥ 0.9. In conclusion, higher urinary AAMA concentrations were associated with increased levels of urinary 8-OHdG in this cohort. Further studies are warranted to determine if there is a causal relationship between acrylamide exposure and oxidative stress.

In utero exposure to environmental lead and manganese and neurodevelopment at 2 years of age.

BACKGROUND AND OBJECTIVE: Manganese, lead, arsenic and mercury are common neurotoxic metals in the environment. Nonetheless, the relationship between prenatal exposure to low doses of neurotoxic metals and neurodevelopment in children is not clear. The objective of this study was to explore the relationship between in utero exposure to environmental neurotoxic metals and neurodevelopment at 2 years of age. METHODS: The population of this study came from the Taiwan Birth Panel Study. We included 230 pairs of non-smoking mothers without any occupational exposure and their singleton full-term children. The information about exposure during pregnancy was obtained using a structured questionnaire, and the manganese, lead, arsenic and mercury levels in umbilical cord blood samples were analyzed using inductively coupled plasma mass spectrometry. We used the Comprehensive Developmental Inventory for Infants and Toddlers (CDIIT) to evaluate the developmental status of each child at 2 years of age, and we examined the association of in utero exposure to environmental metals and neurodevelopment using linear regression models. RESULTS: The median concentrations of manganese, lead, arsenic and mercury in the cord blood samples in this study were 47.90 µg/L (range, 17.88-106.85 µg/L), 11.41 µg/L (range 0.16-43.22 µg/L), 4.05 µg/L (range, 1.50-12.88 µg/L) and 12.17 µg/L (range, 1.53-64.87 µg/L), respectively. After adjusting for maternal age, infant gender, environmental tobacco smoke during pregnancy and after delivery, Home Observation for Measurement of the Environment Inventory results, and arsenic and mercury levels in cord blood, we found that manganese and lead levels above the 75th percentile had a significant adverse association with the overall (ß=-7.03, SE=2.65, P=0.0085), cognitive (ß=-8.19, SE=3.17, P=0.0105), and language quotients (ß=-6.81, SE=2.73, P=0.0133) of the CDIIT. CONCLUSIONS: In utero exposure to environmental manganese and lead may have an adverse association with neurodevelopment at 2 years of age, and there is an interaction effect between the manganese and lead levels in the cord blood that could aggravate the effect.

Septic arthritis caused by Mycobacterium fortuitum and Mycobacterium abscessus in a prosthetic knee joint: case report and review of literature.

Nontuberculous mycobacterium (NTM) is an infrequent cause of prosthetic knee joint infections. Simultaneous infection with different NTM species in a prosthetic knee joint has not been previously reported. A case of prosthetic knee joint infection caused by Mycobacterium abscessus and M. fortuitum is described in this report. The patient was successfully treated with adequate antibiotics and surgery. The clinical features of sixteen previously reported cases of prosthetic knee joint infection caused by NTM are reviewed.

Chronic ingestion of alcohol modulates expression of ubiquitin editing enzyme A20 in lung macrophages.

BACKGROUND: Alcohol abuse is involved in the pathogenesis of multiple organ disorders; the underlying mechanism is incompletely understood. The ubiquitin editing enzyme A20 is involved in regulating activities in the cell. Suppression of A20 is suggested as one factor in the initiation of inflammation. This study investigates the mechanism by which chronic alcohol consumption modulates the levels of ubiquitin editing enzyme A20 in macrophages and further contributes to induce endothelial barrier dysfunction in the lung. METHODS: Mice were gavage-fed with 40% alcohol daily for 0-3 weeks. Airway macrophages were collected by lung lavage. Expression of ubiquitin editing enzyme A20 in isolated macrophages was assessed at both mRNA and protein levels. The endothelial barrier function of the lung was evaluated by the Evans blue method. RESULTS: Mice treated with alcohol for 3 weeks showed an increase in cell infiltration in the lung in response to exposure to peptidoglycan; over 80% of the infiltrated cells were macrophages. Furthermore, we observed that A20 level was suppressed in macrophages of mice treated with alcohol; the levels of tumor necrosis factor, interleukin-6 and nuclear factor kappa B in macrophage were increased. In addition, the endothelial barrier function of the lung was compromised, showing excessive infiltration of Evans blue in the lung indicating lung edema. Pretreatment with synthesized A20 inhibited alcohol-induced lung endothelial barrier dysfunction. CONCLUSIONS: We conclude that chronic alcohol ingestion disturbs the endothelial barrier function in the lung by modulating macrophage properties. Increase in A20 in the cell may have potential for the treatment of inflammatory disorders.

Epidural abscess caused by Haemophilus aphrophilus misidentified as Pasteurella species.

Haemophilus aphrophilus is one of the normal oropharyngeal flora and rarely implicated as a pathogen of spinal infection. A case of H. aphrophilus bacteremia complicated with epidural abscess, psoas muscle abscess, and spondylodiscitis is described in this report. The pathogen was mis-identified as Pasteurella spp. at the very start, and was confirmed by the molecular method. He was successfully treated with adequate antibiotics and surgery. The clinical features of sixteen previously reported cases of spinal infection caused by H. aphrophilus are reviewed.

[Cellular mechanism of heart injury in the early stage of crush injury in rats].

OBJECTIVE: To study cellular mechanism of cardiomyocytes injury in the early stage of crush injury by observing some effects of crush injury rat sera on cultured neonatal rat cardiomyocytes. METHODS: One to three days old neonatal rat cardiomyocytes were cultured in vitro and some effects of crush injury rat sera on beating rate, cell surface area, total protein content, 3H-Leu incorporation, intracellular calcium concentration ([Ca2+]i) and Fos protein expression were observed in cultured rat cardiomyocytes. RESULTS: Compared with normal rat serum group, crush injury rat sera decreased beating rate(beats/min) of cardiomyocytes from 88.3 to 26.4, cell surface area, total protein content, 3H-Leu incorporation, [Ca2+]i (nmol/L) and PI of Fos protein expression were increased. CONCLUSION: Crush injury rat sera suppress cell beating, increase intracellular calcium, induce Fos protein synthesis and cause cell hypertrophy, which may cause cardiac injury in the early stage of rush injury.

[Relationship between DNA degradation and postmortem interval of corrupt corpse].

OBJECTIVE: To study the relationship between DNA degradation and postmortem interval of corrupt corpse. METHODS: By determining the marrow DNA content with histochemical technique and image analysis. RESULTS: The content of marrow DNA decreased gradually with prolongation of postmortem interval, and it evencould be detected till 14 days after death. CONCLUSION: There was a linear relationship between the degradation rate of the nuclear DNA and postmortem interval.

[A quantitative analysis of peptidergic innervation in sinoatrial node in cases of sudden manhood death syndrome].

OBJECTIVE: To study the distribution and proportion of neuropeptide containing nervers in the sinus node in cases of sudden manhood death syndrome (SMDS) and to explore the mechanism of SMDS. METHODS: Immunohistochemical staining and quantitative analysis of neuropeptide Y (NPY) and vasoactive intestinal peptide(VIP) in the sinus node in 6 cases of SMDS and in 12 cases of non-cardiac death(control group) were achieved by LSAB method and computerized image system. RESULTS: As for NPY positive materials, VIP positive materials and the ratio of VIP/NPY in the sinus nodes, there were no significant difference between the control group and SMDS group. CONCLUSION: The mechanism of SMDS and the abnormality of autonomic nervous innervation in the sinoatrial nodes maybe incorrelation.

[The relationship between postmortem degradation of marrow DNA in bosom bone and late postmortem interval estimation].

OBJECTIVE: To study the relationship between degradation of marrow DNA and late postmortem interval (PMI). METHODS: Marrow were left on natural condition for 0,1,3,5,7 day after death respectively, Marrow DNA were detected by using Feulgen staining and computerized image analysis. RESULTS: The content of marrow DNA could be detected till 7 days after death yet. CONCLUSION: The degradation of marrow DNA may be used on estimation the late PMI.