Quiescent hepatic stellate cells induce toxicity and sensitivity to doxorubicin in cancer cells through a caspase-independent cell death pathway: Central role of apoptosis-inducing factor

Hepatocellular carcinoma (HCC) is a major health problem worldwide and in the United States as its incidence has increased substantially within the past two decades. HCC therapy remains a challenge, primarily due to underlying liver disorders such as cirrhosis that determines treatment approach and efficacy. Activated hepatic stellate cells (A-HSCs) are the key cell types involved in hepatic fibrosis/cirrhosis. A-HSCs are important constituents of HCC tumor microenvironment (TME) and support tumor growth, chemotherapy resistance, cancer cell migration, and escaping immune surveillance. This makes A-HSCs an important therapeutic target in hepatic fibrosis/cirrhosis as well as in HCC. Although many studies have reported the role of A-HSCs in cancer generation and investigated the therapeutic potential of A-HSCs reversion in cancer arrest, not much is known about inactivated or quiescent HSCs (Q-HSCs) in cancer growth or arrest. Here we report that Q-HSCs resist cancer cell growth by inducing cytotoxicity and enhancing chemotherapy sensitivity. We observed that the conditioned media from Q-HSCs (Q-HSCCM) induces cancer cell death through a caspase-independent mechanism that involves an increase in apoptosis-inducing factor expression, nuclear localization, DNA fragmentation, and cell death. We further observed that Q-HSCCM enhanced the efficiency of doxorubicin, as measured by cell viability assay. Exosomes present in the conditioned media were not involved in the mechanism, which suggests the role of other factors (proteins, metabolites, or microRNA) secreted by the cells. Identification and characterization of these factors are important in the development of effective HCC therapy.     

The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells

The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic beta-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of beta-cell function. Here, we show that inhibition of AMPK in beta-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the beta-cell.     

Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

Genomic regions associated with microdeletion/microduplication syndromes exhibit extreme diversity of structural variation

Segmental duplications (SDs) are a class of long, repetitive DNA elements whose paralogs share a high level of sequence similarity with each other. SDs mediate chromosomal rearrangements that lead to structural variation in the general population as well as genomic disorders associated with multiple congenital anomalies, including the 7q11.23 (Williams–Beuren Syndrome, WBS), 15q13.3, and 16p12.2 microdeletion syndromes. Population-level characterization of SDs has generally been lacking because most techniques used for analyzing these complex regions are both labor and cost intensive. In this study, we have used a high-throughput technique to genotype complex structural variation with a single molecule, long-range optical mapping approach. We characterized SDs and identified novel structural variants (SVs) at 7q11.23, 15q13.3, and 16p12.2 using optical mapping data from 154 phenotypically normal individuals from 26 populations comprising five super-populations. We detected several novel SVs for each locus, some of which had significantly different prevalence between populations. Additionally, we localized the microdeletion breakpoints to specific paralogous duplicons located within complex SDs in two patients with WBS, one patient with 15q13.3, and one patient with 16p12.2 microdeletion syndromes. The population-level data presented here highlights the extreme diversity of large and complex SVs within SD-containing regions. The approach we outline will greatly facilitate the investigation of the role of inter-SD structural variation as a driver of chromosomal rearrangements and genomic disorders.     

Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g [dry weight] of cells(-1). Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).     

Application of short-form oral health impact profile on elderly Japanese

Objectives: The purpose of this study was to use the oral health impact profile (OHIP‐14) to evaluate the impact of oral disease on the quality of life of a group of independently‐living elderly persons in an urban area of Japan. Subjects: A total of 1244 participants of the Senior Citizen's College, who attended the lectures once a week. They were community‐dwelling, independently‐living people over 60 years of age. Measurements: Japanese version of the short‐form OHIP‐14. Results: Internal reliability for the 14 items overall was very high (Cronbach's α = 0.95). Report of ‘painful aching’ and ‘uncomfortable to eat’ were the two most highly scored items using the mean sum OHIP‐14 score. A multiple logistic regression analysis indicated that the sum OHIP‐14 score had significant associations with self‐assessment of general health, dental status, and a perceived need for dental treatment. However, age, gender, dissatisfaction with financial status or education level was not significantly associated with the sum OHIP‐14. Compared with that of other countries, the items were ranked similarly, whereas the perceived magnitudes of the problems were quite different from other population. Conclusions: The OHIP‐14 in Japanese had a high internal reliability, was significantly associated with dental status and comparable ranking for items when compared with studies from other countries.     

Glutamate transport and cellular glutamine metabolism: regulation in LLC-PK1 vs. LLC-PK1-F+ cell lines

The glutamate (Glu) transporter may modulate cellular glutamine(Gln) metabolism by regulating both the rates of hydrolysis andsubsequent conversion of Glu to -ketoglutarate andNH⁺₄. By delivering Glu, a competitiveinhibitor of Gln for the phosphate-dependent glutaminase (PDG) as wellas an acid-load activator of glutamate dehydrogenase (GDH) flux, thetransporter may effectively substitute extracellularly generated Glufrom the -glutamyltransferase for that derived intracellularly fromGln. We tested this hypothesis in two closely related porcine kidneycell lines, LLC-PK₁ and LLC-PK₁-F⁺,the latter selected to grow in the absence of glucose, relying on Glnas their sole energy source. Both cell lines exhibited PDG suppressionas the result of Glu uptake while disrupting the extracellularL-Glu uptake, withD-aspartate-acceleratedintracellular Glu formation coupled primarily to the ammoniagenicpathway (GDH). Conversely, enhancing the extracellular Glu formationwith p-aminohippurate and Glu uptakesuppressed intracellular Gln hydrolysis whileNH⁺₄ formation from Glu increased. Thus theseresults are consistent with the transporter's dual role in modulatingboth PDG and GDH flux. Interestingly, PDG flux was actually higher inthe Gln-adapted LLC-PK₁-F⁺cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parentalLLC-PK₁ cells, revealing theimportance of both Glu and Gln transport in the modulation of PDG flux.Nevertheless, when studied at physiological Gln concentration, PDG fluxfalls under tight Glu transporter control as Gln uptake decreases,suggesting that cellular Gln metabolism may indeed be under Glutransporter control in vivo.