Autophagy: A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes

The effects of same-session combined exercise training on cardiorespiratory and functional fitness in older adults: a systematic review and meta-analysis

Endurance and strength training are effective strategies for counteracting age-associated reductions in physical performance in older adults, with a combination of both exercise modes recommended to maximise potential fitness benefits. This meta-analysis sought to quantify the effects of same-session combined endurance and strength training on fitness in adults aged over 50 years. Five electronic databases were searched with studies required to include one of the following outcome measures: VO2peak, 6-min walk test (6MWT), 8-ft timed up-and-go (TUG), and 30-s chair stand. Separate random-effects meta-analyses compared combined training with (1) no-exercise control, (2) endurance training, and (3) strength training with probabilistic magnitude-based inferences subsequently applied. Twenty-seven studies involving 1346 subjects with a mean age of 68.8 years (range 54–85 years) were included in the analysis. The meta-analysed effect on VO2peak was a moderately beneficial effect for the combined training compared to no-exercise controls (3.6 mL kg−1 min−1; ± 95% confidence limits 0.8 mL kg−1 min−1) with additional increases for studies with greater proportions of female participants and shorter training interventions. Combined training also had small-to-moderately beneficial effects on VO2peak when compared to endurance training (0.8 mL kg−1 min−1; ± 1.0 mL kg−1 min−1), 30-s chair stand when compared with strength training (1.1 repetitions; ± 0.5 repetitions) and on TUG (0.8 s; ± 0.7 s), 30-s chair stand (2.8 repetitions; ± 1.7 repetitions), and 6MWT (31.5 m; ± 22.4 m) when compared to no-exercise controls. All other comparisons were unclear. Same-session combined training can induce clinically relevant fitness improvements in older adults

A flow adhesion assay to study leucocyte recruitment to human hepatic sinusoidal endothelium under conditions of shear stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this 'adhesion cascade' can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process

Spectroscopic Characterization, Computational Investigation, and Comparisons of ECX

Three newly synthesized [Na+(221-Kryptofix)] salts containing AsCO–, PCO–, and PCS– anions were successfully electrosprayed into a vacuum, and these three ECX– anions were investigated by negative ion photoelectron spectroscopy (NIPES) along with high-resolution photoelectron imaging spectroscopy. For each ECX– anion, a well-resolved NIPE spectrum was obtained, in which every major peak is split into a doublet. The splittings are attributed to spin–orbit coupling (SOC) in the ECX• radicals. Vibrational progressions in the NIPE spectra of ECX– were assigned to the symmetric and the antisymmetric stretching modes in ECX• radicals. The electron affinities (EAs) and SO splittings of ECX• are determined from the NIPE spectra to be AsCO•: EA = 2.414 ± 0.002 eV, SO splitting = 988 cm–1; PCO•: EA = 2.670 ± 0.005 eV, SO splitting = 175 cm–1; PCS•: EA = 2.850 ± 0.005 eV, SO splitting = 300 cm–1. Calculations using the B3LYP, CASPT2, and CCSD(T) methods all predict linear geometries for both the anions and the neutral radicals. The calculated EAs and SO splittings for ECX• are in excellent agreement with the experimentally measured values. The simulated NIPE spectra, which are based on the calculated Franck–Condon factors, and the SO splittings nicely reproduce all of the observed spectral peaks, thus allowing unambiguous spectral assignments. The finding that PCS• has the greatest EA of the three triatomic molecules considered here is counterintuitive based upon simple electronegativity considerations, but this finding is understandable in terms of the movement of electron density from phosphorus in the HOMO of PCO– to sulfur in the HOMO of PCS–. Comparisons of the EAs of PCO• and PCS• with the previously measured EA values for NCO• and NCS• are made and discussed.National Science Foundation (U.S.) (Grant CHE-1362118

Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression

A number of mutations in α4β2-containing (α4β2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the β2 subunit called β2V287L. Two α4β2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(β2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(β2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4β2 to form a high-sensitivity α5α4β2 nAChR. Previous studies suggest that the β2V287L mutation suppresses low-sensitivity α4β2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the β2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The β2V287L mutation reduced the EC50 values of high- and low-sensitivity α4β2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4β2 expression. In contrast, it did not affect the EC50 of α5α4β2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, β2V287L increased the α4β2 response to a sub-saturating ACh concentration (1 μM). Finally, imaging SEP-tagged α5, α4, β2, and β2V287L subunits showed that β2V287L reduced total α4β2 nAChR surface expression, increased the number of β2 subunits per α4β2 receptor, and increased surface α5α4β2 nAChR expression. Thus, the β2V287L mutation alters the subunit composition and sensitivity of α4β2 nAChRs, and increases α5α4β2 surface expression

Delivery of temozolomide and N3-propargyl analog to brain tumors using an apoferritin nanocage

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values 500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies

Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Collagen hydrogels are a rapidly expanding platform in bioengineering and soft materials engineering for novel applications focused on medical therapeutics, medical devices and biosensors. Observations linking microstructure to material properties and function enables rational design strategies to control this space. Visualisation of the microscale organisation of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organisation of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualise and explore materials on the microscale. However, investigation of collagen gel microstructure is difficult without imparting structural changes during preparation and/or observation. Electrons are poorly propagated within liquid-phase materials, limiting the ability of electron microscopy to interrogate hydrated gels. Sample preparation techniques to remove water induce artefactual changes in material microstructure particularly in complex materials such as collagen, highlighting a critical need to develop robust material handling protocols for the imaging of collagen hydrogels. Here a collagen hydrogel is fabricated, and the gel state explored under high-vacuum (10−6Pa) and low-vacuum (80–120Pa) conditions, and in an environmental SEM chamber. Visualisation of collagen fibres is found to be dependent on the degree of sample hydration, with higher imaging chamber pressures and humidity resulting in decreased feature fidelity. Reduction of imaging chamber pressure is used to induce evaporation of gel water content, revealing collagen fibres of significantly larger diameter than observed in samples dehydrated prior to imaging. Rapid freezing and cryogenic handling of the gel material is found to retain a porous 3D structure following sublimation of the gel water content. Comparative analysis of collagen hydrogel materials demonstrates the care needed when preparing hydrogel samples for electron microscopy