Transmission routes maintaining a viral pathogen of steelhead trout within a complex multi‐host assemblage

This is the first comprehensive region wide, spatially explicit epidemiologic analysis of surveillance data of the aquatic viral pathogen infectious hematopoietic necrosis virus (IHNV) infecting native salmonid fish. The pathogen has been documented in the freshwater ecosystem of the Pacific Northwest of North America since the 1950s, and the current report describes the disease ecology of IHNV during 2000–2012. Prevalence of IHNV infection in monitored salmonid host cohorts ranged from 8% to 30%, with the highest levels observed in juvenile steelhead trout. The spatial distribution of all IHNV‐infected cohorts was concentrated in two sub‐regions of the study area, where historic burden of the viral disease has been high. During the study period, prevalence levels fluctuated with a temporal peak in 2002. Virologic and genetic surveillance data were analyzed for evidence of three separate but not mutually exclusive transmission routes hypothesized to be maintaining IHNV in the freshwater ecosystem. Transmission between year classes of juvenile fish at individual sites (route 1) was supported at varying levels of certainty in 10%–55% of candidate cases, transmission between neighboring juvenile cohorts (route 2) was supported in 31%–78% of candidate cases, and transmission from adult fish returning to the same site as an infected juvenile cohort was supported in 26%–74% of candidate cases. The results of this study indicate that multiple specific transmission routes are acting to maintain IHNV in juvenile fish, providing concrete evidence that can be used to improve resource management. Furthermore, these results demonstrate that more sophisticated analysis of available spatio‐temporal and genetic data is likely to yield greater insight in future studies.     

Mechanism of type‐III protein secretion: Regulation of FlhA conformation by a functionally critical charged‐residue cluster

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram‐negative pathogens to export virulence factors into host cells. This mode of protein export is termed type‐III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton‐binding groups that might function in transport. Conserved proton‐binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well‐conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two‐hybrid experiments demonstrate self‐interaction of the domain, and targeted cross‐linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton‐actuated movements in the cytoplasmic domains of FlhA.     

Seasonal metabolic flexibility is correlated with microclimate variation in horned larks and house sparrows

Maximum and minimum metabolic rates in birds are flexible traits and such flexibility can be advantageous in variable climates. The climatic variability hypothesis (CVH) posits that more variable climates should result in greater metabolic flexibility for geographically distinct populations. Whether the CVH applies to sympatric species occupying microclimates differing in variability is unknown. Microclimates of open habitats are likely more variable than those of sheltered habitats. If the CVH extends to microclimates, we expect birds from open habitats to show greater flexibility than those from sheltered habitats. To test this extension of the CVH, we compared seasonal variation in microclimates and metabolic rates for sympatric horned larks Eremophila alpestris, which occupy open habitats, and house sparrows Passer domesticus, which occupy sheltered habitats. We measured operative temperature (T_e, an integrative measure of the thermal environment), summit metabolic rate (M_sum, maximal cold-induced metabolic rate), and basal metabolic rate (BMR, minimal maintenance metabolic rate) in summer and winter. For both winter and summer, daily minimum T_e was similar between open and sheltered habitats but maximum T_e was higher for open habitats. Winter microclimates, however, were colder for open than for sheltered habitats after accounting for convective differences. Both species increased M_sum in winter, but seasonal M_sum flexibility was greater for larks (43%) than for sparrows (31%). Winter increases in BMR were 92.5% and 11% for larks and sparrows, respectively, with only the former attaining statistical significance. Moreover, species * season interactions in general linear models for whole-organism metabolic rates were significant for BMR and showed a similar, although not significant, pattern for M_sum, with greater seasonal metabolic flexibility in horned larks than in house sparrows. These results suggest that extending the CVH to sympatric bird species occupying different microclimates may be valid.     

Structure of human monoamine oxidase B, a drug target for the treatment of neurological disorders.

Monoamine oxidase B (MAO B) is a mitochondrial outermembrane flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. We determined the structure of the human enzyme to 3 A resolution. The enzyme binds to the membrane through a C-terminal transmembrane helix and apolar loops located at various positions in the sequence. The electron density shows that pargyline, an analog of the clinically used MAO B inhibitor, deprenyl, binds covalently to the flavin N5 atom. The active site of MAO B consists of a 420 A(3)-hydrophobic substrate cavity interconnected to an entrance cavity of 290 A(3). The recognition site for the substrate amino group is an aromatic cage formed by Tyr 398 and Tyr 435. The structure provides a framework for probing the catalytic mechanism, understanding the differences between the B- and A-monoamine oxidase isoforms and designing specific inhibitors.     

Fabrication and electromechanical characterization of freestanding asymmetric membranes

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.     

Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.Subject terms: Gynaecological cancer, Preclinical research     

Age group DNA methylation differences in lemon sharks (Negaprion brevirostris): Implications for future age estimation tools

Age information is often non‐existent for most shark populations due to a lack of measurable physiological and morphological traits that can be used to estimate age. Recently, epigenetic clocks have been found to accurately estimate age for mammals, birds, and fish. However, since these clocks rely, among other things, on the availability of reference genomes, their application is hampered in non‐traditional model organisms lacking such molecular resources. The technique known as Methyl‐Sensitive Amplified Polymorphism (MSAP) has emerged as a valid alternative for studying DNA methylation biomarkers when reference genome information is missing, and large numbers of samples need to be processed. Accordingly, the MSAP technique was used in the present study to characterize global DNA methylation patterns in lemon sharks from three different age groups (juveniles, subadults, and adults). The obtained results reveal that, while MSAP analyses lack enough resolution as a standalone approach to infer age in these organisms, the global DNA methylation patterns observed using this technique displayed significant differences between age groups. Overall, these results confer that DNA methylation does change with age in sharks like what has been seen for other vertebrates and that MSAP could be useful as part of an epigenetics pipeline to infer the broad range of ages found in large samples sizes.     

Regulation and function of the mammalian tricarboxylic acid cycle

The tricarboxylic acid (TCA) cycle, otherwise known as the Krebs cycle, is a central metabolic pathway that performs the essential function of oxidizing nutrients to support cellular bioenergetics. More recently, it has become evident that TCA cycle behavior is dynamic, and products of the TCA cycle can be co-opted in cancer and other pathologic states. In this review, we revisit the TCA cycle, including its potential origins and the history of its discovery. We provide a detailed accounting of the requirements for sustained TCA cycle function and the critical regulatory nodes that can stimulate or constrain TCA cycle activity. We also discuss recent advances in our understanding of the flexibility of TCA cycle wiring and the increasingly appreciated heterogeneity in TCA cycle activity exhibited by mammalian cells. Deeper insight into how the TCA cycle can be differentially regulated and, consequently, configured in different contexts will shed light on how this pathway is primed to meet the requirements of distinct mammalian cell states.