Stable Associations Masked by Temporal Variability in the Marine Copepod Microbiome

Copepod-bacteria interactions include permanent and transient epi- and endobiotic associations that may play roles in copepod health, transfer of elements in the food web, and biogeochemical cycling. Microbiomes of three temperate copepod species (Acartia longiremis, Centropages hamatus, and Calanus finmarchicus) from the Gulf of Maine were investigated during the early summer season using high throughput amplicon sequencing. The most prominent stable component of the microbiome included several taxa within Gammaproteobacteria, with Pseudoalteromonas spp. especially abundant across copepod species. These Gammaproteobacteria appear to be promoted by the copepod association, likely benefitting from nutrient enriched microenvironments on copepods, and forming a more important part of the copepod-associated community than Vibrio spp. during the cold-water season in this temperate system. Taxon-specific associations included an elevated relative abundance of Piscirickettsiaceae and Colwelliaceae on Calanus, and Marinomonas sp. in Centropages. The communities in full and voided gut copepods had distinct characteristics, thus the presence of a food-associated microbiome was evident, including higher abundance of Rhodobacteraceae and chloroplast sequences in the transient communities. The observed variability was partially explained by collection date that may be linked to factors such as variable time since molting, gender differences, and changes in food availability and type over the study period. While some taxon-specific and stable associations were identified, temporal changes in environmental conditions, including food type, appear to be key in controlling the composition of bacterial communities associated with copepods in this temperate coastal system during the early summer.     

Measures of Quality of Care for People with HIV: A Scoping Review of Performance Indicators for Primary Care

The healthcare of people with HIV is transitioning from specialty care to the primary healthcare (PHC) system. However, many of the performance indicators used to measure the quality of HIV care pre-date this transition. The goal of this work was to examine how existing HIV care performance indicators measure the comprehensive and longitudinal care offered in a PHC setting. A scoping review consisting of peer-reviewed and grey literature searches was performed. Two reviewers evaluated study eligibility and indicators in documents meeting inclusion criteria were extracted into a database. Indicators were matched to a PHC performance measurement framework to determine their applicability for evaluating quality of care in the PHC setting. The literature search identified 221 publications, of which 47 met inclusion criteria. 1184 indicators were extracted and removal of duplicates left 558 unique indicators. A majority of the 558 indicators fell under the ‘secondary prevention’ (12%) and ‘care of chronic conditions’ (33%) domains when indicators were matched to the PHC performance framework. Despite the imbalance, nearly all performance domains in the PHC framework were populated by at least one indicator with significant concentrations in domains such as patient-provider relationship, patient satisfaction, population and community characteristics, and access to care. Existing performance frameworks for the care of people with HIV provide a comprehensive set of indicators that align well with a PHC performance framework. Nonetheless, some important elements of care, such as patient-reported outcomes, are poorly covered by existing indicators. Advancing our understanding of how the experience of care for people with HIV is impacted by changes in health services delivery, specifically more care within the PHC system, will require performance indicators to capture this aspect of HIV care.     

Massage and stretching reduce spinal reflex excitability without affecting twitch contractile properties

Both stretching and massage can increase range of motion. Whereas the stretching-induced increases in ROM have been attributed to changes in neural and muscle responses, there is no literature investigating the ROM mechanisms underlying the interaction of stretch and massage. The objective of this paper was to evaluate changes in neural and evoked muscle responses with two types of massage and static stretching. With this repeated measures design, 30 s of plantar flexors musculotendinous junction (MTJ) and tapotement (TAP) massage were implemented either with or without 1 min of concurrent stretching as well as a control condition. Measures included the soleus maximum H-reflex/M-wave (H/M) ratio, as well as electromechanical delay (EMD), and evoked contractile properties of the triceps surae. With the exception of EMD, massage and stretch did not significantly alter triceps surae evoked contractile properties. Massage with and without stretching decreased the soleus H/M ratio. Both TAP conditions provided greater H/M ratio depression than MTJ massage while the addition of stretch provided the greatest inhibition. Both massage types when combined with stretching increased the duration of the EMD. In conclusion, MTJ and TAP massage as well as stretching decreased spinal reflex excitability, with TAP providing the strongest suppression. While static stretching prolongs EMD, massage did not affect contractile properties.     

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC₅₀s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99⁸²A, I59⁵⁰V, and V37³²I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC₅₀ of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked β-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.Numerous cytoplasmic and nuclear proteins are post-translationally modified with O-linked β-N-acetylglucosamine (O-GlcNAc).¹ GlcNAcylation is involved in almost all aspects of cellular metabolism (1) and is highly dependent on the nutrient status of the cell (2). The O-GlcNAc modification rivals phosphorylation in both abundance and protein distribution. Recent studies indicate that signaling pathways can be regulated by the interplay of these two modifications at the same or proximal sites on numerous protein substrates (3).Current understanding of the functions of O-GlcNAc and of the function of O-GlcNAcylation and its relationship to phosphorylation is severely hampered by the difficulties in detecting this labile monosaccharide modification. Problems associated with the identification of O-GlcNAc sites include the following. (a) O-GlcNAc is quickly removed by hydrolases during cell lysis. (b) Like phosphorylation, O-GlcNAc is usually present in less than stoichiometric amounts at given sites on protein substrates. (c) O-GlcNAc is readily lost as an oxonium ion during conventional peptide sequence analysis by collision-activated dissociation (CAD) (supplemental Fig. 1). (d) Modified and unmodified forms of the peptide often co-elute during reverse phase HPLC (supplemental Fig. 2), and the preferential ionization of the unmodified peptide suppresses the signal observed for the corresponding O-GlcNAc-modified peptide (supplemental Fig. 2, b and c).Several attempts have been made to enrich samples for O-GlcNAc-modified proteins and peptides. Immunoaffinity purification of O-GlcNAc-modified peptides with an antibody (CTD 110.6) has been largely unsuccessful because of low binding avidity (4). Long, wheat germ agglutinin lectin columns (∼39 ft) provide some enrichment but also bind strongly to complex glycans (5). A mutant galactosyltransferase (GalT1) has been used to label GlcNAcylated proteins with a ketone-containing galactose analog (6). Following proteolytic digestion, O-GlcNAc-modified peptides were biotinylated with hydrazine chemistry, isolated on a column packed with avidin beads, eluted with free biotin, and sequenced by ETD mass spectrometry. Failure to elute peptides with high efficiency from the avidin column and an inability to direct the fragmentation to the peptide backbone limit the usefulness of this approach. Reported here is an enrichment methodology that (a) is highly specific for O-GlcNAc-modified peptides, (b) provides for efficient release of the captured peptides from an affinity support, and (c) facilitates complete characterization of the released peptides by ETD mass spectrometry.     

Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2

Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser(254)) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [(3)H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [(3)H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b - measured both directly by the binding of [(3)H]NBMPR, and indirectly via inhibition of [(3)H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 microM TBB for 48 h decreased both the K(D) and B(max) of [(3)H]NBMPR binding, as well as the V(max) of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser(254), was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser(254) from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane.     

Not putting all their eggs in one basket: bet‐hedging despite extraordinary annual reproductive output of desert tortoises

Bet‐hedging theory makes the counter‐intuitive prediction that, if juvenile survival is low and unpredictable, organisms should consistently reduce short‐term reproductive output to minimize the risk of reproductive failure in the long‐term. We investigated the long‐term reproductive output of an Agassiz's desert tortoise (Gopherus agassizii) population and conformance to a bet‐hedging strategy of reproduction in an unpredictable but comparatively productive environment. Most females reproduced every year, even during periods of low precipitation and poor germination of food plants, and the mean percentage of reproducing females did not differ significantly on an annual basis. Although mean annual egg production (clutch size × clutch frequency) differed significantly among years, mean clutch size and mean clutch frequency remained relatively constant. During an El Niño year, mean annual egg production and mean annual clutch frequency were the highest ever reported for this species. Annual egg production was positively influenced by maternal body size but clutch size and clutch frequency were not. Our long‐term results confirm earlier conclusions based on short‐term research that desert tortoises have a bet‐hedging strategy of producing small clutches almost every year. The risk of long‐term reproductive failure is minimized in unpredictable environments, both through time by annually producing multiple small clutches over a long reproductive lifespan, even in years of low resource availability, and through space by depositing multiple annual clutches in different locations. The extraordinary annual reproductive output of this population appears to be the result of a typically high but unpredictable biomass of annual food plants at the site relative to tortoise habitat in dryer regions. Under the comparatively productive but unpredictable conditions, tortoises conform to predictions of a bet‐hedging strategy of reproduction with relatively small but consistent clutch sizes. Published 2015. This article is a U.S. Government work and is in the public domain in the USA, Biological Journal of the Linnean Society, 2015, 115 , 399–410.     

The many faces of tau

While the microtubule-binding capacity of the protein tau has been known for many years, new functions of tau in signaling and cytoskeletal organization have recently emerged. In this review, we highlight these functions and the potential roles of tau in neurodegenerative disease. We also discuss the therapeutic potential of drugs targeting various aspects of tau biology.