Decreased Proportion of Cytomegalovirus Specific CD8 T-Cells but No Signs of General Immunosenescence in Alzheimer’s Disease

Cytomegalovirus (CMV) has been suggested as a contributing force behind the impaired immune responsiveness in the elderly, with decreased numbers of naïve T-cells and an increased proportion of effector T-cells. Immunological impairment is also implicated as a part of the pathogenesis in Alzheimer’s disease (AD). The aim of this study was to investigate whether AD patients present with a different CMV-specific CD8 immune profile compared to non-demented controls. Blood samples from 50 AD patients and 50 age-matched controls were analysed for HLA-type, CMV serostatus and systemic inflammatory biomarkers. Using multi-colour flow cytometry, lymphocytes from peripheral blood mononuclear cells were analysed for CMV-specific CD8 immunity with MHC-I tetramers A01, A02, A24, B07, B08 and B35 and further classified using CD27, CD28, CD45RA and CCR7 antibodies. Among CMV seropositive subjects, patients with AD had significantly lower proportions of CMV-specific CD8 T-cells compared to controls, 1.16 % vs. 4.13 % (p=0.0057). Regardless of dementia status, CMV seropositive subjects presented with a lower proportion of naïve CD8 cells and a higher proportion of effector CD8 cells compared to seronegative subjects. Interestingly, patients with AD showed a decreased proportion of CMV-specific CD8 cells but no difference in general CD8 differentiation.     

De novo indol-3-ylmethyl glucosinolate biosynthesis,and not long-distance transport,contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.     

Field-based video observations of wild barnacle cyprid behaviour in response to textural and chemical settlement cues

Many marine invertebrate larvae respond behaviourally to environmental settlement cues, yet behaviours are often only inferred from settlement patterns or are limited to laboratory studies. The behaviour of wild cypris larvae of Semibalanus balanoides L. was filmed on settlement tiles in the field. Tiles were of five different textures with a nested treatment of crude conspecific adult extract (AE). The effects of texture and AE on eleven defined behaviours were analysed. Texture affected the gross and net exploratory distances, velocity, acceleration and time spent exploring. AE attracted more cyprids during the first minute of immersion and increased the time spent on surfaces. Relatively few arrivals that either travel far and fast, or exit the surface rapidly, may indicate a lower chance of settlement. An increase in time spent on a surface may increase the probability of being in contact with the surface when the sign stimulus to settle occurs.     

Elevated Plasma Homocysteine in Older Shift‐Workers: A Potential Risk Factor for Cardiovascular Morbidity

There is evidence supporting an association between shift work and cardiovascular morbidity, but the underlying mechanisms are unknown. The present paper investigated the levels of cardiovascular biochemical risk factors in shift‐workers both with (n=26) and without (n=103) sleep complaints, and in day‐workers (n=173) working in the same plant. Blood samples were taken in the morning after an overnight fast and analyzed for homocysteine, C‐reactive protein, and lipid profile. Biochemical data were compared among groups after stratifying workers by age (i.e., <40 and ≥40 yrs). Shift‐workers who complained about sleep disturbances and who were ≥40 years of age had significantly higher levels of homocysteine than did their younger counterparts—shift‐workers who did not complain of sleep disturbances and day‐workers. There were no other between‐group differences in any of the biochemical variables. The results of this investigation demonstrate an association between sleep disturbances in older shift‐workers and mild hyperhomocysteinemia. The elevated homocysteine levels may play a role in the increased rates of cardiovascular morbidity in shift‐workers, and they may have practical implications regarding the nutrition of shift‐workers.     

Seasonal fluctuations of resource abundance and avian feeding guilds across forest–farmland boundaries in tropical Africa

Seasonal fluctuations in climatic factors are expected to increase in future decades. However, little is known about the response of tropical species communities to seasonal fluctuations in climate and resource availability, particularly across different habitat types. We examined the relationship between spatio‐temporal fluctuations in the abundance of fruits and invertebrates and two avian feeding guilds, i.e. frugivores and insectivores, in forest and farmland habitats in western Kenya. Fruits and invertebrates fluctuated substantially throughout the year, but seasonal fluctuations were asynchronous between the two habitat types. Species richness and total abundance of frugivores and insectivores also fluctuated strongly and were closely related to the abundance of their respective resources. Frugivore species richness fluctuated anti‐cyclical in forest and farmland habitats, suggesting that several frugivorous species tracked fruit resources across habitat boundaries. In contrast, insectivorous bird richness fluctuated synchronously in the two habitat types, suggesting a lack of local‐scale movements across habitat boundaries. We conclude that bird communities strongly respond to seasonal fluctuations in resource availability, but responses differ between feeding guilds. While frugivores seem to respond flexibly to seasonal fluctuations, for instance by tracking fruit resources across habitat boundaries, insectivorous birds appear to be more susceptible to the expected increase in seasonal fluctuations in resource availability.     

Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

The ATP-dependent insertion of Mg²⁺ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg²⁺ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity.     

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)–thiosemicarbazide (TSC) or DAMO–antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO–TSC (Z′-factors 0.7–0.8) was determined to be superior to DAMO–antipyrine (Z′-factors 0.5–0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO–TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.     

Design,synthesis and structure–activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.     

Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.