Targeting the ATP-dependent formation of herpesvirus ribonucleoprotein particle assembly as an antiviral approach

Human herpesviruses are responsible for a range of debilitating acute and recurrent diseases, including a number of malignancies. Current treatments are limited to targeting the herpesvirus DNA polymerases, however with emerging viral resistance and little efficacy against the oncogenic herpesviruses, there is an urgent need for new antiviral strategies. Herein we describe a mechanism to inhibit the replication of the oncogenic herpesvirus Kaposi’s sarcoma associated herpesvirus (KSHV), by targeting the ATP-dependent formation of viral ribonucleoprotein particles (vRNPs). We demonstrate that small molecule inhibitors which selectively inhibit the ATPase activity of the cellular human transcription/export complex (hTREX) protein UAP56, result in effective inhibition of vRNP formation, viral lytic replication and infectious virion production. Strikingly, as all human herpesviruses utilize conserved mRNA processing pathways involving hTREX components, we demonstrate the feasibility of this approach for pan-herpesvirus inhibition

ARID3B: A novel regulator of the Kaposi's sarcoma-associated herpesvirus lytic cycle

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle—viral latency and the productive lytic cycle—and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation

In-Situ Biofilm Formation in Hyper Alkaline Environments

Lime manufacture in the UK has resulted in the generation of a number of alkaline sites (>pH 11.0) with complex indigenous microbial populations. Within the present study, retrievable cotton samples were used to investigate the fate of cellulose, the primary carbon source, within three sites aged from ≈25 to 140 years. Following 3 months incubation in situ, biofilms had formed on all cotton samples in these extreme pH conditions; with matrices comprised of carbohydrates, proteins, lipids and eDNA. Biofilms from the older sites contained greater amounts of eDNA, a structural component that aids the production of a denser biofilm. The age of the sites correlated with a shift from polysaccharides composed of β 1,4 and β 1,3 linked sugars to those composed of pyranosyl sugars within the older sites. These changes were reflected in the active biofilm communities which shifted from being Clostridiales dominated in the youngest site to Proteobacteria dominated in the older sites. The study demonstrates that the microbial communities resident in anthropogenic alkaline sites are able to form biofilms at pH values > pH 11.0 and that these biofilms evolve toward Proteobacteria dominated communities employing eDNA and pyranosyl sugar based polysaccharides to build the biofilm matrix

Utilising proteomic approaches to understand oncogenic human herpesviruses

The γ‑herpesviruses Epstein-Barr virus and Kaposi's sarcoma‑associated herpesvirus are successful pathogens, each infecting a large proportion of the human population. These viruses persist for the life of the host and may each contribute to a number of malignancies, for which there are currently no cures. Large‑scale proteomic-based approaches provide an excellent means of increasing the collective understanding of the proteomes of these complex viruses and elucidating their numerous interactions within the infected host cell. These large‑scale studies are important for the identification of the intricacies of viral infection and the development of novel therapeutics against these two important pathogens

Extreme enriched and heterogeneous ⁸⁷Sr/⁸⁶Sr ratios recorded in magmatic plagioclase from the Samoan hotspot

We report the major-element, trace-element, and 87Sr/86Sr compositions of six plagioclase crystals from two Samoan lavas with extreme EM2 isotopic compositions (ALIA-115-18 with whole-rock 87Sr/86Sr of 0.718592, and ALIA-115-21 with whole-rock 87Sr/86Sr of 0.720469). We employed laser-ablation split-stream mass spectrometry (LASS) to simultaneously measure 87Sr/86Sr ratios, major-element concentrations, and trace-element concentrations in the same plagioclase crystal volume. We find that two plagioclase crystals have extreme 87Sr/86Sr heterogeneity in excess of 5000 ppm (where ppm of 87Sr/Sr variability86=106⋅[Sr/8687Srmax−87Sr/86Srmin]/87Sr/86Sravg). In two of the plagioclase crystals, we identify the highest 87Sr/86Sr ratios (0.7224) ever measured in any fresh, mantle-derived ocean island basalt (OIB) or OIB-hosted mineral phase.We find that in 87Sr/86Sr-versus-Sr concentration space, the six plagioclase crystals overlap in a “common component” region with higher 87Sr/86Sr than has been previously identified in whole-rock Samoan lavas or mineral separates. We use the occurrence of olivine mineral inclusions (Fo=74.5±0.8, 2 SD) in the high-87Sr/86Sr zone of one plagioclase crystal to infer the bulk composition (Mg#=46.8±0.8, 2 SD) of the extreme EM2 magma from which the olivine and high-87Sr/86Sr plagioclase crystallized. We argue that a relatively evolved EM2 endmember magma mixed with at least one lower-87Sr/86Sr melt to generate the observed intra-crystal plagioclase isotopic heterogeneity.By inferring that subducted terrigenous sediment gives rise to EM2 signatures in Samoan lavas, we estimate that the quantity of sediment necessary to generate the most-elevated 87Sr/86Sr ratios observed in the Samoan plagioclase is ∼7% of the mantle source. We also estimate that sediment subduction into the mantle over geologic time has generated a sediment domain that constitutes 0.02% of the mass of the mantle, a much lower proportion than required in the EM2 mantle source. Even if subducted sediment is concentrated in large low-shear-velocity provinces (LLSVPs) at the base of the mantle (which constitute up to 7.7% of the mantle's mass), then only 0.25% of the LLSVPs are composed of sediment. This requires that the distribution of subducted sediment in the mantle is heterogeneous, and the high relative abundance of sediment in the Samoan EM2 mantle is an anomalous relic of ancient subduction that has survived convective attenuation

The evolution of alkaliphilic biofilm communities in response to extreme alkaline pH values

Extremes of pH present a challenge to microbial life and our understanding of survival strategies for microbial consortia, particularly at high pH, remains limited. The utilization of extracellular polymeric substances within complex biofilms allows micro-organisms to obtain a greater level of control over their immediate environment. This manipulation of the immediate environment may confer a survival advantage in adverse conditions to biofilms. Within the present study alkaliphilic biofilms were created at pH 11.0, 12.0, or 13.0 from an existing alkaliphilic community. In each pH system, the biofilm matrix provided pH buffering, with the internal pH being 1.0–1.5 pH units lower than the aqueous environment. Increasing pH resulted in a reduced removal of substrate and standing biomass associated with the biofilm. At the highest pH investigated (pH 13.0), the biofilms matrix contained a greater degree of eDNA and the microbial community was dominated by Dietzia sp. and Anaerobranca sp

Selective Affimers Recognise the BCL‐2 Family Proteins BCL‐xL and MCL‐1 through Noncanonical Structural Motifs

The BCL‐2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL‐2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non‐antibody‐binding proteins based on a conserved scaffold) to identify ligands for MCL‐1, BCL‐xL, BCL‐2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL‐2 protein. For anti‐apoptotic targets BCL‐xL and MCL‐1, competitive inhibition of their canonical protein‐protein interactions is demonstrated. Co‐crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug‐bound‐like conformation. These proof‐of‐concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL‐2 family modulators and more generally other protein‐protein interaction inhibitors

Arterial oxygen content is precisely maintained by graded erythrocytotic responses in settings of high/normal serum iron levels, and predicts exercise capacity: an observational study of hypoxaemic patients with pulmonary arteriovenous malformations.

Oxygen, haemoglobin and cardiac output are integrated components of oxygen transport: each gram of haemoglobin transports 1.34 mls of oxygen in the blood. Low arterial partial pressure of oxygen (PaO2), and haemoglobin saturation (SaO2), are the indices used in clinical assessments, and usually result from low inspired oxygen concentrations, or alveolar/airways disease. Our objective was to examine low blood oxygen/haemoglobin relationships in chronically compensated states without concurrent hypoxic pulmonary vasoreactivity.165 consecutive unselected patients with pulmonary arteriovenous malformations were studied, in 98 cases, pre/post embolisation treatment. 159 (96%) had hereditary haemorrhagic telangiectasia. Arterial oxygen content was calculated by SaO2 x haemoglobin x 1.34/100.There was wide variation in SaO2 on air (78.5-99, median 95)% but due to secondary erythrocytosis and resultant polycythaemia, SaO2 explained only 0.1% of the variance in arterial oxygen content per unit blood volume. Secondary erythrocytosis was achievable with low iron stores, but only if serum iron was high-normal: Low serum iron levels were associated with reduced haemoglobin per erythrocyte, and overall arterial oxygen content was lower in iron deficient patients (median 16.0 [IQR 14.9, 17.4]mls/dL compared to 18.8 [IQR 17.4, 20.1]mls/dL, p<0.0001). Exercise tolerance appeared unrelated to SaO2 but was significantly worse in patients with lower oxygen content (p<0.0001). A pre-defined athletic group had higher Hb:SaO2 and serum iron:ferritin ratios than non-athletes with normal exercise capacity. PAVM embolisation increased SaO2, but arterial oxygen content was precisely restored by a subsequent fall in haemoglobin: 86 (87.8%) patients reported no change in exercise tolerance at post-embolisation follow-up.Haemoglobin and oxygen measurements in isolation do not indicate the more physiologically relevant oxygen content per unit blood volume. This can be maintained for SaO2 ≥78.5%, and resets to the same arterial oxygen content after correction of hypoxaemia. Serum iron concentrations, not ferritin, seem to predict more successful polycythaemic responses

Red blood cell glutathione peroxidase activity in female nulligravid and pregnant rats

<p>Abstract</p> <p>Background</p> <p>The alterations of the glutathione peroxidase enzyme complex system occur in physiological conditions such as aging and oxidative stress consequent to strenuous exercise.</p> <p>Methods</p> <p>Authors optimize the spectrophotometric method to measure glutathione peroxidase activity in rat red blood cell membranes.</p> <p>Results</p> <p>The optimization, when applied to age paired rats, both nulligravid and pregnant, shows that pregnancy induces, at seventeen d of pregnancy, an increase of both reactive oxygen substance concentration in red blood cells and membrane glutathione peroxidase activity.</p> <p>Conclusion</p> <p>The glutathione peroxidase increase in erythrocyte membranes is induced by systemic oxidative stress long lasting rat pregnancy.</p