Identification of viral genomic elements responsible for rabies virus neuroinvasiveness

Attenuated tissue culture-adapted and natural street rabies virus (RV) strains differ greatly in their neuroinvasiveness. To identify the elements responsible for the ability of an RV to enter the CNS from a peripheral site and to cause lethal neurological disease, we constructed a full-length cDNA clone of silver-haired bat-associated RV (SHBRV) strain 18 and exchanged the genes encoding RV proteins and genomic sequences of this highly neuroinvasive RV strain with those of a highly attenuated nonneuroinvasive RV vaccine strain (SN0). Analysis of the recombinant RV (SB0), which was recovered from SHBRV-18 cDNA, indicated that this RV is phenotypically indistinguishable from WT SHBRV-18. Characterization of the chimeric viruses revealed that in addition to the RV glycoprotein, which plays a predominant role in the ability of an RV to invade the CNS from a peripheral site, viral elements such as the trailer sequence, the RV polymerase, and the pseudogene contribute to RV neuroinvasiveness. Analyses also revealed that neuroinvasiveness of an RV correlates inversely with the time necessary for internalization of RV virions and with the capacity of the virus to grow in neuroblastoma cells.     

Diabetes-Related Factors and the Effects of Ticagrelor Plus Aspirin in the THEMIS and THEMIS-PCI Trials

BackgroundTHEMIS (The Effect of Ticagrelor on Health Outcomes in Diabetes Mellitus Patients Intervention Study) (n = 19,220) and its pre-specified THEMIS-PCI (The Effect of Ticagrelor on Health Outcomes in Diabetes Mellitus Patients Intervention Study-Percutaneous Coronary Intervention) (n = 11,154) subanalysis showed, in individuals with type 2 diabetes mellitus (median duration 10.0 years; HbA_1c 7.1%) and stable coronary artery disease without prior myocardial infarction (MI) or stroke, that ticagrelor plus aspirin (compared with placebo plus aspirin) produced a favorable net clinical benefit (composite of all-cause mortality, MI, stroke, fatal bleeding, and intracranial bleeding) if the patients had a previous percutaneous coronary intervention.ObjectivesIn these post hoc analyses, the authors examined whether the primary efficacy outcome (cardiovascular death, MI, stroke: 3-point major adverse cardiovascular events [MACE]), primary safety outcome (Thrombolysis In Myocardial Infarction–defined major bleeding) and net clinical benefit varied with diabetes-related factors.MethodsOutcomes were analyzed across baseline diabetes duration, HbA_1c, and antihyperglycemic medications.ResultsIn THEMIS, the incidence of 3-point MACE increased with diabetes duration (6.7% for ≤5 years, 11.1% for >20 years) and HbA_1c (6.4% for ≤6.0%, 11.8% for >10.0%). The relative benefits of ticagrelor plus aspirin on 3-point MACE reduction (hazard ratio [HR]: 0.90; p = 0.04) were generally consistent across subgroups. Major bleeding event rate (overall: 1.6%) did not vary by diabetes duration or HbA_1c and was increased similarly by ticagrelor across all subgroups (HR: 2.32; p < 0.001). These findings were mirrored in THEMIS-PCI. The efficacy and safety of ticagrelor plus aspirin did not differ by baseline antihyperglycemic therapy. In THEMIS-PCI, but not THEMIS, ticagrelor generally produced favorable net clinical benefit across diabetes duration, HbA_1c, and antihyperglycemic medications.ConclusionTicagrelor plus aspirin yielded generally consistent and favorable net clinical benefit across the diabetes-related factors in THEMIS-PCI but not in the overall THEMIS population.     

Hyperspectral interference tomography of nacre

Structural characterization of biologically formed materials is essential for understanding biological phenomena and their enviro-nment, and for generating new bio-inspired engineering concepts. For example, nacre—the inner lining of some mollusk shells—encodes local environmental conditions throughout its formation and has exceptional strength due to its nanoscale brick-and-mortar structure. This layered structure, comprising alternating transparent aragonite (CaCO₃) tablets and thinner organic polymer layers, also results in stunning interference colors. Existing methods of structural characterization of nacre rely on some form of cross-sectional analysis, such as scanning or transmission electron microscopy or polarization-dependent imaging contrast (PIC) mapping. However, these techniques are destructive and too time- and resource-intensive to analyze large sample areas. Here, we present an all-optical, rapid, and nondestructive imaging technique—hyperspectral interference tomography (HIT)—to spatially map the structural parameters of nacre and other disordered layered materials. We combined hyperspectral imaging with optical-interference modeling to infer the mean tablet thickness and its disorder in nacre across entire mollusk shells from red and rainbow abalone (Haliotis rufescens and Haliotis iris) at various stages of development. We observed that in red abalone, unexpectedly, nacre tablet thickness decreases with age of the mollusk, despite roughly similar appearance of nacre at all ages and positions in the shell. Our rapid, inexpensive, and nondestructive method can be readily applied to in-field studies.

Complex optical phenomena can emerge from a variety of biological or bio-inspired processes, from arrays of colors in peacocks (1) and other birds (2), butterflies (3), and opals (4), to the metal-like sheen of herring (5) and unique polarization-dependent properties of jewel beetles (6) and Pollia fruit (7). Nacre, or mother-of-pearl, is a prominent biologically formed mineral structure found throughout our oceans. It lines the inside of the shells formed by many mollusks, including bivalves, cephalopods, and gastropods. It features brilliant iridescent colors (Fig. 1) and is studied and emulated in part because of its outstanding mechanical performance (8, 9). The striking, colorful appearance of nacre has been a source of scientific curiosity since the days of Brewster (10), Rayleigh (11), and Raman (12, 13), and is the product of optical interference resulting from multiple interface reflections as light propagates through its stratified structure comprising stacks of transparent polygonal aragonite tablets (CaCO₃) interspersed with organic polymer (chitin and proteins) layers (14–16) (Fig. 1A). Nacre is one of seven mollusk shell structures (17). In the nacre structure, the aragonite tablets are typically 5 to 10 μm in diameter and hundreds of nanometers thick [200 to 1,100 nm across all shells, and 250 to 500 nm in red abalone (18)], while the organic sheets are an order of magnitude thinner (14, 16, 19). In columnar nacre formed by gastropods like abalone and snails (Fig. 1), co-oriented tablets are stacked on top of one another, while in sheet nacre formed by bivalves like pearl oysters and pen shells, co-oriented tablets are staggered diagonally (18) (see Movie S1 for an animation showing how co-oriented tablets are stacked in columnar nacre). Despite the significant structural and formation–mechanism differences, the thicknesses of tablets and organic layers are similar in columnar and sheet nacre, and so are the optical and mechanical behavior (20). The resulting palette of colors is primarily dependent on the nacre tablet thickness and the viewing angle, and the optical response that yields these colors can be understood as that of a Bragg reflector (21) with disorder in the layer thicknesses, where the optical band gaps are determined by the thicknesses of the transparent layers (5, 22, 23). Thus, the spectrum of light reflected from a nacre surface encodes information about its physical structure (Fig. 1 B–D).Open in a separate windowFig. 1.(A) Nacre, the colorful iridescent inner lining of some mollusk shells. Here, the red abalone, or H. rufescens, shell features columnar nacre, which comprises thousands of layers of polygonal aragonite tablets interspersed with organic sheets. (B) A close-up photograph of the nacre surface shows a variety of colors and nonuniformities. (C and D) Given a broadband white light source illuminating nacre at a fixed angle of incidence, variations in color are observed due to the difference in average thickness of aragonite tablets comprising nacre. (E) Hyperspectral interference tomography (HIT) setup: A hyperspectral camera collects predominantly specular reflectance data across a sample illuminated by a collimated source at a fixed angle of incidence from the normal to the sample (θ). The reflected light is polarized using a wire-grid polarizer. (F) A color photograph of a region of nacre that was analyzed. (G) Map of the mean tablet thickness (MTT) obtained using HIT, overlaid on a grayscale rendering of the photograph in F. Highlighted in red is a 5 × 5-mm region used to analyze the ontogeny of nacre in Fig. 4. The region around this area was masked off using opaque tape, which is highlighted with the dashed white box.Understanding and characterizing the structure of nacre and other biomaterials have deep and surprising implications. For example, the average thickness of the tablets comprising ancient nacre can be used as a proxy for local ocean temperatures at the time of nacre formation, enabling paleoclimatology spanning hundreds of millions of years (18, 24, 25). The structure of nacre is also an inspiration for engineered materials thousands of times stronger than the constituent materials (15, 26, 27). To that end, new techniques have been developed to probe and understand the structure of nacre, such as polarization-dependent imaging contrast (PIC) mapping using X-ray absorption near-edge structure spectroscopy combined with photoemission electron spectromicroscopy (18, 28, 29), or X-ray nanotomography (30). However, these characterization techniques such as cross-sectional electron microscopy result in the destruction of the sample and are time-consuming and costly.Here, we present a method for rapid, nondestructive, and large-scale structural characterization of disordered and nonuniform stratified thin-film materials and apply it to the analysis of nacre. Our all-optical method employs hyperspectral imaging combined with thin-film modeling to extract nacre mean tablet thicknesses (MTTs) and tablet degree of disorder (σ)—defined as the standard deviation of the thicknesses—across large areas (Fig. 1 E–G). This characterization method is designated as hyperspectral interference tomography (HIT). We used HIT to map the structure of mollusk shell nacre across many stages of development and identified a previously unexplored relationship between the age of the organism and the structure of the nacre layer. We investigated two particular species of nacre-forming mollusks, Haliotis rufescens (red abalone) and Haliotis iris (paua, or rainbow abalone; data only in SI Appendix), for which the aragonite tablet thicknesses lie within a range of 250 to 500 nm (18, 31); however, the method is applicable to any other transparent layered structure of animal, plant, geologic, or synthetic origin.     

In Vitro Resistance Studies with Bacteria That Exhibit Low Mutation Frequencies: Prediction of “Antimutant” Linezolid Concentrations Using a Mixed Inoculum Containing both Susceptible and Resistant Staphylococcus aureus

Bacterial resistance studies using in vitro dynamic models are highly dependent on the starting inoculum that might or might not contain spontaneously resistant mutants (RMs). To delineate concentration-resistance relationships with linezolid-exposed Staphylococcus aureus, a mixed inoculum containing both susceptible cells and RMs was used. An RM selected after the 9th passage of the parent strain (MIC, 2 μg/ml) on antibiotic-containing media (RM9; MIC, 8 μg/ml) was chosen for the pharmacodynamic studies, because the mutant prevention concentration (MPC) of linezolid against the parent strain in the presence of RM9 at 10² (but not at 10⁴) CFU/ml did not differ from the MPC value determined in the absence of the RMs. Five-day treatments with twice-daily linezolid doses were simulated at concentrations either between the MIC and MPC or above the MPC. S. aureus RMs (resistant to 2× and 4× MIC but not 8× and 16× MIC) were enriched at ratios of the 24-h area under the concentration-time curve (AUC₂₄) to the MIC that provide linezolid concentrations between the MIC and MPC for 100% (AUC₂₄/MIC, 60 h) and 86% (AUC₂₄/MIC, 120 h) of the dosing interval. No such enrichment occurred when linezolid concentrations were above the MIC and below the MPC for a shorter time (37% of the dosing interval; AUC₂₄/MIC, 240 h) or when concentrations were consistently above the MPC (AUC₂₄/MIC, 480 h). These findings obtained using linezolid-susceptible staphylococci supplemented with RMs support the mutant selection window hypothesis. This method provides an option to delineate antibiotic concentration-resistance relationships with bacteria that exhibit low mutation frequencies.     

RANKL Expression Is Increased in Circulating Mononuclear Cells of Patients with Calcific Aortic Stenosis

We aimed to investigate whether the expression of the OPG/RANK/RANKL triad in peripheral blood mononuclear cells (PBMC) and circulating levels of markers of ectopic mineralization (OPG, FGF-23, PPi) are modified in patients with calcific aortic valve disease (CAVD). We found that patients affected by CAVD (n?=?50) had significantly higher circulating levels of OPG as compared to control individuals (p?=?0.003). No differences between the two groups were found in FGF-23 and PPi levels. RANKL expression was higher in the PBMC from CAVD patients (p?=?0.018) and was directly correlated with the amount of valve calcification (p?=?0.032). In vitro studies showed that treatment of valve interstitial cells (VIC) with RANKL plus phosphate was followed by increase in matrix mineralization (p?=?0.001). In conclusion, RANKL expression is increased in PBMC of patients with CAVD, is directly correlated with the degree of valve calcification, and promotes pro-calcific differentiation of VIC.     

Incidence of HCC in chronic hepatitis C patients with advanced hepatic fibrosis who achieved SVR following DAAs: A prospective study

Liver cirrhosis is an important risk factor for hepatocellular carcinoma. The reported annual incidence of HCC is about 3%‐8% in CHC cirrhotic patients. Based on the Cochrane systematic review, there was no clear evidence, on the long‐term clinical effects of DAAs in patients achieving SVR, as regard liver cirrhosis‐related HCC incidence. The aim of the study was to determine the incidence of HCC in chronic hepatitis C patients genotype IV with liver cirrhosis and advanced liver fibrosis after achieving SVR following DAA treatment in a prospective large cohort of HCV patients with long follow‐up. This was a prospective observational cohort study including 2372 CHC patients with advanced liver fibrosis or cirrhosis receiving DAA therapy in outpatient clinics at the Egyptian Liver Research Institute and Hospital since January 2015. Liver fibrosis was assessed using transient elastography. Abdominal ultrasonography and AFP measurement were done at baseline and follow‐up visits every 6 months, in addition to triphasic abdominal MSCT when needed. Patients were followed up after achieving SVR12 for at least 12 months. HCC developed in 109 cases during the follow‐up period (mean 23.60 ± 8.25 months). Overall HCC incidence was 2.338/100 PY, 95% CI = 1.942‐2.814. In patients with cirrhosis, the incidence of HCC was 2.917/100 PY, 95% CI = 2.407‐3.535, while in patients with advanced liver fibrosis the incidence of HCC was 0.664/100 PY, 95% CI = 0.333‐1.326. In conclusion, the incidence of HCC was reduced in chronic hepatitis C genotype 4 patients with liver cirrhosis (F4) and advanced hepatic fibrosis (F3) who achieved SVR following DAA therapy.     

Human genetic variation altering anthrax toxin sensitivity

The outcome of exposure to infectious microbes or their toxins is influenced by both microbial and host genes. Some host genes encode defense mechanisms, whereas others assist pathogen functions. Genomic analyses have associated host gene mutations with altered infectious disease susceptibility, but evidence for causality is limited. Here we demonstrate that human genetic variation affecting capillary morphogenesis gene 2 (CMG2), which encodes a host membrane protein exploited by anthrax toxin as a principal receptor, dramatically alters toxin sensitivity. Lymphoblastoid cells derived from a HapMap Project cohort of 234 persons of African, European, or Asian ancestry differed in sensitivity mediated by the protective antigen (PA) moiety of anthrax toxin by more than four orders of magnitude, with 99% of the cohort showing a 250-fold range of sensitivity. We find that relative sensitivity is an inherited trait that correlates strongly with CMG2 mRNA abundance in cells of each ethnic/geographical group and in the combined population pool (P = 4 × 10(-11)). The extent of CMG2 expression in transfected murine macrophages and human lymphoblastoid cells affected anthrax toxin binding, internalization, and sensitivity. A CMG2 single-nucleotide polymorphism (SNP) occurring frequently in African and European populations independently altered toxin uptake, but was not statistically associated with altered sensitivity in HapMap cell populations. Our results reveal extensive human diversity in cell lethality dependent on PA-mediated toxin binding and uptake, and identify individual differences in CMG2 expression level as a determinant of this diversity. Testing of genomically characterized human cell populations may offer a broadly useful strategy for elucidating effects of genetic variation on infectious disease susceptibility.     

Mixotrophic basis of Atlantic oligotrophic ecosystems

Oligotrophic subtropical gyres are the largest oceanic ecosystems, covering >40% of the Earth's surface. Unicellular cyanobacteria and the smallest algae (plastidic protists) dominate CO(2) fixation in these ecosystems, competing for dissolved inorganic nutrients. Here we present direct evidence from the surface mixed layer of the subtropical gyres and adjacent equatorial and temperate regions of the Atlantic Ocean, collected on three Atlantic Meridional Transect cruises on consecutive years, that bacterioplankton are fed on by plastidic and aplastidic protists at comparable rates. Rates of bacterivory were similar in the light and dark. Furthermore, because of their higher abundance, it is the plastidic protists, rather than the aplastidic forms, that control bacterivory in these waters. These findings change our basic understanding of food web function in the open ocean, because plastidic protists should now be considered as the main bacterivores as well as the main CO(2) fixers in the oligotrophic gyres.