Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.     

Correction to: Who is the ocean? Preface to the future seas 2030 special issue

Reviews in Fish Biology and Fisheries - A correction to this paper has been published: https://doi.org/10.1007/s11160-021-09657-9     

Do Insectivorous Birds use Volatile Organic Compounds from Plants as Olfactory Foraging Cues? Three Experimental Tests

Some insectivorous birds orient towards insect‐defoliated trees even when they do not see the foliar damage or the herbivores. There are, however, only a few studies that have examined the mechanisms behind this foraging behaviour. Previous studies suggest that birds can use olfactory foraging cues (e.g. volatile organic compounds (VOCs) emitted by defoliated plants), indirect visual cues or a combination of the two sensory cues. VOCs from insect‐defoliated plants are known to attract natural enemies of herbivores, and researchers have hypothesized that VOCs could also act as olfactory foraging cues for birds. We conducted three experiments across a range of spatial scales to test this hypothesis. In each experiment, birds were presented with olfactory cues and their behavioural responses or foraging outcomes were observed. In the first experiment, two different VOC blends, designed to simulate the volatile emissions of mountain birch (Betula pubescens ssp. czerepanovii) after defoliation by autumnal moth (Epirrita autumnata) larvae, were used in behavioural experiments in aviaries with pied flycatchers (Ficedula hypoleuca). The second experiment was a field‐based trial of bird foraging efficiency; the same VOC blends were applied to mountain birches, silver birches (B. pendula) and European white birches (B. pubescens) with plasticine larvae attached to the trees to serve as artificial prey for birds and provide a means to monitor predation rate. In the third experiment, the attractiveness of silver birch saplings defoliated by autumnal moth larvae versus intact controls was tested with great tits (Parus major) and blue tits (Cyanistes caeruleus) in an aviary. Birds did not orient towards either artificial or real trees with VOC supplements or towards herbivore‐damaged saplings when these saplings and undamaged alternatives were hidden from view. These findings do not support the hypothesis that olfactory foraging cues are necessary in the attraction of birds to herbivore‐damaged trees.     

Investigation of mercury concentrations in fur of phocid seals using stable isotopes as tracers of trophic levels and geographical regions

Recent studies have shown that the complementary analysis of mercury (Hg) concentrations and stable isotopic ratios of nitrogen (δ¹⁵N) and carbon (δ¹³C) can be useful for investigating the trophic influence on the Hg exposure and accumulation in marine top predators. In this study, we propose to evaluate the interspecies variability of Hg concentrations in phocids from polar areas and to compare Hg bioaccumulation between both hemispheres. Mercury concentrations, δ¹⁵N and δ¹³C were measured in fur from 85 individuals representing 7 phocidae species, a Ross seal (Ommatophoca rossii), Weddell seals (Leptonychotes weddellii), crabeater seals (Lobodon carcinophagus), harbour seals (Phoca vitulina), grey seals (Halichoerus grypus), ringed seals (Pusa hispida) and a bearded seal (Erignathus barbatus), from Greenland, Denmark and Antarctica. Our results showed a positive correlation between Hg concentrations and δ¹⁵N values among all individuals. Seals from the Northern ecosystems displayed greater Hg concentrations, δ¹⁵N and δ¹³C values than those from the Southern waters. Those geographical differences in Hg and stable isotopes values were likely due to higher environmental Hg concentrations and somewhat greater number of steps in Arctic food webs. Moreover, dissimilarities in feeding habits among species were shown through δ¹⁵N and δ¹³C analysis, resulting in an important interspecific variation in fur Hg concentrations. A trophic segregation was observed between crabeater seals and the other species, resulting from the very specific diet of krill of this species and leading to the lowest observed Hg concentrations.     

Predicting Particle Size During Fluid Bed Granulation Using Process Measurement Data

In this study, a new concept for particle size prediction during the fluid bed granulation is presented. Using the process measurements data obtained from a design of experimental study, predictive partial least squares models were developed for spraying and drying phases. Measured and calculated process parameters from an instrumented fluid bed granulation environment were used as explaining factors, whereas an in-line particle size data determined by spatial filtering technique were used as response. Modeling was carried out by testing all possible combinations of two to six process parameters (factors) of the total of 41 parameters. Eleven batches were used for model development and four batches for model testing. The selected models predicted particle size (d ₅₀) well, especially during the spraying phase (Q ² = 0.86). While the measured in-line d ₅₀ data were markedly influenced by different process failures, e.g., impaired fluidization activity, the predicted data remained more consistent. This introduced concept can be applied in fluid bed granulation processes if the granulation environment is soundly instrumented and if reliable real-time particle size data from the design of experiment batches are retrieved for the model development.     

Simple and inexpensive immunoassay-based diagnostic tests

Simple and inexpensive yet sensitive and robust diagnostic tests are critically needed for resource-poor settings to enable timely diagnosis and effective use of limited health care resources. Current tests are often too expensive, too slow, or have compromised clinical performance, and they often require health care professional to perform the test. In addition, most assays are not intended to be used in extreme environmental conditions, but their performance may be affected by high temperatures and humidity often encountered in resource-poor settings. This review provides an overview of current immunoassay technologies and their advantages and limitations with respect to their feasibility to resource-poor settings. Future trends of immunoassay development for decentralized testing are also discussed. Homogeneous assays as such are out of the scope of this article because they are generally not yet sensitive enough or otherwise less feasible for inexpensive rapid diagnostic tests.     

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.     

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.     

Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex

The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.