A central role for microvillous receptor presentation in leukocyte adhesion under flow

AbstractLeukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation (“tethering”), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking

Quantitative Chemically-Specific Coherent Diffractive Imaging of Buried Interfaces using a Tabletop EUV Nanoscope

Characterizing buried layers and interfaces is critical for a host of applications in nanoscience and nano-manufacturing. Here we demonstrate non-invasive, non-destructive imaging of buried interfaces using a tabletop, extreme ultraviolet (EUV), coherent diffractive imaging (CDI) nanoscope. Copper nanostructures inlaid in SiO2 are coated with 100 nm of aluminum, which is opaque to visible light and thick enough that neither optical microscopy nor atomic force microscopy can image the buried interfaces. Short wavelength (29 nm) high harmonic light can penetrate the aluminum layer, yielding high-contrast images of the buried structures. Moreover, differences in the absolute reflectivity of the interfaces before and after coating reveal the formation of interstitial diffusion and oxidation layers at the Al-Cu and Al-SiO2 boundaries. Finally, we show that EUV CDI provides a unique capability for quantitative, chemically-specific imaging of buried structures, and the material evolution that occurs at these buried interfaces, compared with all other approaches.Comment: 12 pages, 8 figure

Triosephosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis

To address the source of infection in humans and public health importance of Giardia duodenalis parasites from animals, nucleotide sequences of the triosephosphate isomerase (TPI) gene were generated for 37 human isolates, 15 dog isolates, 8 muskrat isolates, 7 isolates each from cattle and beavers, and 1 isolate each from a rat and a rabbit. Distinct genotypes were found in humans, cattle, beavers, dogs, muskrats, and rats. TPI and small subunit ribosomal RNA (SSU rRNA) gene sequences of G. microti from muskrats were also generated and analyzed. Phylogenetic analysis on the TPI sequences confirmed the formation of distinct groups. Nevertheless, a major group (assemblage B) contained most of the human and muskrat isolates, all beaver isolates, and the rabbit isolate. These data confirm that G. duodenalis from certain animals can potentially infect humans and should be useful in the detection, differentiation, and taxonomy of Giardia spp

Potential Antiviral Options against SARS-CoV-2 Infection

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19

Comparison of Bone and Renal Effects In HIV-infected Adults Switching to Abacavir or Tenofovir Based Therapy in a Randomized Trial

Our objective was to compare the bone and renal effects among HIV-infected patients randomized to abacavir or tenofovir-based combination anti-retroviral therapy.In an open-label randomized trial, HIV-infected patients were randomized to switch from zidovudine/lamivudine (AZT/3TC) to abacavir/lamivudine (ABC/3TC) or tenofovir/emtricitabine (TDF/FTC). We measured bone mass density (BMD) and bone turnover biomarkers (osteocalcin, osteocalcin, procollagen type 1 N-terminal propeptide (P1NP), alkaline phosphatase, type I collagen cross-linked C-telopeptide (CTx), and osteoprotegerin). We assessed renal function by estimated creatinine clearance, plasma cystatin C, and urinary levels of creatinine, albumin, cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL). The changes from baseline in BMD and renal and bone biomarkers were compared across study arms.Of 40 included patients, 35 completed 48 weeks of randomized therapy and follow up. BMD was measured in 33, 26, and 27 patients at baseline, week 24, and week 48, respectively. In TDF/FTC-treated patients we observed significant reductions from baseline in hip and lumbar spine BMD at week 24 (-1.8% and -2.5%) and week 48 (-2.1% and -2.1%), whereas BMD was stable in patients in the ABC/3TC arm. The changes from baseline in BMD were significantly different between study arms. All bone turnover biomarkers except osteoprotegerin increased in the TDF/FTC arm compared with the ABC/3TC arm, but early changes did not predict subsequent loss of BMD. Renal function parameters were similar between study arms although a small increase in NGAL was detected among TDF-treated patients.Switching to TDF/FTC-based therapy led to decreases in BMD and increases in bone turnover markers compared with ABC/3TC-based treatment. No major difference in renal function was observed.Clinicaltrials.gov NCT00647244

Tonic Shock Induces Detachment of Giardia lamblia

The single-celled organism Giardia lamblia colonizes the small intestine of a wide variety of hosts, including humans. Giardiasis infections can cause severe gastrointestinal symptoms and pose a major health concern in the developing world. Giardia are known to attach robustly to a variety of surfaces, but the conditions that influence this attachment are not known. In this study, we examined the behavior of attached Giardia parasites exposed to rapid changes in solution properties, like those Giardia might encounter in the intestine. After systematically varying media concentration and composition, we found that only one solution property caused rapid detachment of Giardia cells: tonicity, which is a measure of the total concentration of solutes in the solution that are unable to pass through a semi-permeable membrane (here, the cell membrane of Giardia). We found similar results for Giardia initially attached to monolayers of intestinal cells. Giardia cells remaining attached after a change in tonicity are able to adapt to the change, highlighting the general ability of this organism to weather normal changes in the intestinal environment. We propose that Giardia's susceptibility to large changes in tonicity could be explored as a possible new route for treatment of giardiasis

An overview of the cutaneous porphyrias

This is an overview of the cutaneous porphyrias. It is a narrative review based on the published literature and my personal experience; it is not based on a formal systematic search of the literature. The cutaneous porphyrias are a diverse group of conditions due to inherited or acquired enzyme defects in the porphyrin–haem biosynthetic pathway. All the cutaneous porphyrias can have (either as a consequence of the porphyria or as part of the cause of the porphyria) involvement of other organs as well as the skin. The single commonest cutaneous porphyria in most parts of the world is acquired porphyria cutanea tarda, which is usually due to chronic liver disease and liver iron overload. The next most common cutaneous porphyria, erythropoietic protoporphyria, is an inherited disorder in which the accumulation of bile-excreted protoporphyrin can cause gallstones and, rarely, liver disease. Some of the porphyrias that cause blistering (usually bullae) and fragility (clinically and histologically identical to porphyria cutanea tarda) can also be associated with acute neurovisceral porphyria attacks, particularly variegate porphyria and hereditary coproporphyria. Management of porphyria cutanea tarda mainly consists of visible-light photoprotection measures while awaiting the effects of treating the underlying liver disease (if possible) and treatments to reduce serum iron and porphyrin levels. In erythropoietic protoporphyria, the underlying cause can be resolved only with a bone marrow transplant (which is rarely justifiable in this condition), so management consists particularly of visible-light photoprotection and, in some countries, narrowband ultraviolet B phototherapy. Afamelanotide is a promising and newly available treatment for erythropoietic protoporphyria and has been approved in Europe since 2014

Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism