The vascular phenotype in Pseudoxanthoma elasticum and related disorders: contribution of a genetic disease to the understanding of vascular calcification

Vascular calcification is a complex and dynamic process occurring in various physiological conditions such as aging and exercise or in acquired metabolic disorders like diabetes or chronic renal insufficiency. Arterial calcifications are also observed in several genetic diseases revealing the important role of unbalanced or defective anti- or pro-calcifying factors. Pseudoxanthoma elasticum (PXE) is an inherited disease (OMIM 264800) characterized by elastic fiber fragmentation and calcification in various soft conjunctive tissues including the skin, eyes, and arterial media. The PXE disease results from mutations in the ABCC6 gene, encoding an ATP-binding cassette transporter primarily expressed in the liver, kidneys suggesting that it is a prototypic metabolic soft-tissue calcifying disease of genetic origin. The clinical expression of the PXE arterial disease is characterized by an increased risk for coronary (myocardial infarction), cerebral (aneurysm and stroke), and lower limb peripheral artery disease. However, the structural and functional changes in the arterial wall induced by PXE are still unexplained. The use of a recombinant mouse model inactivated for the Abcc6 gene is an important tool for the understanding of the PXE pathophysiology although the vascular impact in this model remains limited to date. Overlapping of the PXE phenotype with other inherited calcifying diseases could bring important informations to our comprehension of the PXE disease

A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug-antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR. DVP linkers were also used to efficiently re-bridge both monomeric and dimeric protein systems, demonstrating their potential utility for general protein modification, protein stabilisation or the development of other protein-conjugate therapeutics.AstraZeneca, Cambridge Trusts, EPSRC, BBSRC, Royal Society, MR

Analysis of HER2 genomic binding in breast cancer cells identifies a global role in direct gene regulation.

HER2 is a transmembrane receptor tyrosine kinase, which plays a key role in breast cancer due to a common genomic amplification. It is used as a marker to stratify patients in the clinic and is targeted by a number of drugs including Trastuzumab and Lapatinib. HER2 has previously been shown to translocate to the nucleus. In this study, we have explored the properties of nuclear HER2 by analysing the binding of this protein to the chromatin in two breast cancer cell lines. We find genome-wide re-programming of HER2 binding after treatment with the growth factor EGF and have identified a de novo motif at HER2 binding sites. Over 2,000 HER2 binding sites are found in both breast cancer cell lines after EGF treatment, and according to pathway analysis, these binding sites were enriched near genes involved in protein kinase activity and signal transduction. HER2 was shown to co-localise at a small subset of regions demarcated by H3K4me1, a hallmark of functional enhancer elements and HER2/H3K4me1 co-bound regions were enriched near EGF regulated genes providing evidence for their functional role as regulatory elements. A chromatin bound role for HER2 was verified by independent methods, including Proximity Ligation Assay (PLA), which confirmed a close association between HER2 and H3K4me1. Mass spectrometry analysis of the chromatin bound HER2 complex identified EGFR and STAT3 as interacting partners in the nucleus. These findings reveal a global role for HER2 as a chromatin-associated factor that binds to enhancer elements to elicit direct gene expression events in breast cancer cells

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

The NTI-tss device for the therapy of bruxism, temporomandibular disorders, and headache – Where do we stand? A qualitative systematic review of the literature

<p>Abstract</p> <p>Background</p> <p>The NTI-tss device is an anterior bite stop, which, according to the manufacturer, is indicated for the prevention and treatment of bruxism, temporomandibular disorders (TMDs), tension-type headaches, and migraine. The aim of this systematic review was to appraise the currently available evidence regarding the efficacy and safety of the NTI-tss splint.</p> <p>Methods</p> <p>We performed a systematic search in nine electronic databases and in NTI-tss-associated websites (last update: December 31, 2007). The reference lists of all relevant articles were perused. Five levels of scientific quality were distinguished. Reporting quality of articles about randomized controlled trials (RCTs) was evaluated using the Jadad score. To identify adverse events, we searched in the identified publications and in the MAUDE database.</p> <p>Results</p> <p>Nine of 68 relevant publications reported about the results of five different RCTs. Two RCTs concentrated on electromyographic (EMG) investigations in patients with TMDs and concomitant bruxism (Baad-Hansen et al 2007, Jadad score: 4) or with bruxism alone (Kavaklı 2006, Jadad score: 2); in both studies, compared to an occlusal stabilization splint the NTI-tss device showed significant reduction of EMG activity. Two RCTs focused exclusively on TMD patients; in one trial (Magnusson et al 2004, Jadad score: 3), a stabilization appliance led to greater improvement than an NTI-tss device, while in the other study (Jokstad et al 2005, Jadad score: 5) no difference was found. In one RCT (Shankland 2002, Jadad score: 1), patients with tension-type headache or migraine responded more favorably to the NTI-tss splint than to a bleaching tray. NTI-tss-induced complications related predominantly to single teeth or to the occlusion.</p> <p>Conclusion</p> <p>Evidence from RCTs suggests that the NTI-tss device may be successfully used for the management of bruxism and TMDs. However, to avoid potential unwanted effects, it should be chosen only if certain a patient will be compliant with follow-up appointments. The NTI-tss bite splint may be justified when a reduction of jaw closer muscle activity (e.g., jaw clenching or tooth grinding) is desired, or as an emergency device in patients with acute temporomandibular pain and, possibly, restricted jaw opening.</p

Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%–50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8–92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity