Antibody avidity and multi-specificity combined to confer protection against SARS-CoV-2 and resilience against viral escape

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs), and in deployment by the need for high doses. In this study, we leverage the MULTI-specific, multi-Affinity antiBODY (Multabody, MB) platform, derived from the human apoferritin protomer, to drive the multimerization of antibody fragments and generate exceptionally potent and broad SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity for the core of these engineered antibody-like molecules at 2.1 [A] resolution. We demonstrate that neutralization potency improvements of the MB over corresponding IgGs translates into superior in vivo protection: in the SARS-CoV-2 mouse challenge model, comparable in vivo protection was achieved for the MB delivered at 30x lower dose compared to the corresponding IgGs. Furthermore, we show how MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding IgGs lose their ability to neutralize potently. Multiple mAb specificities could also be combined into a single MB molecule to expand the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Dual inhibition of vacuolar ATPase and TMPRSS2 is required for complete blockade of SARS-CoV-2 entry into cells

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using non-specific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

A new high-pressure technique for the measurement of low frequency seismic attenuation using cyclic torsional loading.

We report a new technique for torsional testing of materials under giga-pascal pressures, which uses a shearing module in a large-volume Paris-Edinburgh press in combination with high-resolution fast radiographic x-ray imaging. The measurement of the relative amplitude and phase lag between the cyclic displacement in the sample and a standard material (Al2O3) provides the effective shear modulus and attenuation factor for the sample. The system can operate in the 0.001-0.01 Hz frequency range and up to 5 GPa and 2000 K although high-temperature measurements may be affected by grain growth and plastic strain. Preliminary experimental results on San Carlos olivine are in quantitative agreement with previously reported Q-1 factors at lower pressure. This cyclic torsional loading method opens new directions to quantify the viscoelastic properties of minerals/rocks at seismic frequencies and under pressure-temperature conditions relevant to the Earth's mantle for a better interpretation of seismological data.

Microelectromechanical Transducer to Monitor High-Doses of Nuclear Irradiation.

This paper reports the design, fabrication and measured performance of a passive microelectromechanical transducer for the wireless monitoring of high irradiation doses in nuclear environments. The sensing device is composed of a polymer material (high-density polyethylene) sealed inside a cavity. Subjected to ionizing radiation, this material releases various gases, which increases the pressure inside the cavity and deflects a dielectric membrane. From the measurement of the deflection, the variation of the applied pressure can be estimated, and, in turn, the dose may be determined. The microelectromechanical structure can also be used to study and validate the radiolysis properties of the polymer through its gas emission yield factor. Measurement of the dielectric membrane deflection is performed here to validate on the one hand the required airtightness of the cavity exposed to doses about 4 MGy and on the other hand, the functionality of the fabricated dosimeter for doses up to 80 kGy. The selection of appropriate materials for the microelectromechanical device is discussed, and the outgassing properties of the selected high-density polyethylene are analysed. Moreover, the technological fabrication process of the transducer is detailed.

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

Acoustofluidic rotational tweezing enables high-speed contactless morphological phenotyping of zebrafish larvae.

Modern biomedical research and preclinical pharmaceutical development rely heavily on the phenotyping of small vertebrate models for various diseases prior to human testing. In this article, we demonstrate an acoustofluidic rotational tweezing platform that enables contactless, high-speed, 3D multispectral imaging and digital reconstruction of zebrafish larvae for quantitative phenotypic analysis. The acoustic-induced polarized vortex streaming achieves contactless and rapid (~1 s/rotation) rotation of zebrafish larvae. This enables multispectral imaging of the zebrafish body and internal organs from different viewing perspectives. Moreover, we develop a 3D reconstruction pipeline that yields accurate 3D models based on the multi-view images for quantitative evaluation of basic morphological characteristics and advanced combinations of metrics. With its contactless nature and advantages in speed and automation, our acoustofluidic rotational tweezing system has the potential to be a valuable asset in numerous fields, especially for developmental biology, small molecule screening in biochemistry, and pre-clinical drug development in pharmacology.

Multivalency transforms SARS-CoV-2 antibodies into broad and ultrapotent neutralizers

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus Disease 2019 (COVID-19), has caused a global pandemic. Antibodies are powerful biotherapeutics to fight viral infections; however, discovery of the most potent and broadly acting clones can be lengthy. Here, we used the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 x 10-14 M were achieved as a result of up to 10,000-fold potency enhancements. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and Ig-like in vivo bioavailability. This MULTi-specific, multi-Affinity antiBODY (Multabody; or MB) platform contributes a new class of medical countermeasures against COVID-19 and an efficient approach to rapidly deploy potent and broadly-acting therapeutics against infectious diseases of global health importance. One Sentence Summarymultimerization platform transforms antibodies emerging from discovery screens into potent neutralizers that can overcome SARS-CoV-2 sequence diversity.

Loss-of-function mutations in MRAP2 are pathogenic in hyperphagic obesity with hyperglycemia and hypertension.

The G-protein-coupled receptor accessory protein MRAP2 is implicated in energy control in rodents, notably via the melanocortin-4 receptor1. Although some MRAP2 mutations have been described in people with obesity1-3, their functional consequences on adiposity remain elusive. Using large-scale sequencing of MRAP2 in 9,418 people, we identified 23 rare heterozygous variants associated with increased obesity risk in both adults and children. Functional assessment of each variant shows that loss-of-function MRAP2 variants are pathogenic for monogenic hyperphagic obesity, hyperglycemia and hypertension. This contrasts with other monogenic forms of obesity characterized by excessive hunger, including melanocortin-4 receptor deficiency, that present with low blood pressure and normal glucose tolerance4. The pleiotropic metabolic effect of loss-of-function mutations in MRAP2 might be due to the failure of different MRAP2-regulated G-protein-coupled receptors in various tissues including pancreatic islets.

Contactless, programmable acoustofluidic manipulation of objects on water.

Contact-free manipulation of small objects (e.g., cells, tissues, and droplets) using acoustic waves eliminates physical contact with structures and undesired surface adsorption. Pioneering acoustic-based, contact-free manipulation techniques (e.g., acoustic levitation) enable programmable manipulation but are limited by evaporation, bulky transducers, and inefficient acoustic coupling in air. Herein, we report an acoustofluidic mechanism for the contactless manipulation of small objects on water. A hollow-square-shaped interdigital transducer (IDT) is fabricated on lithium niobate (LiNbO3), immersed in water and used as a sound source to generate acoustic waves and as a micropump to pump fluid in the ±x and ±y orthogonal directions. As a result, objects which float adjacent to the excited IDT can be pushed unidirectionally (horizontally) in ±x and ±y following the directed acoustic wave propagation. A fluidic processor was developed by patterning IDT units in a 6-by-6 array. We demonstrate contactless, programmable manipulation on water of oil droplets and zebrafish larvae. This acoustofluidic-based manipulation opens avenues for the contactless, programmable processing of materials and small biosamples.

Polyketide Synthase Plays a Conserved Role in Otolith Formation.

Otoliths (ear stones) are biomineralized complexes essential for the balancing and hearing function of the inner ears in fish. Their formation is controlled by a genetically programmed biological process that is yet to be defined. We have isolated and characterized a spontaneous genetic mutant zebrafish with a complete absence of otoliths, named no otolith 1 (not1). not1 mutants are unable to develop otoliths during embryonic stages and fail to respond to acoustic stimuli, indicating an inner ear defect. We identified a deleterious mutation (G239R) that altered a highly conserved amino acid residue in the zebrafish ortholog of type I polyketide synthase (pks1) to underlie these phenotypes and showed that expression of the polyketide synthase gene of Japanese medaka fish could rescue the otolith deficiency in not1 mutant zebrafish. Our finding highlights a critical and conserved role of type I polyketide synthase in the initiation of otolith formation. Given the functional homology between otoliths in teleost fish and otoconia in mammals and humans, not1 mutants provide a new animal model for the study of human otoconia-related diseases.

In-Situ Wireless Pressure Measurement Using Zero-Power Packaged Microwave Sensors.

This paper reports the indoor wireless measurement of pressure from zero-power (or passive) microwave (24 GHz) sensors. The sensors are packaged and allow the remote measurement of overpressure up to 2.1 bars. Their design, fabrication process and packaging are detailed. From the measurement of sensor scattering parameters, the outstanding sensitivity of 995 MHz/bar between 0.8 and 2.1 bars was achieved with the full-scale measurement range of 1.33 GHz. Moreover, the 3D radar imagery technique was applied for the remote interrogation of these sensors in electromagnetic reverberant environments. The full-scale dynamic range of 4.9 dB and the sensitivity of 4.9 dB/bar between 0.7 and 1.7 bars were achieved with radar detection in a highly reflective environment. These measurement results demonstrate for the first time the ability of the radar imagery technique to interrogate fully passive pressure sensors in electromagnetic reverberant environments.

A novel NEUROG3 mutation in neonatal diabetes associated with a neuro-intestinal syndrome.

Neonatal diabetes mellitus (NDM) is a rare form of non-autoimmune diabetes usually diagnosed in the first 6 months of life. Various genetic defects have been shown to cause NDM with diverse clinical presentations and variable severity. Among transcriptional factor genes associated with isolated or syndromic NDM, a few cases of homozygous mutations in the NEUROG3 gene have been reported, all mutated patients presenting with congenital malabsorptive diarrhea with or without diabetes at a variable age of onset from early life to childhood. Through a targeted next-generation sequencing assay for monogenic diabetes genes, we aimed to search for pathogenic deleterious mutation in a Turkish patient with NDM, severe malabsorptive diarrhea, neurointestinal dysplasia and other atypical features. In this patient, we identified a novel homozygous nonsense mutation (p.Q4*) in NEUROG3. The same biallelic mutation was found in another affected family member. Of note, the study proband presents with abnormalities of the intrahepatic biliary tract, thyroid gland and central nervous system, which has never been reported before in NEUROG3 mutation carriers. Our findings extend the usually described clinical features associated with NEUROG3 deficiency in humans, and question the extent to which a complete lack of NEUROG3 expression may affect pancreas endocrine function in humans.

[Orthodontics and Alexis Schange in 1841]. / L'orthodontie d'Alexis Schange en 1841.

INTRODUCTION: Alexis Schange was one of the first practitioners able to use accurate casts of the dental arches thanks to the invention of the impression-tray by Delabarre. Consequently, his diagnoses were more precise and his appliances fitted better than those of his predecessors. MATERIALS AND METHOD: The author will first outline the status of Orthodontics prior to Schange and the advent of the first casts, before describing Schange's contribution to Orthodontics. DISCUSSION: In 1841, Schange published his "Handbook of Dental Uprighting", a clear and pedagogical work and undoubtedly the best over the many years when Orthodontics was exclusively European.

[Optimization of the final lingual occlusion: an analysis]. / Optimisation de l'occlusion statique linguale terminale : une analyse.

INTRODUCTION: The objectives of orthodontic treatment are to achieve a functional, aesthetic and sustainable occlusion. However, its analysis is often limited to the study of its buccal side, easy to check in mouth. Yet, the lingual occlusion is also of paramount importance. MATERIAL AND METHOD: After calling to mind the ideal static objectives of treatment, described by some authors and defined by some scientific societies, this article studies the different supports for the analysis of lingual static occlusion and proposes a new protocol for the carving of orthodontic casts. RESULTS: The lingual occlusion end-of-treatment objectives lack details, whether by the scientific societies or literature, although we possess simple ways to study them. DISCUSSION: The lingual part of occlusion is rarely studied in our daily practice, particularly because its analysis cannot be achieved by a direct intraoral examination. However, it is of major importance in the success and stability of our treatments.

A truncating mutation in CEP55 is the likely cause of MARCH, a novel syndrome affecting neuronal mitosis.

BACKGROUND: Hydranencephaly is a congenital anomaly leading to replacement of the cerebral hemispheres with a fluid-filled cyst. The goals of this work are to describe a novel autosomal-recessive syndrome that includes hydranencephaly (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly (MARCH)); to identify its genetic cause(s) and to provide functional insight into pathomechanism. METHODS: We used homozygosity mapping and exome sequencing to identify recessive mutations in a single family with three affected fetuses. Immunohistochemistry, RT-PCR and imaging in cell lines, and zebrafish models, were used to explore the function of the gene and the effect of the mutation. RESULTS: We identified a homozygous nonsense mutation in CEP55 segregating with MARCH. Testing the effect of this allele on patient-derived cells indicated both a reduction of the overall CEP55 message and the production of a message that likely gives rise to a truncated protein. Suppression or ablation of cep55l in zebrafish embryos recapitulated key features of MARCH, most notably renal dysplasia, cerebellar hypoplasia and craniofacial abnormalities. These phenotypes could be rescued by full-length but not truncated human CEP55 message. Finally, we expressed the truncated form of CEP55 in human cells, where we observed a failure of truncated protein to localise to the midbody, leading to abscission failure and multinucleated daughter cells. CONCLUSIONS: CEP55 loss of function mutations likely underlie MARCH, a novel multiple congenital anomaly syndrome. This association expands the involvement of centrosomal proteins in human genetic disorders by highlighting a role in midbody function.

[The birth of Edgewise or the last and best Angle's mechanisms]. / La naissance de l'Edgewise ou le dernier et le meilleur mécanisme d'Angle.

INTRODUCTION: Mechanical forces applied to the teeth constitute a not-insignificant feature of orthodontic treatment. Edgewise, the most commonly used type of mechanics, was introduced 88 years ago and has become a standard worldwide. The invention of Edgewise by E.H. Angle in 1928 is an event in the history of orthodontics which deserves to be recalled. MATERIALS AND METHODS: Starting with the initial search for this method and leading up to a presentation of the technique itself, this paper provides an overview of the history of Angle mechanics. RESULTS: In order to demonstrate the first mechanical system to function in the three planes of space, Angle provided precise diagrams and instructions for use while insisting, on several occasions, that his mechanics was not complicated and that careful study should allow users to overcome any potential difficulties. DISCUSSION: The mechanism as devised by Angle, who died shortly after launching his invention, was only used for a short length of time. However, all subsequently invented therapeutic techniques incorporated the mechanical principles underpinning Edgewise, paying tribute in this way to a great invention.

What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.

[The prehistory of orthodontics]. / La préhistoire de l'orthodontie.

Orthodontics came into being in 1728. Previously, practitioners were at a loss when confronted with crooked teeth. A Latin philosopher had an ingenious flash of orthodontic inspiration. Other authors were content to either extract the malposed teeth or to modify their shape. However, interest in an approach to preventive orthodontics had now begun.