Bacterial Biomarkers of the Oropharyngeal and Oral Cavity during SARS-CoV-2 Infection.

(1) Background: Individuals with COVID-19 display different forms of disease severity and the upper respiratory tract microbiome has been suggested to play a crucial role in the development of its symptoms. (2) Methods: The present study analyzed the microbial profiles of the oral cavity and oropharynx of 182 COVID-19 patients compared to 75 unaffected individuals. The samples were obtained from gargle screening samples. 16S rRNA amplicon sequencing was applied to analyze the samples. (3) Results: The present study shows that SARS-CoV-2 infection induced significant differences in bacterial community assemblages, with Prevotella and Veillonella as biomarkers for positive-tested people and Streptococcus and Actinomyces for negative-tested people. It also suggests a state of dysbiosis on the part of the infected individuals due to significant differences in the bacterial community in favor of a microbiome richer in opportunistic pathogens. (4) Conclusions: SARS-CoV-2 infection induces dysbiosis in the upper respiratory tract. The identification of these opportunistic pathogenic biomarkers could be a new screening and prevention tool for people with prior dysbiosis.

Long-Term Consequences of COVID-19 in Predominantly Immunonaive Patients: A Canadian Prospective Population-Based Study.

Background: Lingering symptoms are frequently reported after acute SARS-CoV-2 infection, a condition known as post-COVID-19 condition (PCC). The duration and severity of PCC in immunologically naïve persons remain unclear. Furthermore, the long-term consequences of these chronic symptoms on work and mental health are poorly documented. Objective: To determine the outcome, the risk factors, and the impact on work and mental health associated with post-COVID-19 symptoms. Methods: This prospective population-based study assessed acute COVID-19 symptoms and their evolution for up to nine months following infection. Individuals aged 18 years and older with COVID-19 in three Canadian regions between 1 November 2020 and 31 May 2021 were recruited. Participants completed a questionnaire that was either administered by trained student investigators over the phone or self-administered online. Results: A total of 1349 participants with a mean age of 46.6 ± 16.0 years completed the questionnaire. Participants were mostly unvaccinated at the time of their COVID-19 episode (86.9%). Six hundred and twenty-two participants (48.0%) exhibited one symptom or more, at least three months post-COVID-19. Among participants with PCC, 23.0% to 37.8% experienced fatigue at the time of survey. Moreover, 6.1% expressed psychological distress. Risk factors for PCC and fatigue included female sex (OR = 1.996), higher number of symptoms (OR = 1.292), higher severity of episode (OR = 3.831), and having a mental health condition prior to the COVID-19 episode (OR = 5.155). Conclusions: In this multicenter cohort study, almost half (47%) of the participants reported persistent symptoms >3 months after acute infection. Baseline risk factors for PCC include female sex, number and severity of symptoms during acute infection, and a previous diagnosis of mental health disorder. Having PCC negatively impacted health-related quality of life and these patients were more likely to exhibit psychological distress, as well as fatigue.

Real-Time Sensing with Multiplexed Optomechanical Resonators.

Nanoelectromechanical resonators have been successfully used for a variety of sensing applications. Their extreme resolution comes from their small size, which strongly limits their capture area. This leads to a long analysis time and the requirement for large sample quantity. Moreover, the efficiency of the electrical transductions commonly used for silicon resonators degrades with increasing frequency, limiting the achievable mechanical bandwidth and throughput. Multiplexing a large number of high-frequency resonators appears to be a solution, but this is complex with electrical transductions. We propose here a route to solve these issues, with a multiplexing scheme for very high-frequency optomechanical resonators. We demonstrate the simultaneous frequency measurement of three silicon microdisks fabricated with a 200 mm wafer large-scale process. The readout architecture is simple and does not degrade the sensing resolutions. This paves the way toward the realization of sensors for multiparametric analysis with an extremely low limit of detection and response time.

Optomechanical mass spectrometry.

Nanomechanical mass spectrometry has proven to be well suited for the analysis of high mass species such as viruses. Still, the use of one-dimensional devices such as vibrating beams forces a trade-off between analysis time and mass resolution. Complex readout schemes are also required to simultaneously monitor multiple resonance modes, which degrades resolution. These issues restrict nanomechanical MS to specific species. We demonstrate here single-particle mass spectrometry with nano-optomechanical resonators fabricated with a Very Large Scale Integration process. The unique motion sensitivity of optomechanics allows designs that are impervious to particle position, stiffness or shape, opening the way to the analysis of large aspect ratio biological objects of great significance such as viruses with a tail or fibrils. Compared to top-down beam resonators with electrical read-out and state-of-the-art mass resolution, we show a three-fold improvement in capture area with no resolution degradation, despite the use of a single resonance mode.

Optomechanical resonating probe for very high frequency sensing of atomic forces.

Atomic force spectroscopy and microscopy are invaluable tools to characterize nanostructures and biological systems. State-of-the-art experiments use resonant driving of mechanical probes, whose frequency reaches MHz in the fastest commercial instruments where cantilevers are driven at nanometer amplitude. Stiffer probes oscillating at tens of picometers provide a better access to short-range interactions, yielding images of molecular bonds, but they are little amenable to high-speed operation. Next-generation investigations demand combining very high frequency (>100 MHz) with deep sub-nanometer oscillation amplitude, in order to access faster (below microsecond) phenomena with molecular resolution. Here we introduce a resonating optomechanical atomic force probe operated fully optically at a frequency of 117 MHz, two decades above cantilevers, with a Brownian motion amplitude four orders below. Based on Silicon-On-Insulator technology, the very high frequency probe demonstrates single-pixel sensing of contact and non-contact interactions with sub-picometer amplitude, breaking open current limitations for faster and finer force spectroscopy.

Enhanced all-optical cavity-tuning using graphene.

All-optical tuning of the resonance of an optical cavity is used to realise optical signal-processing including modulation, switching, and signal-routing. The tuning of optical resonance is dictated by the two primary effects induced by optical absorption: charge-carrier-generation and heat-generation. Since these two effects shift the resonance in opposite directions in a pure silicon-on-insulator (SOI) micro-ring resonator as well as in a graphene-on-SOI system, the efficiency and the dynamic range of all-optical resonance-tuning is limited. In this work, in a graphene-oxide-silicon waveguide system, we demonstrate an exceptional resonance-tuning-efficiency of 300 p m/m W (0.055 π/m W), with a large dynamic range of 1.2 n m (0.22 π) from linear resonance to optical bistability. The dynamics of the resonance-tuning indicates that the superior resonance-tuning is due to large linear-absorption-induced thermo-optic effect. Competing free-carrier dispersion is suppressed as a result of the large separation between graphene and the silicon core. This work reveals new ways to improve the performance of graphene-on-waveguide systems in all-optical cavity-tuning, low-frequency all-optical modulation, and switching.

Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators.

Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100.

Single-particle mass spectrometry with arrays of frequency-addressed nanomechanical resonators.

One of the main challenges to overcome to perform nanomechanical mass spectrometry analysis in a practical time frame stems from the size mismatch between the analyte beam and the small nanomechanical detector area. We report here the demonstration of mass spectrometry with arrays of 20 multiplexed nanomechanical resonators; each resonator is designed with a distinct resonance frequency which becomes its individual address. Mass spectra of metallic aggregates in the MDa range are acquired with more than one order of magnitude improvement in analysis time compared to individual resonators. A 20 NEMS array is probed in 150 ms with the same mass limit of detection as a single resonator. Spectra acquired with a conventional time-of-flight mass spectrometer in the same system show excellent agreement. We also demonstrate how mass spectrometry imaging at the single-particle level becomes possible by mapping a 4-cm-particle beam in the MDa range and above.

First Imported Case of Chikungunya Virus Infection in a Travelling Canadian Returning from the Caribbean.

This is the first Canadian case of Chikungunya virus (CHIKV) infection reported in a traveller returning from the Caribbean. Following multiple mosquito bites in Martinique Island in January 2014, the patient presented with high fever, headaches, arthralgia on both hands and feet, and a rash on the trunk upon his return to Canada. Initial serological testing for dengue virus infection was negative. Support therapy with nonsteroidal anti-inflammatory drugs was administered. The symptoms gradually improved 4 weeks after onset with residual arthralgia and morning joint stiffness. This clinical feature prompted the clinician to request CHIKV virus serology which was found to be positive for the presence of IgM and neutralizing antibodies. In 2014, over four hundred confirmed CHIKV infection cases were diagnosed in Canadian travellers returning from the Caribbean and Central America. Clinical suspicion of CHIKV or dengue virus infections should be considered in febrile patients with arthralgia returning from the recently CHIKV endemic countries of the Americas.

Frequency fluctuations in silicon nanoresonators.

Frequency stability is key to the performance of nanoresonators. This stability is thought to reach a limit with the resonator's ability to resolve thermally induced vibrations. Although measurements and predictions of resonator stability usually disregard fluctuations in the mechanical frequency response, these fluctuations have recently attracted considerable theoretical interest. However, their existence is very difficult to demonstrate experimentally. Here, through a literature review, we show that all studies of frequency stability report values several orders of magnitude larger than the limit imposed by thermomechanical noise. We studied a monocrystalline silicon nanoresonator at room temperature and found a similar discrepancy. We propose a new method to show that this was due to the presence of frequency fluctuations, of unexpected level. The fluctuations were not due to the instrumentation system, or to any other of the known sources investigated. These results challenge our current understanding of frequency fluctuations and call for a change in practices.

Evaluation of alginate purification methods: effect on polyphenol, endotoxin, and protein contamination.

Alginate, a polysaccharide extracted from brown seaweed, is widely used for the microencapsulation of islets of Langerhans, allowing their transplantation without immunosuppression. This natural polymer is known to be largely contaminated. The implantation of islets encapsulated using unpurified alginate leads to the development of fibrotic cell overgrowth around the microcapsules and normalization of the blood glucose is restricted to a very short period if it is achieved at all. Several research groups have developed their own purification method and obtained relatively good results. No comparative evaluation of the efficiencies of these methods has been published. We conducted an evaluative study of five different alginate preparations: a pharmaceutical-grade alginate in its raw state, the same alginate after purification according to three different published methods, and a commercially available purified alginate. The results showed that all purification methods reduced the amounts of known contaminants, that is, polyphenols, endotoxins, and proteins, although with varying efficiencies. Increased viscosity of alginate solutions was observed after purification of the alginates. Despite a general efficiency in decreasing contamination levels, all of the purified alginates contained relatively high residual amounts of protein contaminants. Because proteins may be immunogenic, these residual proteins may have a role in persisting microcapsule immunogenicity.

Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers.

Microencapsulation in semi-permeable membranes protects transplanted cells against immune destruction. Microcapsule strength is critical. We describe a method to microencapsulate living cells in alginate-poly-L-lysine (PLL)-alginate membranes with covalent links between adjacent layers of microcapsule membranes, while preserving the desired membrane molecular weight cut-off (MWCO) and microencapsulated cell viability. A heterobifunctional photoactivatable cross-linker, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) was used. The N-hydroxysuccinimide ester group of ANB-NOS was covalently linked to PLL. Islets of Langerhans were immobilized in alginate beads, incubated in PLL-ANB-NOS and again in alginate. Upon illumination with UVA, covalent links were created between the phenyl azide residue of ANB-NOS and alginate from both the core bead and the outer coating. Covalently linked microcapsules remained intact after 3 years in a strong alkaline buffer (pH 12), whereas standard microcapsules disappeared within 45 s in the same solution. A standardized mechanical stress broke 22-fold more standard than covalently linked microcapsules. The MWCO and microencapsulated cell viability were similar with standard and covalently linked microcapsules. These microcapsules, extremely resistant to chemical and mechanical stresses, will be useful in numerous applications.

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells.

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.

Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38.

Activation of the tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) by VEGF leads to the activation of stress-activated protein kinase (SAPK)2/p38 and then to actin polymerization and reorganization into stress fibers in endothelial cells. In turn, this triggers endothelial cell migration. Yet, nothing is known about the molecular mechanisms that couple VEGFR2 to SAPK2/p38. Here, we found that VEGF increased by twofold the activity of the small GTPase Cdc42 and that the expression of two different constitutively active forms of Cdc42 (Cdc42 V12 and Cdc42 L61) led to a marked increase in the formation of stress fibers that was sensitive to SAPK2/p38 inhibition by SB203580. Moreover, the expression of a dominant-negative form of Cdc42 (Cdc42 N17) inhibited the activation of SAPK2/p38 and of its direct target MAP kinase-activated protein kinase 2. These results indicate that Cdc42 is upstream of SAPK2/p38 in response to the activation of VEGFR2 by VEGF. In contrast, we found that neither RhoA nor Rac was involved in the SAPK2/p38-mediated actin reorganization induced by VEGF. Using a site-specific mutant of the major autophosphorylation site Y1214 on VEGFR2, we found that the mutant Y1214F inhibited the activation of both Cdc42 and SAPK2/p38 in response to VEGF. We conclude that phosphorylation of Y1214 on VEGFR2 is required to trigger the sequential activation of Cdc42 and SAPK2/p38 and to drive the SAPK2/p38-mediated actin remodeling in stress fibers in endothelial cells exposed to VEGF.