3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials.

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pKa of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

Molecular dynamics simulations of the spike trimeric ectodomain of the SARS-CoV-2 Omicron variant: structural relationships with infectivity, evasion to immune system and transmissibility.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron is currently the most prevalent SARS-CoV-2 variant worldwide. Herein, we calculated molecular dynamics simulations of the trimeric spikeWT and SpikeBA.1 for 300 ns. Our results show that SpikeBA.1 has more conformational flexibility than SpikeWT. Our principal component analysis (PCA) allowed us to observe a broader spectrum of different conformations for SpikeBA.1, mainly at N-terminal domain (NTD) and receptor-binding domain (RBD). Such increased flexibility could contribute to decreased neutralizing antibody recognition of this variant. Our molecular dynamics data show that the RBDBA.1 easily visits an up-conformational state and the prevalent D614G mutation is pivotal to explain molecular dynamics results for this variant because to lost hydrogen bonding interactions between the residue pairs K854SC/D614SC, Y837MC/D614MC, K835SC/D614SC, T859SC/D614SC. In addition, SpikeBA.1 residues near the furin cleavage site are more flexible than in SpikeWT, probably due to P681H and D614G substitutions. Finally, dynamical cross-correlation matrix (DCCM) analysis reveals that D614G and P681H may allosterically affect the cleavage site S1/S2. Conversely, S2' site may be influenced by residues located between NTD and RBD of a neighboring protomer of the SpikeWT. Such communication may be lost in SpikeBA.1, explaining the changes of the cell tropism in the viral infection. In addition, the movements of the NTDWT and NTDBA.1 may modulate the RBD conformation through allosteric effects. Taken together, our results explain how the structural aspects may explain the observed gains in infectivity, immune system evasion and transmissibility of the Omicron variant.Communicated by Ramaswamy H. Sarma.

Molecular Dynamics Analysis of Fast-Spreading Severe Acute Respiratory Syndrome Coronavirus 2 Variants and Their Effects on the Interaction with Human Angiotensin-Converting Enzyme 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving with mutations in the spike protein, especially in the receptor-binding domain (RBD). The failure of public health measures in some countries to contain the spread of the disease has given rise to novel viral variants with increased transmissibility. However, key questions about how quickly the variants can spread remain unclear. Herein, we performed a structural investigation using molecular dynamics simulations and determined dissociation constant (K D) values using surface plasmon resonance assays of three fast-spreading SARS-CoV-2 variants, alpha, beta, and gamma, as well as genetic factors in host cells that may be related to the viral infection. Our results suggest that the SARS-CoV-2 variants facilitate their entry into the host cell by moderately increased binding affinities to the human ACE2 receptor, different torsions in hACE2 mediated by RBD variants, and an increased spike exposure time to proteolytic enzymes. We also found that other host cell aspects, such as gene and isoform expression of key genes for the infection (ACE2, FURIN, and TMPRSS2), may have few contributions to the SARS-CoV-2 variant infectivity. In summary, we concluded that a combination of viral and host cell factors allows SARS-CoV-2 variants to increase their abilities to spread faster than the wild type.

Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern: A Perspective for Emerging More Transmissible and Vaccine-Resistant Strains.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) are constantly threatening global public health. With no end date, the pandemic persists with the emergence of novel variants that threaten the effectiveness of diagnostic tests and vaccines. Mutations in the Spike surface protein of the virus are regularly observed in the new variants, potentializing the emergence of novel viruses with different tropism from the current ones, which may change the severity and symptoms of the disease. Growing evidence has shown that mutations are being selected in favor of variants that are more capable of evading the action of neutralizing antibodies. In this context, the most important factor guiding the evolution of SARS-CoV-2 is its interaction with the host's immune system. Thus, as current vaccines cannot block the transmission of the virus, measures complementary to vaccination, such as the use of masks, hand hygiene, and keeping environments ventilated remain essential to delay the emergence of new variants. Importantly, in addition to the involvement of the immune system in the evolution of the virus, we highlight several chemical parameters that influence the molecular interactions between viruses and host cells during invasion and are also critical tools making novel variants more transmissible. In this review, we dissect the impacts of the Spike mutations on biological parameters such as (1) the increase in Spike binding affinity to hACE2; (2) bound time for the receptor to be cleaved by the proteases; (3) how mutations associate with the increase in RBD up-conformation state in the Spike ectodomain; (4) expansion of uncleaved Spike protein in the virion particles; (5) increment in Spike concentration per virion particles; and (6) evasion of the immune system. These factors play key roles in the fast spreading of SARS-CoV-2 variants of concern, including the Omicron.

Potential Well in Poincaré Recurrence.

From a physical/dynamical system perspective, the potential well represents the proportional mass of points that escape the neighbourhood of a given point. In the last 20 years, several works have shown the importance of this quantity to obtain precise approximations for several recurrence time distributions in mixing stochastic processes and dynamical systems. Besides providing a review of the different scaling factors used in the literature in recurrence times, the present work contributes two new results: (1) For ϕ-mixing and ψ-mixing processes, we give a new exponential approximation for hitting and return times using the potential well as the scaling parameter. The error terms are explicit and sharp. (2) We analyse the uniform positivity of the potential well. Our results apply to processes on countable alphabets and do not assume a complete grammar.

Femtosecond laser micromachining of an optofluidics-based monolithic whispering-gallery mode resonator coupled to a suspended waveguide.

A monolithic lab-on-a-chip fabricated by femtosecond laser micromachining capable of label-free biosensing is reported. The device is entirely made of fused silica, and consists of a microdisk resonator integrated inside a microfluidic channel. Whispering gallery modes are excited by the evanescent field of a circular suspended waveguide, also incorporated within the channel. Thermal annealing is performed to decrease the surface roughness of the microstructures to a nanometric scale, thereby reducing intrinsic losses and maximizing the Q-factor. Further, thermally-induced morphing is used to position, with submicrometric precision, the suspended waveguide tangent to the microresonator to enhance the spatial overlap between the evanescent field of both optical modes. With this fabrication method and geometry, the alignment between the waveguide and the resonator is robust and guaranteed at all instances. A maximum sensitivity of 121.5 nm/RIU was obtained at a refractive index of 1.363, whereas near the refractive index range of water-based solutions the sensitivity is 40 nm/RIU. A high Q-factor of 105 is kept throughout the entire measurement range.

Temperature Stability and Spectral Tuning of Long Period Fiber Gratings Fabricated by Femtosecond Laser Direct Writing.

Long period fiber gratings (LPFGs) were fabricated in a standard single mode fiber (SMF-28e) through femtosecond (fs) laser direct writing. LPFGs with longer and shorter periods were fabricated, which allows coupling from the fundamental core mode to lower and higher order asymmetric cladding modes (LP1,6 and LP1,12, respectively). For the grating periods of 182.7 and 192.5 µm, it was verified that the LP1,12 mode exhibits a TAP at approximately 1380 and 1448 nm in air and water, respectively. Characterization of the LPFGs subjected to high-temperature thermal treatment was accomplished. Fine-tuning of the resonance band's position and thermal stability up to 600 °C was shown. The temperature sensitivity was characterized for the gratings with different periods and for different temperature ranges. A maximum sensitivity of -180.73, and 179.29 pm/°C was obtained for the two resonances of the 182.7 µm TAP LPFG, in the range between 250 and 600 °C.

Influence of exposure time to saliva and antioxidant treatment on bond strength to enamel after tooth bleaching: an in situ study

Objectives: This study evaluated the influence of different exposure times to saliva in situ in comparison with an antioxidant treatment on composite resin bond strength to human enamel restored after tooth bleaching. Material and Methods: Forty human teeth specimens measuring 5x5 mm were prepared and randomly allocated into 5 groups with 8 specimens each: Gct (control group, restored on unbleached enamel); Gbl (restored immediately after bleaching); Gsa (bleached, treated with 10% sodium ascorbate gel for 60 min and restored); G7d (bleached, exposed to saliva in situ for 7 days and restored); and G14d (bleached, exposed to saliva in situ for 14 days and restored). Restored samples were cut into 0.8 mm2 sticks that were tested in microtensile. Specimens were microscopically analyzed and failure modes were classified as adhesive, cohesive, or mixed. Pretest and cohesive failures were not considered in the statistical analysis, which was performed with one-way ANOVA and Tukey's post-hoc test (α=0.05), with the dental specimen considered as the experimental unit. Results: Mean bond strength results found for Gbl in comparison with Gct indicated that bleaching significantly reduced enamel adhesiveness (P<0.01). However, no statistically significant differences were found between Gct, Gsa and G7d (P>0.05). Bond strength found for G14d was significantly higher than for Gsa (P<0.01). Fractures modes were predominantly of a mixed type. Conclusions: Bonding strength to bleached enamel was immediately restored with the application of sodium ascorbate and exposure to human saliva in situ for at least 7 days. Best results were obtained with exposure to human saliva in situ for 14 days. Treatment with sodium ascorbate gel for 60 min may be recommended in cases patients cannot wait for at least 7 days for adhesive techniques to be performed. .

Influence of exposure time to saliva and antioxidant treatment on bond strength to enamel after tooth bleaching: an in situ study.

OBJECTIVES: This study evaluated the influence of different exposure times to saliva in situ in comparison with an antioxidant treatment on composite resin bond strength to human enamel restored after tooth bleaching. MATERIAL AND METHODS: Forty human teeth specimens measuring 5x5 mm were prepared and randomly allocated into 5 groups with 8 specimens each: Gct (control group, restored on unbleached enamel); Gbl (restored immediately after bleaching); Gsa (bleached, treated with 10% sodium ascorbate gel for 60 min and restored); G7d (bleached, exposed to saliva in situ for 7 days and restored); and G14d (bleached, exposed to saliva in situ for 14 days and restored). Restored samples were cut into 0.8 mm2 sticks that were tested in microtensile. Specimens were microscopically analyzed and failure modes were classified as adhesive, cohesive, or mixed. Pretest and cohesive failures were not considered in the statistical analysis, which was performed with one-way ANOVA and Tukey's post-hoc test (α=0.05), with the dental specimen considered as the experimental unit. RESULTS: Mean bond strength results found for Gbl in comparison with Gct indicated that bleaching significantly reduced enamel adhesiveness (P<0.01). However, no statistically significant differences were found between Gct, Gsa and G7d (P>0.05). Bond strength found for G14d was significantly higher than for Gsa (P<0.01). Fractures modes were predominantly of a mixed type. CONCLUSIONS: Bonding strength to bleached enamel was immediately restored with the application of sodium ascorbate and exposure to human saliva in situ for at least 7 days. Best results were obtained with exposure to human saliva in situ for 14 days. Treatment with sodium ascorbate gel for 60 min may be recommended in cases patients cannot wait for at least 7 days for adhesive techniques to be performed.

A new optics-based gastroesophageal reflux probe.

This work was carried out with the purpose of developing a new method of gastroesophageal reflux (GOR) detection. It is based on the emission of a light beam to the inferior part of the oesophagus and on the detection and analysis of the corresponding reflected light intensity. The optical properties of the oesophageal lumen are then used to identify the GOR episodes, solving, in this way, the existing drawback of using pH probes that fail in the cases where GOR episodes are neutral or short duration acid. The necessary instrumentation for the application of this new technique, including the probe itself and its associated optics, was developed. The result is a low-cost portable instrument, based on the Microchip microcontroller PIC16C77, with enough flexibility to be used in other biomedical applications. This new simple apparatus only needs an adequate light source - diode laser - and an adequate photosensor - photodiode - to make the interface to the probe that guides the light to and from oesophagus. Our results show the capability of this new technique to make the identification of GOR episodes.