Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Evidence that Maackia amurensis seed lectin (MASL) exerts pleiotropic actions on oral squamous cells with potential to inhibit SARS-CoV-2 infection and COVID-19 disease progression.

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 125 million confirmed COVID-19 cases that have caused over 2.7 million deaths worldwide as of March 2021. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface "spike" protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. In addition, we report that MASL also inhibits SARS-CoV-2 infection of kidney epithelial cells in culture. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream RT-PCR testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log 10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on vero-E6 cell lines was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, preservation, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

A Semisynthetic Kanglemycin Shows In Vivo Efficacy against High-Burden Rifampicin Resistant Pathogens.

Semisynthetic rifamycin derivatives such as rifampicin (Rif) are first line treatments for tuberculosis and other bacterial infections. Historically, synthetic modifications made to the C-3/C-4 region of the rifamycin naphthalene core, like those seen in Rif, have yielded the biggest improvements in pharmacological properties. However, modifications found in natural product rifamycin congeners occur at other positions in the structure. The kanglemycins (Kangs) are a family of rifamycin congeners with a unique collection of natural modifications including a dimethylsuccinic acid appended to their polyketide backbone. These modifications confer activity against the single most common clinically relevant Rif resistance (RifR) mutation in the antibiotic's target, the bacterial RNA polymerase (RNAP). Here we evaluate the in vivo efficacy of Kang A, the parent compound in the Kang family, in a murine model of bacterial peritonitis/sepsis. We then set out to improve its potency by combining its natural tailoring modifications with semisynthetic derivatizations at either its acid moiety or in the C-3/C-4 region. A collection of C-3/C-4 benzoxazino Kang derivatives exhibit improved activity against wild-type bacteria, and acquire activity against the second most common clinically relevant RifR mutation. The semisynthetic analogue 3'-hydroxy-5'-[4-isobutyl-1-piperazinyl] benzoxazino Kang A (Kang KZ) protected mice against infection with either Rif sensitive MRSA or a highly virulent RifRStaphylococcus  aureus strain in a neutropenic peritonitis/sepsis model and led to reduced bacterial burdens. The compounds generated in this study may represent promising candidates for treating RifR infections.

In Vitro Comparative Evaluation of Frictional Resistance of Connecticut New Arch Wires, Stainless Steel and Titanium Molybdenum Alloy Archwires Against Different Brackets.

Background and objectives Friction between the bracket and archwire during sliding mechanics is of great concern in orthodontics, as it reduces the effectiveness of the orthodontic appliance and slows down tooth movement. The aim of this study was to evaluate frictional resistance of stainless steel (SS), titanium molybdenum alloy (TMA), and Connecticut new arch (CNA) wires against SS and ceramic brackets. The surface textures of the brackets and wires were also evaluated by scanning electron microscopy (SEM) before and after testing. Method A total of 180 premolar brackets of SS (ORMCO Corp., Orange, CA) and 180 ceramic (3M Unitek, Maplewood, MN) with a 0.022-inch slot and 180 SS, TMA, and CNA wires of 0.017 x 0.025 inches and 0.019 x 0.025 inches were tested. The SS brackets and ceramic brackets were bonded onto the SS bar with cyanoacrylate adhesive with the help of a jig. The wire assembly was vertically mounted and clamped to the jaws of the universal testing machine with 10 N load cell, and friction was measured along with other readings. The surface roughness of brackets and wires were examined using SEM in 200 X magnification before and after testing. Results TMA wire showed the greatest frictional force compared to SS and CNA wire. The frictional force was greater in the 0.019 x 0.025-inch wire compared to the 0.017 x 0.025-inch wires. The highest frictional force was noted in the SS bracket and 0.019 x 0.02-inch TMA wire combination. A statistically significant difference was not seen between the SS bracket and 0.019 x 0.025-inch SS wire and the 0.019 x 0.025-inch CNA wire combinations. SEM showed that the TMA archwire had the roughest surface area compared to SS and CNA wires, and the ceramic bracket had more surface roughness than the SS bracket. Conclusion CNA wire demonstrated frictional resistance similar to the SS wire. CNA wire can be used instead of TMA wire because of its better range of action, high spring back, and less frictional resistance for space closure in sliding mechanics.

Effect of Bracket Slot and Archwire Dimension on Posterior Tooth Movement in Sliding Mechanics: A Three-dimensional Finite Element Analysis.

Introduction Space closure by molar protraction has always been a challenge in orthodontic treatment due to larger root surface area which requires additional anchorage. Ideally, there should be little or no tipping. However, the protraction forces, being occlusal and buccal to the centre of resistance (CR) of the tooth, cause tipping and rotations. Aim The aim of the study was to assess the effect of bracket slot and archwire dimensions on posterior tooth movement during space closure in sliding mechanics and evaluate the length of power arm to bring about translatory movement of teeth using three-dimensional finite element analysis. Materials and methods A model of the maxillary teeth was created and converted to a finite element format through a meshing software, Hypermesh. Two three-dimensional models, each with a combination of 0.017"× 0.022" archwire in 0.018" slot (model 1) and 0.019"×0.025" archwire in 0.022" slot (model 2), were generated. Power arms of different lengths were attached to the first molar. Miniscrew was inserted between the canine and first premolar. Results In model one, the power arm of 10-mm height provided controlled tooth movement than the one with 6 mm height, and in model two, power arms of both 6-mm and 10-mm height produced controlled tooth movement. Conclusions As the force was raised apically from the slot, more translation was observed. Power arm of 6-mm height can be used due to anatomic limitation of the vestibule.