Thermal stability of diagnostic targets plasmodium falciparum histidine rich protein II and plasmodium lactate dehydrogenase in rapid detection.

During the COVID-19 epidemic, blood samples are usually processed at 56 to attenuate the virus before pathogen detection. 71 blood samples of malaria patients reported by Shanghai Center for Disease Control and Prevention in 2017-2019 were collected, including 38 with Plasmodium falciparum infection, 8 P. malariae, 11 P. ovale and 14 P. vivax. The effect of inactivation on the thermal stability of P. falciparum histidine rich protein II (PfHRPII) and Plasmodium lactate dehydrogenase (pLDH) in blood samples was assessed before and after incubation at 56 for 30 min using the rapid diagnostic test (RDT) kit. The results showed that among the 38 P. falciparum T1-positive (PfHRPII) blood samples before heat treatment, 35 samples remained to be T1-positive (92.11%, 35/38, chi2=3.123, P>0.05) after heat treatment;while 54 blood samples (26 P. falciparum, 6 P. vivax, 10 P. ovale and 12 P. vivax) that were T2-positive (pLDH) before heat treatment turned to be T2-negative (positive rate 0, 0/54, chi2=87.755, P<0.01) after heat treatment. It was demonstrated that PfHRPII is stable during incubation at 56 for 30 min, while pLDH is unstable and degraded or inactivated during the heating. Therefore, the detection results of P. falciparum will not be affected by RDT, but diagnosis of the parasites other than P. falciparum in blood samples may be missed.Copyright © 2021, National Institute of Parasitic Diseases. All rights reserved.

Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors.

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MProWT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is ∼400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (Tm), respectively, relative to MProWT. The H41A mutation results in a 20-fold increase in the dissociation constant (Kd), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and Tm, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. Structures of the four inhibitor complexes with MPro1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MProWT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MProWT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MProC145A also results in a significant, but smaller, increase in Kd and decrease in ΔG and Tm, relative to NMV.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

ThermalProGAN: A sequence-based thermally stable protein generator trained using unpaired data.

MOTIVATION: The synthesis of proteins with novel desired properties is challenging but sought after by the industry and academia. The dominating approach is based on trial-and-error inducing point mutations, assisted by structural information or predictive models built with paired data that are difficult to collect. This study proposes a sequence-based unpaired-sample of novel protein inventor (SUNI) to build ThermalProGAN for generating thermally stable proteins based on sequence information. RESULTS: The ThermalProGAN can strongly mutate the input sequence with a median number of 32 residues. A known normal protein, 1RG0, was used to generate a thermally stable form by mutating 51 residues. After superimposing the two structures, high similarity is shown, indicating that the basic function would be conserved. Eighty four molecular dynamics simulation results of 1RG0 and the COVID-19 vaccine candidates with a total simulation time of 840[Formula: see text]ns indicate that the thermal stability increased. CONCLUSION: This proof of concept demonstrated that transfer of a desired protein property from one set of proteins is feasible. Availability and implementation: The source code of ThermalProGAN can be freely accessed at https://github.com/markliou/ThermalProGAN/ with an MIT license. The website is https://thermalprogan.markliou.tw:433. Supplementary information: Supplementary data are available on Github.

Assessing the thermal stability of toolmarks casting materials through optical comparison microscopy, virtual comparison microscopy and quantitative similarity scores.

Toolmarks, particularly those found on bulky, inaccessible or immovable items, can be recovered by casting. To allow for subsequent comparative examinations, the casting material, typically polysiloxanes or silicones, must be able to capture and preserve fine details within a toolmark accurately. To study the stability of such details after exposure to heat, toolmark casts were heated at either 60 °C for 2 h, or 90 °C for 1 h. These casts were subsequently compared to casts that had not been exposed to heat, using traditional optical comparison microscopy, as well as virtual comparison microscopy. Digitised toolmark signatures were also extracted from the casts and compared pairwise to obtain quantitative similarity scores based on cross-correlation, consecutive matching striae and Mann-Whitney U-statistic. Our results show that the fine surface details captured on all four commercial toolmark casting materials tested herein remained stable after exposure to heat. This study shows that the above heating protocols are viable viral inactivation methods for toolmark casts that are potentially contaminated with human coronaviruses, such as SARS-CoV-2. Our findings also apply to other scenarios, such as for casts that were left in a vehicle parked under the sun.

Amino‐terminated hyperbranched polymer functionalized graphene oxide with in situ trapped silver nanoparticles for high‐performance antibacterial nonwoven fabric

Antibacterial fabric with high thermal stability and mechanical strength is important for personalized protection, especially under the background of coronavirus pandemic (COVID‐19). This paper presents a facile approach toward high‐efficient antibacterial polypropylene spunbonded nonwoven fabrics (SNFs), which are decorated by a composite of graphene oxide embedded with silver nanoparticles (AgNPs/GO) through dip‐coating and in situ reduction effect of pre‐introduced amino‐terminated hyperbranched polymer (HBP‐NH2). Typically, HBP‐NH2 was grafted onto the GO nanosheets, then silver ions were trapped and self‐reduced by the HBP‐NH2 to generate silver nanoparticles decorated GO. The produced AgNPs are uniformly dispersed on the GO with a size of 13 nm. As an antibacterial coating, the Ag/GO composite could tightly wrap the SNFs fibers through the dip‐padding method, capable of enhancing the thermal stability and mechanical property of SNFs. The treated SNFs exhibited excellent antibacterial activities (~99.9%) against both Echerisia coli and Staphylococcus aureus, promising important potential for biomedical and personal protection applications.

A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529).

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have risen to dominance, which contains far more mutations in the spike protein in comparison to previously reported variants, compromising the efficacy of most existing vaccines or therapeutic monoclonal antibodies. Nanobody screened from high-throughput naïve libraries is a potential candidate for developing preventive and therapeutic antibodies. Methods: Four nanobodies specific to the SARS-CoV-2 wild-type receptor-binding domain (RBD) were screened from a naïve phage display library. Their affinity and neutralizing activity were evaluated by surface plasmon resonance assays, surrogate virus neutralization tests, and pseudovirus neutralization assays. Preliminary identification of the binding epitopes of nanobodies by peptide-based ELISA and competition assay. Then four multivalent nanobodies were engineered by attaching the monovalent nanobodies to an antibody-binding nanoplatform constructed based on the lumazine synthase protein cage nanoparticles isolated from the Aquifex aeolicus (AaLS). Finally, the differences in potency between the monovalent and multivalent nanobodies were compared using the same methods. Results: Three of the four specific nanobodies could maintain substantial inhibitory activity against the Omicron (B.1.1.529), of them, B-B2 had the best neutralizing activity against the Omicron (B.1.1.529) pseudovirus (IC50 = 1.658 µg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC50 = 0.653 µg/mL). The results of peptide-based ELISA indicated that LS-B-B2 might react with the linear epitopes in the SARS-CoV-2 RBD conserved regions, which would clarify the mechanisms for the maintenance of potent neutralization of Omicron (B.1.1.529) preliminary. Conclusion: Our study indicated that the AaLS could be used as an antibody-binding nanoplatform to present nanobodies on its surface and improve the potency of nanobodies. The multivalent nanobody LS-B-B2 may serve as a potential agent for the neutralization of SARS-CoV-2 variants.

Research progress on adenovirus vector vaccines

Adenovirus（Ad） vectors have been widely used as gene delivery vehicles in gene therapy studies since Ad does not integrate into the host genome, thus the risk of insertion mutation is very low.Ad vectors induce immune responses and have relatively high thermal stability, which make them potential vaccine vectors.The outbreak of coronavirus disease 2019（COVID-19） has drawn more attention to the application of Ad vector vaccines.Vaccination is still the most economical and effective means to prevent and control infectious diseases, including COVID-19, and a variety of Ad vector vaccines have been developed.In this review, we describe the basic characteristics, immune mechanism, clinical application and research progress of Ad vectors.

Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Development of a thermostable SARS-CoV-2 variant-based bivalent protein vaccine with cross-neutralizing potency against Omicron subvariants.

SARS-CoV-2 variants have posed significant challenges to the hopes of using ancestral strain-based vaccines to address the risk of breakthrough infection by variants. We designed and developed a bivalent vaccine based on SARS-CoV-2 Alpha and Beta variants (named SCTV01C). SCTV01C antigens were stable at 25 ^oC for at least 6 months. In the presence of a squalene-based oil-in-water adjuvant SCT-VA02B, SCTV01C showed significant protection efficacy against antigen-matched Beta variant, with favorable safety profiles in rodents. Notably, SCTV01C exhibited cross-neutralization capacity against Omicron subvariants (BA.1, BA.1.1, BA.2, BA.3, and BA.4/5) in mice, superior to a WT (D614G)-based vaccine, which reinforced our previously published findings that SCTV01C exhibited broad-spectrum neutralizing potencies against over a dozen pre-Omicron variants and the Omicron BA.1 variant. In summary, variant-based multivalent protein vaccine could be a platform approach to address the challenging issues of emerging variants, vaccine hesitancy and the needs of affordable and thermal stable vaccines.

Half face mask with regulation thermal stability system for continuous air flow: Stabimask

This article presents the design and prototyping of an electronic mask as personal protective equipment for the virus pandemic known as COVID - 19. Needs were identified such as: tightness and comfort. Requirements for use for long periods of time;this was considered for the design, also it has an adjustable ventilation system. The mask was simulated and validated with Solidworks Flow Simulation software, in addition a PID control model was implemented, thereby, it was shown that enough flow is generated to vary the temperature in a range of 20 to 37.2 ° C inside the mask. The design considers an outlet duct and an inlet duct with filters that prevent the entry of polluting particles, providing adequate protection. The prototype was made by 3D printing, And the thermal stability was achieved with the implementation of the temperature regulation system. The results obtained were validated, and they allow to future research to provide greater efficiency to masks. © 2022 ACM.

Development of COVID-19 Detection Using SPR Sensors: A Preliminary Result

Coronavirus disease or COVID-19 is a virus from the Coronaviridae family that has caused pandemics throughout the world since the end of 2019. The virus spreads ten times faster through human interaction than SARS-CoV. The RNA sequence of COVID-19 has a 79.5% similarity with SARS-CoV. Fast and specific detection of COVID-19 is needed so that patient detection can be done quickly and accurately. One method that can be developed as a COVID-19 biosensor is aptamers-based biosensors. The aptamer is an artificial oligo nucleic acid that can specifically bind to target molecules. The aptamer is easily and chemically modifiable for increasing stability and reducing toxicity. It shows a comparable affinity for the target virus and better thermal stability than monoclonal antibodies. This advantage makes aptamer a promising candidate in diagnostic and detection applications. The goal of this research is to use an RNA aptamer as the specific recognition element in a portable surface plasmon resonance (SPR) biosensor for the detection of COVID-19 in humans. An aptamer RNA 1 COVID-19 was designed using the COVID-19 sequence from GISAID using the in silico method. End of 3’ aptamer RNA 1 was modified with dithiol. And Then, the aptamer was immobilized on the gold nanoparticle sensor surface via Cysteine-dithiol binding. The RNA solution, that had been extracted from swab samples, was diluted ten times before being used as a sample. The immobilized aptamer RNA 1 captured COVID-19 in RNA solution, causing an increase in refraction index (r.u). An aptamer RNA 1 was found to bind RNA virus of COVID-19 where the positive sample of COVID-19 has refraction index (r.u) between 3 r.u – 10 r.u for various Ct values.

NANOBIOPHYSICS OF NEW TYPE CORONAVIRUS

The Covid-19 pandemic has swept across the world, causing a never seen burden on our health care systems and challenging biomedical research to give appropriate answers to the epidemic. Modern, one-particle biophysical methods ensure special insight to the characteristics of the cause of the epidemic, the SARS-CoV-2. The virus carries a crown-like layer of spike proteins, which plays a fundamental role in the process of infection. The topography structure and mechanical characteristics of native virions have been determined by atomic force microscopy. Spike proteins form a dynamic surface due to their flexibility and motility. Virions are surprisingly resistant to mechanical compression, and their structure is able to recover after mechanical perturbation. The global structure of the virus is resistant to heat effect, but spike proteins dissociate from the surface with higher temperatures. The mechanical and dynamic characteristics of SARS-CoV-2 contribute to its virulence. The applied one-particle biophysical methods play an important role in understanding and fighting with the more common virus infections. © 2022 Literatura Medica Publishing House. All rights reserved.

Thermal stability of diagnostic targets plasmodium falciparum histidine rich protein Ⅱ and plasmodium lactate dehydrogenase in rapid detection

During the COVID-19 epidemic, blood samples are usually processed at 56 ℃ to attenuate the virus before pathogen detection. 71 blood samples of malaria patients reported by Shanghai Center for Disease Control and Prevention in 2017-2019 were collected, including 38 with Plasmodium falciparum infection, 8 P. malariae, 11 P. ovale and 14 P. vivax. The effect of inactivation on the thermal stability of P. falciparum histidine rich protein Ⅱ (PfHRPⅡ) and Plasmodium lactate dehydrogenase (pLDH) in blood samples was assessed before and after incubation at 56 ℃ for 30 min using the rapid diagnostic test (RDT) kit. The results showed that among the 38 P. falciparum T1-positive (PfHRPⅡ) blood samples before heat treatment, 35 samples remained to be T1-positive (92.11%, 35/38, χ2=3.123, P>0.05) after heat treatment;while 54 blood samples (26 P. falciparum, 6 P. vivax, 10 P. ovale and 12 P. vivax) that were T2-positive (pLDH) before heat treatment turned to be T2-negative (positive rate 0, 0/54, χ2=87.755, P<0.01) after heat treatment. It was demonstrated that PfHRPⅡ is stable during incubation at 56 ℃ for 30 min, while pLDH is unstable and degraded or inactivated during the heating. Therefore, the detection results of P. falciparum will not be affected by RDT, but diagnosis of the parasites other than P. falciparum in blood samples may be missed. © 2021, National Institute of Parasitic Diseases. All rights reserved.

Experimental investigations into abrasive flow machining (AFM) of 3D printed ABS and PLA parts

PurposeThe surface roughness of additively manufactured parts is usually found to be high. This limits their use in industrial and biomedical applications. Therefore, these parts required post-processing to improve their surface quality. The purpose of this study is to finish three-dimensional (3D) printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) parts using abrasive flow machining (AFM).Design/methodology/approachA hydrogel-based abrasive media has been developed to finish 3D printed parts. The developed abrasive media has been characterized for its rheology and thermal stability using sweep tests, thermogravimetric analysis (TGA) and differential thermal analysis (DTA). The ABS and PLA cylindrical parts have been prepared using fused deposition modeling (FDM) and finished using AFM. The experiments were designed using Taguchi (L9 OA) method. The effect of process parameters such as extrusion pressure (EP), layer thickness (LT) and abrasive concentration (AC) was investigated on the amount of material removed (MR) and percentage improvement in surface roughness (%ΔRa).FindingsThe developed abrasive media was found to be effective for finishing FDM printed parts using AFM. The microscope images of unfinished and finished showed a significant improvement in surface topography of additively manufactures parts after AFM. The results reveal that AC is the most significant parameter during the finishing of ABS parts. However, EP and AC are the most significant parameters for MR and %ΔRa, respectively, during the finishing of PLA parts.Practical implicationsThe FDM technology has applications in the biomedical, electronics, aeronautics and defense sectors. PLA has good biodegradable and biocompatible properties, so widely used in biomedical applications. The ventilator splitters fabricated using FDM have a profile similar to the shape used in the present study.Research limitations/implicationsThe present study is focused on finishing FDM printed cylindrical parts using AFM. Future research may be done on the AFM of complex shapes and freeform surfaces printed using different additive manufacturing (AM) techniques.Originality/valueAn abrasive media consists of xanthan gum, locust bean gum and fumed silica has been developed and characterized. An experimental study has been performed by combining printing parameters of FDM and finishing parameters of AFM. A comparative analysis in MR and %ΔRa has been reported between 3D printed ABS and PLA parts.

Corrigendum: Characterization of SARS-CoV-2 Variants N501Y.V1 and N501Y.V2 Spike on Viral Infectivity.

[This corrects the article DOI: 10.3389/fcimb.2021.720357.].

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern.

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha's small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

Vaccine cold chain management and cold storage technology to address the challenges of vaccination programs

The outbreaks of infectious diseases that spread across countries have generally existed for centuries. An example is the occurrence of the COVID-19 pandemic in 2020, which led to the loss of lives and economic depreciation. One of the essential ways of handling the spread of viruses is the discovery and administration of vaccines. However, the major challenges of vaccination programs are associated with the vaccine cold chain management and cold storage facilities. This paper discusses how vaccine cold chain management and cold storage technology can address the challenges of vaccination programs. Specifically, it examines different systems for preserving vaccines in either liquid or frozen form to help ensure that they are not damaged during distribution from manufacturing facilities. Furthermore, A vaccine is likely to provide very low efficacy when it is not properly stored. According to preliminary studies, the inability to store vaccine properly is partly due to the incompetency of many stakeholders, especially in technical matters. The novelty of this study is to thoroughly explore cold storage technology for a faster and more comprehensive vaccine distribution hence it is expected to be one of the reference and inspiration for stakeholders.

Melting and Re-Freezing Leads to Irreversible Changes in the Morphology and Molecular-Level Dynamics of Pfizer-BioNTech COVID-19 Vaccine.

Background and Objectives: As an mRNA-based vaccine, the Pfizer-BioNTech COVID-19 vaccine has stringent cold storage requirements to preserve functionality of the mRNA active ingredient. To this end, lipid components of the vaccine formulation play an important role in stabilizing and protecting the mRNA molecule for long-term storage. The purpose of the current study was to measure molecular-level dynamics as a function of temperature in the Pfizer-BioNTech COVID-19 vaccine to gain microscopic insight into its thermal stability. Materials and Methods: We used quasielastic and inelastic neutron scattering to probe (1) the vaccine extracted from the manufacturer-supplied vials and (2) unperturbed vaccine in the original manufacturer-supplied vials. The latter measurement was possible due to the high penetrative power of neutrons. Results: Upon warming from the low-temperature frozen state, the vaccine in its original form exhibits two-step melting, indicative of a two-phase morphology. Once the melting is completed (above 0 °C), vaccine re-freezing cannot restore its original two-phase state. This observation is corroborated by the changes in the molecular vibrational spectra. The molecular-level mobility measured in the resulting single-phase state of the re-frozen vaccine greatly exceeds the mobility measured in the original vaccine. Conclusions: Even a brief melting (above 0 °C) leads to an irreversible alteration of the two-phase morphology of the original vaccine formulation. Re-freezing of the vaccine results in a one-phase morphology with much increased molecular-level mobility compared to that in the original vaccine, suggesting irreversible deterioration of the vaccine's in-storage stability. Neutron scattering can be used to distinguish between the vibrational spectra characteristic of the original and deteriorated vaccines contained in the unperturbed original manufacturer-supplied vials.

Structure-Based Primer Design Minimizes the Risk of PCR Failure Caused by SARS-CoV-2 Mutations.

The coronavirus disease 2019 (COVID-19) has caused and is still causing tremendous damage to the global economy and human health. Qualitative reverse transcription-PCR (RT-qPCR) is the golden standard for COVID-19 test. However, the SARS-CoV-2 variants may not only make vaccine less effective but also evade RT-qPCR test. Here we suggest an innovative primer design strategy for the RT-qPCR test of SARS-CoV-2. The principle is that the primers should be designed based on both the nucleic acid sequence and the structure of the protein encoded. The three nucleotides closest to the 3' end of the primer should be the codon which encodes the tryptophan in the structure core. Based on this principle, we designed a pair of primers targeting the nucleocapsid (N) gene. Since tryptophan is encoded by only one codon, any mutation that occurs at this position would change the amino acid residue, resulting in an unstable N protein. This means that this kind of SARS-CoV-2 variant could not survive. In addition, both our data and previous reports all indicate that the mutations occurring at other places in the primers do not significantly affect the RT-qPCR result. Consequently, no SARS-CoV-2 variant can escape detection by the RT-qPCR kit containing the primers designed based on our strategy.