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The coronavirus disease 2019 (COVID-19) pandemic has highlighted the importance of developing novel vaccines. An ideal vaccine should trigger an intense immune reaction without causing significant side effects. In this study we found that substitution of tryptophan located in the cores of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein structures with certain smaller amino acids resulted in variants with melting temperatures of 33–37 °C. An enzyme activity assay indicated that the proteolytic activity of the main proteinase (3CLpro) decreased sharply when the environmental temperature exceeded the melting temperature, implying that other protein variants may lose most of their functions under the same conditions. This finding suggests that a virus variant containing engineered proteins with melting temperatures of 33–37 °C may only be functional in the upper respiratory tract where the temperature is about 33 °C, but will be unable to invade internal organs, which maintain temperatures above 37 °C, thus making it possible to construct temperature-sensitive attenuated vaccines.

Toxoplasma gondii (T. gondii) is widely spread around the world, which can cause serious harm to immunosuppressed patients. Currently, the commercial test kits are poor at assessing T. gondii infection and vaccine effectiveness, making an urgent need to exploit effective enzyme-linked immunosorbent assay with great performance to compensate for this deficiency. Here, the TgIMP1 recombinant protein was expressed in E. coli BL(21) cells. The TgIMP1 was purified with affinity chromatography and the reactivity was retained with anti-TgIMP1 antibodies. The TgIMP1 was then used to develop an indirect ELISA (IMP1-iELISA) and the reaction conditions of IMP1-iELISA were optimized. As a result, the cut-off value was determined to be 0.2833 by analyzing the OD450nm values of forty T. gondii-negative sera. The coefficient of variation of 6 T. gondii-positive sera within and between runs were both less than 10%. The IMP1-iELISA was non-cross-reactive with the sera of cytomegalovirus, herpes virus, rubella virus, Cryptosporidium spp., Theileria spp., Neospora spp. and Plasmodium spp.. Furthermore, the sensitivity and specificity of IMP1-iELISA were 98.9% and 96.7%, respectively, based on testing 150 serum samples. The results suggest that this IMP1-iELISA is specific, sensitive, repeatable and can be applied to the detection of T. gondii infections in the medical and health industries.

Abstract For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence.

Toxoplasma gondii (T. gondii) is an opportunistic parasite that can infect the central nervous system (CNS), causing severe toxoplasmosis and behavioral cognitive impairment. Mortality is high in immunocompromised individuals with toxoplasmosis, most commonly due to reactivation of infection in the CNS. There are still no effective vaccines and drugs for the prevention and treatment of toxoplasmosis. There are five developmental stages for T. gondii to complete life cycle, of which the tachyzoite and bradyzoite stages are the key to the acute and chronic infection. In this study, to better understanding of how T. gondii interacts with the host CNS at different stages of infection, we constructed acute and chronic infection models of T. gondii in astrocytes, and used label-free proteomics to detect the proteome changes before and after infection, respectively. A total of 4676 proteins were identified, among which 163 differentially expressed proteins (fold change ≥ 1.5 or ≤ 0.67 and p-value ≤ 0.05) including 109 up-regulated proteins and 54 down-regulated proteins in C8-TA vs C8 group, and 719 differentially expressed proteins including 495 up-regulated proteins and 224 down-regulated proteins in C8-BR vs C8-TA group. After T. gondii tachyzoites infected astrocytes, differentially expressed proteins were enriched in immune-related biological processes to promote the formation of bradyzoites and maintain the balance of T. gondii, CNS and brain. After T. gondii bradyzoites infected astrocytes, the differentially expressed proteins up-regulated the host's glucose metabolism, and some up-regulated proteins were strongly associated with neurodegenerative diseases. These findings not only provide new insights into the psychiatric pathogenesis of T. gondii, but also provide potential targets for the treatment of acute and chronic Toxoplasmosis.

Gardnerella overgrowth is the primary cause of bacterial vaginosis (BV), a common vaginal infection with incidences as high as 23–29% worldwide. Here, we studied the pathogenicity, drug resistance, and prevalence of varying Gardnerella spp. We isolated 20 Gardnerella strains from vaginal samples of 31 women in local China. Ten strains were then selected via phylogenetic analysis of cpn60 and vly gene sequences to carry out genome sequencing and comparative genomic analysis. Biofilm-formation, sialidase, and antibiotic resistance activities of the strains were characterized. All strains showed striking heterogeneity in genomic structure, biofilm formation and drug resistance. Two of the ten strains, JNFY3 and JNFY15, were classified as Gardnerella swidsinskii and Gardnerella piotii, respectively, according to their phenotypic characteristics and genome sequences. In particular, seven out of the ten strains exhibited super resistance (≥ 128 μg/mL) to metronidazole, which is the first line of treatment for BV in China. Based on the biochemical and genomic results of the strains, we proposed a treatment protocol of prevalent Gardnerella strains in local China, which provides the basis for accurate diagnosis and therapy.

Diaporthe spp. are critical plant pathogens that cause wood cankers, wilt, dieback, and fruit rot in a wide variety of economic plant hosts and are regarded as one of the most acute threats faced by the kiwifruit industry worldwide. Diaporthe phragmitis NJD1 is a highly pathogenic isolate of soft rot of kiwifruit. Here, we present a high-quality genome-wide sequence of D. phragmitis NJD1 that was assembled into 28 contigs containing a total size of 58.33 Mb and N50 length of 3.55 Mb. These results lay a solid foundation for understanding host-pathogen interaction and improving disease management strategies.

Gardnerella vaginalis is the main pathogen that causes bacterial vaginosis. In the healthy vaginal microecological environment of a woman, the lactobacilli produce lactate and hydrogen peroxide to inhibit the growth of pathogens such as G. vaginalis. The lack of lactobacilli results in a high pH and low hydrogen peroxide in the vagina which facilitate G. vaginalis growth, leading to the imbalance of the vaginal microecology. In this study, lactate and hydrogen peroxide were added to a G. vaginalis culture medium to simulate the co-culture of the lactobacilli and G. vaginalis, and then the genes related to the stress response of G. vaginalis were identified using transcriptomics and proteomics. It was indicated that, among all the upregulated genes, most of them encoded transporters associated with the efflux of harmful substances, and the majority of the downregulated genes were related to the biofilm formation and epithelial cell adhesion. This study may help find new drug targets for G. vaginalis for the development of novel therapies for bacterial vaginosis.

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.

Summary: Sensing stressful conditions and adjusting the cellular metabolism to adapt to the environment are essential activities for bacteria to survive in variable situations. Here, we describe a stress-related protein, YdiU, and characterize YdiU as an enzyme that catalyzes the covalent attachment of uridine-5′-monophosphate to a protein tyrosine/histidine residue, an unusual modification defined as UMPylation. Mn2+ serves as an essential co-factor for YdiU-mediated UMPylation. UTP and Mn2+ binding converts YdiU to an aggregate-prone state facilitating the recruitment of chaperones. The UMPylation of chaperones prevents them from binding co-factors or clients, thereby impairing their function. Consistent with the recent finding that YdiU acts as an AMPylator, we further demonstrate that the self-AMPylation of YdiU padlocks its chaperone-UMPylation activity. A detailed mechanism is proposed based on the crystal structures of Apo-YdiU and YdiU-AMPNPP-Mn2+ and on molecular dynamics simulation models of YdiU-UTP-Mn2+ and YdiU-UTP-peptide. In vivo data demonstrate that YdiU effectively protects Salmonella from stress-induced ATP depletion through UMPylation.

We study the stationary Stokes system with variable coefficients in the whole space, a half space, and on bounded Lipschitz domains. In the whole and half spaces, we obtain a priori Ẇq1-estimates for any q ∈ [2,∞) when the coefficients are merely measurable functions in one fixed direction. For the system on bounded Lipschitz domains with a small Lipschitz constant, we obtain a Wq1-estimate and prove the solvability for any q ∈ (1,∞) when the coefficients are merely measurable functions in one direction and have locally small mean oscillations in the orthogonal directions in each small ball, where the direction is allowed to depend on the ball.

We consider degenerate parabolic and elliptic equations of second order with $C^1$ and $C^2$ coefficients. Error bounds for certain types of finite-difference schemes are obtained.

We are concerned with interior and global gradient estimates for solutions to a class of singular quasilinear elliptic equations with measure data, whose prototype is given by the p-Laplace equation -- Δp u = μ with p ∈ (1,2). The cases when p ∈ (2 - 1/n, 2) and p ∈ (3n-2/2n-1, 2 - 1/n] were studied in Duzaar and Mingione [J. Funct. Anal. 259 (2010), 379-418] and Nguyen and Phuc [J. Funct. Anal. 278 (2020), art. 108391] respectively. In this paper, we improve the results of Nguyen and Phuc and address the open case when p ∈ (1, 3n-2/2n-1]. Interior and global modulus of continuity estimates of the gradients of solutions are also established. [ABSTRACT FROM AUTHOR]

We present a priori estimates and unique solvability results in the mixed-norm Lebesgue spaces for the kinetic Kolmogorov-Fokker-Planck (KFP) equation in divergence form. The leading coefficients are bounded uniformly nondegenerate with respect to the velocity variable v and satisfy a vanishing mean oscillation (VMO) type condition. We consider the L2 case separately and treat more general equations, which include the relativistic KFP equation. This paper is a continuation of our previous work on Lp estimates for KFP equations in nondivergence form. [ABSTRACT FROM AUTHOR]

In this paper, we investigate pointwise time analyticity of solutions to nonlocal parabolic equations in the settings of ℝd and a complete Riemannian manifold M. On the one hand, in ℝd, we prove that any solution u=u(t,x) to ..., where ... is a nonlocal operator of order α, is time analytic in (0,1] if u satisfies the growth condition ... for any ... and ... . We also obtain pointwise estimates for ..., where ... is the fractional heat kernel. Furthermore, under the same growth condition, we show that the mild solution is the unique solution. On the other hand, in a manifold M, we also prove the time analyticity of the mild solution under the same growth condition and the time analyticity of the fractional heat kernel when M satisfies the Poincaré inequality and the volume doubling condition. Moreover, we also study the time and space derivatives of the fractional heat kernel in Rd using the method of Fourier transform and contour integrals. We find that when ..., the fractional heat kernel is time analytic at t=0 when x≠0, which differs from the standard heat kernel. As corollaries, we obtain a sharp solvability condition for the backward nonlocal parabolic equations and time analyticity of some nonlinear nonlocal parabolic equations with power nonlinearity of order p . These results are related to those in [9] and [22], which deal with local equations. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global development. Rapid and accurate diagnosis is critical for containing the pandemic and treating patients in time. As the gold standard for SARS-CoV-2 diagnosis, the qualitative reverse transcription-PCR (RT-qPCR) test has long been criticized for its long detection time. In this study, we optimized the primers and probes targeting SARS-CoV-2 ORF1ab and N gene designed by the Chinese Center for Disease Control and Preventions (CDC) to increase their Tm values to meet the optimal elongation temperature of Taq DNA polymerase, thus greatly shortened the elongation time. The higher elongation temperature in turn narrowed the temperature range of the reaction and saved more time. In addition, by shortening the distance between the fluorophore at the 5' end and the quencher in the middle we got a probe with higher signal-to-noise ratio. Finally, by using all these measures and optimized RT-qPCR program we successfully reduced the time (nucleic acid extraction step is not included) for nucleic acid test from 74 min to 26 min. [ABSTRACT FROM AUTHOR]