QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site—the FCS, loop length, and glycosylation—are required for efficient SARS-CoV-2 replication and pathogenesis.

SARS-CoV-2 emerged in late 2019 and has caused the largest pandemic since the 1918 influenza outbreak (1). An unusual feature of SARS-CoV-2 is the presence of a furin cleavage site (FCS) in its spike protein (2). The CoV spike is a trimer of spike proteins composed of the S1 and S2 subunits, responsible for receptor binding and membrane fusion, respectively (1). After receptor binding, the spike protein is proteolytically cleaved at the S1/S2 and S2′ sites to activate the fusion machinery. For SARS-CoV-2, the spike protein contains a novel cleavage motif recognized by the host cell furin protease (PRRAR) directly upstream of the S1/S2 cleavage site that facilitates cleavage prior to virion release from the producer cell. This FCS, not found in other group 2B CoVs, plays a key role in spike processing, infectivity, and pathogenesis as shown by our group and others (3, 4).Importantly, another novel amino acid motif, QTQTN, is found directly upstream of the FCS. This QTQTN motif, also absent in other group 2B CoVs, is often deleted and has been pervasive in cultured virus stocks of the alpha, beta, and delta variants (5–8). In addition, the QTQTN deletion has been observed in a small subset of patient samples as well (9–11). Because this deletion has been frequently identified, we set out to characterize it and determine whether it has consequences for viral replication and virulence. Using our infectious clone (12, 13), we demonstrated that the loss of this motif attenuates SARS-CoV-2 replication in respiratory cells in vitro and pathogenesis in hamsters. The QTQTN deletion results in reduced spike cleavage and diminished capacity to use serine proteases on the cell surface for entry. Importantly, mutations of glycosylation-enabling residues in the QTQTN motif results in similar replication attenuation despite intact spike processing. Together, our results highlight elements in the SARS-CoV-2 spike in addition to the FCS that contribute to increased replication and pathogenesis.     

Discovery of 2-(4-cyano-3-trifluoromethylphenyl amino)-4-(4-quinazolinyloxy)-6-piperazinyl(piperidinyl)-s-triazines as potential antibacterial agents

A series of 2-(4-cyano-3-trifluoromethylphenyl amino)-4-(4-quinazolinyloxy)-6-piperazinyl(piperidinyl)-s-triazines have been synthesized in this study by a simple and efficient synthetic protocol. The synthetic route to final piperazinyl s-triazines involved two nucleophilic substitution reactions of 4-amino-2-trifluoromethyl-benzonitrile and 4-hydroxyquinazoline with 2,4,6-trichloro-1,3,5-triazine resulting in 2,4-disubstituted-6-chloro-1,3,5-triazine derivative to introduce the piperazinyl or piperidinyl functionality. The structures of the compounds were elucidated with the aid of IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR spectroscopy, and elemental analysis. The antimicrobial activity of the compounds was tested against eight bacteria (Staphylococcus aureus MTCC 96, Bacillus cereus MTCC 619, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 741, Klebsiella pneumoniae MTCC 109, Salmonella typhi MTCC 733, Proteus vulgaris MTCC 1771, Shigella Flexneria MTCC 1457) and four fungi (Aspergillus niger MTCC 282, Aspergillus fumigatus MTCC 343, Aspergillus clavatus MTCC 1323, and Candida albicans MTCC 183). The title compounds were also investigated for their antituberculosis activity against MTB H37 R_V strain using BACTEC MGIT and L. J. agar dilution method. The bioassay results showed that compounds 5d, 5n, 5p, 5s, and 5t demonstrated 99% inhibition at the MIC of 6.25?μg/ml, equivalent to standard drug pyrazinamide.     

<Emphasis Type=Italic>Aphelenchus avenae</Emphasis> (Nematoda: Aphelenchidae) under the rhizosphere of <Emphasis Type=Italic>Brassica napus</Emphasis>

Soil samples under the rhizosphere of Brasicca napus were collected from three localities (Bílé Podolí, Prague, Kylešovice). All three localities were positive for the presence of Aphelenchus avenae. Morphological and molecular features of A. avenae are presented in this research note.     

Determination of Genetic Diversity of the Morinda tinctoria Population in Historical Mandore Garden

Morinda tinctoria (Rubiaceae) commonly called as ‘Indian Mulberry’ is one of the avenue trees of historically important Mandore garden planted anthropogenically in the past. Objective of this study was to determine the genetic diversity of M. tinctoria population at Mandore garden using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeat Markers. A total of 97 bands were produced from 10 random amplified polymorphic DNA primers (49 bands) and 8 inter simple sequence repeat primers (48 bands). No polymorphic bands were obtained in any accessions using the two methods, which strongly suggested that M. tinctoria of this region appear as identical clones. Monomorphic pattern revealed the clonal plantation of M. tinctoria in this historical garden. This is the first report on use of molecular markers to infer the ancient plantation history. Consequences of genetically monomorphic population under global climate changes are discussed and conservation strategy is proposed.     

Primary Chondrosarcoma of Male Breast: A Rare Case

Sarcomas of the breast are relatively rare and account for 1% of all primary malignant tumors of the breast. Pure and primary chondrosarcoma of the male breast is an extremely rare tumor. It may arise either from the breast stroma itself or from underlying bone or cartilage. Differential diagnoses include cystosarcoma phyllodes and breast metaplastic carcinoma with chondroid differentiation.     

社区护理人员灾害准备的研究进展

对国内外社区护理人员灾害准备模式、存在的问题、影响因素、培训现状及测量工具进行综述。提出应建立完善的社区护理人员灾害准备培训体系,建立适合我国国情的社区护理人员灾害应对准备模式并建立社区护理人员灾害准备测量工具的区域性常模,旨在为今后提高社区护理人员的灾害准备度提供参考依据。