SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

Differential cell counts using center-point networks achieves human-level accuracy and efficiency over segmentation

Differential cell counts is a challenging task when applying computer vision algorithms to pathology. Existing approaches to train cell recognition require high availability of multi-class segmentation and/or bounding box annotations and suffer in performance when objects are tightly clustered. We present differential count network (“DCNet”), an annotation efficient modality that utilises keypoint detection to locate in brightfield images the centre points of cells (not nuclei) and their cell class. The single centre point annotation for DCNet lowered burden for experts to generate ground truth data by 77.1% compared to bounding box labeling. Yet centre point annotation still enabled high accuracy when training DCNet on a multi-class algorithm on whole cell features, matching human experts in all 5 object classes in average precision and outperforming humans in consistency. The efficacy and efficiency of the DCNet end-to-end system represents a significant progress toward an open source, fully computationally approach to differential cell count based diagnosis that can be adapted to any pathology need.

COUP-TFI specifies the medial entorhinal cortex identity and induces differential cell adhesion to determine the integrity of its boundary with neocortex

Development of cortical regions with precise, sharp, and regular boundaries is essential for physiological function. However, little is known of the mechanisms ensuring these features. Here, we show that determination of the boundary between neocortex and medial entorhinal cortex (MEC), two abutting cortical regions generated from the same progenitor lineage, relies on COUP-TFI (chicken ovalbumin upstream promoter–transcription factor I), a patterning transcription factor with graded expression in cortical progenitors. In contrast with the classical paradigm, we found that increased COUP-TFI expression expands MEC, creating protrusions and disconnected ectopic tissue. We further developed a mathematical model that predicts that neuronal specification and differential cell affinity contribute to the emergence of an instability region and boundary sharpness. Correspondingly, we demonstrated that high expression of COUP-TFI induces MEC cell fate and protocadherin 19 expression. Thus, we conclude that a sharp boundary requires a subtle interplay between patterning transcription factors and differential cell affinity.

SARS-CoV-2 Spreads through Cell-to-Cell Transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While ACE2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the variants of concern (VOC) B.1.1.7 and B.1.351 have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccine sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

Bronchoalveolar Lavage Fluid Cytology in Healthy Cynomolgus Macaques

Bronchoalveolar lavage, or BAL, is a minimally invasive procedure frequently used for clinical and non-clinical research, allowing studies of the respiratory system. Macaques are the most widely used non-human primate models in biomedical research. However, very little information is available in the literature concerning BAL cytology in macaques. The purpose of this study was to establish BAL reference values and document an atlas of BAL cytology from healthy cynomolgus macaques. BALs were obtained from 30 macaques and BAL fluid differential cell counts based on 400 nucleated cells/BAL sample were performed by a board-certified clinical pathologist. Results were analyzed using Reference Value Advisor macroinstructions and the effect of blood and oropharyngeal contaminations was investigated. Overall, nucleated cells interval percentages in BAL fluids were 55.8 to 93.7 for macrophages, 1.8 to 37.1 for lymphocytes, 0.4 to 8.7 for neutrophils, and 0.4 to 9.8 for eosinophils. Mild oropharyngeal contamination did not affect BAL differential cell counts, whilst a slight but significant increase of the percentage of lymphocytes was observed in samples with mild blood contamination. Mucus and variable numbers of ciliated epithelial cells were commonly present. Rarely, multinucleated macrophages and mastocytes were also observed. The reference intervals established in this study provide a useful baseline for the assessment of BAL cytological data in cynomolgus macaques.

The microtubule-associated protein WDL4 modulates auxin distribution to promote apical hook opening in Arabidopsis

The unique apical hook in dicotyledonous plants protects the shoot apical meristem and cotyledons when seedlings emerge through the soil. Its formation involves differential cell growth under the coordinated control of plant hormones, especially ethylene and auxin. Microtubules are essential players in plant cell growth that are regulated by multiple microtubule-associated proteins (MAPs). However, the role and underlying mechanisms of MAP-microtubule modules in differential cell growth are poorly understood. In this study, we found that the previously uncharacterized Arabidopsis MAP WAVE-DAMPENED2-LIKE4 (WDL4) protein plays a positive role in apical hook opening. WDL4 exhibits a temporal expression pattern during hook development in dark-grown seedlings that is directly regulated by ethylene signaling. WDL4 mutants showed a delayed hook opening phenotype while overexpression of WDL4 resulted in enhanced hook opening. In particular, wdl4-1 mutants exhibited stronger auxin accumulation in the concave side of the apical hook. Furthermore, the regulation of the auxin maxima and trafficking of the auxin efflux carriers PIN-FORMED1 (PIN1) and PIN7 in the hook region is critical for WDL4-mediated hook opening. Together, our study demonstrates that WDL4 positively regulates apical hook opening by modulating auxin distribution, thus unraveling a mechanism for MAP-mediated differential plant cell growth.

The Sequential Action of MIDA9/PP2C.D1, PP2C.D2, and PP2C.D5 Is Necessary to Form and Maintain the Hook After Germination in the Dark

During seedling etiolation after germination in the dark, seedlings have closed cotyledons and form an apical hook to protect the meristem as they break through the soil to reach the surface. Once in contact with light, the hook opens and cotyledons are oriented upward and separate. Hook development in the dark after seedling emergence from the seed follows three distinctly timed and sequential phases: formation, maintenance, and eventual opening. We previously identified MISREGULATED IN DARK9 (MIDA9) as a phytochrome interacting factor (PIF)-repressed gene in the dark necessary for hook development during etiolated growth. MIDA9 encodes the type 2C phosphatase PP2C.D1, and pp2c-d1/mida9 mutants exhibit open hooks in the dark. Recent evidence has described that PP2C.D1 and other PP2C.D members negatively regulate SMALL AUXIN UP RNA (SAUR)-mediated cell elongation. However, the fundamental question of the timing of PP2C.D1 action (and possibly other members of the PP2C.D family) during hook development remains to be addressed. Here, we show that PP2C.D1 is required immediately after germination to form the hook. pp2c.d1/mida9 shows reduced cell expansion in the outer layer of the hook and, therefore, does not establish the differential cell growth necessary for hook formation, indicating that PP2C.D1 is necessary to promote cell elongation during this early stage. Additionally, genetic analyses of single and high order mutants in PP2C.D1, PP2C.D2, and PP2C.D5 demonstrate that the three PP2C.Ds act collectively and sequentially during etiolation: whereas PP2C.D1 dominates hook formation, PP2C.D2 is necessary during the maintenance phase, and PP2C.D5 acts to prevent opening during the third phase together with PP2C.D1 and PP2C.D2. Finally, we uncover a possible connection of PP2C.D1 levels with ethylene physiology, which could help optimize hook formation during post-germinative growth in the dark.