The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.

Recurrent MI is common in patients with coronary artery disease and associates with high mortality. Here we developed a surgical mouse model in which two subsequent MIs affect different left ventricular regions in the same mouse. Recurrent MI was induced by ligating the left circumflex followed by the left anterior descending branch of the coronary artery. We characterized the resulting ischemia by whole-heart fluorescent coronary angiography after optical organ clearing and by cardiac MRI. We report that a first MI induces bone marrow "memory" via a circulating signal, thereby affecting hematopoietic factor expression in bone marrow macrophages. This altered the organism's reaction to subsequent events. Inspite at least similar extent of injury reported by blood troponin, recurrent MI caused reduced emergency hematopoiesis and less leukocytosis than a first MI. Consequently, fewer leukocytes migrated to the ischemic myocardium. The hematopoietic response to lipopolysaccharide was also mitigated after a previous MI. Our data suggest that hematopoietic and innate immune responses are shaped by a preceding MI. ! 2 ! 3 ! 4 S. Cremer and M.J. Schloss developed and validated the mouse model of recurrent MI. C. Vinegoni developed fluorescent coronary angiography. S. Cremer, M.J. Schloss, S. Zhang, D. Rohde and C.Vinegoni performed experiments and collected, analyzed and discussed data. G. Wojtkiewicz and S. Schmitt performed imaging experiments and collected data. S. Cremer, F. Swirski and M. Nahrendorf conceived experiments and discussed results and strategy. M. Nahrendorf conceived, designed and directed the study. S. Cremer and M. Nahrendorf wrote the manuscript, which was revised and approved by all authors. ! 13 ! 28 Supplementary Figure 2. HSPC numbers 10 days after MI. (A) Experimental outline. Mice had LAD MI 10 days before bone marrow analysis. (B) Dot plots and (C) quantification of HSPC in bone marrow of mice with MI compared to naive mice. (D) Numbers of mature leukocytes in bone marrow. *p<0.05, n=5 Student's t-test. Data are mean ± s.e.m.. ! 29 Supplementary Figure 3. Troponin after a first versus recurrent MI. (A) Experimental design. (B) Troponin levels after LAD ligation as the first compared to recurrent MI. n=5-9. ! 30 Supplementary Figure 4. Macrophage gating. (A) Gating strategy for bone marrow macrophages with antibody directed against F4/80 and (B) isotype control. (C) Histograms in red depicting the expression of MHCII, Cx3cr1, CD169 and Vcam1 in Gr1 hi monocytes and bone marrow macrophages. Isotype controls are shown in blue. ! 31 Supplementary Video 1: MRI short axis cine loop of LCX infarct. Delayed enhancement MRI short axis views of a mouse with LCX MI demonstrating injury of posterolateral left ventricular wall. Supplementary Video 2: MRI short axis cine loop after recurrent MI (LCX followed by LAD ligation). Delayed enhancement MRI short axis views of the same mouse as in supplementary video 1, now also showing injury of the anterior left ventricular wall.! 32

Purpose: To apply a novel visible and near-infrared optical coherence tomography (vnOCT) in the dexamethasone-induced ocular hypertension mouse model, and test the capability of four optical markers, peripapillary retinal nerve fiber layer (RNFL) thickness, total retinal blood flow, VN ratio and hemoglobin oxygen saturation (sO2), in detecting retinal ganglion cell (RGC) damage in association with ocular hypertension.Methods: Twelve mice (C57BL/6J) were separated into a control (n=6) and a dexamethasone group (n=6) receiving twice daily saline or dexamethasone eye drops, respectively, for 7 weeks. Intraocular pressure (IOP) measurements were taken at baseline and weekly. Optical measurements by vnOCT were longitudinally taken at baseline, 4 weeks and 7 weeks. Following week 7, ex vivo RGC counting was performed by immunostaining. Results:The dexamethasone group showed a measurable rise in IOP by week 2. Despite the IOP differences between the dexamethasone and control groups, there was not a statistical difference in RNFL thickness or total blood flow over 7 weeks. The dexamethasone group did show an increase in retinal arteriovenous sO2 difference (A-V sO2) that was significant at week 4 and 7. The RNFL VN ratio showed a significant decrease at week 4 and 7 in dexamethasone group associated with a decreased RGC count.Conclusions: RNFL VN ratio and A-V sO2 are capable of detecting early retinal alterations in the dexamethasone-induced ocular hypertension mouse model. Data analysis suggests VN ratio and A-V sO2 are correlated with RGC loss secondary to ocular hypertension, while being independent of IOP.

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