Generalized Ricci Curvature Bounds for Three Dimensional Contact Subriemannian manifolds

Measure contraction property is one of the possible generalizations of Ricci curvature bound to more general metric measure spaces. In this paper, we discover sufficient conditions for a three dimensional contact subriemannian manifold to satisfy this property.Comment: 49 page

Comparison of cobinamide to hydroxocobalamin in reversing cyanide physiologic effects in rabbits using diffuse optical spectroscopy monitoring.

Our purpose is to compare cobinamide to hydroxocobalamin in reversing cyanide (CN)-induced physiologic effects in an animal model using diffuse optical spectroscopy (DOS). Cyanide poisoning is a major threat worldwide. Cobinamide is a novel molecule that can bind two molecules of cyanide, has a much higher binding affinity than hydroxocobalamin, and is more water soluble. We investigated the ability of equimolar doses of cobinamide and hydroxocobalamin to reverse the effects of cyanide exposure in an animal model monitored continuously by DOS. Cyanide toxicity was induced in 16 New Zealand white rabbits by intravenous infusion. Animals were divided into three groups: controls (n=5) received saline following cyanide, hydroxocobalamin (N=6) following cyanide, and cobinamide (N=5) following cyanide. Cobinamide caused significantly faster and more complete recovery of oxy- and deoxyhemoglobin concentrations in cyanide-exposed animals than hydroxocobalamin- or saline-treated animals, with a recovery time constant of 13.8+/-7.1 min compared to 75.4+/-25.1 and 76.4+/-42.7 min, for hydroxocobalamin- and saline-treated animals, respectively (p<0.0001). This study indicates that cobinamide more rapidly and completely reverses the physiologic effects of cyanide than equimolar doses of cobalamin at the dose used in this study, and CN effects and response can be followed noninvasively using DOS

iPep-SARS2-E in vitro validation.

(A) Electron microscopic image of iPep-SARS2-E-treated Vero-E6 cells at 24 h post-infection. Arrowheads, small particles found in the nuclear envelope. Scale bar, 1 μm. (B) Experimental design to examine whether intracellular particles are infectious in Vero-E6 cells treated with iPep-SARS2-E. (C) Result of quantitative endpoint titration assay used to quantify intracellular virus particles of PBS- and iPep-SARS2-E (10 μM)-treated Vero-E6 cells. There is a significant reduction of infectivity in iPep-SARS2-E though still infectious. Each well (cell plating at 24 h, 2,500 harvested cells onto 4 × 104 uninfected fresh cells per a well that were seeded the night before) was scored based on infectivity compared to virus controls with zero indicating no infection and 100 indicating complete infection (CPE). Student’s t test was used (**** P D) Western blots of Spike and GAPDH proteins in PBS- and iPep-SARS2-E (10 μM)-treated Vero-E6 cells at 24 h post-infection with SARS-CoV-2 WA1 (MOI, 0.10, 24 h), suggesting the effect of iPep-SARS2-E on Spike expression and/or stability. (E–H) qPCR of SARS-CoV-2 N (E), E (F), JUN/AP-1 expression (G) of PBS (n = 6)- and iPep-SARS2-E (10 μM, n = 6)-treated Vero-E6 cells comparing to non-infected cells (n = 6) at 48 h post-infection. The expression of these genes was normalized to a house-keeping gene, GAPDH. One-way ANOVA with Tukey’s multiple comparisons test was used (**** P P P H) qPCR of SARS2-CoV-2 N expression of PBS (n = 6)- and iPep-SARS2-E (10 μM, n = 6)-treated Vero-E6 cell culture supernatant (sup) at 48 h post-infection. Student’s t test was used (**** P I) Representative confocal fluorescent images of Vero-E6 cells treated with PBS or iPep-SARS2-E at 48 h post-infection. SARS-CoV-2 N antibody (red) and Hoechst 33258 dye (blue, for nucleus) were used with antibodies of subcellular organelle markers (green): BiP for endoplasmic reticulum (ER), ERGIC-53 for ER Golgi inter compartment (ERGIC), and LAMP1 for lysosome. Scale bar, 5 μm. The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. (PDF)</p

Alignment of human coronavirus envelope proteins.

Envelope protein sequence alignment of SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. CLUSTALW 2.1 multiple sequence alignment software is used to obtain the alignment. (PDF)</p

Characterization of iPep-SARS2-E.

(A) Immunoprecipitation of 2E protein using Ni column and HEK 293T cells transfected using 2E-YFP with or without 6xHis-MY18-2ED (His-MY18). Anti-GFP antibody was used to blot 2E-YFP protein bands. (B) Representative epi-fluorescence image of NIH 3T3 cells co-transfected with 2E-mKate2 (red) and His-MY18 plasmids. Anti-His tag antibody conjugated to Alexa Fluor 488 (green) and Hoechst 33258 dye (blue, nucleus) were used after cell fixation. White arrowheads, co-localization of red and green fluorescence. Scale bar, 5 μm. (C) Representative SARS2-E currents in 2E-PM-expressing HEK 293S cells mock-transfect and co-transfected with MY18-2ED peptide construct. (D) MY18-2ED significantly blocked 2E currents. Student’s t test was used (*** P E) Representative immunoblot images of HEK 293T cells transfected with SARS2-E fused with YFP (2E-YFP) and 48 h treated with 10 μM TAT-MY18-2ED or MY18-WT peptides (negative control). Anti-GFP (for 2E-YFP, top) and GAPDH antibodies (as loading control, bottom) were used. Putative aggregates of 2E-YFP proteins (&) were observed even though Urea-based lysis buffer was used for the sample preparation. #, nonspecific bands around 40 and 50 kDa according to the YFP blotting (S3B Fig). The whole blotting image of GAPDH is shown in S3A Fig. (F and G) Quantification of 2E-YFP monomeric form (F) and aggregates (G) of HEK 293T cells transfected with 2E-YFP and treated using TAT-MY18-2ED (n = 4) or MY18-WT peptides (n = 4). Student’s t test was used (*** P H) Representative immunoblot images of HEK 293T cells transfected with YFP plasmid and 48 h treated with TAT-MY18-2ED (10 μM, n = 3) and MY18-WT peptides (10 μM, n = 3, a negative control) and non-treated cells (n = 3, another negative control). Anti-GFP (for YFP, top) and GAPDH antibodies (as loading control, bottom) were used. The whole blotting images and quantification are shown in S3B and S3C Fig, respectively. (I) Schematic representation of the molecular mechanism underlying the effect of MY18 peptide on 2E protein and lysosomal function. The images are prepared using BioRender: Top, 2E induces deacidification in lysosome (Fig 1B and 1C); bottom, MY18 peptides binds 2E proteins (Fig 3A and 3B), resulting in 2E inhibition (Fig 3C and 3D) and restored lysosomal activity (Figs 1B, 1C and S2A) and 2E protein reduction (Fig 3E–3G). The data underlying this figure can be found in S1 Data. All the graphs in the figure are mean ± SD. HEK, human embryonic kidney; NIH, National Institute of Health.</p

iPep-SARS2-E in vivo test using intranasal administration.

(A) Schematic representative of mouse intranasal administration of iPep-SARS2-E. Red box demonstrates the nasal tissue region harvested for the following fluorescent imaging. The image is from BioRender software. (B) Representative fluorescent and bright field images of nasal tissues isolated from mice administrated intranasally with PBS or Alexa594-conjugated iPep-SARS2-E peptide (TAT-MY18-2ED-A594, 10 μM, 2 h). After isolating the tissues, the samples were washing using PBS 3 times, and the fluorescent and bright field images were taken by a fluorescent stereoscope. Scale bar, 1 mm. (C) Experimental design for the iPep-SARS2-E safety test in vivo. (D) There is no significant difference the effects on body weight among iPep-SARS2-E-treated (n = 6), non-treated (n = 5), and PBS-treated Balb/c mouse groups (n = 5). One-way ANOVA with Tukey’s multiple comparisons was used at each day. (E, F) There were no significant differences in Cxcl12 (E) and C5a (F) among iPep-SARS2-E-treated (n = 5), non-treated (n = 4), and PBS-treated mice (n = 4). One-way ANOVA with Tukey’s multiple comparisons was used (n.s., not significant). (G) Experimental design for the iPep-SARS2-E test in vivo using intranasal administration. (H) iPep-SARS2-E prevents body weight loss in SARS-CoV-2 MA10-infected Balb/c mice (5.0 × 10^4 PFU/mouse). Student’s t test was used at each day (** P P I, J) Representative immunoblots of SARS-CoV-2 E (2E, I) and mouse Gapdh proteins (J) in SARS-CoV-2 MA10-infected mouse lung tissues with PBS or iPep-SARS2-E treatment. (K) iPep-SARS2-E peptides significantly reduced the protein expression of 2E in MA10-infected Balb/c mouse lung tissues (PBS, n = 4; iPep-SARS2-E, n = 4). Student’s t test was used (* P S1 Data. All the graphs in the figure are mean ± SD. (PDF)</p

The effect of iPep-SARS2-E on SARS2-E expression.

(A) Representative GAPDH immunoblot image of HEK 293T cells transfected with SARS2-E fused with YFP (2E-YFP) and treated with 10 μM TAT-MY18-2ED or MY18-WT (negative control). The image between 30 and 40 kDa is used in Fig 3E. (B) Representative immunoblot images of HEK 293T cells transfected with YFP plasmid and treated with 10 μM TAT-MY18-2ED or MY18-WT (negative control) for 48 h. Anti-GFP (for YFP, top) and GAPDH antibodies (as loading control, bottom) were used. The short- and long-exposure film images are shown. #, nonspecific bands around 40 and 50 kDa are found in the cell lysate. The band images are used in Fig 3H. (C) Quantification of YFP protein expression of HEK 293T cells transfected using YFP plasmid non-treated (n = 3) and treated with TAT-MY18-2ED (n = 3) or MY18-WT peptides (n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used (n.s., not significant). The data underlying this figure can be found in S1 Data. The graph in the figure is mean ± SD. (PDF)</p

Permeability of iPep-SARS2-E.

(A) Representative fluorescent and bright field images of time-course cell-penetrating test using Alexa Fluor 594(A594)-conjugated iPep-SARS2-E peptides, A594-TAT-MY18-2ED (amino-terminal conjugation, N-term, 10 μM, bottom), and TAT-MY18-2ED-A594 (carboxyl-terminal, C-term, 10 μM, top) in NIH 3T3 cells after the incubation started. Scale bar, 50 μm. (B) Quantification of red fluorescence-positive cells treated with the A594-conjugated peptides for the peptide cell-penetrating “on” kinetics (mean ± SD). The data underlying this figure can be found in S1 Data. (C) Experimental design for the peptide stability, “off” kinetics, quantification. (D) Representative fluorescent and bright field images after washout of A594-conjugated TAT-MY18-2ED peptide (C-term version) in NIH 3T3 cells. White arrowheads, fluorescent puncta. Scale bar, 50 μm. (PDF)</p

Raw datasets of the experiments.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.</div