The annotation of six cluster N Mycobacterium smegmatis phages (Kevin1, Nenae, Parmesanjohn, ShrimpFriedEgg, Smurph, and SpongeBob) reveals regions of genomic diversity, particularly within the central region of the genome. The genome of Kevin1 includes two orphams (genes with no similarity to other phage genes), with one predicted to encode an AAA-ATPase.

Published studies indicate that TMEM184A is a heparin receptor that interacts with and transduces stimulation from heparin in vascular cells. Previous studies have indicated that heparin increases endothelial nitric oxide synthase (eNOS) activity in bovine endothelial cells. However, the precise mechanism remains unknown. In this study, we investigated the impact of heparin treatment and TMEM184A on eNOS’s activation and the role of eNOS in heparin signaling in the cloned A7r5 rat vascular smooth muscle cell line and confirmed results in endothelial cells. We employed a combination of TMEM184A knockdown A7r5 cells along with transient eNOS knockdown and enzyme inhibitor strategies. The results indicate that heparin induces phosphorylation of eNOS. eNOS can be immunoprecipitated with TMEM184A and is internalized to the perinuclear region in a TMEM184A-dependent manner in response to heparin. We also examined how heparin treatment leads to phosphorylation of eNOS and confirmed that TMEM184A and Ca2+ were required to mediate heparin-elicited eNOS phosphorylation. Evidence supporting the involvement of transient receptor potential cation channel subfamily V member 4 with TMEM184A in this eNOS activation process is also presented.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

Morpholino (MO) knockdown (KD) of the novel vascular heparin receptor, Transmembrane protein 184A (TMEM184A), increases endothelial cell (EC) proliferation rates, leading to disorganized and stunted vessel outgrowth in the regenerating zebrafish caudal fin. A similar vascular phenotype is observed in the developing zebrafish embryo, where TMEM184A KD reduces the number and length of intersegmental vessels (ISVs), as well as total Vascular Endothelial Cadherin (VEC) protein in ISV ECs. The Heparan Sulfate Proteoglycan Syndecan‐4 (Sdcn‐4) is required for VEC internalization in angiogenesis and interacts with membrane proteins to promote cellular signaling and molecular processes through interactions with its attached heparan sulfate (HS) chains. Since HS is nearly identical to heparin in structure and both TMEM184A and Sdcn‐4 appear to be required for stable angiogenesis, we asked whether TMEM184A and Sdcn‐4 interact and function in the same pathway to promote vascular sprouting and regeneration. Using MO knockdown of TMEM184A and Sdcn‐4 in the regenerating zebrafish caudal fin, we demonstrate that co‐injected subthreshold concentrations of both MOs synergize to produce the same vascular defect. In cells, we find that immunostaining of TMEM184A, Sdcn‐4, and VEC show trimeric colocalization at the cell surface, that in vesicle puncta they colocalize with the recycling marker Rab5a, and that TMEM184A precipitates Sdcn‐4 in whole cell lysate. In TMEM184A overexpression (OE) backgrounds and in ECs treated with heparin, immunostaining of VEC show increased expression levels in adherens junctions. Collectively, these results suggest that TMEM184A and Sdcn‐4 function in the same pathway to promote stable angiogenic repair, that TMEM184A interacts with both Syndecan‐4 and VEC in ECs, that they form a complex that is potentially recycled at the membrane surface, and that TMEM184A overexpression or activation stabilizes VEC in adherens junctions. Further study is required to determine whether TMEM184A and Syndecan‐4 function together to regulate VEC trafficking and promote angiogenesis or whether TMEM184A expression impacts on VEC membrane stability are distinct from the TMEM184A–Sdcn‐4 molecular mechanism that regulates angiogenic repair.

Heparin is a known anticoagulant that, with cellular uptake, contributes to a variety of physiological conditions, including experimentally observed decreases in vascular cell proliferation and inflammation. The fact that heparin is endocytosed and decreases cellular responses rather than blocking them entirely suggested a receptor mediated model, which has been previously identified in our laboratory as TMEM184A. Our data suggest that TMEM184A associates with vascular endothelial‐cadherin (VE‐Cadherin) and heparan sulfate proteoglycans (HSPGS), modulating mechanotransduction events in response to heparin treatment and fluid shear stress (FSS), which replicates the stress induced by blood flow under physiological conditions. TMEM184A seems to play a role in trafficking HSPGs such as syndecan‐1 (SDC1) and glypican‐1 (GPC1), both of which have been characterized as mechanosensing proteoglycans. In recent studies it has been demonstrated that VE‐Cadherin may interact with HS chains. Data in the literature have shown that when HS chains are degraded by Heparinase III treatment, the strain induced decrease in VE‐Cadherin is abolished, indicating that the HSPGS are crucial in sensing the strain. Given these findings, as well as our preliminary evidence that TMEM184A associates with HSPGs, we hypothesize that TMEM184A acts as an interface in the mediation of mechanosignaling events on the extracellular surface of adherens junctions. Co‐immunoprecipitation pull down of TMEM184A followed by subsequent western blotting and immunostaining showed both VE‐Cadherin and TMEM184A in the bound supernatant, indicating that the two may colocalize at endothelial adherens junctions and associate in a mechanosensing complex. Using a GFP‐TMEM184A plasmid construct to overexpress TMEM184A in bovine aortic endothelial cells (BAOECS), our research team observed increased surface levels of VE‐Cadherin in adherens junctions under static flow conditions. These overexpressing cells were subjected to FSS, which increases TMEM184A associations with HSPGs. Our team observed an additive increase in VE‐Cadherin in adherens junctions when compared to overexpression alone in static conditions. An additional experiment done to elucidate the role of endogenous heparin (HSPGs) revealed that BAOECs washed with a concentrated salt buffer to remove HPSG‐protein interactions demonstrated decreased colocalization of VE‐Cadherin, TMEM184A, and various vesicle associated membrane proteins. The addition of heparin rescued these colocalization events, implicating HSPGs in this complex. Our ongoing work aims to elucidate how associations of TMEM184A with VE‐cadherin and HSPGs facilitate activation of mechanically induced signals and mediate the trafficking of adherens junctions’ surface proteins. Support or Funding Information HL54269 to LJLK