Lee, D;Helal, Z;Kim, J;Hunt, A;Barbieri, A;Tocco, N;Frasca, S;Kerr, K;Hyeon, J;Chung, D;Risatti, G;
| DOI: 10.3390/v13112141
We report the first detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a 3-month-old dog in Connecticut that died suddenly and was submitted to the state veterinary diagnostic laboratory for postmortem examination. Viral RNA was detected in multiple organs of the dog by reverse transcription real time-PCR (RT-qPCR). Negative and positive sense strands of viral RNA were visualized by in situ hybridization using RNAscope technology. Complete genome sequencing and phylogenetic analysis of the hCoV-19/USA/CT-CVMDL-Dog-1/2021 (CT_Dog/2021) virus were conducted to identify the origin and lineage of the virus. The CT_Dog/2021 virus belonged to the GH/B1.2. genetic lineage and was genetically similar to SARS-CoV-2 identified in humans in the U.S. during the winter of 2020-2021. However, it was not related to other SARS-CoV-2 variants identified from companion animals in the U.S. It contained both the D614G in spike and P323L in nsp12 substitutions, which have become the dominant mutations in the United States. The continued sporadic detections of SARS-CoV-2 in companion animals warrant public health concerns about the zoonotic potential of SARS-CoV-2 and enhance our collective understanding of the epidemiology of the virus.
Feigin, CY;Moreno, JA;Ramos, R;Mereby, SA;Alivisatos, A;Wang, W;van Amerongen, R;Camacho, J;Rasweiler, JJ;Behringer, RR;Ostrow, B;Plikus, MV;Mallarino, R;
PMID: 36961889 | DOI: 10.1126/sciadv.ade7511
Lateral flight membranes, or patagia, have evolved repeatedly in diverse mammalian lineages. While little is known about patagium development, its recurrent evolution may suggest a shared molecular basis. By combining transcriptomics, developmental experiments, and mouse transgenics, we demonstrate that lateral Wnt5a expression in the marsupial sugar glider (Petaurus breviceps) promotes the differentiation of its patagium primordium. We further show that this function of Wnt5a reprises ancestral roles in skin morphogenesis predating mammalian flight and has been convergently used during patagium evolution in eutherian bats. Moreover, we find that many genes involved in limb development have been redeployed during patagium outgrowth in both the sugar glider and bat. Together, our findings reveal that deeply conserved genetic toolkits contribute to the evolutionary transition to flight in mammals.
Journal of clinical pathology
Humphries, MP;Bingham, V;Abdullah Sidi, F;Craig, S;Lara, B;El-Daly, H;O'Doherty, N;Maxwell, P;Lewis, C;McQuaid, S;Lyness, J;James, J;Snead, DRJ;Salto-Tellez, M;
PMID: 36717223 | DOI: 10.1136/jcp-2022-208525
Interrogation of immune response in autopsy material from patients with SARS-CoV-2 is potentially significant. We aim to describe a validated protocol for the exploration of the molecular physiopathology of SARS-CoV-2 pulmonary disease using multiplex immunofluorescence (mIF).The application of validated assays for the detection of SARS-CoV-2 in tissues, originally developed in our laboratory in the context of oncology, was used to map the topography and complexity of the adaptive immune response at protein and mRNA levels.SARS-CoV-2 is detectable in situ by protein or mRNA, with a sensitivity that could be in part related to disease stage. In formalin-fixed, paraffin-embedded pneumonia material, multiplex immunofluorescent panels are robust, reliable and quantifiable and can detect topographic variations in inflammation related to pathological processes.Clinical autopsies have relevance in understanding diseases of unknown/complex pathophysiology. In particular, autopsy materials are suitable for the detection of SARS-CoV-2 and for the topographic description of the complex tissue-based immune response using mIF.
The Journal of physiology
Peltekian, L;Gasparini, S;Fazan, FS;Karthik, S;Iverson, G;Resch, JM;Geerling, JC;
PMID: 37291801 | DOI: 10.1113/JP283169
In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.
DETERMINATION OF SINGLE NUCLEOTIDE POLYMORPHISM (RS566926) OF WNT5A IN NONSYNDROMIC CLEFT LIP AND PALATE IN A PAKISTANI POPULATION
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Anjum, R;Mehmood, S;Nagi, A;Shahzad, M;Chuadhry, S;
| DOI: 10.1016/j.oooo.2021.03.042
Background Orofacial clefts are the most common birth defects affecting 1 in 750 live births worldwide. Various genetic loci to be involved in nonsyndromic cleft lip and palate has been identified with a variation among populations. Wnt5a is expressed in the frontonasal prominence and maxillary process, which fuse to form the primary palate. Therefore, its dysregulation can lead to certain birth defects along with other diseases. Single nucleotide polymorphism (rs566926) in Wnt5A shows a significant association with nonsyndromic cleft lip and palate in Brazilian and European American populations. Objective The aim of the present study was to describe single nucleotide polymorphism (SNP; rs566926) in patients with nonsyndromic cleft lip and palate in a Pakistani population. Methods This study was conducted on 120 patients with nonsyndromic cleft lip and palate. Demographics and phenotypes were noted. Blood samples were collected in ethylenediaminetetraacetic acid vials. DNA was extracted followed by conventional polymerase chain reaction. SNP (566926) was determined by Sanger sequencing. Data were analyzed using NCBI Blast and SPSS (24.0). Results The mean age of n = 30 patients was 51.33 ± 61.33 months. Sixty percent were male and 40% were female. Regarding cleft types, 70% were both cleft lip and palate, 26% cleft lip only, and 3.3% cleft palate only. Heterozygous polymorphism (T/G) was seen in 33.3% of patients with both cleft lip and palate with bilateral involvement and heterozygous polymorphism (T) was seen in 16.6%. Conclusions SNP in the WNT5A gene is associated with cleft lip and palate, supporting its involvement in pathogenesis of cleft lip and palate. Further studies are recommended to determine the role of Wnt5a genes during craniofacial development.
Choudhary, S;Kanevsky, I;Yildiz, S;Sellers, RS;Swanson, KA;Franks, T;Rathnasinghe, R;Munoz-Moreno, R;Jangra, S;Gonzalez, O;Meade, P;Coskran, T;Qian, J;Lanz, TA;Johnson, JG;Tierney, CA;Smith, JD;Tompkins, K;Illenberger, A;Corts, P;Ciolino, T;Dormitzer, PR;Dick, EJ;Shivanna, V;Hall-Ursone, S;Cole, J;Kaushal, D;Fontenot, JA;Martinez-Romero, C;McMahon, M;Krammer, F;Schotsaert, M;García-Sastre, A;
PMID: 35128980 | DOI: 10.1177/01926233211072767
Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.
Mizutani M, Wu JC, Nusse R.
PMID: - | DOI: 10.1161/JAHA.115.002457
Background The adult mammalian heart responds to cardiac injury by formation of persistent fibrotic scar that eventually leads to heart failure. In contrast, the neonatal mammalian heart reacts to injury by the development of transient fibrotic tissue that is eventually replaced by regenerated cardiomyocytes. How fibrosis occurs in the neonatal mammalian heart remains unknown. To start elucidating the molecular underpinnings of neonatal cardiac fibrosis, we investigated Wnt signaling in the neonatal heart after cryoinjury.
Methods and Results Using expression of the Wnt target gene Axin2 as an indicator of Wnt/β‐catenin signaling activation, we discovered that epicardial cells in the ventricles are responsive to Wnt in the uninjured neonatal heart. Lineage‐tracing studies of these Wnt‐responsive epicardial cells showed that they undergo epithelial‐to‐mesenchymal transition and infiltrate into the subepicardial space and exhibit fibroblast phenotypes after injury. In addition, we showed that—similar to adult ischemic injury—neonatal cryoinjury results in activation of Wnt signaling in cardiac fibroblasts near injured areas. Furthermore, through in situ hybridization of all 19 Wnt ligands in injured neonatal hearts, we observed upregulation of Wnt ligands (Wnt2b, Wnt5a, and Wnt9a) that had not been implicated in the adult cardiac injury response.
Conclusions These results demonstrate that cryoinjury in neonatal heart leads to the formation of fibrotic tissue that involves Wnt‐responsive epicardial cells undergoing epithelial‐to‐mesenchymal transition to give rise to fibroblasts and activation of Wnt signaling in resident cardiac fibroblasts.
Diffuse trophoblast damage is the hallmark of SARS-CoV-2-associated fetal demise
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Garrido-Pontnou, M;Navarro, A;Camacho, J;Crispi, F;Alguacil-Guillén, M;Moreno-Baró, A;Hernandez-Losa, J;Sesé, M;Ramón Y Cajal, S;Garcia Ruíz, I;Serrano, B;Garcia-Aguilar, P;Suy, A;Ferreres, JC;Nadal, A;
PMID: 34006935 | DOI: 10.1038/s41379-021-00827-5
Placental pathology in SARS-CoV-2-infected pregnancies seems rather unspecific. However, the identification of the placental lesions due to SARS-CoV-2 infection would be a significant advance in order to improve the management of these pregnancies and to identify the mechanisms involved in a possible vertical transmission. The pathological findings in placentas delivered from 198 SARS-CoV-2-positive pregnant women were investigated for the presence of lesions associated with placental SARS-CoV-2 infection. SARS-CoV-2 infection was investigated in placental tissues through immunohistochemistry, and positive cases were further confirmed by in situ hybridization. SARS-CoV-2 infection was also investigated by RT-PCR in 33 cases, including all the immunohistochemically positive cases. Nine cases were SARS-CoV-2-positive by immunohistochemistry, in situ hybridization, and RT-PCR. These placentas showed lesions characterized by villous trophoblast necrosis with intervillous space collapse and variable amounts of mixed intervillous inflammatory infiltrate and perivillous fibrinoid deposition. Such lesions ranged from focal to massively widespread in five cases, resulting in intrauterine fetal death. Two of the stillborn fetuses showed some evidence of SARS-CoV-2 positivity. The remaining 189 placentas did not show similar lesions. The strong association between trophoblastic damage and placenta SARS-CoV-2 infection suggests that this lesion is a specific marker of SARS-CoV-2 infection in placenta. Diffuse trophoblastic damage, massively affecting chorionic villous tissue, can result in fetal death associated with COVID-19 disease.
Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte
Cardiovascular diabetology
D'Onofrio, N;Scisciola, L;Sardu, C;Trotta, MC;De Feo, M;Maiello, C;Mascolo, P;De Micco, F;Turriziani, F;Municinò, E;Monetti, P;Lombardi, A;Napolitano, MG;Marino, FZ;Ronchi, A;Grimaldi, V;Hermenean, A;Rizzo, MR;Barbieri, M;Franco, R;Campobasso, CP;Napoli, C;Municinò, M;Paolisso, G;Balestrieri, ML;Marfella, R;
PMID: 33962629 | DOI: 10.1186/s12933-021-01286-7
About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.
Bräuninger, H;Stoffers, B;Fitzek, ADE;Meißner, K;Aleshcheva, G;Schweizer, M;Weimann, J;Rotter, B;Warnke, S;Edler, C;Braun, F;Roedl, K;Scherschel, K;Escher, F;Kluge, S;Huber, TB;Ondruschka, B;Schultheiss, HP;Kirchhof, P;Blankenberg, S;Püschel, K;Westermann, D;Lindner, D;
PMID: 34647998 | DOI: 10.1093/cvr/cvab322
Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). MACE-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset "Heart Cell Atlas" and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis.MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The GO term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response.This study reveals, that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte-destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.Cardiac injury can be documented in COVID-19, regardless the direct cardiac virus infection and is known to be associated with outcome. However, the direct virus infection of the myocardium leads to transcriptomic alterations and might therefore additionally contribute to pathophysiological processes in COVID-19. Therefore, consequences of cardiac virus infection need to be investigated in future studies, since they might also contribute to long-term effects in case of survival.
Tissue factor upregulation is associated with SARS-CoV-2 in the lungs of COVID-19 patients
Journal of thrombosis and haemostasis : JTH
Subrahmanian, S;Borczuk, A;Salvatore, S;Fung, KM;Merrill, JT;Laurence, J;Ahamed, J;
PMID: 34236752 | DOI: 10.1111/jth.15451
A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis.We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19.We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs.Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2 = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P < .001) higher than control lungs. Fibrin thrombi and thrombi positive for platelet factor 4 (PF4) were found in close proximity to regions expressing TF in COVID-19 ARDS lung, and correlated with TF expression (fibrin, R2 = .52, P < .001; PF4, R2 = .59, P < .001).These data suggest that upregulation of TF expression is associated with thrombus formation in COVID-19 lungs and could be a key therapeutic target. Correlation of TF expression with SARS-CoV-2 in lungs of COVID-19 patients also raises the possibility of direct TF induction by the virus.
WNT signaling in pre-granulosa cells is required for ovarian folliculogenesis and female fertility
Development (Cambridge, England)
Habara, O;Logan, CY;Kanai-Azuma, M;Nusse, R;Takase, HM;
PMID: 33914868 | DOI: 10.1242/dev.198846
In mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless (Wls) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of β-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.
