Immunofluorescence studies on the expression of the SARS-CoV-2 receptors in human term placenta

Until September 2021, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2; COVID-19) pandemic caused over 217 million infections and over 4.5 million deaths. In pregnant women the risk factors for the need of intensive care treatment are generally the same as in the overall population. Of note, COVID-19+ women deliver earlier than COVID-19- women, and the risk for severe neonatal and perinatal morbidity and mortality is significantly higher. The probability and pathways of vertical transmission of the virus from the pregnant woman to the fetus are highly controversial. Recent data have shown that 54 (13%) of 416 neonates born to COVID-19-positive women were infected. Here, we investigated term placentas collected before the SARS-CoV-2 pandemic and studied the main COVID-19 receptors ACE2, TMPRSS2, as well as NRP1. We performed qPCR and immunofluorescence on cryosections in combination with markers for syncytiotrophoblast, endothelial cells, macrophages and stromal cells. The qPCR studies showed expression of both the truncated delta form of ACE2, which does not bind the COVID-19 spike protein, and the long form. The ACE2 antibody used does not distinguish between the two forms. We did not observe expression of the canonical SARS-CoV-2 entry machinery on syncytio- and cytotrophoblast. ACE2 and TMPRSS2 are co-expressed in a subpopulation of stromal cells, which in part are CD68-positive macrophages. NRP1 is localized to endothelial cells. In sum, the term placenta is not an organ that directly favors vertical transmission of COVID-19, however, microtraumas and placentitis may weaken its barrier function.

Macrophage Activation in the Dorsal Root Ganglion in Rats Developing Autotomy after Peripheral Nerve Injury

Autotomy, self-mutilation of a denervated limb, is common in animals after peripheral nerve injury (PNI) and is a reliable proxy for neuropathic pain in humans. Understanding the occurrence and treatment of autotomy remains challenging. The objective of this study was to investigate the occurrence of autotomy in nude and Wistar rats and evaluate the differences in macrophage activation and fiber sensitization contributing to the understanding of autotomy behavior. Autotomy in nude and Wistar rats was observed and evaluated 6 and 12 weeks after sciatic nerve repair surgery. The numbers of macrophages and the types of neurons in the dorsal root ganglion (DRG) between the two groups were compared by immunofluorescence studies. Immunostaining of T cells in the DRG was also assessed. Nude rats engaged in autotomy with less frequency than Wistar rats. Autotomy symptoms were also relatively less severe in nude rats. Immunofluorescence studies revealed increased macrophage accumulation and activation in the DRG of Wistar rats. The percentage of NF200+ neurons was higher at 6 and 12 weeks in Wistar rats compared to nude rats, but the percentage of CGRP+ neurons did not differ between two groups. Additionally, macrophages were concentrated around NF200-labeled A fibers. At 6 and 12 weeks following PNI, CD4+ T cells were not found in the DRG of the two groups. The accumulation and activation of macrophages in the DRG may account for the increased frequency and severity of autotomy in Wistar rats. Our results also suggest that A fiber neurons in the DRG play an important role in autotomy.

Amyloidoses – pathogenesis, classification, diagnosis

Amyloidoses – also known as amyloidosis or betafibrillosis – a diverse group of diseases in which amorphous protein with a changed conformational structure is deposited extracellularly, leading to the failure of many organs. The basic classifications of amyloidoses take into account: the type of precursor protein, the division into generalized (systemic) amyloidoses, in which amyloid deposits accumulate in many organs, vessel walls and connective tissue (e.g. AL amyloidosis) and local (localized) amyloidoses – limited to only one organ (e.g. corneal amyloidosis) as well as congenital and acquired diseases. Symptoms of amyloidosis are non-specific and not very characteristic, moreover, their severity depends on the type of disease and organ involvement. The diagnosis of amyloidosis should be considered in patients with heart failure without coronary artery disease, with neuropathy, or proteinuria or hepatomegaly of unclear origin. Diagnosis of amyloidosis is based on the evaluation of tissue biopsy samples and the presence of abnormal proteins, i.e. amyloid, or on the fibrillary evaluation confirmation of the filamentous nature of amyloid deposits using electron microscopy. The next step is differential diagnosis and amyloid differential identification, which is based on immunohistochemical and immunofluorescence studies using labeled antibodies. The "gold standard" used in typing amyloidosis and identifying an amyloidogenic protein is mass spectrometry. Laboratory tests are used to assess organ involvement, which is the basis of the prognostic classification.

The human sperm basal body is a complex centrosome important for embryo pre-implantation development

The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis has not been directly addressed so far. Using proteomics and immunofluorescence studies we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetically activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.

Deciphering the mechanoresponsive role of β-catenin in keratoconus epithelium

Keratoconus (KC) is a corneal dystrophy characterized by progressive ectasia that leads to severe visual impairment and remains one of the leading indications for corneal transplantation. The etiology is believed to be multifactorial and alterations have been documented in the biomechanical, biochemical and ultrastructural characteristics of the cornea. While the exact site of disease origin is still debated, changes in the corneal epithelium are believed to occur even before the disease is clinically manifested. In this study we investigate the possible role of β-catenin as mechanotransducer in KC corneal epithelium. The sheets of corneal epithelium removed from keratoconic eyes when they underwent collagen crosslinking as a therapeutic procedure were used for this study. The healthy corneal epithelium of patients undergoing Laser Refractive Surgery for the correction of their refractive error, served as controls. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyse the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, and Rac123. Co-immunoprecipitation of β-catenin followed by mass spectrometry of KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. Histology data reported breaks in the Bowman’s layer in KC patients. We hypothesize that these breaks expose the epithelium to the keratoconic corneal stroma, which, is known to have a decreased elastic modulus and that β-catenin acts as a mechanotransducer that induces structural changes such as loss of polarity (Syntaxin3) and barrier function (ZO1) through membrane delocalization. The results of our study strongly suggest that β-catenin could be a putative mechanotransducer in KC epithelium, thus supporting our hypothesis.

Mechanisms of hyperexcitability in Alzheimer’s disease hiPSC-derived neurons and cerebral organoids vs isogenic controls

Human Alzheimer’s disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However, the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity, we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids, we found increased excitatory bursting activity, which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability.

Deciphering the mechanoresponsive role of β-catenin in Keratoconus epithelium

Keratoconus (KC) a disease with established biomechanical instability of the corneal stroma, is an ideal platform to identify key proteins involved in mechanosensing. This study aims to investigate the possible role of β-catenin as mechanotransducer in KC epithelium. KC patients were graded as mild, moderate or severe using Amsler Krumeich classification. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyze the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, Rac123. Co-immunoprecipitation (Co-IP) of β-catenin followed by mass spectrometry of mild KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. We observed down regulation of E-cadherin, α-catenin, ZO1 and upregulation of Cyclin D1, α-actinin and RhoA in KC corneal epithelium. β-catenin Co-IP from mild KC epithelium identified new interacting partners such as StAR-related lipid transfer protein3, Dynamin-1-like protein, Cardiotrophin-1,Musculin, Basal cell adhesion molecule and Protocadherin Fat 1.β-catenin localization was altered in KC which was validatedin vitro, using control corneal epithelium grown on different substrate stiffness. β-catenin localization is dependent upon the elastic modulus of the substrate and acts as mechanotransducer by altering its interaction and regulating the barrier function in corneal epithelium.