Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports

Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphology and functionality. However, due to the optical properties of the filter materials and the cells’ columnar shape, cell quality is hard to assess via light microscopy. In this study, we aim to evaluate transepithelial electrical resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 w to achieve diverse culture qualities, and TEER was measured before processing samples for histology. Culture quality was scored using morphological criteria (presence of cilia, confluence and cell polarity). We furthermore analyzed the correlation between cellular height (as a measure of apical–basal polarization) and TEER in fully differentiated routine cultures (biological variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm2. Only 5 % of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. We unveiled a highly significant (P < 0.0001) negative linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical–basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium

Parents' future visions for their autistic transition-age youth: hopes and expectations

Researchers have documented that young adults with autism spectrum disorder have poor outcomes in employment, post-secondary education, social participation, independent living, and community participation. There is a need to further explore contributing factors to such outcomes to better support successful transitions to adulthood. Parents play a critical role in transition planning, and parental expectations appear to impact young adult outcomes for autistic individuals. The aim of this study was to explore how parents express their future visions (i.e. hopes and expectations) for their autistic transition-age youth. Data were collected through focus groups and individual interviews with 18 parents. Parents' hopes and expectations focused on eight primary domains. In addition, parents often qualified or tempered their stated hope with expressions of fears, uncertainty, realistic expectations, and the perceived lack of guidance. We discuss our conceptualization of the relations among these themes and implications for service providers and research.Accepted manuscrip

Sex-specific computational models of the spontaneously hypertensive rat kidneys: factors affecting nitric oxide bioavailability

Sex-specific computational models of the spontaneously hypertensive rat kidneys: factors affecting nitric oxide bioavailability. Am J Physiol Renal Physiol 313: F174 –F183, 2017. First published March 29, 2017; doi:10.1152/ajprenal.00482.2016.—The goals of this study were to 1) develop a computational model of solute transport and oxygenation in the kidney of the female spontaneously hypertensive rat (SHR), and 2) apply that model to investigate sex differences in nitric oxide (NO) levels in SHR and their effects on medullary oxygenation and oxidative stress. To accomplish these goals, we first measured NO synthase (NOS) 1 and NOS3 protein expression levels in total renal microvessels of male and female SHR. We found that the expression of both NOS1 and NOS3 is higher in the renal vasculature of females compared with males. To predict the implications of that finding on medullary oxygenation and oxidative stress levels, we developed a detailed computational model of the female SHR kidney. The model was based on a published male kidney model and represents solute transport and the biochemical reactions among O2, NO, and superoxide (O2 ) in the renal medulla. Model simulations conducted using both male and female SHR kidney models predicted significant radial gradients in interstitial fluid oxygen tension (PO2) and NO and O2 concentration in the outer medulla and upper inner medulla. The models also predicted that increases in endothelial NO-generating capacity, even when limited to specific vascular segments, may substantially raise medullary NO and PO2 levels. Other potential sex differences in SHR, including O2 production rate, are predicted to significantly impact oxidative stress levels, but effects on NO concentration and PO2 are limited.This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-106102 to A. T. Layton, and by American Heart Association Grant 14GRNT20480199 to J. C. Sullivan. (R01-DK-106102 - National Institute of Diabetes and Digestive and Kidney Diseases; 14GRNT20480199 - American Heart Association)Accepted manuscrip

CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate

Does Inflation Provide Natural Initial Conditions for the Universe?

If our universe underwent inflation, its entropy during the inflationary phase was substantially lower than it is today. Because a low-entropy state is less likely to be chosen randomly than a high-entropy one, inflation is unlikely to arise through randomly-chosen initial conditions. To resolve this puzzle, we examine the notion of a natural state for the universe, and argue that it is a nearly-empty spacetime. If empty space has a small vacuum energy, however, inflation can begin spontaneously in this background. This scenario explains why a universe like ours is likely to have begun via a period of inflation, and also provides an origin for the cosmological arrow of time.Comment: Submitted to Gravity Research Foundation Essay Competition; based on hep-th/041027

T Cell Regulation of Antibody Responses to Infection and Immunization

T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 infection, SARS-CoV-2 vaccination, and influenza A virus infection. Even though Tfh-independent antibodies to SARS-CoV-2 had evidence of reduced somatic hypermutation, they were still high-affinity, durable, and reactive against diverse spike-derived epitopes and were capable of neutralizing both homologous SARS-CoV-2 and the B.1.351 (beta) variant of concern. Indeed, we found by epitope mapping and BCR sequencing that Tfh cells focused the B cell response and therefore, in the absence of Tfh cells, a more diverse clonal repertoire was maintained. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GC-derived antibodies that might compensate for GCs damaged by viral inflammation.Furthermore, we sought to reconcile the roles of Tfh cell-derived IL-4 as both a pro-survival factor for highly proliferative GC B cells as well as a switch factor for IgE and IgG1. Due to its potent effects on B cell proliferation and differentiation, IL-4 is considered a canonical Tfh cell cytokine, produced even during antimicrobial responses that elicit little IgG1 and no IgE. However, given that IL-4 is also a switch factor that is sufficient for IgE induction, this raises the question of how Tfh cells produce IL-4 during type 1 immune responses without aberrantly inducing IgE. We first clarified the role of Tfh cell-derived IL-4 during type 1 immune responses, finding that it was required for IgG1 switching in response to immunization with lipopolysaccharide and haptenated protein antigen as well as influenza A virus infection; however, GC B cell formation and plasmablast differentiation were unaffected by the loss of IL-4 from Tfh cells. In addition, we found that Tfh cells during type 1 immune responses generated minimal IL-4 protein, with levels of IL-4 tightly regulated by both transcriptional and post-transcriptional mechanisms. These data support the role of Tfh cell-derived IL-4 as a rheostat for the appropriate induction of IgG1 versus IgE antibodies during type 1 and type 2 immune responses, rather than as a pro-survival factor for GC B cells. Finally, when public health officials raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating COVID-19 at the start of the pandemic, we sought to determine whether and how NSAIDs could affect COVID-19 pathogenesis. NSAIDs affect the production of prostaglandins, which play diverse biological roles in homeostasis and inflammatory responses. Thus, it is plausible that NSAIDs could affect COVID-19 pathogenesis in multiple ways, including modifying expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2; regulating replication of SARS-CoV-2 in host cells; and modulating the immune response to SARS-CoV-2. We found that NSAID treatment had no effect on ACE2 expression, viral entry, or viral replication. However, NSAIDs did affect the immune response to SARS-CoV-2 by impairing the production of proinflammatory cytokines as well as early neutralizing antibodies. Our findings therefore indicate that NSAID treatment may affect COVID-19 outcomes by dampening the inflammatory response and the production of protective antibodies, which also has implications for NSAID use during SARS-CoV-2 vaccination