A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types.

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

A direct comparison of mouse and human intestinal development using epithelial gene expression patterns.

BACKGROUND: Preterm infants are susceptible to unique pathology due to their immaturity. Mouse models are commonly used to study immature intestinal disease, including necrotizing enterocolitis (NEC). Current NEC models are performed at a variety of ages, but data directly comparing intestinal developmental stage equivalency between mice and humans are lacking. METHODS: Small intestines were harvested from C57BL/6 mice at 3-4 days intervals from birth to P28 (n = 8 at each age). Preterm human small intestine samples representing 17-23 weeks of completed gestation were obtained from the University of Pittsburgh Health Sciences Tissue Bank, and at term gestation during reanastamoses after resection for NEC (n = 4-7 at each age). Quantification of intestinal epithelial cell types and messenger RNA for marker genes were evaluated on both species. RESULTS: Overall, murine and human developmental trends over time are markedly similar. Murine intestine prior to P10 is most similar to human fetal intestine prior to viability. Murine intestine at P14 is most similar to human intestine at 22-23 weeks completed gestation, and P28 murine intestine is most similar to human term intestine. CONCLUSION: Use of C57BL/6J mice to model the human immature intestine is reasonable, but the age of mouse chosen is a critical factor in model development.

Tumor necrosis factor induces developmental stage-dependent structural changes in the immature small intestine.

BACKGROUND: Premature infants are commonly subject to intestinal inflammation. Since the human small intestine does not reach maturity until term gestation, premature infants have a unique challenge, as either acute or chronic inflammation may alter the normal development of the intestinal tract. Tumor necrosis factor (TNF) has been shown to acutely alter goblet cell numbers and villus length in adult mice. In this study we tested the effects of TNF on villus architecture and epithelial cells at different stages of development of the immature small intestine. METHODS: To examine the effects of TNF-induced inflammation, we injected acute, brief, or chronic exposures of TNF in neonatal and juvenile mice. RESULTS: TNF induced significant villus blunting through a TNF receptor-1 (TNFR1) mediated mechanism, leading to loss of villus area. This response to TNFR1 signaling was altered during intestinal development, despite constant TNFR1 protein expression. Acute TNF-mediated signaling also significantly decreased Paneth cells. CONCLUSIONS: Taken together, the morphologic changes caused by TNF provide insight as to the effects of inflammation on the developing intestinal tract. Additionally, they suggest a mechanism which, coupled with an immature immune system, may help to explain the unique susceptibility of the immature intestine to inflammatory diseases such as NEC.

Host defense peptides human ß defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of preterm infant morbidity and mortality. Treatment for NEC is limited and non-targeted, which makes new treatment and prevention strategies critical. Host defense peptides (HDPs) are essential components of the innate immune system and have multifactorial mechanisms in host defense. LL-37 and hBD2 are two HDPs that have been shown in prior literature to protect from neonatal sepsis-induced mortality or adult inflammatory bowel disease, respectively. Therefore, this article sought to understand if these two HDPs could influence NEC severity in murine preclinical models. NEC was induced in P14-16 C57Bl/6 mice and HDPs were provided as a pretreatment or treatment. Both LL-37 and hBD2 resulted in decreased NEC injury scores as a treatment and hBD2 as a pretreatment. Our data suggest LL-37 functions through antimicrobial properties, while hBD2 functions through decreases in inflammation and improvement of intestinal barrier integrity.

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels.

Acid sensing ion channels (ASICs), Ca(2+) and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.

The association between dietary fiber and infertility among US Women: The National Health and Nutrition Examination Survey, 2013-2018.

Introduction: Background: studies have shown that dietary factors are linked to female infertility, but the relation between dietary fiber consumption and infertility has not been proven. The purpose of this research was to investigate whether there is an independent association between dietary fiber intake and infertility in American women. Material and methods: a secondary analysis of the National Health and Nutrition Examination Survey (NHANES) dataset has been conducted, covering three cycles from 2013 to 2018. A total of 3,497 participants were included in the data analysis. The independent and dependent variables of interest were dietary fiber intake and infertility. Covariates included sociodemographic, questionnaire, diet, and physical examination data. Multiple logistic regression and sensitivity analyses were performed to investigate the relationship of dietary fiber intake with infertility. Results: each additional increase in log10 dietary fiber consumption was associated with a 32 % lower risk of infertility (OR, 0.68; 95 % CI, 0.48-0.96). The outcome is still robust in the minimally as well as the fully adjusted model. The possibility of a nonlinear association between dietary fiber intake and infertility was ruled out by the GAM model and smooth curve fitting. The results showed that there is an inverse linear correlation between dietary fiber intake and infertility. Conclusions: the association between intake of dietary fiber and infertility is linear, and increasing dietary fiber intake may be beneficial for lower infertility.

Introducción: Antecedentes: el consumo de fibra dietética es un factor importante en la infertilidad femenina. El objetivo del estudio fue investigar si existe una relación independiente entre el consumo de fibra dietética y la infertilidad en las mujeres estadounidenses. Material y métodos: se realizó un análisis secundario del conjunto de datos de la encuesta nacional de salud y nutrición (NHANES), que abarcó tres ciclos entre 2013 y 2018. Se incluyeron 3497 participantes para el análisis de datos. Las variables independientes y dependientes asociadas fueron la ingesta de fibra dietética y la infertilidad. Las covariables incluyeron datos sociodemográficos, de los cuestionarios, dietéticos y médicos. Se realizó un análisis de regresión logística multivariada y un análisis de sensibilidad para determinar la relación entre el consumo de fibra dietética y la infertilidad. Resultados: cada aumento log10 en el consumo de fibra dietética se asoció con una reducción del 32 % en el riesgo de infertilidad (OR: 0,68; intervalo de confianza del 95 %: 0,48-0,96). En el modelo mínimo y totalmente ajustado, los resultados siguen siendo robustos. El modelo GAM y el ajuste de curvas suavizadas descartaron la posibilidad de una relación no lineal entre la ingesta de fibra dietética y la infertilidad. Los resultados muestran una correlación lineal negativa entre la ingesta de fibra dietética y la infertilidad. Conclusiones: se observó una relación lineal entre la ingesta de fibra dietética y la infertilidad. El aumento de la ingesta de fibra dietética redujo la incidencia de la infertilidad.

Dextran-stabilized Fe-Mn bimetallic oxidase-like nanozyme for total antioxidant capacity assay of fruit and vegetable food.

The total antioxidant capacity (TAC) has become increasingly vital for evaluating antioxidant food quality in the field of healthcare. Herein, a convenient and sensitive method for TAC assay was proposed based on the absorbance difference of reaction systems between various antioxidants existed in food and Dex-FeMnzyme/oxTMB. Under the optimum condition, the limit of detection (LOD) of the colorimetric sensor was 1.17 µM with the linear concentration range from 1 µM to 30 µM. The analysis results demonstrated the excellent feasibility of practical application in fruit and vegetable food, which offered a new avenue for the establishment of colorimetric biosensors.

Acid-sensing ion channels interact with and inhibit BK K+ channels.

Acid-sensing ion channels (ASICs) are neuronal non-voltage-gated cation channels that are activated when extracellular pH falls. They contribute to sensory function and nociception in the peripheral nervous system, and in the brain they contribute to synaptic plasticity and fear responses. Some of the physiologic consequences of disrupting ASIC genes in mice suggested that ASIC channels might modulate neuronal function by mechanisms in addition to their H(+)-evoked opening. Within ASIC channel's large extracellular domain, we identified sequence resembling that in scorpion toxins that inhibit K(+) channels. Therefore, we tested the hypothesis that ASIC channels might inhibit K(+) channel function by coexpressing ASIC1a and the high-conductance Ca(2+)- and voltage-activated K(+) (BK) channel. We found that ASIC1a associated with BK channels and inhibited their current. Reducing extracellular pH disrupted the association and relieved the inhibition. BK channels, in turn, altered the kinetics of ASIC1a current. In addition to BK, ASIC1a inhibited voltage-gated Kv1.3 channels. Other ASIC channels also inhibited BK, although acidosis-dependent relief of inhibition varied. These results reveal a mechanism of ion channel interaction and reciprocal regulation. Finding that a reduced pH activated ASIC1a and relieved BK inhibition suggests that extracellular protons may enhance the activity of channels with opposing effects on membrane voltage. The wide and varied expression patterns of ASICs, BK, and related K(+) channels suggest broad opportunities for this signaling system to alter neuronal function.

Feeding Formula Eliminates the Necessity of Bacterial Dysbiosis and Induces Inflammation and Injury in the Paneth Cell Disruption Murine NEC Model in an Osmolality-Dependent Manner.

Necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in preterm infants. Formula feeding is a risk factor for NEC and osmolality, which is increased by the fortification that is required for adequate growth of the infant, has been suggested as a potential cause. Our laboratory has shown that Paneth cell disruption followed by induction of dysbiosis can induce NEC-like pathology in the absence of feeds. We hypothesized adding formula feeds to the model would exacerbate intestinal injury and inflammation in an osmolality-dependent manner. NEC-like injury was induced in 14-16 day-old C57Bl/6J mice by Paneth cell disruption with dithizone or diphtheria toxin, followed by feeding rodent milk substitute with varying osmolality (250-1491 mOsm/kg H2O). Animal weight, serum cytokines and osmolality, small intestinal injury, and cecal microbial composition were quantified. Paneth cell-disrupted mice fed formula had significant NEC scores compared to controls and no longer required induction of bacterial dysbiosis. Significant increases in serum inflammatory markers, small intestinal damage, and overall mortality were osmolality-dependent and not related to microbial changes. Overall, formula feeding in combination with Paneth cell disruption induced NEC-like injury in an osmolality-dependent manner, emphasizing the importance of vigilance in designing preterm infant feeds.

Fetal exposure to maternal inflammation interrupts murine intestinal development and increases susceptibility to neonatal intestinal injury.

Fetal exposure to chorioamnionitis can impact the outcomes of the developing fetus both at the time of birth and in the subsequent neonatal period. Infants exposed to chorioamnionitis have a higher incidence of gastrointestinal (GI) pathology, including necrotizing enterocolitis (NEC); however, the mechanism remains undefined. To simulate the fetal exposure to maternal inflammation (FEMI) induced by chorioamnionitis, pregnant mice (C57BL/6J, IL-6-/-, RAG-/- or TNFR1-/-) were injected intraperitoneally on embryonic day (E)15.5 with lipopolysaccharide (LPS; 100 µg/kg body weight). Pups were delivered at term, and reared to postnatal day (P)0, P7, P14, P28 or P56. Serum and intestinal tissue samples were collected to quantify growth, inflammatory markers, histological intestinal injury, and goblet and Paneth cells. To determine whether FEMI increased subsequent susceptibility to intestinal injury, a secondary dose of LPS (100 µg/kg body weight) was given on P5, prior to tissue harvesting on P7. FEMI had no effect on growth of the offspring or their small intestine. FEMI significantly decreased both goblet and Paneth cell numbers while simultaneously increasing serum levels of IL-1ß, IL-10, KC/GRO (CXCL1 and CXCL2), TNF and IL-6. These alterations were IL-6 dependent and, importantly, increased susceptibility to LPS-induced intestinal injury later in life. Our data show that FEMI impairs normal intestinal development by decreasing components of innate immunity and simultaneously increasing markers of inflammation. These changes increase susceptibility to intestinal injury later in life and provide novel mechanistic data to potentially explain why preterm infants exposed to chorioamnionitis prior to birth have a higher incidence of NEC and other GI disorders.

Loss of murine Paneth cell function alters the immature intestinal microbiome and mimics changes seen in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.

Lipopolysaccharide-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood.

PROBLEM: Premature birth complicates 10%-12% of deliveries. Infection and inflammation are the most common etiologies and are associated with increased offspring morbidity and mortality. We hypothesize that lipopolysaccharide (LPS)-induced maternal inflammation causes direct placenta injury and subsequent injury to the fetal intestine. METHOD OF STUDY: Pregnant C57Bl6 mice were injected intraperitoneally on day 15.5 with 100 µg/kg LPS or saline. Maternal serum, amniotic fluid, placental samples, and ileal samples of offspring were obtained assessed for inflammation and/or injury. Maternal placental ultrasounds were performed. Placental DNA was isolated for microbiome analysis. RESULTS: Maternal injection with LPS caused elevated IL-1ß, IL-10, IL-6, KC-GRO, and TNF. Placental tissue showed increased IL-1ß, IL-6, and KC-GRO and decreased IL-10, but no changes were observed in amniotic fluid. Placental histology demonstrated LPS-induced increases in mineralization and necrosis, but no difference in placental blood flow. Most placentas had no detectable microbiome. Exposure to maternal LPS induced significant injury to the ilea of the offspring. CONCLUSION: Lipopolysaccharide causes a maternal inflammatory response that is mirrored in the placenta. Placental histology demonstrates structural changes; however, placental blood flow is preserved. LPS also induces an indirect intestinal injury in the offspring that lasts beyond the neonatal period.

Paneth-cell-disruption-induced necrotizing enterocolitis in mice requires live bacteria and occurs independently of TLR4 signaling.

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in premature infants. Both human surgical specimens and animal models suggest a potential involvement of Paneth cells in NEC pathogenesis. Paneth cells play critical roles in epithelial homeostasis, innate immunity and host-microbial interactions. Yet, the complex interplay between Paneth cell disruption, epithelial barrier dysfunction and microbial-driven inflammation remains unclear in the immature intestine. In this study, mucosal intestinal injury consistent with human NEC was induced in postnatal day 14-16 (P14-P16) mice by disrupting Paneth cells, followed by gavage with Klebsiella pneumonia. Mucosal injury was determined by histology, serum cytokine levels and epithelial barrier dysfunction. Toll-like receptor 4 (TLR4) activation was examined using protein expression, gene expression, and TLR4-/- mice. Finally, the role of bacteria was evaluated using heat-killed bacteria, conditioned media, Bacillus cereus and cecal slurries. We found that live bacteria were required to induce injury; however, TLR4 activation was not required. NEC induced by Paneth cell disruption results in altered localization of tight junction proteins and subsequent loss of barrier function. Prior research has shown a requirement for TLR4 activation to induce NEC-like damage. However, many infants develop NEC in the absence of Gram-negative rod bacteremia, raising the possibility that alternative pathways to intestinal injury exist. In this study, we show a previously unknown mechanism for the development of intestinal injury equivalent to that seen in human NEC and that is not dependent on TLR4 pathways. These data are congruent with the new hypothesis that NEC may be the consequence of several disease processes ending in a final common inflammatory pathway.

The association between dietary fiber and infertility among US women: The National Health and Nutrition Examination Survey, 2013-2018 / La relación entre la fibra dietética y la infertilidad femenina en los Estados unidos: encuesta nacional de salud y nutrición, 2013-2018

Background: studies have shown that dietary factors are linked to female infertility, but the relation between dietary fiber consumption and infertility has not been proven. The purpose of this research was to investigate whether there is an independent association between dietary fiber intake and infertility in American women. Material and methods: a secondary analysis of the National Health and NUTRITION Examination Survey (NHANES) dataset has been conducted, covering three cycles from 2013 to 2018. A total of 3,497 participants were included in the data analysis. The independent and dependent variables of interest were dietary fiber intake and infertility. Covariates included sociodemographic, questionnaire, diet, and physical examination data. Multiple logistic regression and sensitivity analyses were performed to investigate the relationship of dietary fiber intake with infertility. Results: each additional increase in log10 dietary fiber consumption was associated with a 32 % lower risk of infertility (OR, 0.68; 95 % CI, 0.48-0.96). The outcome is still robust in the minimally as well as the fully adjusted model. The possibility of a nonlinear association between dietary fiber intake and infertility was ruled out by the GAM model and smooth curve fitting. The RESULTS showed that there is an inverse linear correlation between dietary fiber intake and infertility. Conclusions: the association between intake of dietary fiber and infertility is linear, and increasing dietary fiber intake may be beneficial for lower infertility. (AU)

Antecedentes: el consumo de fibra dietética es un factor importante en la infertilidad femenina. El objetivo del estudio fue investigar si existe una relación independiente entre el consumo de fibra dietética y la infertilidad en las mujeres estadounidenses. Material y métodos: se realizó un análisis secundario del conjunto de datos de la encuesta nacional de salud y nutrición (NHANES), que abarcó tres ciclos entre 2013 y 2018. Se incluyeron 3497 participantes para el análisis de datos. Las variables independientes y dependientes asociadas fueron la ingesta de fibra dietética y la infertilidad. Las covariables incluyeron datos sociodemográficos, de los cuestionarios, dietéticos y médicos. Se realizó un análisis de regresión logística multivariada y un análisis de sensibilidad para determinar la relación entre el consumo de fibra dietética y la infertilidad. Resultados: cada aumento log10 en el consumo de fibra dietética se asoció con una reducción del 32 % en el riesgo de infertilidad (OR: 0,68; intervalo de confianza del 95 %: 0,48-0,96). En el modelo mínimo y totalmente ajustado, los resultados siguen siendo robustos. El modelo GAM y el ajuste de curvas suavizadas descartaron la posibilidad de una relación no lineal entre la ingesta de fibra dietética y la infertilidad. Los resultados muestran una correlación lineal negativa entre la ingesta de fibra dietética y la infertilidad. Conclusiones: se observó una relación lineal entre la ingesta de fibra dietética y la infertilidad. El aumento de la ingesta de fibra dietética redujo la incidencia de la infertilidad. (AU)

Evaluation of hematologic variables in newborn C57/BL6 mice up to day 35.

BACKGROUND: Hematologic variables are often analyzed in animal analogs during the investigation of complex disease etiologies such as necrotizing enterocolitis. However, reference intervals (RI) can vary depending on animal strain, age, and sampling site. Reference intervals have been published for adult C57BL/6J mice, but not newborn C57BL/6J mice. OBJECTIVES: The purpose of the present study was to determine hematologic RI in newborn C57BL/6J mice up to day 35. METHODS: C57BL/6J mice founders from The Jackson Laboratory were bred at the University of Iowa. Blood samples were obtained via facial vein sampling at postnatal days 0 (p0), p7, p14, p21, p28, or young adulthood (p35). CBCs were determined with the Sysmex XT-2000iV analyzer within 30 minutes of blood collection at a 1:10 dilution. Statistics were determined using nonparametric methods following ASVCP guidelines. RESULTS: Hematologic RI were determined for each of the 6 groups (n = 247, n ≥ 39 per group). Significantly higher values for HGB, RBC, and PLT counts were observed with advancing developmental age. Total WBC counts remained relatively stable during the first 35 days of life. However, WBC differential counts were dominated by neutrophils and lymphocytes in the younger mice, with a trend toward a lymphocytic leukogram on day 35. CONCLUSIONS: These results illustrate the dynamic changes in hematologic variables during murine development after birth. Utilization of age-specific RI is advised when evaluating data derived from experimental perinatal mouse models.

Intrauterine Growth Restriction Alters Mouse Intestinal Architecture during Development.

Infants with intrauterine growth restriction (IUGR) are at increased risk for neonatal and lifelong morbidities affecting multiple organ systems including the intestinal tract. The underlying mechanisms for the risk to the intestine remain poorly understood. In this study, we tested the hypothesis that IUGR affects the development of goblet and Paneth cell lineages, thus compromising the innate immunity and barrier functions of the epithelium. Using a mouse model of maternal thromboxane A2-analog infusion to elicit maternal hypertension and resultant IUGR, we tested whether IUGR alters ileal maturation and specifically disrupts mucus-producing goblet and antimicrobial-secreting Paneth cell development. We measured body weights, ileal weights and ileal lengths from birth to postnatal day (P) 56. We also determined the abundance of goblet and Paneth cells and their mRNA products, localization of cellular tight junctions, cell proliferation, and apoptosis to interrogate cellular homeostasis. Comparison of the murine findings with human IUGR ileum allowed us to verify observed changes in the mouse were relevant to clinical IUGR. At P14 IUGR mice had decreased ileal lengths, fewer goblet and Paneth cells, reductions in Paneth cell specific mRNAs, and decreased cell proliferation. These findings positively correlated with severity of IUGR. Furthermore, the decrease in murine Paneth cells was also seen in human IUGR ileum. IUGR disrupts the normal trajectory of ileal development, particularly affecting the composition and secretory products of the epithelial surface of the intestine. We speculate that this abnormal intestinal development may constitute an inherent "first hit", rendering IUGR intestine susceptible to further injury, infection, or inflammation.

Simultaneous disruption of mouse ASIC1a, ASIC2 and ASIC3 genes enhances cutaneous mechanosensitivity.

Three observations have suggested that acid-sensing ion channels (ASICs) might be mammalian cutaneous mechanoreceptors; they are structurally related to Caenorhabditis elegans mechanoreceptors, they are localized in specialized cutaneous mechanosensory structures, and mechanical displacement generates an ASIC-dependent depolarization in some neurons. However, previous studies of mice bearing a single disrupted ASIC gene showed only subtle or no alterations in cutaneous mechanosensitivity. Because functional redundancy of ASIC subunits might explain limited phenotypic alterations, we hypothesized that disrupting multiple ASIC genes would markedly impair cutaneous mechanosensation. We found the opposite. In behavioral studies, mice with simultaneous disruptions of ASIC1a, -2 and -3 genes (triple-knockouts, TKOs) showed increased paw withdrawal frequencies when mechanically stimulated with von Frey filaments. Moreover, in single-fiber nerve recordings of cutaneous afferents, mechanical stimulation generated enhanced activity in A-mechanonociceptors of ASIC TKOs compared to wild-type mice. Responses of all other fiber types did not differ between the two genotypes. These data indicate that ASIC subunits influence cutaneous mechanosensitivity. However, it is unlikely that ASICs directly transduce mechanical stimuli. We speculate that physical and/or functional association of ASICs with other components of the mechanosensory transduction apparatus contributes to normal cutaneous mechanosensation.

Mini-thin filaments regulated by troponin-tropomyosin.

Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, "mini-thin filaments" were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin-tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1-3)-TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 +/- 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin-troponin composition indicated one troponin-tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin-troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation.