In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth

The apical ectodermal ridge (AER) in the vertebrate limb is required for limb outgrowth and patterning. To investigate the role BMP ligands expressed in the AER play in limb development we selectively inactivated both Bmp2 and Bmp4 in this tissue. The autopods of mice lacking both of these genes contained extra digits, digit bifurcations and interdigital webbing due to a decrease in programmed cell death and an increase in cell proliferation in the underlying mesoderm. Upon removal of Bmp2 and Bmp4 in the AER, no defects in proximal-distal patterning were observed. At the molecular level, removal of Bmp2 and Bmp4 in the AER caused an increase in Fgf expression, which correlated with an increase in both the width and length of the AER. Investigation of Engrailed-1 (En1) expression in the AER of limb buds in which Bmp2 and Bmp4 had been removed indicated that En1 expression was absent from this tissue. Our data suggests that AER expression of Bmp2 and Bmp4 is required for digit and dorsal-ventral patterning but surprisingly not for limb outgrowth.     

Hyperpolarization of murine small caliber mesenteric arteries by activation of endothelial proteinase-activated receptor 2

Activation of endothelial proteinase-activated receptor 2 (PAR-2) relaxes vascular smooth muscle (VSM) and causes hypotension by nitric oxide (NO)-prostanoid-dependent and -independent mechanisms. We investigated whether endothelium-dependent hyperpolarization of VSM was the mechanism whereby resistance caliber arteries vasodilated independently of NO. VSM membrane potentials and isometric tension were measured concurrently to correlate the electrophysiological and mechanical changes in murine small caliber mesenteric arteries. In uncontracted arteries, the PAR-2 agonist, SLIGRL-NH2 (0.1 to 10 micromol/L), hyperpolarized the VSM membrane potential only in endothelium-intact arterial preparations. This response was unaltered by treatment of arteries with inhibitors of NO synthases (L-NAME), soluble guanylyl cyclase (ODQ), and cyclooxygenases (indomethacin). L-NAME, ODQ, and indomethacin also failed to inhibit SLIGRL-NH2-induced hyperpolarization and of cirazoline-contracted mesenteric arteries. However, in blood vessels that were depolarized and contracted with 30 mmol/L KCl, the effects of the SLIGRL-NH2 on membrane potential and tension were not observed. SLIGRL-NH2-induced hyperpolarization and relaxation was inhibited completely by the combination of apamin plus charybdotoxin, but only partially inhibited after treatment with the combination of barium plus ouabain, suggesting an important role for SKCa and IKCa channels and a lesser role for Kir channels and Na+/K+ ATPases in the hyperpolarization response. We concluded that activation of endothelial PAR-2 hyperpolarized the vascular smooth muscle (VSM) cells of small caliber arteries, without requiring the activation of NO synthases, cyclooxygenases, or soluble guanylyl cyclase. Indeed, this hyperpolarization may be a primary mechanism for PAR-2-induced hypotension in vivo.     

Endogenous opiates inhibit gastric acid secretion induced by central administration of Thyrotropin-Releasing Hormone (TRH)

Thyrotropin-releasing hormone (TRH) administered intraventricularly (ICV) to rats causes a dose-dependent increase in gastric acid secretion over a range of 0.01 μg to 10 μg in the pyloris ligated rat. The maximum increase in gastric acid secretion occurs in the first hour. This effect of TRH is not mediated by its metabolites, histidyl-proline diketopiperazine or pyroglutamyl-histidyl-proline (acid TRH). β-endorphin, D-alanine-methionine-enkephalin and the leucine-enkephalin precursor, dynorphin, all inhibit TRH-induced gastric acid secretion. Bombesin, which reduces basal gastric acid secretion had no effect on TRH-induced secretion.     

Prevalence and typing of HPV DNA in atypical squamous cells in pregnant women

OBJECTIVE: To determine the prevalence and typing of HPV DNA in pregnant women with a diagnosis of atypical squamous cells (ASC) and to assess whether pregnancy-related changes contribute to the diagnosis of ASC. STUDY DESIGN: HPV testing was performed on residual specimens from the ThinPrep Pap test (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) in pregnant women diagnosed as ASC (study group, n = 105), low and high grade squamous intraepithelial lesion (LSIL and HSIL) (positive control, n = 33) and negative for epithelial cell abnormality (negative control, n = 20). All cases were reviewed by 2 cytopathologists to obtain consensus diagnoses using the Bethesda System 2001 criteria. The study group cases were further subcategorized into ASC of undetermined significance (ASCUS, n = 99) and ASC cannot exclude HSIL (ASC-H, n = 6). HPV testing was also performed on an ASC control group consisting of 68 consecutive ASC cases in nonpregnant women, matched by age. RESULTS: Mean patient age was 23.7 years for the study group and 25.6 years for the ASC control group. HPV DNA was detected in 88.6% of cases in the study group, including 87.9% of ASC-US and 100% of ASC-H cases. Of the HPV positive cases, 79.6%, 4.3%, 5.4% and 10.8% had high-risk, mixed high- and low-risk, low-risk and unknown HPV types, respectively. The most frequent HPV types detected were: types 52 (31.2%), 16 (15.1%), 39 (11.8%), 53 (10.8%), and 18 and 58 (9.7% each). Multiple viral types were detected in 43.0% of cases. The prevalence of HPV DNA in the positive and negative controls in pregnant women was 100% and 55%, respectively. HPV DNA was detected in 83.8% of the ASC control group. CONCLUSION: Regardless of pregnancy-related changes, the prevalence of HPV DNA in pregnant women (88.6%) was similar to that found in ASC in nonpregnant women of the same reproductive-age group (83.8%), and the high-risk types accounted for the vast majority of cases (83.9%). These findings demonstrate that pregnancy-related changes do not contribute to the diagnosis of ASC in this subset of women. Furthermore, the high HPV DNA prevalence in reproductive-age women (< 40 years) suggests that HPV testing may have limited utility in effective management of these patients.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Airway branching has conserved needs for local parasympathetic innervation but not neurotransmission

Background

Parasympathetic signaling has been inferred to regulate epithelial branching as well as organ regeneration and tumor development. However, the relative contribution of local nerve contact versus secreted signals remains unclear. Here, we show a conserved (vertebrates to invertebrates) requirement for intact local nerves in airway branching, persisting even when cholinergic neurotransmission is blocked.

Results

In the vertebrate lung, deleting enhanced green fluorescent protein (eGFP)-labeled intrinsic neurons using a two-photon laser leaves adjacent cells intact, but abolishes branching. Branching is unaffected by similar laser power delivered to the immediately adjacent non-neural mesodermal tissue, by blocking cholinergic receptors or by blocking synaptic transmission with botulinum toxin A. Because adjacent vasculature and epithelial proliferation also contribute to branching in the vertebrate lung, the direct dependence on nerves for airway branching was tested by deleting neurons in Drosophila embryos. A specific deletion of neurons in the Drosophila embryo by driving cell-autonomous RicinA under the pan-neuronal elav enhancer perturbed Drosophila airway development. This system confirmed that even in the absence of a vasculature or epithelial proliferation, airway branching is still disrupted by neural lesioning.

Conclusions

Together, this shows that airway morphogenesis requires local innervation in vertebrates and invertebrates, yet neurotransmission is dispensable. The need for innervation persists in the fly, wherein adjacent vasculature and epithelial proliferation are absent. Our novel, targeted laser ablation technique permitted the local function of parasympathetic innervation to be distinguished from neurotransmission.

     

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.