Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme.

Ion channels are essential for proper morphogenesis of the craniofacial skeleton. However, the molecular mechanisms underlying this phenomenon are unknown. Loss of the Kcnj2 potassium channel disrupts Bone Morphogenetic Protein (BMP) signaling within the developing palate. BMP signaling is essential for the correct development of several skeletal structures, including the palate, though little is known about the mechanisms that govern BMP secretion. We introduce a tool to image the release of bone morphogenetic protein 4 (BMP4) from mammalian cells. Using this tool, we show that depolarization induces BMP4 release from mouse embryonic palate mesenchyme cells in a calcium-dependent manner. We show native transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day (E) 9.5 to 13.5 mice. Disruption of Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Together, these data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and changes in intracellular calcium for mammalian craniofacial morphogenesis. SUMMARY: We show that embryonic palate mesenchyme cells undergo transient changes in intracellular calcium. Depolarization of these cells induces BMP4 release suggesting that ion channels are a node in BMP4 signaling.

Endoplasmic Reticulum Calcium Mediates Drosophila Wing Development.

Background: The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning. However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and bone morphogenetic protein (BMP/Dpp) release for Drosophila wing development. Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology. Ion channels impact development of several tissues and organisms in which BMP signaling is essential. In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin. Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum (ER) Ca++ release into the cytoplasm to regulate the release of BMP. Materials and Methods: To test this hypothesis, we reduced expression of four proteins that control ER calcium, Stromal interaction molecule 1 (Stim), Calcium release-activated calcium channel protein 1 (Orai), SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), small conductance calcium-activated potassium channel (SK), and Bestrophin 2 (Best2) using RNAi and documented wing phenotypes. We use live imaging to study calcium and Dpp release within pupal wings and larval wing discs. Additionally, we employed immunohistochemistry to characterize Small Mothers Against Decapentaplegic (SMAD) phosphorylation downstream of the BMP/Dpp pathway following RNAi knockdown. Results: We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced BMP/Dpp release. Conclusion: Our results suggest control of ER calcium homeostasis is required for BMP/Dpp release, and Drosophila wing development.

Intrauterine exposure to nicotine through maternal vaping disrupts embryonic lung and skeletal development via the Kcnj2 potassium channel.

Smoking cigarettes during pregnancy is associated with adverse effects on infants including low birth weight, defective lung development, and skeletal abnormalities. Pregnant women are increasingly turning to vaping [use of electronic (e)-cigarettes] as a perceived safer alternative to cigarettes. However, nicotine disrupts fetal development, suggesting that like cigarette smoking, nicotine vaping may be detrimental to the fetus. To test the impact of maternal vaping on fetal lung and skeletal development in mice, pregnant dams were exposed to e-cigarette vapor throughout gestation. At embryonic day (E)18.5, vape exposed litter sizes were reduced, and some embryos exhibited growth restriction compared to air exposed controls. Fetal lungs were collected for histology and whole transcriptome sequencing. Maternally nicotine vaped embryos exhibited histological and transcriptional changes consistent with impaired distal lung development. Embryonic lung gene expression changes mimicked transcriptional changes observed in adult mouse lungs exposed to cigarette smoke, suggesting that the developmental defects may be due to direct nicotine exposure. Fetal skeletons were analyzed for craniofacial and long bone lengths. Nicotine directly binds and inhibits the Kcnj2 potassium channel which is important for bone development. The length of the maxilla, palatal shelves, humerus, and femur were reduced in vaped embryos, which was further exacerbated by loss of one copy of the Kcnj2 gene. Nicotine vapor exposed Kcnj2KO/+ embryos also had significantly lower birth weights than unexposed animals of either genotype. Kcnj2 mutants had severely defective lungs with and without vape exposure, suggesting that potassium channels may be broadly involved in mediating the detrimental developmental effects of nicotine vaping. These data indicate that intrauterine nicotine exposure disrupts fetal lung and skeletal development likely through inhibition of Kcnj2.

Single cell sequencing of the mouse anterior palate reveals mesenchymal heterogeneity.

BACKGROUND: Cleft palate is one of the most prevalent birth defects. Mice are useful for studying palate development because of their morphological and genetic similarities to humans. In mice, palate development occurs between embryonic days (E)11.5 to 15.5. Single cell transcriptional profiles of palate cell populations have been a valuable resource for the craniofacial research community, but we lack a single cell transcriptional profile for anterior palate at E13.5, at the transition from proliferation to shelf elevation. RESULTS: A detailed single cell RNA sequencing analysis reveals heterogeneity in expression profiles of the cell populations of the E13.5 anterior palate. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA FISH) reveals epithelial populations segregate into layers. Mesenchymal populations spatially segregate into four domains. One of these mesenchymal populations expresses ligands and receptors distinct from the rest of the mesenchyme, suggesting that these cells have a unique function. RNA velocity analysis shows two terminal cell states that contribute to either the proximal or distal palatal regions emerge from a single progenitor pool. CONCLUSION: This single cell resolution expression data and detailed analysis from E13.5 anterior palate provides a powerful resource for mechanistic insight into secondary palate morphogenesis for the craniofacial research community.

Evaluation of thiol/disulfide homeostasis in rheumatoid arthritis and disease activity.

BACKGROUND: It has been suggested that the deterioration in the antioxidant defense system due to thiols may cause the pro-oxidant/antioxidant imbalance seen in rheumatoid arthritis (RA). This study was conducted to evaluate thiol/disulfide (-SH/-SS) homeostasis in patients with RA compared to healthy controls, and to validate the limited number of studies examining the relationship between Disease Activity Score-28 (DAS28) and thiol parameters. METHOD: A total of 100 individuals (mean age: 46.3 ± 12.03) consisting of 86 females and 14 males were included in the RA group, and a total of 100 individuals (mean age: 43.3 ± 10.96 years) consisting of 78 females and 22 males were included in the control group. DAS28 was used to assess RA disease activity. -SH/-SS homeostasis parameters were measured using the automated spectrophotometric method described by Erel and Neselioglu. RESULTS: While native thiol (-SH) (p:0.001), total thiol (-SH + -SS) (p < 0.0001) levels and -SH\(-SS + -SH) ratio (p: 0.018) were lower in the RA group compared to the healthy controls, disulfide (-SS) level (p: 0.005)), -SS\-SH (p: 0.001) and -SS\(-SS + -SH) (p: 0.002) ratios were found to be higher. In the control group and the group in remission (defined by DAS28 < 2.6), the median values of -SH (p:0.002) and -SS + -SH (p:0.0008) were found to be significantly higher, and the median value of -SS (p: 0.001) was found to be lower compared to the other DAS28 groups. While a negative correlation was found between DAS28 and -SH (r: -0.243, p: 0.007), a positive correlation was found between DAS28 and -SS (r: 0.316, p: 0.0003), -SS\-SH (r:0.229, p: 0.002) and -SS\(-SS + -SH) (r: 0.285, p: 0.0009). CONCLUSIONS: The plasma thiol antioxidant pool was decreased in RA compared to healthy controls and those with active disease compared to those in remission.

Ion Channel Contributions to Morphological Development: Insights From the Role of Kir2.1 in Bone Development.

The role of ion channels in neurons and muscles has been well characterized. However, recent work has demonstrated both the presence and necessity of ion channels in diverse cell types for morphological development. For example, mutations that disrupt ion channels give rise to abnormal structural development in species of flies, frogs, fish, mice, and humans. Furthermore, medications and recreational drugs that target ion channels are associated with higher incidence of birth defects in humans. In this review we establish the effects of several teratogens on development including epilepsy treatment drugs (topiramate, valproate, ethosuximide, phenobarbital, phenytoin, and carbamazepine), nicotine, heat, and cannabinoids. We then propose potential links between these teratogenic agents and ion channels with mechanistic insights from model organisms. Finally, we talk about the role of a particular ion channel, Kir2.1, in the formation and development of bone as an example of how ion channels can be used to uncover important processes in morphogenesis. Because ion channels are common targets of many currently used medications, understanding how ion channels impact morphological development will be important for prevention of birth defects. It is becoming increasingly clear that ion channels have functional roles outside of tissues that have been classically considered excitable.

Seroepidemiology of Toxoplasma gondii infection in free-range chickens (Gallus domesticus) of Central Ethiopia.

We performed a seroepidemiological study of Toxoplasma gondii infection in free-range chickens from October 2012 to May 2013. We used cross-sectional two-stage cluster sampling to collect blood samples from wing veins of 601 chickens from central Ethiopia. T. gondii-specific antibodies were assayed by modified agglutination test (MAT). We collected information about risk factors by questionnaire and used univariable and multivariable logistic regression to assess risk factors. An overall seroprevalence of 30·5% [95% confidence interval (CI) 26·27-34·14] and 54·2% (95% CI 47·06-61·36) was found at animal- and flock-level, respectively. The MAT end titre of seropositive chickens (n = 183) were 1 : 60 in 46, 1 : 180 in 28, 1 : 540 in 29, ⩾1 : 1620 in 48, 1 : 6000 in 22, 1 : 18,000 in five, 1 : 54,000 in one, and ⩾1 : 162,000 in four. Animal-level risk factors identified using multivariable logistic regression model were: midland altitude [odds ratio (OR) 2·53, 95% CI 1·12-5·72], cross and exotic breeds (OR 3·17, 95% CI 1·39-7·23), increased age of chickens (OR 2·32, 95% CI 1·19-4·49), extensive management (OR 6·92, 95% CI 1·34-35·86) and the presence of cats (OR 2·08, 95% CI 1·20-3·61). Similarly, flock-level risk factors were midland altitude (OR 3·62, 95% CI 1·31-9·99) and the presence of cats (OR 1·19-4·94). The knowledge of the local people about the health risk of cats to humans and animals is poor. Housing and management of cats and chickens are also poor. The widespread presence of T. gondii infection in free-range chickens of Central Ethiopia provides suggestive evidence for the high level of contamination of the living environment of people with T. gondii oocysts. Meat from free-range chickens might be an important source of infection for humans. Altitude, breed, age, management and presence of cats are independent predictors of seropositivity. Education of farmers about toxoplasmosis and further studies to elucidate the burden of toxoplasmosis in animals and humans warrants consideration.

Detection of circulating antigens and parasite specific antibodies in filariasis.

In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.

Evaluation of Bactimos wettable powder, granules and briquets against mosquito larvae in Malaysia.

There was high mortality in late larval instars of Aedes albopictus (Skuse) from laboratory and field populations in the 24 h after application of three Bactimos formulations of Bacillus thuringiensis H-14. Mortalities were higher and residual effects longer in field populations than in laboratory ones. Briquets were the most effective formulation (mortality 96-100% after five weeks; 76-92% after eight weeks). Culex quinquefasciatus Say larvae were tested only against the briquet formulation. In the laboratory, 100% mortality of late instars persisted for six weeks and dropped to 48-88% after eight weeks. In the field, late instars were reduced by 62-87% after 24 h and 69-72% after one week compared to increases in an untreated population of 160% and 176% respectively.

Susceptibility of non-fermentative gram-negative organisms to ureidopenicillins and quinolones.

The susceptibility of non-fermentative Gram-negative bacteria (excluding Pseudomonas aeruginosa) to ureidopenicillins and new quinolones was investigated. The ureidopenicillins were not active against the strains except against Pseudomonas species, 90% of which were inhibited by 64 mg/l. The new quinolones, particularly ciprofloxacin, had excellent activity (MIC range 0.06-8 mg/l) against these strains which included those resistant to beta-lactams and aminoglycosides. This suggested a promising alternative group of compounds to be used in chemotherapy of infections caused by non-fermenters.

The role of some cyclorrhaphan flies as carriers of human helminths in Malaysia.

The role of some adult flies (Diptera: Cyclorrhapha) as carriers of helminth parasites of man was studied at four sites in Malaysia: a refuse dump, where no helminth-positive flies were detected, and in three peri-domestic situations where four species of flies carried up to three types of nematodes. The dominant fly species Chrysomya megacephala (Fabricius) carried eggs of the roundworm Ascaris lumbricoides L., the pinworm Trichuris trichiura (L.) and hookworm on the adult external body surface and in the gut lumen, in association with Bukit Lanjan aborigines. Chrysomya rufifacies (Macquart) and Sarcophaga spp. also had Ascaris lumbricoides and Trichurus trichiura eggs in their gut contents. Human helminths were not recovered from Lispe leucospila (Wiedemann), Lucilia cuprina (Wiedemann) or the housefly Musca domestica L. In an urban slum area of Kuala Lumpur city, filariform larvae identified as the hookworm Necator americanus (Stiles) occurred in the intestines of the face-fly Musca sorbens Wiedemann (22 larvae per 100 flies) and of Chrysomya megacephala (4.5 larvae per 100 flies). This concentration of apparently infective N. americanus in M. sorbens, a fly which often breeds in faeces and browses on human skin, could have transmission potential.

Evaluation of some adhesives for collecting Musca domestica and Chrysomya megacephala adults or mosquito larvae in sticky traps.

1. Seven types of water-insoluble adhesives were evaluated in sticky traps for collecting adults of Musca domestica L. and Chrysomya megacephala (Fabricius) or mosquito larvae (Aedes aegypti (L.) and Culex quinquefasciatus Say). 2. Adhesive viscosity affected the tackiness of the glues and this determined their trapping efficiency in air or water. 3. From the 'Hyvis' range of adhesives tested, 'Hyvis 200' was most effective for trapping adult flies. 4. With 24 h exposure to fourth instar Ae.aegypti larvae in tapwater, submerged plates coated with 'Hyvis 10', 'Hyvis 30' or 'Hyvis 200' formulations trapped the majority of larvae. In polluted water the highest rates of trapping were 17.3% of Ae.aegypti and 18.7% of Cx quinquefasciatus with 'Hyvis 200'. Floating traps were consistently less productive than submerged traps under laboratory conditions. 5. In a heavily polluted natural breeding-site of Cx quinquefasciatus, floating traps were more productive than submerged sticky traps with four of seven adhesives tested, the most efficient being 'Hyvis 200' (4.2 mosquitoes per hour) and Hyvis:polyethylene 90:10 (4.5/h). Despite the relative inefficiency of aquatic traps, emergent adults, pupae and second to fourth instars of larvae were collected quickly from the habitat.