Blocking hedgehog signaling after ablation of the dorsal neural tube allows regeneration of the cardiac neural crest and rescue of outflow tract septation

Cardiac neural crest cells (CNCC) migrate into the caudal pharynx and arterial pole of the heart to form the outflow septum. Ablation of the CNCC results in arterial pole malalignment and failure of outflow septation, resulting in a common trunk overriding the right ventricle. Unlike preotic cranial crest, the postotic CNCC do not normally regenerate. We applied the hedgehog signaling inhibitor, cyclopamine (Cyc), to chick embryos after CNCC ablation and found normal heart development at day 9 suggesting that the CNCC population was reconstituted. We ablated the CNCC, and labeled the remaining neural tube with DiI/CSRE and applied cyclopamine. Cells migrated from the neural tube in the CNCC-ablated, cyclopamine-treated embryos but not in untreated CNCC-ablated embryos. The newly generated cells followed the CNCC migration pathways, expressed neural crest markers and supported normal heart development. Finally, we tested whether reducing hedgehog signaling caused redeployment of the dorsal–ventral axis of the injured neural tube, allowing generation of new neural crest-like cells. The dorsal neural tube marker, Pax7, was maintained 12 h after CNCC ablation with Cyc treatment but not in the CNCC-ablated alone. This disruption of dorsal–ventral neural patterning permits a new wave of migratory cardiac neural crest-like cells.     

N',2-diphenylquinoline-4-carbohydrazide based NK3 receptor antagonists

A new class of potent NK3R antagonists based on the N',2-diphenylquinoline-4-carbohydrazide core is described. In an ex vivo assay in gerbil, the lead compound 2g occupies receptors within the CNS following oral dosing (Occ(90) 30 mg/kg po; plasma Occ(90) 0.95 microM) and has good selectivity and promising PK properties.     

Comparison of West African and Congo Basin Monkeypox Viruses in BALB/c and C57BL/6 Mice

Although monkeypox virus (MPXV) studies in wild rodents and non-human primates have generated important knowledge regarding MPXV pathogenesis and inferences about disease transmission, it might be easier to dissect the importance of virulence factors and correlates of protection to MPXV in an inbred mouse model. Herein, we compared the two clades of MPXV via two routes of infection in the BALB/c and C57BL/6 inbred mice strains. Our studies show that similar to previous animal studies, the Congo Basin strain of MPXV was more virulent than West African MPXV in both mouse strains as evidenced by clinical signs. Although animals did not develop lesions as seen in human MPX infections, localized signs were apparent with the foot pad route of inoculation, primarily in the form of edema at the site of inoculation; while the Congo Basin intranasal route of infection led to generalized symptoms, primarily weight loss. We have determined that future studies with MPXV and laboratory mice would be very beneficial in understanding the pathogenesis of MPXV, in particular if used in in vivo imaging studies. Although this mouse model may not suffice as a model of human MPX disease, with an appropriate inbred mouse model, we can unravel many unknown aspects of MPX pathogenesis, including virulence factors, disease progression in rodent hosts, and viral shedding from infected animals. In addition, such a model can be utilized to test antivirals and the next generation of orthopoxvirus vaccines for their ability to alter the course of disease.     

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.     

Establishment of the black-tailed prairie dog (Cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high- and low-dose monkeypox virus challenges

The 2003 monkeypox virus (MPXV) outbreak and subsequent laboratory studies demonstrated that the black-tailed prairie dog is susceptible to MPXV infection and that the ensuing rash illness is similar to human systemic orthopoxvirus (OPXV) infection, including a 7- to 9-day incubation period and, likely, in some cases a respiratory route of infection; these features distinguish this model from others. The need for safe and efficacious vaccines for OPVX in areas where it is endemic or epidemic is important to protect an increasingly OPXV-naïve population. In this study, we tested current and investigational smallpox vaccines for safety, induction of anti-OPXV antibodies, and protection against mortality and morbidity in two MPXV challenges. None of the smallpox vaccines caused illness in this model, and all vaccinated animals showed anti-OPXV antibody responses and neutralizing antibody. We tested vaccine efficacy by challenging the animals with 10⁵ or 10⁶ PFU Congo Basin MPXV 30 days postvaccination and evaluating morbidity and mortality. Our results demonstrated that vaccination with either Dryvax or Acambis2000 protected the animals from death with no rash illness. Vaccination with IMVAMUNE also protected the animals from death, albeit with (modified) rash illness. Based on the results of this study, we believe prairie dogs offer a novel and potentially useful small animal model for the safety and efficacy testing of smallpox vaccines in pre- and postexposure vaccine testing, which is important for public health planning.     

Norovirus disease: changing epidemiology and host susceptibility factors

Noroviruses cause the majority of acute viral gastroenteritis cases that occur worldwide. The increased recognition of noroviruses as the cause of outbreaks and sporadic disease is due to the recent availability of improved norovirus-specific diagnostics. Transmission of these viruses is facilitated by their high prevalence in the community, shedding of infectious virus particles from asymptomatic individuals and the high stability of the virus in the environment. Currently, the spectrum of clinical disease and the understanding of host susceptibility factors are changing. Cases of chronic norovirus gastroenteritis have been observed in transplant recipients and unusual clinical presentations have been recognized in otherwise healthy adults that are under physical stress. Recently, noroviruses were found to bind to gut-expressed carbohydrates, leading to a correlation between a person's genetically determined carbohydrate expression and their susceptibility to Norwalk virus infection. Greater community surveillance and further investigation of carbohydrate receptor-binding properties could provide further insights into norovirus transmission, susceptibility and pathogenesis, and should aid in developing vaccines and antiviral therapies for this common viral disease.     

Features of Child Food Insecurity after the 2010 Haiti Earthquake: Results from Longitudinal Random Survey of Households

Background

Recent commentary on the health consequences of natural disasters has suggested a dearth of research on understanding the antecedents prior to the disaster that are associated with health consequences after the disaster. Utilizing data from a two-wave panel survey of Port-au-Prince, Haiti, conducted just prior to and six weeks after the January 2010 earthquake, we test factors prior to the quake hypothesized to be associated with food insecurity after the quake.

Methods

Using random Global Positioning System (GPS) sampling, we re-interviewed 93.1% (N = 1732) of the original 1,800 households interviewed in 2009. Respondents were queried with regard to mortalities, injuries, food security, housing, and other factors after the quake.

Findings

Child food insecurity was found to be common on all three indices of food security (17.2%–22.6%). Additionally, only 36.5% of school-aged children were attending school prior to the quake. Findings suggest that prior schooling was associated with a substantial reduction on food insecurity indices (OR 0.62–0.75). Findings further suggest that several household characteristics were associated with food insecurity for children. Prior chronic/acute illnesses, poor living conditions, remittances from abroad, primary respondent mental health, and histories of criminal and other human rights violations committed against family members prior to the quake were associated with food insecurity after the earthquake. Earned household income after the quake was only associated with one of the measures of food insecurity.

Interpretation

Food insecurity for children was common after the quake. Those households vulnerable on multiple dimensions prior to the quake were also vulnerable to food insecurity after the quake. Remittances from abroad were leading protective factors for food security. Because Haiti is well known for the potentiality of both hurricanes and earthquakes, reconstruction and redevelopment should focus on ameliorating potential vulnerabilities to poor outcomes in these natural disasters.     

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain >90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.     

Genetic interrelationships of saccharolytic Clostridium botulinum types B, E and F and related clostridia as revealed by small-subunit rRNA gene sequences

Abstract The phylogenetic interrelationships of saccharolytic C. botulinum types B, E and F together with eleven other saccharolytic clostridia were examined by 16S rRNA gene sequencing. Comparative analysis of the sequence data revealed that the saccharolytic C. botulinum types B, E and F were highly related and represents a single genetic group. Strains of C. barati and C. butyricum that produce botulinal neurotoxin revealed almost 100% 16S rRNA sequence identity with their respective non-toxigenic counterparts and were phylogenetically distinct from saccharolytic C. botulinum (types B, E and F). Proteolytic C. botulinum type F was shown to be phylogenetically remote from the saccharolytic C. botulinum group. The implications of the sequence data for the taxonomy of the C. botulinum complex are discussed.     

Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C‐terminal α‐helix (α9) of the proapoptotic Bcl‐2 family protein BAX. The isolated α9 fragment (residues 173–192) is also competent to disrupt model membranes, and the structures of its membrane‐associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two‐dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β‐sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.