Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins ^1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references ^{1-6,7 ,8}).     

Protein 4.1B contributes to the organization of peripheral myelinated axons

Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber.     

Pandemic influenza due to pH1N1/2009 virus: estimation of infection burden in Reunion Island through a prospective serosurvey, austral winter 2009

Background

To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season.

Methodology/Principal Findings

A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive.

Conclusions

Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.     

Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B

Background

The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Results

We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)⁺ paranode-type, as well as a Caspr2⁺ and Kv1⁺ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr⁺ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.

Conclusions

α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.     

Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.     

Influence of colostrum intake on piglet survival and immunity

Colostrum intake from birth to 24 h after the onset of parturition (T24) was estimated for 526 piglets from 40 litters. Plasma concentrations of immunoglobulin G (IgG), lactate, glucose and cortisol were determined at T24 for six piglets per litter. Plasma IgG concentration was also assayed at weaning (28 days) on the same piglets. Rectal temperature was measured at T24 on all piglets. Mortality was recorded until weaning and comparisons were made between piglets that died before weaning and those that were still alive at weaning. The piglets that died before weaning had lower birth weight, lower colostrum intake, lower weight gain between birth and T24, and had a lower rectal temperature, higher plasma cortisol concentration and lower plasma IgG and glucose concentrations at T24 than piglets still alive at weaning. In addition, a higher proportion of piglets that died before weaning had difficulty taking their first breath after birth and were affected by splayleg. Considering all piglets, colostrum intake was positively related to rectal temperature and plasma glucose concentration and negatively related to plasma cortisol concentration at T24. Plasma IgG concentration at T24 was explained by colostrum intake, IgG concentration in the ingested colostrum, birth weight and birth rank (P<0.0001). Plasma IgG concentration at weaning was related to plasma IgG concentration at T24 (r=0.54; P<0.0001) and to colostrum intake (r=0.32; P<0.0001). Finally, body weight was explained by colostrum intake, birth weight and age until 6 weeks of age (P<0.0001). These results show that colostrum intake is the main determinant of piglet survival through provision of energy and immune protection and has potential long-term effects on piglet growth and immunity.     

Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Cost effectiveness of fibrosis assessment prior to treatment for chronic hepatitis C patients

Background and Aims

Chronic hepatitis C (HCV) is a liver disease affecting over 3 million Americans. Liver biopsy is the gold standard for assessing liver fibrosis and is used as a benchmark for initiating treatment, though it is expensive and carries risks of complications. FibroTest is a non-invasive biomarker assay for fibrosis, proposed as a screening alternative to biopsy.

Methods

We assessed the cost-effectiveness of FibroTest and liver biopsy used alone or sequentially for six strategies followed by treatment of eligible U.S. patients: FibroTest only; FibroTest with liver biopsy for ambiguous results; FibroTest followed by biopsy to rule in; or to rule out significant fibrosis; biopsy only (recommended practice); and treatment without screening. We developed a Markov model of chronic HCV that tracks fibrosis progression. Outcomes were expressed as expected lifetime costs (2009 USD), quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios (ICER).

Results

Treatment of chronic HCV without fibrosis screening is preferred for both men and women. For genotype 1 patients treated with pegylated interferon and ribavirin, the ICERs are $5,400/QALY (men) and $6,300/QALY (women) compared to FibroTest only; the ICERs increase to $27,200/QALY (men) and $30,000/QALY (women) with the addition of telaprevir. For genotypes 2 and 3, treatment is more effective and less costly than all alternatives. In clinical settings where testing is required prior to treatment, FibroTest only is more effective and less costly than liver biopsy. These results are robust to multi-way and probabilistic sensitivity analyses.

Conclusions

Early treatment of chronic HCV is superior to the other fibrosis screening strategies. In clinical settings where testing is required, FibroTest screening is a cost-effective alternative to liver biopsy.     

Experimental study of early olfactory neuron differentiation and nerve formation using quail-chick chimeras

For the formation of a functional olfactory system, the key processes are neuronal differentiation, including the expression of one or the other olfactory receptors, the correct formation of the nerve and organization of periphero-central connections. These processes take place during embryonic development starting from early stages. Consequently, avian embryos afford an attractive model to study these mechanisms. Taking advantage of species-specific equipment of olfactory receptors genes in different bird species, interspecific avian chimeras were set up by grafting early chick olfactory placodes in same stage quail embryos. Their analysis was performed using different complementary approaches. In situ hybridisation using probes to different chick olfactory receptor (COR) genes indicated that the choice of expression of an olfactory receptor by a neuron is independent of the environment of the olfactory placode and of interactions with the central nervous system. Futhermore, a chick olfactory receptor gene subgroup (COR3 ), absent in the host genome, was expressed by neurons from the graft. The question was then raised of the consequences of such heterospecific differentiation on axonal projections and fiber convergence. The DiI labeling of olfactory fibres in chimeras revealed anomalies in the formation of the nerve from the chick graft. In agreement with the hypothesis of olfactory receptor (OR) involvement in axonal guidance and periphero-central synapse organisation, the presence of migrating cells and axonal fibres from the graft, expressing foreign ORs and having different interactions with the host environment than the host fibres and migrating cells, might explain these anomalies.