HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence.

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

High-contrast, synchronous volumetric imaging with selective volume illumination microscopy.

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

Pre-processing visualization of hyperspectral fluorescent data with Spectrally Encoded Enhanced Representations.

Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER's enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis.

Znf385C mediates a novel p53-dependent transcriptional switch to control timing of facial bone formation.

Jaw formation involves an intricate series of molecular events, whereby a chondrogenic scaffold precedes osteogenesis. The mechanisms coupling timing of cartilage maturation to onset of bone differentiation are poorly understood, particularly for neural crest-derived bones of the head. Here we present a novel zebrafish gene/protein-trap Citrine-fusion line that reveals transient expression of the zinc-finger protein Znf385C in maturing chondrocytes of the jaw. Functional analysis shows that loss of Znf385C disrupts a distinct peak of p21(cip1/waf1) expression in the chondrocytes, as well as causes premature ossification of the zebrafish jaw. We find that Znf385C is expressed as two splice variants which act differentially to activate p21(cip1/waf1) and/or interact with p53 in subcellular compartments. Taken together, the results suggest that Znf385C acts as a developmental switch for p53 function that modulates cell cycle arrest of chondrocytes and regulates timing of jaw cartilage maturation and ossification.

Evolutionarily conserved role for SoxC genes in neural crest specification and neuronal differentiation.

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

Neuroanatomical profiles of deafness in the context of native language experience.

The study of congenitally deaf adult humans provides an opportunity to examine neuroanatomical plasticity resulting from altered sensory experience. However, attributing the source of the brain's structural variance in the deaf is complicated by the fact that deaf individuals also differ in their language experiences (e.g., sign vs spoken), which likely influence brain anatomy independently. Although the majority of deaf individuals in the United States are born to hearing parents and are exposed to English, not American Sign Language (ASL) as their first language, most studies on deafness have been conducted with deaf native users of ASL (deaf signers). This raises the question of whether observations made in deaf signers can be generalized. Using a factorial design, we compared gray (GMV) and white (WMV) matter volume in deaf and hearing native users of ASL, as well as deaf and hearing native users of English. Main effects analysis of sensory experience revealed less GMV in the deaf groups combined (compared with hearing groups combined) in early visual areas and less WMV in a left early auditory region. The interaction of sensory experience and language experience revealed that deaf native users of English had fewer areas of anatomical differences than did deaf native users of ASL (each compared with their hearing counterparts). For deaf users of ASL specifically, WMV differences resided in language areas such as the left superior temporal and inferior frontal regions. Our results demonstrate that cortical plasticity resulting from deafness depends on language experience and that findings from native signers cannot be generalized.

Zebrafish stem/progenitor factor msi2b exhibits two phases of activity mediated by different splice variants.

The Musashi (Msi) family of RNA-binding proteins is important in stem and differentiating cells in many species. Here, we present a zebrafish gene/protein trap line gt(msi2b-citrine)(ct) (57) (a) that expresses a Citrine fusion protein with endogenous Msi2b. Our results reveal two phases of Msi2b expression: ubiquitous expression in progenitor cells in the early embryo and later, tissue-specific expression in differentiating cells in the olfactory organ, pineal gland, and subpopulations of neurons in the central nervous system (CNS). Interestingly, this division between early and late phases is paralleled by differential expression of msi2b alternative splicing products. Whereas the full-length and long variant v3 Msi2b predominate at early stages, the later expression of variants in differentiating tissues appears to be tissue specific. Using the gt(msi2b-citrine)(ct) (57) (a), we characterized tissue-specific expression of Msi2b with cellular resolution in subsets of differentiating cells in the olfactory organ, pineal gland, CNS, and ventral neural tube. By performing transcription activator-like effectors nuclease-mediated biallelic genome editing or morpholino knockdown of Msi2b in zebrafish, our results show that early inactivation of Msi2b results in severe embryonic defects including hypertrophy of the ventricles and shortening of the body, consistent with an important role in cell proliferation and survival. Moreover, specific inactivation of Msi2b full-length indicates that this species is essential for the early role of Msi2b. This line provides a valuable tool both for live imaging of the endogenous Msi2b at subcellular resolution and manipulation of Msi2b-expressing cells.

Laminin ß1a controls distinct steps during the establishment of digestive organ laterality.

Visceral organs, including the liver and pancreas, adopt asymmetric positions to ensure proper function. Yet the molecular and cellular mechanisms controlling organ laterality are not well understood. We identified a mutation affecting zebrafish laminin ß1a (lamb1a) that disrupts left-right asymmetry of the liver and pancreas. In these mutants, the liver spans the midline and the ventral pancreatic bud remains split into bilateral structures. We show that lamb1a regulates asymmetric left-right gene expression in the lateral plate mesoderm (LPM). In particular, lamb1a functions in Kupffer's vesicle (KV), a ciliated organ analogous to the mouse node, to control the length and function of the KV cilia. Later during gut-looping stages, dynamic expression of Lamb1a is required for the bilayered organization and asymmetric migration of the LPM. Loss of Lamb1a function also results in aberrant protrusion of LPM cells into the gut. Collectively, our results provide cellular and molecular mechanisms by which extracellular matrix proteins regulate left-right organ morphogenesis.

Cortical plasticity for visuospatial processing and object recognition in deaf and hearing signers.

Experience-dependent plasticity in deaf participants has been shown in a variety of studies focused on either the dorsal or ventral aspects of the visual system, but both systems have never been investigated in concert. Using functional magnetic resonance imaging (fMRI), we investigated functional plasticity for spatial processing (a dorsal visual pathway function) and for object processing (a ventral visual pathway function) concurrently, in the context of differing sensory (auditory deprivation) and language (use of a signed language) experience. During scanning, deaf native users of American Sign Language (ASL), hearing native ASL users, and hearing participants without ASL experience attended to either the spatial arrangement of frames containing objects or the identity of the objects themselves. These two tasks revealed the expected dorsal/ventral dichotomy for spatial versus object processing in all groups. In addition, the object identity matching task contained both face and house stimuli, allowing us to examine category-selectivity in the ventral pathway in all three participant groups. When contrasting the groups we found that deaf signers differed from the two hearing groups in dorsal pathway parietal regions involved in spatial cognition, suggesting sensory experience-driven plasticity. Group differences in the object processing system indicated that responses in the face-selective right lateral fusiform gyrus and anterior superior temporal cortex were sensitive to a combination of altered sensory and language experience, whereas responses in the amygdala were more closely tied to sensory experience. By selectively engaging the dorsal and ventral visual pathways within participants in groups with different sensory and language experiences, we have demonstrated that these experiences affect the function of both of these systems, and that certain changes are more closely tied to sensory experience, while others are driven by the combination of sensory and language experience.

Developing methods to compare low-education community-based and university-based survey teams.

We compared the quality of low-education community-based survey teams to college educated graduate students. Our approach was to develop methods, conduct a pilot survey, and report lessons. Community and university teams conducted surveys from non-overlapping random samples of addresses at a public housing development in Boston, Massachusetts. The two types of teams make a similar number of attempts (122 and 124, respectively), and there was no statistically significant difference between the teams in terms of response rate or amount of missing data. Similarly, there was no statistically significant difference in refusal rate or in responses to questions in the survey. There was, however, evidence that the community teams used data tracking forms improperly. This study suggests that it is possible to study the relative quality of community and university-based teams in terms of data collection. The findings also suggest that the two types of teams may be roughly comparable.

A correção da mordida cruzada posterior unilateral com desvio funcional melhora a assimetria facial? / Does the correction of a functional posterior unilateral crossbite improve the facial asymmetry?

OBJETIVO: avaliar, através de fotografias clínicas de 30 pacientes, as alterações ocorridas na assimetria labial presente em pacientes portadores de mordida cruzada posterior unilateral funcional (MCPUF), após a correção ortodôntica. MÉTODOS: para quantificar essas modificações, foram mensuradas as áreas dos quadrantes labiais antes (T1) e após o tratamento (T2). Os lábios superiores e inferiores foram subdivididos em 4 quadrantes e as suas áreas foram medidas e expressas como percentagem da sua área total. A quantidade de correção da assimetria foi obtida calculando a diferença no percentual da área entre os quadrantes do lábio em T1 e T2. RESULTADOS: os resultados demonstraram haver uma melhora significativa na assimetria observada inicialmente nestes pacientes do lado onde o desvio postural da mandíbula se apresentava.

AIM: To evaluate, analyzing clinical photographs of 30 patients, the changes occurred, after orthodontic correction, on the labial asymmetry in patients with unilateral posterior functional crossbite. METHODS: To quantify this changes, the area of the labial quadrants and their surface area were measured before (T1) and after (T2) the treatment. Upper and lower lips were subdivided in 4 quadrants, and their surface area were measured and expressed in percentage of the total surface area. The degree of correction was obtained by calculating the difference in the percentile of the surface area among the quadrants in T1 and T2. RESULTS: The results showed a significant improvement of the asymmetry on the mandibular shift side.

Phonology and reading: a response to Wang, Trezek, Luckner, and Paul.

Four critical responses to an article, "The Role of Phonology and Phonologically Related Skills in Reading Instruction for Students Who Are Deaf or Hard of Hearing" (Wang, Trezek, Luckner, & Paul, 2008), are presented. Issue is taken with the conclusions of the article by Wang and colleagues regarding the "necessary" condition of phonological awareness for the development of reading skills among deaf readers. Research findings (not cited by Wang and colleagues) are pointed out that reveal weak correlations between phonemic awareness and reading comprehension, and stronger correlations between other variables such as overall language skill and early exposure to a visual language.

Phonological awareness and short-term memory in hearing and deaf individuals of different communication backgrounds.

Previous work in deaf populations on phonological coding and working memory, two skills thought to play an important role in the acquisition of written language skills, have focused primarily on signers or did not clearly identify the subjects' native language and communication mode. In the present study, we examined the effect of sensory experience, early language experience, and communication mode on the phonological awareness skills and serial recall of linguistic items in deaf and hearing individuals of different communicative and linguistic backgrounds: hearing nonsigning controls, hearing users of ASL, deaf users of ASL, deaf oral users of English, and deaf users of cued speech. Since many current measures of phonological awareness skills are inappropriate for deaf populations on account of the verbal demands in the stimuli or response, we devised a nonverbal phonological measure that addresses this limitation. The Phoneme Detection Test revealed that deaf cuers and oral users, but not deaf signers, performed as well as their hearing peers when detecting phonemes not transparent in the orthography. The second focus of the study examined short-term memory skills and found that in response to the traditional digit span as well as an experimental visual version, digit-span performance was similar across the three deaf groups, yet deaf subjects' retrieval was lower than that of hearing subjects. Our results support the claim (Bavelier et al., 2006) that lexical items processed in the visual-spatial modality are not as well retained as information processed in the auditory channel. Together these findings show that the relationship between working memory, phonological coding, and reading may not be as tightly interwoven in deaf students as would have been predicted from work conducted in hearing students.

Rationale for referring class II patients for early orthodontic treatment

A tendência de indicacão de tratamento precoce (antes de surto de crescimento) para casos de Classe II de Angle tem sido observada, embora não haja evidência científica para embasar tal decisão. O objetivo deste estudo foi identificar as vantagens e desvantagens da indicacão e quais aparelhos são usados no tratamento. Foram enviados questionários (n=192) com a documentacão completa de um paciente Classe II para dois professores de cada curso de especializacão em Ortodontia no Brasil, os quais 107 foram respondidos. Os resultados demonstraram que os aparelhos mais usados foram: Extra-oral (80,4 por cento), Thurow (50,0 por cento) e Bionator (44,4 por cento). Foram citados como maiores vantagens: aumento de auto-estima do paciente (78,5 por cento) e a reducão da incidência de trauma nos dentes incisivos (63,6 por cento), por outro lado a principal desvantagem citada foi a saturacão de cooperacão do paciente (73,8 por cento). Considerando o tratamento precoce de Classe II não houve o consenso se a correcão deveria ser realizada em duas ou em uma única fase e nem a selecão do aparelho a ser utilizado. Porém os ortodontistas deveriam levar em consideracão o estado psicológico do paciente, a severidade do caso clínico e a susceptibilidade a trauma dos incisivos superiores. É importante que os responsáveis sejam esclarecidos quanto à necessidade do tratamento precoce.

Avaliação do efeito de forças ortopédicas no tratamento da maloclusão de classe III esquelética / Evaluation of orthopedic force effect in treatment of skeletal class III malocclusion

Apesar da baixa incidência da maloclusão de Classe III esquelética em população caucasiana, para muitos Ortodontistas, este tipo de maloclusão é estremamente difícil de se tratar apenas com a Ortodontia convencional. Muitas vezes há a necessidade de complementação cirúrgica durante o tratamento. Por esta razão, o diagnóstico e o tratamento geram grandes polêmicas pela sua complexidade e dificuldade na obtenção de estabilidade. Diante da maloclusão de Classe III nas crianças, a intervenção em idade precoce (antes do surto de crescimento) é indicata. A estabilidade é ainda questionável com o uso de mentoneira, a combinação de mentoneira e máscara facial pode ser um método favorável quando a criança apresenta retrusão maxilar e protrusão mandibular. A máscara facial é o instrumento mais efetivo diante do tratamento de maloclusão de Classe III esquelética com a retrusão maxilar e padrão de crescimento hipodivergente. Um dos pontos mais importantes para obtenção de estabilidade a longo prazo é a sobrecorreção, quando se trata com a expansão palatina e a máscara facial

Rationale for referring class II patients for early orthodontic treatment.

The tendency of indicating early treatment (before growth spurt) when dealing with Angle Class II cases has been noticed, although there is no definite scientific evidence to justify such decision. The aim of this study was to identify the advantages and disadvantages to this approach and which appliances are used for this purpose. For that purpose, a questionnaire containing full records of a Class II patient was sent to two professors of each Orthodontic graduate program in Brazil (n=96, total 192), from which 107 were properly answered. Results demonstrated that the most used appliances were the headgear (80.4%), maxillary splint (50%) and Bionator (44.4%). The benefits most often quoted were increase of patient self-esteem (78.5%) and reduction in the incidence of incisors trauma (63.6%), while the main disadvantage was saturation of patient compliance (73.8%). Considering early Class II treatment, there is still no unanimity as to treating in one or two stages or in selection of appliances. However, the orthodontists should consider the physical situation of the patient, severity of cases and susceptibility of trauma to the maxillary incisors. It is crucial that updated information is given to parents, in order to justify this approach.