Transcriptomic and macroscopic architectures of intersubject functional variability in human brain white-matter

Intersubject variability is a fundamental characteristic of brain organizations, and not just “noise”. Although intrinsic functional connectivity (FC) is unique to each individual and varies across brain gray-matter, the underlying mechanisms of intersubject functional variability in white-matter (WM) remain unknown. This study identified WMFC variabilities and determined the genetic basis and macroscale imaging in 45 healthy subjects. The functional localization pattern of intersubject variability across WM is heterogeneous, with most variability observed in the heteromodal cortex. The variabilities of heteromodal regions in expression profiles of genes are related to neuronal cells, involved in synapse-related and glutamic pathways, and associated with psychiatric disorders. In contrast, genes overexpressed in unimodal regions are mostly expressed in glial cells and were related to neurological diseases. Macroscopic variability recapitulates the functional and structural specializations and behavioral phenotypes. Together, our results provide clues to intersubject variabilities of the WMFC with convergent transcriptomic and cellular signatures, which relate to macroscale brain specialization.

Application of RNA subcellular fraction estimation method to explore RNA localization regulation

RNA localization is involved in multiple biological processes. Recent advances in subcellular fractionation-based sequencing approaches uncovered localization pattern on a global scale. Most of existing methods adopt relative localization ratios (such as ratios of separately normalized transcripts per millions of different subcellular fractions without considering the difference in total RNA abundances in different fractions), however, absolute ratios may yield different results on the preference to different cellular compartment. Experimentally, adding external Spike-in RNAs to different fractionation can be used to obtain absolute ratios. In addition, a spike-in independent computational approach based on multiple linear regression model can also be used. However, currently, no custom tool is available. To solve this problem, we developed a method called subcellular fraction abundance estimator to correctly estimate relative RNA abundances of different subcellular fractionations. The ratios estimated by our method were consistent with existing reports. By applying the estimated ratios for different fractions, we explored the RNA localization pattern in cell lines and also predicted RBP motifs that were associated with different localization patterns. In addition, we showed that different isoforms of same genes could exhibit distinct localization patterns. To conclude, we believed our tool will facilitate future subcellular fractionation-related sequencing study to explore the function of RNA localization in various biological problems.

Interplay Between the N-Terminal Domains of Arabidopsis Starch Synthase 3 Determines the Interaction of the Enzyme With the Starch Granule

The elongation of the linear chains of starch is undertaken by starch synthases. class 3 of starch synthase (SS3) has a specific feature: a long N-terminal region containing starch binding domains (SBDs). In this work, we analyze in vivo the contribution of these domains to the localization pattern of the enzyme. For this purpose, we divided the N-terminal region of Arabidopsis SS3 in three domains: D1, D2, and D3 (each of which contains an SBD and a coiled-coil site). Our analyses indicate that the N-terminal region is sufficient to determine the same localization pattern observed with the full-length protein. D2 binds tightly the polypeptide to the polymer and it is necessary the contribution of D1 and D3 to avoid the polypeptide to be trapped in the growing polymer. The localization pattern of Arabidopsis SS3 appears to be the result of the counterbalanced action of the different domains present in its N-terminal region.

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

ABSTRACT The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.

Visualization of intestinal infections with astro- and sapovirus in mink (Neovison vison) kits by in situ hybridization

Clarification of the infection microbiology remains a challenge in the pre-weaning diarrhea (PWD) syndrome in farmed mink (Neovison vison). Duodenal, jejunal and colon sections from 36 mink kits with PWD were systematically examined by chromogen in situ hybridization (CISH) targeting two incriminated viruses: Mink astrovirus and mink sapovirus. Using the RNAscope® 2.5 HD Duplex Assay astrovirus and sapovirus were visualized and simultaneously demonstrated in the gut tissue. Both viruses infect enterocytes in the small intestine with a specific localization pattern; astrovirus affects the two apical thirds of the villi, whereas sapovirus generally affects the basal parts of the villi. Furthermore, we demonstrated that astrovirus in mink does not target the goblet cells. This is the first time astro- and calicivirus have been visualized in mink kit gut tissue, and these findings might be important in clarification of the impact of these viruses in the PWD syndrome.

Resolving pronominal reference in local contexts: a referent selection task

Studies on global and local discourse have shown that sign languages indeed allow for occurrence of referentially unanchored pronominal index (ıx) signs referring to non-present antecedents to appear in ambiguous contexts. In local contexts, resolving the reference of these expressions has been suggested to either depend on a modality-specific anaphora resolution mechanism (localization) or on the next mention bias (first/second mention preference) influencing the salience of the referents. This paper presents a two-alternative forced choice referent selection task investigating the impact of a default localization pattern on the interpretation of referentially unanchored pronominal ix sign, in the local discourse with two competing antecedents. To do that, comparative response data was collected from right- and left-handed signers of German Sign Language (DGS) and Turkish Sign Language (TİD). Results provide evidence for second-mention or object preference for pronominal ix in both languages. In addition, the default localization pattern is identified only in restricted environments (i.e., with reciprocal verbs) to resolve pronominal reference. This modality-specific means is shown to be subject to variation across two unrelated sign languages under investigation. -----------------------------------------------------------------------------RESOLVENDO A REFERÊNCIA PRONOMINAL EM CONTEXTOS LOCAIS: UMA TAREFA DE SELEÇÃO DE REFERENTESEstudos existentes sobre discursos globais e locais mostram que as línguas de sinais claramente permitem que os sinais de apontação (ıx) pronominais referenciais não-ancorados referindo-se a antecedentes ausentes possam ocorrer em contextos ambíguos. Em contextos locais, sugere-se que a resolução da referência dessas expressões dependeriam tanto de um mecanismo de resolução anafórica específico da modalidade visual (localização), bem como de uma tendência de que a próxima menção (uma preferência pela primeira/segunda menção) influenciasse na saliência dos referentes que serão retomados. Este artigo apresenta uma tarefa de seleção de referentes feita obrigatoriamente a partir de duas alternativas que teve por objetivo investigar o impacto do padrão de localização default na interpretação do sinal de apontação pronominal referencial não-ancorado no discurso local, tendo como competidores, dois potenciais antecedentes. Para realizar tal tarefa, comparamos os dados obtidos das respostas de sinalizadores destros e canhotos da Língua de Sinais Alemã (DGS) e da Língua de Sinais Turca (TİD). Os resultados evidenciam que ıx pronominal retoma preferencialmente o segundo referente mencionado ou o objeto em ambas as línguas. Ainda, identificamos que o padrão de localização default só resolve a referência pronominal em ambientes restritos (ex: com verbos recíprocos). Mostramos que este modo de resolver a referência pronominal é específico da modalidade visual e está sujeito à variação paramétrica nas duas línguas de sinais investigadas, que não possuem relação de familiaridade.---Original em inglês.

The morphogenetic protein CotE drives exosporium formation by positioning CotY and ExsY during sporulation of Bacillus cereus

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates interactions of spores with their environment, modulates spore adhesion and germination and could be implicated in pathogenesis. The exosporium is composed of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, and surrounded by a glycoprotein hairy nap. The morphogenetic protein CotE is necessary for Bacillus cereus exosporium integrity, but how CotE directs exosporium assembly remains unknown. Here, we followed the localization of SNAP-tagged CotE, -CotY and -ExsY during B. cereus sporulation, using super-resolution fluorescence microscopy and evidenced interactions among these proteins. CotE, CotY and ExsY are present as complexes at all sporulation stages and follow a similar localization pattern during endospore formation that is reminiscent of the localization of Bacillus subtilis CotE. We show that B. cereus CotE drives the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process as previously proposed but it is performed through the formation of two caps, as observed during B. subtilis coat morphogenesis. It appears that a general principle governs the assembly of the spore surface layers of Bacillaceae.

Records of RNA locations in living yeast revealed through covalent marks

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3′ terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.

Alternating dynamics of oriC, SMC and MksBEF in segregation of Pseudomonas aeruginosa chromosome

Condensins are essential for global chromosome organization in diverse bacteria. Atypically, Pseudomonas aeruginosa encodes condensins from two superfamilies, SMC-ScpAB and MksBEF. We report that the two proteins play specialized roles in chromosome packing and segregation and are synthetically lethal with ParB. Inactivation of SMC or MksB asymmetrically affected global chromosome layout, its timing of segregation and sometimes triggered a chromosomal inversion. Localization pattern was also unique to each protein. SMC clusters colocalized with oriC throughout cell cycle except shortly after origin duplication, whereas MksB clusters emerged at cell quarters shortly prior to oriC duplication and stayed there even after cell division. Relocation of the proteins was abrupt and coordinated with oriC dynamic. These data reveal that the two condensins asymmetrically play dual roles in chromosome maintenance by organizing it and mediating its segregation. Furthermore, the choreography of condensins and oriC relocations suggest an elegant mechanism for the birth and maturation of chromosomes.ImportanceMechanisms that define the chromosome as a structural entity remain unknown. A key element in this process are condensins, which globally organize chromosomes and contribute to their segregation. This study characterized condensin and chromosome dynamics in Pseudomonas aeruginosa, which harbors condensins from two major protein superfamilies, SMC and MksBEF. The study revealed that both proteins asymmetrically play a dual role in chromosome maintenance by spatially organizing the chromosomes and guiding their segregation but can substitute for each other in some activities. The timing of chromosome, SMC and MksBEF relocation was highly ordered and interdependent revealing causative relationships in the process. Moreover, MksBEF was found to produce clusters at the site of chromosome replication that survived cell division and remained in place until chromosome replication was complete. Overall, these data delineate the functions of condensins from the SMC MksBEF superfamilies, reveal the existence of a chromosome organizing center and suggest a mechanism that might explain the biogenesis of chromosomes.