Hi-C techniques: from genome assemblies to transcription regulation.

The invention of chromosome-conformation capture (3C) techniques, in particular the key method Hi-C providing genome-wide information about chromatin contacts, revolutionized the way we study the three-dimensional (3D) organization of the nuclear genome and how it impacts transcription, replication and DNA repair. Since the frequency of chromatin contacts between pairs of genomic segments predictably relates to the distance in the linear genome, the Hi-C information has also proved useful for scaffolding genomic sequences. Here, we review recent enhancements in experimental procedures of Hi-C and its various derivatives such as Micro-C, HiChIP, and Capture Hi-C. We assess advantages and limitations of the techniques, and present examples of their use in recent plant studies. We also report on progress in computational tools used in assembling genome sequences.

Identification and functional characterization of the sugarcane (Saccharum spp.) AMT2-type ammonium transporter ScAMT3;3 revealed a presumed role in shoot ammonium remobilization.

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4 +), the sugarcane's preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4 + and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N-NH4 + in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4 + transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g-1 root DW h-1). We were able to infer that ScAMT3;3 plays a presumed role in NH4 + source-sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Holocentromeres can consist of merely a few megabase-sized satellite arrays.

The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.

Helical coiling of metaphase chromatids.

Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.

Consistencies and contradictions in different polymer models of chromatin architecture.

Genetic information is stored in very long DNA molecules, which are folded to form chromatin, a similarly long polymer fibre that is ultimately organised into chromosomes. The organisation of chromatin is fundamental to many cellular functions, from the expression of the genetic information to cell division. As a long polymer, chromatin is very flexible and may adopt a myriad of shapes. Globally, the polymer physics governing chromatin dynamics is very well understood. But chromatin is not uniform and regions of it, with chemical modifications and bound effectors, form domains and compartments through mechanisms not yet clear. Polymer models have been successfully used to investigate these mechanisms to explain cytological observations and build hypothesis for experimental validation. Many different approaches to conceptualise chromatin in polymer models can be envisioned and each reflects different aspects. Here, we compare recent approaches that aim at reproducing prominent features of interphase chromatin organisation: the compartmentalisation into eu- and heterochromatin compartments, the formation of a nucleolus, chromatin loops and the rosette and Rabl conformations of interphase chromosomes. We highlight commonalities and contradictions that point to a modulation of the mechanisms involved to fine degree. Consolidating models will require the inclusion of yet hidden or neglected parameters.

A simple model explains the cell cycle-dependent assembly of centromeric nucleosomes in holocentric species.

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.

Role of sphingomyelin on the interaction of the anticancer drug gemcitabine hydrochloride with cell membrane models.

The fight against drug resistance in chemotherapy requires a molecular-level understanding of the drug interaction with cell membranes, which today is feasible with membrane models. In this study, we report on the interaction of gemcitabine (GEM), a pyrimidine nucleoside antimetabolite used to treat pancreatic cancer, with Langmuir films that mimic healthy and cancerous cell membranes. The cell membrane models were made with eight compositions of a quaternary mixture containing 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), sphingomyelin (SM), and cholesterol (CHOL). The relative concentration of SM was increased so that four of these compositions represented cancerous cells. GEM was found to increase the mean molecular area, also increasing their surface elasticity, with stronger interactions being observed for membranes corresponding to cancerous cells. More specifically, GEM penetrated deepest in the membrane with the highest SM concentration (40 mol%), as inferred from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). This finding was confirmed with molecular dynamics simulations that also indicated how GEM approaches the membrane, which could be useful for guiding the design of drug delivery systems. The experimental and simulation results are consistent with the preferential attachment of GEM onto cancerous cells and highlight the role of SM on drug-cell interactions.

Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity.

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.

N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs.

Disorders of N-linked glycosylation are increasingly reported in the literature. However, the targets that are responsible for the associated developmental and physiological defects are largely unknown. Bone morphogenetic proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. Here, we investigate the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). We identify conserved N-glycosylated sites and describe the effect of mutating these residues on BMP pathway activity in Drosophila Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, we provide evidence that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.

Computer simulations reveal changes in the conformational space of the transcriptional regulator MosR upon the formation of a disulphide bond and in the collective motions that regulate its DNA-binding affinity.

M. tuberculosis oxidation sense Regulator (MosR) is a transcriptional regulator from Mycobacterium tuberculosis. It senses the environment oxidation and regulates the expression of a secreted oxidoreductase, thus defending the bacilli against oxidative stress from the phagosome. While most of the members of the Multiple antibiotics resistance Regulator (MarR) family are ligand-responsive, MosR may dissociate from its DNA site upon formation of an intrachain disulphide bond. However, the structure of MosR in its oxidized state is not known, and it is not clear how the formation of this disulphide bond would lead to the conformational changes required for dissociation of the DNA. Nonetheless, MosR presents two crystallographically resolved conformations in its reduced state: bound and unbound to DNA. We managed to simulate MosR unbound to the DNA, both in the presence and in the absence of the disulphide bond. Our results indicate that this disulphide bond precludes the N-terminal residues from adopting a conformation that stands in-between the helix α1 and the DNA binding domain (DBD) from the other chain. Once this conformation is achieved in the reduced state, this DBD detaches from the dimerization domain and becomes more flexible, being able to perform motions with higher amplitude and higher degree of collectivity. Only then, MosR may achieve a conformation where its recognition helices fit into the major grooves of its DNA site. The analysis of the collective motions performed by MosR, during the different situations sampled by the molecular dynamics (MDs), was only possible by the method of filtering harmonic modes with specific frequencies. The frequency of the collective motions performed by the DBD of MosR in the reduced state to achieve a DNA-binding conformation is in the range of 20 to 50 MHz, but it may be associated to more sporadic events since it requires the combination of a suitable conformation of the N-terminal residues.

NMR structures and molecular dynamics simulation of hylin-a1 peptide analogs interacting with micelles.

Antimicrobial peptides are recognized candidates with pharmaceutical potential against epidemic emerging multi-drug resistant bacteria. In this study, we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to determine the unknown structure and evaluate the interaction with dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles with three W6 -Hylin-a1 analogs antimicrobial peptides (HyAc, HyK, and HyD). The HyAc, HyK, and HyD bound to DPC micelles are all formed by a unique α-helix structure. Moreover, all peptides reach the DPC micelles' core, which thus suggests that the N-terminal modifications do not influence the interaction with zwiterionic surfaces. On the other hand, only HyAc and HyK peptides are able to penetrate the SDS micelle core while HyD remains always at its surface. The stability of the α-helical structure, after peptide-membrane interaction, can also be important to the second step of peptide insertion into the membrane hydrophobic core during permeabilization. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Correlação entre Padrões de Crescimento Facial e Terceiros Molares Inclusos / Correlation between Facial Growth Patterns Andincluded Third Molars

Objetivos: Analisar a prevalência dos padrões de crescimento facial e sua correlação com terceiros molares inclusos. Materiais e Métodos: A amostra, composta por 40 pacientes, foi obtida em um centro de radiologia odontológica, a partir deum banco de dados de arquivos de 800 pacientes, obedecendo a critérios de inclusão como: idade acima de 19 anos; presença de telerradiografia em norma lateral e uma radiografia panorâmica, obtidas na mesma época; presença de, no mínimo, um terceiro molar incluso com ápices dentários completamente fechados; permanência de todos os elementos dentários no lado do hemi-arco do elemento incluso; ausência de tratamento ortodôntico prévio. As telerradiografias foram utilizadas para a análise cefalométrica, utilizando-se os ângulos FMA, SN.GoMe e SN.Gn, interpretados de acordo com o padrão USP de análise cefalométrica e comparados com as normas préestabelecidas por essa análise, para a classificação dos padrões faciais em braquicefálico, mesocefálico ou dolicocefálico. A presença e a distribuição dos terceiros molares inclusos foram observadas com a utilização de radiografias panorâmicas. A correlação entre os ângulos dos padrões faciais e os terceiros molares inclusos foi verificada através do teste de Spearman (p<0,05). Dos pacientes pesquisados, 40% apresentaram perfil dolicocefálico, 35% mesocefálico e 25% braquicefálico. Conclusão:Não se constatou nenhuma significância para a correlação dos padrões faciais, nos três ângulos avaliados, com o número de terceiros molares inclusos. (AU)

Objective: This study analyzes the prevalence of facial growth patterns and their possible correlation with included third molars. Material and Methods: The sample consisted of 40 patients from a file database of 800 patients, referred to a dental radiology center. The following inclusion criteria were considered: 19 years or older; records including a lateral teleradiograph and a panoramic radiograph obtained at the same time; presence of at least one included third molar with completely closed apexes; presence of all teeth on the hemiarch of the included tooth; no history of orthodontic treatment. Teleradiographs were used for cephalometric analysis considering the angles FMA, SN.GoMe and SN.Gn, interpreted according to the USP standard analysis and compared with pre-established standards for the classification of facial patterns (brachycephalic, mesocephalic or dolichocephalic). The presence and distribution of third molars were observed using panoramic radiographs. The correlations between the angles of the facial patterns and the presence of included third molars was analyzed using Spearman's test (p<0.05). Conclusion: The patients' profiles identified were as follows: 40% dolichocephalic, 35% mesocephalic and 25% brachycephalic. No significant correlation was found between the facial patterns ­ for all the three angles analyzed ­ and the number of included third molars. (AU)

Resistência ao Cisalhamento de Braquetes Colados com Dois Tipos de Agentes de União e Expostos à Ação de Bebidas Ácidas / Shear Bond Strength of Orthodontic Brackets Bond with Two Types of Bonding Agents and Exposed to Acidic Beverages

Objetivos: Avaliar a influência de bebidas ácidas na resistência de união de braquetes ortodônticos colados com os agentes resinosos Transbond™ PLUS Color Change (TPCC) e Ortholink™ e quantificar o Índice de Remanescente Adesivo (IRA) após a descolagem do braquete, a fim de constatar qual compósito apresenta maior resistência à tensão cisalhante e o local onde ocorreu a falha adesiva, respectivamente. Materiais e Métodos: Noventa e seis incisivos bovinos tiveram braquetes colados em sua superfície vestibular e foram divididos em dois grupos (n=48), de acordo com o agente de união utilizado: I) TPCC e II)Ortholink. Em seguida, cada grupo foi separado em quatro subgrupos (n=12), de acordo com a substância na qual as amostras seriam imersas: S1) Coca-cola®, S2) Suco de laranja Del Valle®, S3) Gatorade® e S4) saliva artificial (controle). As amostras dos subgrupos S1, S2 e S3 foram submetidas a três ciclos corrosivos diários de 5 minutos, com intervalos de 2 horas, durante 30 dias, sendo sempre recolocadas em saliva artificial até que um novo ciclo fosse iniciado. Ao fim deste período, foram submetidas ao ensaio mecânico de cisalhamento e à avaliação do IRA. Resultados: Não foram encontradas diferenças estatisticamente significantes para as resinas entre si e entre os subgrupos, nos dois parâmetros testados (p>0,05). Conclusão: Não houve influência das substâncias ácidas testadas sobre a força de adesão dos compósitos utilizados, possuindo ambos resistências semelhantes, e que tanto a interface resina/ braquete quanto braquete/dente apresentaram falhas adesivas. (AU)

Objective: To evaluate the influence of acidic beverageson the bond strength of orthodontic brackets bonded with the resinous agents Transbond ™ PLUS Color Change (TPCC) and Ortholink ™.The Adhesive Remnant Index (ARI) after bracket removal was further quantified, in order to determine which composite has a higher resistance to shear stress and where there was adhesive failure. Material and Methods: Ninety-six bovine incisors had brackets bonded on their vestibular surfaces and were divided into two groups (n = 48), according to the bonding agent used: I) TPCC and II) Ortholink. Then, each group was divided into four subgroups (n = 12) in accordance with the substance inwhich the samples would be immersed: S1) Coca-Cola®, S2) Orange juice Del Valle®, S3) Gatorade® and S4) artificial saliva (control). Samples of subgroups S1, S2 and S3 were subjected to three corrosive cycles daily for 5 minutes with 2 hours intervals for 30 days, each being replaced in artificial saliva until a new cycle was started. At the end of this period, sampleswere subjected to mechanical shear test and the ARI assessment. Results: No statistically significant differences were found betweenthe compositesand between the subgroups for the two parameters tested (P>0.05). Conclusion: There was no influence of acidic beverageson the bond strength of the composites, asboth showedsimilar resistance, and the resin/bracket interfaces as well asthe bracket/tooth interfaces showed adhesive failures. (AU)

alphaPS1betaPS integrin receptors regulate the differential distribution of Sog fragments in polarized epithelia.

Bone morphogenetic proteins (BMPs) have important functions during epithelial development. In Drosophila, extracellular Short gastrulation (Sog) limits the action of the BMP family member Decapentaplegic (Dpp). We have shown that Integrin receptors regulate Sog activity and distribution during pupal wing development to direct placement of wing veins. Here, we show that Integrins perform a similar function in the follicular epithelium, impacting Dpp function during oogenesis and embryonic development. As reported for the wing, this effect is specific to mew, which codes for alphaPS1 integrin. Sog is subject to cleavage by metalloproteases, generating fragments with different properties. We also show that Integrins regulate the distribution of C- and N-terminal Sog fragments in both epithelia, suggesting they may regulate the quality of BMP outputs. Our data indicate that alphaPS1betaPS integrin receptors regulate the amount and type of Sog fragments available for diffusion in the extracellular space during oogenesis and pupal wing development.