Inhibition of basal FGF receptor signaling by dimeric Grb2.

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.

Canonical Typicality under General Quantum Channels.

With the control of increasingly complex quantum systems, the relevant degrees of freedom we are interested in may not be those traditionally addressed by statistical quantum mechanics. Here, we employ quantum channels to define generalized subsystems, capturing the pertinent degrees of freedom, and obtain their associated canonical state. We show that the generalized subsystem description from almost any microscopic pure state of the whole system will behave similarly to its corresponding canonical state. Such canonical typicality behavior depends on the entropy of the channel used to define the generalized subsystem.

Detection and molecular characterization of a novel mitovirus associated with Passiflora edulis Sims.

Mitoviruses are cryptic capsidless viruses belonging to the family Mitoviridae that replicate and are maintained in the mitochondria of fungi. Complete mitovirus-like sequences were recently assembled from plant transcriptome data and plant leaf tissue samples. Passion fruit (Passiflora spp.) is an economically important crop for numerous tropical and subtropical countries worldwide, and many virus-induced diseases impact its production. From a large-scale genomic study targeting viruses infecting Passiflora spp. in Brazil, we detected a de novo-assembled contig with similarity to other plant-associated mitoviruses. The contig is ∼2.6 kb long, with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRP). This contig has been named "passion fruit mitovirus-like 1" (PfMv1). An alignment of the predicted amino acid sequence of the RdRP of PfMv1 and those of other plant-associated mitoviruses revealed the presence of the six conserved motifs of mitovirus RdRPs. PfMv1 has 79% coverage and 50.14% identity to Humulus lupulus mitovirus 1. Phylogenetic analysis showed that PfMV1 clustered with other plant-associated mitoviruses in the genus Duamitovirus. Using RT-PCR, we detected a PfMv1-derived fragment, but no corresponding DNA was identified, thus excluding the possibility that this is an endogenized viral-like sequence. This is the first evidence of a replicating mitovirus associated with Passiflora edulis, and it should be classified as a member of a new species, for which we propose the name "Duamitovirus passiflorae".

Novel plant-associated genomoviruses from the Brazilian Cerrado biome.

The discovery rate of new plant viruses has increased due to studies involving high-throughput sequencing (HTS), particularly for single-stranded DNA viruses of the family Genomoviridae. We carried out an HTS-based survey of genomoviruses in a wide range of native and exotic trees grown in the Brazilian Cerrado biome, and the complete genome sequences of two novel members of the family Genomoviridae from two distinct genera were determined. Specific primers were designed to detect these genomoviruses in individual samples. A new gemykolovirus (Tecoma stans associated gemykolovirus) was detected in Tecoma stans, and a new gemykibivirus (Ouratea duparquetiana associated gemykibivirus) was detected in Ouratea duparquetiana. A gemykrogvirus related to Gila monster associated gemykrogvirus (80% pairwise identity) was also detected in foliar samples of Trembleya parviflora. Our pilot study paves the way for a better characterization of this diverse collection of genomoviruses as well as their interactions with the associated tree species.

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding.

The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a one-dimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

Virus classification based on in-depth sequence analyses and development of demarcation criteria using the Betaflexiviridae as a case study.

Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the family Betaflexiviridae in the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.

First description of a multisystemic and lethal SARS-CoV-2 variant of concern P.1 (Gamma) infection in a FeLV-positive cat.

BACKGROUND: Phylogenetic studies indicate bats as original hosts of SARS-CoV-2. However, it remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. METHODS: In this study, we analyzed the first fatal case of a SARS-CoV-2 and FeLV co-infection in an eight-year-old male cat. We carried out a clinical evaluation and several laboratory analyses. RESULTS: As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to the animal's death. RT-qPCR analysis showed a SARS-CoV-2 as the causative agent. The virus was detected in several organs, indicating a multisystemic infection. The virus was found in a high load in the trachea, suggesting that the animal may have contribute to the transmission of the virus. The whole-genome sequencing revealed an infection by SARS-CoV-2 Gamma VOC (P.1), and any mutations indicating host adaptation were observed. CONCLUSION: Our data show that FeLV-positive cats are susceptible to SARS-CoV-2 infection and raise questions about the potential of immunocompromised FeLV-positive cats to act as a reservoir for SARS-CoV-2 new variants.

Magnetic alignment of rhodamine/magnetite dual-labeled microtubules probed with inverted fluorescence microscopy.

Molecular machines, as exemplified by the kinesin and microtubule system, are responsible for molecular transport in cells. The monitoring of the cellular machinery has attracted much attention in recent years, requiring sophisticated techniques such as optical tweezers, and dark field hyperspectral and fluorescence microscopies. It also demands suitable procedures for immobilization and labeling with functional agents such as dyes, plasmonic nanoparticles and quantum dots. In this work, microtubules were co-polymerized by incubating a tubulin mix consisting of 7 biotinylated tubulin to 3 rhodamine tubulin. Rhodamine provided the fluorescent tag, while biotin was the anchoring group for receiving streptavidin containing species. To control the microtubule alignment and consequently, the molecular gliding directions, functionalized iron oxide nanoparticles were employed in the presence of an external magnet field. Such iron oxide nanoparticles, (MagNPs) were previously coated with silica and (3-aminopro-pyl)triethoxysilane (APTS) and then modified with streptavidin (SA) for linking to the biotin-functionalized microtubules. In this way, the binding has been successfully performed, and the magnetic alignment probed by Inverted Fluorescence Microscopy. The proposed strategy has proved promising, as tested with one of the most important biological structures of the cellular machinery.

Tobamoviruses of two new species trigger resistance in pepper plants harbouring functional L alleles.

Tobamoviruses are often referred to as the most notorious viral pathogens of pepper crops. These viruses are not transmitted by invertebrate vectors, but rather by physical contact and seeds. In this study, pepper plants displaying mild mottle and mosaic symptoms were sampled in four different regions of Peru. Upon double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, seven samples cross-reacted weakly with antibodies against pepper mild mottle virus (PMMoV), suggesting the presence of tobamoviruses. When employing RT-PCR, conserved primers amplified cDNA fragments of viruses from two putative new tobamovirus species in the samples. The complete genome of two representative isolates were, therefore, sequenced and analysed in silico. These viruses, which were tentatively named yellow pepper mild mottle virus (YPMMoV) and chilli pepper mild mottle virus (CPMMoV), shared highest nucleotide genome sequence identities of 83 and 85 % with bell pepper mottle virus (BpeMV), respectively. Mechanical inoculation of indicator plants with YPMMoV and CPMMoV isolates did not show any obvious differences in host ranges. These viruses were also inoculated mechanically on pepper plants harbouring different resistance L alleles to determine their pathotypes. Pepper plants carrying unfunctional L alleles (L0) to tobamoviruses were infected by all isolates and presented differential symptomatology for YPMMoV and CPMMoV. On the other hand, pepper plants carrying L1, L2, L3 and L4 alleles were resistant to all isolates, indicating that these viruses belong to pathotype P0.

Nanopore sequencing of tomato mottle leaf distortion virus, a new bipartite begomovirus infecting tomato in Brazil.

During a survey in a tomato field in Luziânia (Goiás State, Brazil), a single plant with mottling, chlorotic spots, and leaf distortion was found. A new bipartite begomovirus sequence was identified using nanopore sequence technology and confirmed by Sanger sequencing. The highest nucleotide sequence identity match of the DNA-A component (2596 bases) was 81.64% with tomato golden leaf deformation virus (HM357456). Due to the current species demarcation criterion of 91% nucleotide sequence identity for DNA-A, we propose this virus to be a new member of the genus Begomovirus, named "tomato mottle leaf distortion virus".

H-NS uses an autoinhibitory conformational switch for environment-controlled gene silencing.

As an environment-dependent pleiotropic gene regulator in Gram-negative bacteria, the H-NS protein is crucial for adaptation and toxicity control of human pathogens such as Salmonella, Vibrio cholerae or enterohaemorrhagic Escherichia coli. Changes in temperature affect the capacity of H-NS to form multimers that condense DNA and restrict gene expression. However, the molecular mechanism through which H-NS senses temperature and other physiochemical parameters remains unclear and controversial. Combining structural, biophysical and computational analyses, we show that human body temperature promotes unfolding of the central dimerization domain, breaking up H-NS multimers. This unfolding event enables an autoinhibitory compact H-NS conformation that blocks DNA binding. Our integrative approach provides the molecular basis for H-NS-mediated environment-sensing and may open new avenues for the control of pathogenic multi-drug resistant bacteria.

Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen and one of the leading causes of nosocomial infections. Moreover, the species can cause severe infections in cystic fibrosis patients, in burnt victims and cause disease in domestic animals. The control of these infections is often difficult due to its vast repertoire of mechanisms for antibiotic resistance. Phage therapy investigation with P. aeruginosa bacteriophages has aimed mainly the control of human diseases. In the present work, we have isolated and characterized a new bacteriophage, named Pseudomonas phage BrSP1, and investigated its host range against 36 P. aeruginosa strains isolated from diseased animals and against P. aeruginosa ATCC strain 27853. RESULTS: We have isolated a Pseudomonas aeruginosa phage from sewage. We named this virus Pseudomonas phage BrSP1. Our electron microscopy analysis showed that phage BrSP1 had a long tail structure found in members of the order Caudovirales. "In vitro" biological assays demonstrated that phage BrSP1 was capable of maintaining the P. aeruginosa population at low levels for up to 12 h post-infection. However, bacterial growth resumed afterward and reached levels similar to non-treated samples at 24 h post-infection. Host range analysis showed that 51.4% of the bacterial strains investigated were susceptible to phage BrSP1 and efficiency of plating (EOP) investigation indicated that EOP values in the strains tested varied from 0.02 to 1.72. Analysis of the phage genome revealed that it was a double-stranded DNA virus with 66,189 bp, highly similar to the genomes of members of the genus Pbunavirus, a group of viruses also known as PB1-like viruses. CONCLUSION: The results of our "in vitro" bioassays and of our host range analysis suggested that Pseudomonas phage BrSP1 could be included in a phage cocktail to treat veterinary infections. Our EOP investigation confirmed that EOP values differ considerably among different bacterial strains. Comparisons of complete genome sequences indicated that phage BrSP1 is a novel species of the genus Pbunavirus. The complete genome of phage BrSP1 provides additional data that may help the broader understanding of pbunaviruses genome evolution.

An isolate of sweet potato chlorotic stunt virus from Brazil with a distinct genome organization.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae), is an economically important pathogen of sweet potato. In the present work, the nucleotide sequences of two RNA segments of SPCSV (isolate SPCSV-UNB-01) were determined by MiSeq Illumina sequencing of samples of sweet potato plants grafted onto Ipomoea setosa. A comparative analysis of the genome organization of SPCSV-UNB-01 and other SPCSV sequences showed that RNA1 was lacking p22, and p5.1 and that p5.2. was absent in RNA2, indicating a unique genomic pattern. SPCSV-UNB-01 contained longer p6 and p5 regions, with little similarity to orthologous sequences. Sequence comparison did not reveal any previously identified functional domains within these open reading frames (ORFs). No recombination or rearrangement events were detected. Phylogenetic analysis suggested the possibility of separate entries of SPCSV into South America based on the genetic distance between SPCSV-UNB-01 and the Peruvian isolate m2-47. Samples from northeastern Brazil (State of Pernambuco) were positive for SPCSV when tested using specific primers for the major coat protein (CP) gene. This is the first full-length genome sequence of SPCSV-UNB-01 from Brazil.

Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE-088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE-088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE-088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing.

The Tomato chlorotic spot virus (TCSV) was first reported in the 1980s, having its occurrence limited to Brazil and Argentina. Due to an apparent mild severity in the past, molecular studies concerning TCSV were neglected. However, TCSV has disseminated over the USA and Caribbean countries. In Dominican Republic TCSV has been recently reported on important cultivated crops such as pepper and beans. In this work, we provide the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. In addition, three dsRNA viruses from different virus families were identified coinfecting these plants Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). Phylogenetic analysis showed that the Dominican Republic TCSV isolate has a close relationship with other TCSV isolates and a reassortant isolate between TCSV and Groundnut ringspot virus (GRSV), all found in USA. BPEV, STV and PCV-2 isolates from Dominican Republic were close related to corresponding American isolates. The possible biological implications of these virus-mixed infections are discussed.

Extraction of Dysprosium Ions with DTPA Functionalized Superparamagnetic Nanoparticles Probed by Energy Dispersive X-ray Fluorescence and TEM/High-Angle Annular Dark Field Imaging.

The extraction of dysprosium (Dy3+) ions from aqueous solution was carried out successfully, using magnetite (Fe3O4) nanoparticles functionalized with diethylenetriaminepentaacetic acid (MagNP@DTPA). The process was monitored by energy dispersive X-ray fluorescence spectroscopy, as a function of concentration, proceeding according to a Langmuir isotherm with an equilibrium constant of 2.57 × 10-3 g(MagNP) L-1 and a saturation limit of 63.2 mgDy/gMagNP. The presence of paramagnetic Dy3+ ions attached to the superparamagnetic nanoparticles led to an overall decrease of magnetization. By imaging the nanoparticles surface using scanning transmission electron microscopy equipped with high resolution elemental analysis, it was possible to probe the binding of the Dy3+ ions to DTPA, and to show their distribution in a region of negative magnetic field gradients. This finding is coherent with the observed decrease of magnetization, associated with the antiferromagnetic coupling between the lanthanide ions and the Fe3O4 core.

A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

Complete genome sequence of a putative new secovirus infecting yam (Dioscorea) plants.

The complete genome sequence of a new virus infecting yam plants exhibiting mosaic symptom in Brazil was determined. The genome of this virus is composed of two molecules of positive-sense RNAs of 5979 and 3809 nucleotides in length, excluding the poly(A) tails. One large open reading frame (ORF) in each genomic segment (RNA1-ORF1 and RNA2-ORF2) was predicted. The highest amino acid sequence similarity in the Pro-Pol core region of RNA1 and the CP region of RNA2 was observed with chocolate lily virus A (a putative member of the family Secoviridae), with 54.6 and 27.7 % identity, respectively. This virus is thus likely to be a new member of the family Secoviridae, and we propose the tentative name "dioscorea mosaic-associated virus" (DMaV) for this virus.

Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is distantly related to other genomovirus. It was thus classified in a putative new genus, for which the name "Gemybolavirus" is proposed. These new genomoviruses are tentatively named "Bemisia associated gemybolavirus AdDF", and "Bemisia associated gemycircularvirus AdO".

Erratum to: Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in Central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is related to those of the genus Gemyduguivirus. These new genomoviruses are tentatively named bemisia-associated genomovirus AdO and bemisia-associated genomovirus AdDF.