title: Interface size drives cotranslational assembly of protein complexes

abstract: Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this efficiency is by seeding assembly pathways during translation via the cotranslational assembly of subunits. While recent evidence suggests that such cotranslational assembly is widespread, little is known about the properties of protein complexes associated with the phenomenon. Here, using a combination of proteome-specific protein complex structures and publicly available ribosome profiling data, we show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, we compared the sizes of first and last translated interfaces of heteromers in bacterial, yeast, and human complexes. We detect a clear preference in N-terminal interfaces, which will be translated first and thus more likely to form cotranslationally, to be larger than C-terminal interfaces, suggesting that large interfaces have evolved as a means to maximise the chance of successful cotranslational subunit binding.

category: molecular biology


title: Large protein complex interfaces have evolved to promote cotranslational assembly


title: A modular molecular framework for quickly estimating the binding affinity of the spike protein of SARS-CoV-2 variants for ACE2, in presence of mutations at the spike receptor binding domain.

abstract: The rapid spread of new SARS-CoV-2 variants needs the development of rapid tools for predicting the affinity of the mutated proteins responsible for the infection, i.e., the SARS-CoV-2 spike protein, for the human ACE2 receptor, aiming to understand if a variant can be more efficient in invading host cells. Here we show how our computational pipeline, previously used for studying SARS-CoV-2 spike receptor binding domain (RBD)/ACE2 interactions and pre-/post-fusion conformational changes, can be used for predicting binding affinities of the human ACE2 receptor for the spike protein RBD of the characterized infectious variants of concern/interest B.1.1.7-UK (carrying the mutations N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417N/T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 variant (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Furthermore, we searched for ACE2 structurally related proteins that might be involved in interactions with the SARS-CoV-2 spike protein, in those tissues showing low ACE2 expression, revealing two new proteins, THOP1 and NLN, deserving to be investigated for their possible inclusion in the group of host-cell entry factors responsible for host-cell SARS-CoV-2 invasion and immunity response.

category: biochemistry


title: Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis.

abstract: The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology, viral replication or cytokine expression were observed between lean and obese groups. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection is inadequate for performing nutrition studies, and caution should be used when interpreting resulting data.

category: pathology


title: The fate of Hepatitis E virus capsid protein is regulated by an Arginine-Rich Motif

abstract:
Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation, promoting regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by cellular proteases, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Author summaryHepatitis E virus (HEV) is the major cause of acute viral hepatitis worldwide. Although infection with HEV is usually self-resolving, it can cause up to 30% mortality in pregnant women in the third trimester. There is no specific treatment nor universal vaccine to fight against HEV. In our study, we focused on the HEV ORF2 capsid protein which is produced as different forms that perform distinct functions in the HEV lifecycle. The infectious ORF2i form is the structural component of virions, while the other forms likely act as an immune decoy. Herein, we deciphered the molecular determinants of ORF2 multifunctionality. We identified an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region that controls the subcellular localization, the fate and functions of ORF2 forms. It also promotes ORF2-host cell interactions. Our observations highlight the ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle.

category: microbiology


title: An Arginine-Rich Motif in the ORF2 Capsid Protein Regulates the Hepatitis E Virus Lifecycle and Interactions with the Host Cell


title: Molecular basis and evolutionary cost of a novel phenotype of macrolides/lincosamides resistance in Staphylococcus haemolyticus

abstract: Staphylococcus haemolyticus (S. haemolyticus) is a coagulase-negative Staphylococcus and has become one of the primary pathogens of nosocomial infections. After a long period using the antibiotics against the infections caused by S. haemolyticus, S. haemolyticus developed several resistance phenotypes of macrolides and lincosamides. Here, we acquired four S. haemolyticus clinical isolates, of which three isolates demonstrated antibiotic resistance as reported previously, while one was resistant to both erythromycin and clindamycin without erythromycin induction. Such novel phenotype, known as constitutive Macrolides-Lincosamides-Streptogramins (MLS) resistance, was reported in other bacteria, but it was found for the first time in S. haemolyticus. To further investigate such behavior, we confirmed that the deletion of the methyltransferase ErmC upstream leader peptide was critical to cause constitutive MLS resistance based on the analysis of potential antibiotic resistance genes via whole-genome sequencing and experimental verification. In addition, we discovered that the continuous expression of ErmC may inhibit the growth of S. haemolyticus, which turned out to be the cost with no MLS pressure. Taken together, this study firstly reported the constitutive MLS resistance in S. haemolyticus, and provided new ideas for using macrolides and lincosamides in clinical treatment.


title: Diversity, taxonomy and evolution of archaeal viruses of the class Caudoviricetes

abstract: The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counter defense mechanisms, illuminating common strategies of virus-host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered four distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine and anoxic environments.


title: Hydrophobic mismatch effect is a key factor in protein transport on the Tat pathway

abstract: The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.


title: Hydrophobic mismatch is a key factor in protein transport on the Tat pathway


title: A new twist in ABC transporter mediated multidrug resistance - Pdr5 is a drug/proton co-transporter

abstract: The two major efflux pump systems are involved in multidrug resistance (MDR): (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ABC transporters that are involved in MDR and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of transport and specificity remain elusive. Here, we provide electrophysiological data on reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.


title: TatA and TatB generate a hydrophobic mismatch that is important for function and assembly of the Tat translocon in Escherichia coli

abstract: The Tat system has the unique purpose to translocate folded proteins across energy-transducing membranes. It occurs in bacteria and archaea, as well as in eukaryotic organelles of bacterial origin. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was clearly tolerated. Defects of shortenings or extensions were enhanced by simultaneous mutations in TatA and TatB, indicating partial compensations of mutations in TatA by wild type TatB or vice versa. Length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residues-length is important for Tat component interactions and translocon assembly. To also address potential effects on TatA associations, we characterized these by metal tagging transmission electron microscopy and carried out molecular dynamics simulations. In these simulations, interacting short TMHs of larger TatA assemblies were thinning the membrane together with laterally aligned tilted APHs that generated a deep V-shaped groove. The conserved length of 12 hydrophobic residues may thus not only be important for translocon interactions, but also for a membrane destabilization during Tat transport. If this is the case, the specific short length could be a compromise between functionality and proton leakage minimization.


title: Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression

abstract: Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13 enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.

category: bioengineering


title: Quadratic and adaptive computations yield an efficient representation of song in Drosophila auditory receptor neurons

abstract:
Sensory neurons encode information using multiple nonlinear and dynamical transformations. For instance, auditory receptor neurons in Drosophila adapt to the mean and the intensity of the stimulus, change their frequency tuning with sound intensity, and employ a quadratic nonlinearity. While these computations are considered advantageous in isolation, their combination can lead to a highly ambiguous and complex code that is hard to decode. Combining electrophysiological recordings and computational modelling, we investigate how the different computations found in auditory receptor neurons in Drosophila combine to encode behaviorally-relevant acoustic signals like the courtship song.

The computational model consists of a quadratic filter followed by a divisive normalization stage and reproduces population neural responses to artificial and natural sounds. For general classes of sounds, like band-limited noise, the representation resulting from these highly nonlinear computations is highly ambiguous and does not allow for a recovery of information about the frequency content and amplitude pattern. However, for courtship song, the code is simple and efficient: The quadratic filter improves the representation of the song envelope while preserving information about the songs fine structure across intensities. Divisive normalization renders the presentation of the song envelope robust to the relatively slow fluctuations in intensity that arise during social interactions, while preserving information about the species-specific fast fluctuations of the envelope.

Overall, we demonstrate how a sensory system can benefit from adaptive and nonlinear computations while minimizing concomitant costs arising from ambiguity and complexity of readouts by adapting the code for behaviorally-relevant signals.

category: neuroscience


title: The globular C1q receptor is required for epidermal growth factor receptor signaling during Candida albicans infection

abstract:
During oropharyngeal candidiasis, Candida albicans activates the epidermal growth factor receptor (EGFR), which induces oral epithelial cells to both endocytose the fungus and synthesize proinflammatory mediators that orchestrate the host immune response. To elucidate the signaling pathways that are stimulated when C. albicans interacts with EGFR, we analyzed the proteins that associate with EGFR when C. albicans infects human oral epithelial cells. We identified 1214 proteins that were associated with EGFR in C. albicans-infected cells. We investigated the function of seven of these proteins that either showed increased association with EGFR in response to C. albicans or that mediated the interaction of other microbial pathogens with epithelial cells. Among these proteins, EGFR was found to associate with WW domain-binding protein 2, toll-interacting protein, interferon-induced transmembrane protein 3, and the globular C1q receptor (gC1qR) in viable epithelial cells. Each of these proteins was required for maximal endocytosis of C. albicans and they all regulated fungal-induced production of IL-1{beta} and/or IL-8, either positively or negatively. gC1qR functioned as a key coreceptor with EGFR. Interacting with the C. albicans Als3 invasin, gC1qR was required for the fungus to stimulate both EGFR and the ephrin type-A receptor 2. The combination of gC1qR and EGFR was necessary for maximal endocytosis of C. albicans and secretion of IL-1{beta}, IL-8, and GM-CSF. Thus, this work provides an atlas of proteins that associate with EGFR and identifies several that play a central role in the response of human oral epithelial cells to C. albicans infection.

IMPORTANCEOral epithelial cells play a key role in the pathogenesis of oropharyngeal candidiasis. In addition to being target host cells for C. albicans adherence and invasion, they secrete proinflammatory cytokines and chemokines that recruit T cells and activated phagocytes to foci of infection. It is known that C. albicans activates EGFR on oral epithelial cells, which induces these cells to endocytose the organism and stimulates them to secrete proinflammatory mediators. To elucidate the EGFR signaling pathways that govern these responses, we analyzed the epithelial cell proteins that associate with EGFR in C. albicans-infected epithelial cells. We identified four proteins that physically associate with EGFR and that regulate different aspects of the epithelial response to C. albicans. One of these is gC1qR, which is required for C. albicans to activate EGFR, induce endocytosis, and stimulate the secretion of proinflammatory mediators, indicating that gC1qR functions as a key co-receptor with EGFR.


title: Chemical structure elucidation of the carotenoids in the extremely radiation-resistant bacterium Deinococcus radiodurans R1

abstract: The main carotenoid in the extremely radiation-resistant bacterium Deinococcus radiodurans is deinoxanthin. In this paper, based on UV-Vis, MS/MS spectra as well as the chemical properties, the other main two carotenoids in this bacterium were identified as ((all-E)-1-hydroxy-3,4-didehydro-1,2-dihydro-{beta},{Psi}-carotene- 4-one-1), and ((all-E)-1-hydroxy-3,4-didehydro-1,2,2,3-quahydro-{beta},{Psi}-carotene- 4-one-1), respectively. The fully structure elucidation for the carotenoids should help to gain a better understanding of the radiation-resistant mechanisms in this remarkable bacterium.


title: Age and infectious dose significantly affect disease progression after RHDV2 infection in naive domestic rabbits

abstract: Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations; wild rabbits threaten over 300 native species and result in economic impacts in excess of $200 million AUD to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 hours and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naive rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.


title: Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse

abstract: How distinct cell fates are manifested by direct lineage ancestry from bipotent progenitors, or by specification of individual cell types within a field of cells is a key question for understanding the emergence of tissues. The interplay between skeletal muscle progenitors and associated connective tissues cells provides a model for examining how muscle functional units are established. Most craniofacial structures originate from the vertebrate-specific neural crest cells except in the dorsal portion of the head, where they arise from cranial mesoderm. Here, using multiple lineage-traced single cell RNAseq, advanced computational methods and in situ analyses, we identify Myf5+ bipotent progenitors that give rise to both muscle and juxtaposed connective tissue. Following this bifurcation, muscle and connective tissue cells retain complementary signalling features and maintain spatial proximity. Interruption of upstream myogenic identity shifts muscle progenitors to a connective tissue fate. Interestingly, Myf5-derived connective tissue cells, which adopt a novel regulatory signature, were not observed in ventral craniofacial structures that are colonised by neural crest cells. Therefore, we propose that an ancestral program gives rise to bifated muscle and connective tissue cells in skeletal muscles that are deprived of neural crest.

category: developmental biology


title: Climate change and the complex dynamics of green spruce aphid--spruce plantation interactions

abstract: Aphids can have a significant impact on the growth and commercial yield of spruce plantations. Here we develop a mechanistic deterministic mathematical model for the dynamics of the green spruce aphid (Elatobium abietum Walker) growing on Sitka spruce (Picea sitchensis (Bong.) Carr.). These grow in a northern British climate in managed plantations, with planting, thinning and a 60-year rotation. Aphid infestation rarely kills the tree but can reduce growth by up to 55%. We used the Edinburgh Forest Model (efm) to simulate spruce tree growth. The aphid sub-model is described in detail in an appendix. The only environmental variable which impacts immediately on aphid dynamics is air temperature which varies diurnally and seasonally. The efm variables that are directly significant for the aphid are leaf area and phloem nitrogen and carbon. Aphid population predictions include dying out, annual, biennual and other complex patterns, including chaos. Predicted impacts on plantation yield of managed forests can be large and variable, as has been observed; they are also much affected by temperature, CO2 concentration and other climate variables. However increased CO2 concentration appears to ameliorate the severity of the effects of increasing temperatures coupled to worsening aphid infestations on plantation yield.

category: ecology


title: Tannic Acid Inhibits α-Synuclein Amyloid Fibril Formation via Binding to the Monomer N-terminal Domain

abstract: -Synuclein (S) is an intrinsically disordered protein (IDP) that aggregates into amyloid fibrils during the progression of Parkinsons Disease and other synucleinopathies. The N-terminal domain (residues 1-60) is now understood to play a critical role in the initial nucleation of aggregation, as well as a pivotal role in the monomer-fibril interaction underlying amyloid seeding. Here we report on the interaction between S and the polyphenol tannic acid (TA), where a combination of solution NMR, atomic force microscopy (AFM), and ThT assays have identified that TA targets the S N-terminal domain to inhibit amyloid fibril formation in a pH dependent manner. This work highlights the importance of targeting the N-terminus of S to arrest fibril formation, and suggests the importance of including polyphenolic moieties in future amyloid inhibitors.


title: A robust method for performing 1H-NMR lipidomics in embryonic zebrafish

abstract: The embryonic zebrafish is an ideal system for lipid analyses with relevance to many areas of bioscience research, including biomarkers and therapeutics. Research in this area has been hampered by difficulties in extracting, identifying and quantifying lipids. We employed 1H-NMR at 700MHz to profile lipids in developing zebrafish embryos. The optimal method for lipidomics in embryonic zebrafish incorporated rapid lipid extraction using chloroform and an environment without oxygen depletion. Pools of 10 embryos gave the most acceptable signal-to-noise ratio, and the inclusion of chorions in the sample had no significant effect on lipid abundances. Embryos, bisected into cranial (head and yolk sac) and caudal (tail) regions, were compared by principal component analysis and analysis of variance. The lipid spectra (including lipid annotation) are available in the public repository MetaboLights (MTBLS2396).


title: Comparison of linear combination modeling strategies for GABA-edited MRS at 3T

abstract:
PurposeJ-difference-edited spectroscopy is a valuable approach for the in vivo detection of {gamma}-aminobutyric-acid (GABA) with MRS. A recent expert consensus article recommends linear combination modeling (LCM) of edited MRS but does not give specific details of implementation. This study explores different modeling strategies to adapt LCM for GABA-edited MRS.

Methods61 medial parietal lobe GABA-edited MEGA-PRESS spectra from a recent 3T multi-site study were modeled using 102 different strategies combining six different approaches to account for co-edited macromolecules, three modeling ranges, three baseline knot spacings, and the use of basis sets with or without homocarnosine. The resulting GABA and GABA+ estimates (quantified relative to total creatine), the residuals at different ranges, SDs and CVs, and Akaike information criteria, were used to evaluate the models performance.

ResultsSignificantly different GABA+ and GABA estimates were found when a well-parameterized MM3co basis function was included in the model. The mean GABA estimates were significantly lower when modeling MM, while the CVs were similar. A sparser spline knot spacing led to lower variation in the GABA and GABA+ estimates, and a narrower modeling range - only including the signals of interest - did not substantially improve or degrade modeling performance. Additionally, results suggest that LCM can separate GABA and the underlying co-edited MM3co. Incorporating homocarnosine into the modeling did not significantly improve variance in GABA+ estimates.

ConclusionGABA-edited MRS is most appropriately quantified by LCM with a well-parameterized co-edited MM3co basis function with a constraint to the non-overlapped MM0.93, in combination with a sparse spline knot spacing (0.55 ppm) and a modeling range between 0.5 and 4 ppm.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/445817v3_ufig1.gif" ALT="Figure 1">
View larger version (19K):
org.highwire.dtl.DTLVardef@129ce60org.highwire.dtl.DTLVardef@1ac3797org.highwire.dtl.DTLVardef@1759d69org.highwire.dtl.DTLVardef@b190a3_HPS_FORMAT_FIGEXP  M_FIG C_FIG 102 strategies to model GABA-edited MRS with linear combination modeling were evaluated to quantify GABA and GABA+ in Osprey. Significantly different GABA and GABA+ estimates were found when a well-parameterized macro-molecule at 3 ppm was included. The findings suggest that linear combination modeling needs to be adapted for quantification of GABA-edited MRS.


title: Decomposing age effects in EEG alpha power

abstract: Increasing life expectancy is prompting the need to understand how the brain changes during healthy aging. Research utilizing Electroencephalography (EEG) has found that the power of alpha oscillations decrease from adulthood on. However, non-oscillatory (aperiodic) components in the data may confound results and thus require re-investigation of these findings. The present report aims at analyzing a pilot and two additional independent samples (total N = 533) of resting-state EEG from healthy young and elderly individuals. A newly developed algorithm will be utilized that allows the decomposition of the measured signal into aperiodic and aperiodic-adjusted signal components. By using multivariate sequential Bayesian updating of the age effect in each signal component, evidence across the datasets will be accumulated. It is hypothesized that previously reported age-related alpha power differences will disappear when absolute power is adjusted for the aperiodic signal component. Consequently, age-related differences in the intercept and slope of the aperiodic signal component are expected. Importantly, using a battery of neuropsychological tests, we will assess how the previously reported relationship between cognitive functions and alpha oscillations changes when taking the aperiodic signal into account; this will be done on data of the young and aged individuals separately. The aperiodic signal components and adjusted alpha parameters could potentially offer a promising biomarker for cognitive decline, thus finally the test-retest reliability of the aperiodic and aperiodic-adjusted signal components will be assessed.


title: Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques

abstract:
SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.

Author SummarySARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.


title: MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway

abstract:
Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MK2 and MK3 were selected for further analysis. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation (a) inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during the late phase of CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of this drug against CHIKV. Additionally, CMPD1 also inhibited HSV1 and SARS CoV2-19 infection in vitro. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as key targets for developing effective anti-CHIKV strategies in future.

Author summaryChikungunya virus has been a dreaded disease from the first time it occurred in 1952 Tanzania. Since then it has been affecting the different parts of the world at different time periods in large scale. It is typically transmitted to humans by bites of Aedes aegypti and Aedes albopictus mosquitoes. Although, studies have been undertaken to combat the disease still there are no effective strategies like vaccines or antivirals against it. Therefore it is essential to understand the virus and host interaction to overcome this hurdle. In this study two host factors MK2 and MK3 have been taken into consideration to see how they regulate the multiplication of the virus. The in vitro experiments demonstrated that inhibition of MK2 and MK3 restricted viral infection Further, it was observed that this is due to the blocking of lamellipodium formation by modifying the factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Besides, decreased disease score as well as better survivability was noticed in the in vivo experiments with mice. Therefore, MK2 and MK3 could be considered as the key targets for controlling CHIKV infection.


title: Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host

abstract:
Trypanosoma brucei ssp., unicellular parasites causing human and animal trypanosomiasis, are transmitted between mammals by tsetse flies. Periodic changes in variant surface glycoproteins (VSG), which form the parasite coat in the mammal, allow them to evade the host immune response. Different isolates of T. brucei show heterogeneity in their repertoires of VSG genes and have single nucleotide polymorphisms and indels that can impact on genome editing. T. brucei brucei EATRO1125 (AnTaR1 serodeme) is an isolate that is used increasingly often because it is pleomorphic in mammals and fly transmissible, two characteristics that have been lost by the most commonly used laboratory stocks. We present a genome assembly of EATRO1125, including contigs for the intermediate and mini-chromosomes that serve as repositories of VSG genes. In addition, de novo transcriptome assemblies were performed using Illumina sequences from tsetse-derived trypanosomes. Reads of 150 bases enabled closely related members of multigene families to be discriminated. This revealed that the transcriptome of midgut-derived parasites is dynamic, starting with the expression of high affinity hexose transporters and glycolytic enzymes and then switching to proline uptake and catabolism. These changes resemble the transition from early to late procyclic forms in culture. Further metabolic reprogramming, including upregulation of tricarboxylic acid cycle enzymes, occurs in the proventriculus. Many transcripts upregulated in the salivary glands encode surface proteins, among them 7 metacyclic VSGs, multiple BARPs and GCS1/HAP2, a marker for gametes. A novel family of transmembrane proteins, containing polythreonine stretches that are predicted to be O-glycosylation sites, was also identified. Finally, RNA-Seq data were used to create an optimised annotation file with 5 and 3 untranslated regions accurately mapped for 9302 genes. We anticipate that this will be of use in identifying transcripts obtained by single cell sequencing technologies.

AUTHOR SUMMARYTrypanosoma brucei ssp. are single-celled parasites that cause two tropical diseases: sleeping sickness in humans and nagana in domestic animals. Parasites survive in the host bloodstream because they periodically change their surface coats and also because they can switch from slender dividing forms to stumpy non-dividing forms. The latter can be transmitted to their second host, the tsetse fly. Although closely related, different geographical isolates differ in their repertoire of surface coats and have small, but important differences in their DNA sequences. In addition, laboratory strains that are transferred between mammals by needle passage lose the ability to produce stumpy forms and to infect flies. The isolate T. b. brucei EATRO1125 is often used for research as it produces stumpy forms and is fly transmissible. We provide an assembly of the genome of this isolate, including part of the repertoire of coat proteins, and a detailed analysis of the genes that the parasites express as they establish infection and progress through the fly. This has provided new insights into trypanosome biology. The combined genomic (DNA) and transcriptomic (RNA) data will be useful resources for the trypanosome research community.


title: Histone deacetylase 8 interacts with the GTPase SmRho1 in Schistosoma mansoni

abstract:
BACKGROUNDSchistosoma mansoni histone deacetylase 8 (SmHDAC8) is a privileged target for drug discovery. Invalidation of its transcription by RNAi leads to impaired survival of the worms in infected mice and its inhibition causes cell apoptosis and death. To determine why it is a promising therapeutic target the study of the currently unknown cellular signaling pathways involving this enzyme is essential. Protein partners of SmHDAC8 have been identified by yeast two-hybrid (Y2H) cDNA library screening and by mass spectrometry (MS) analysis. Among these partners we characterized SmRho1, the schistosome orthologue of human RhoA GTPase, which is involved in the regulation of the cytoskeleton. In this work, we validated the interaction between SmHDAC8 and SmRho1 and explored the role of the lysine deacetylase in cytoskeletal regulation.

METHODOLOGY/PRINCIPAL FINDINGSWe characterized two isoforms of SmRho1, SmRho1.1 and SmRho1.2. Co-IP/Mass Spectrometry analysis identified SmRho1 partner proteins and we used two heterologous expression systems (Y2H assay and Xenopus laevis oocytes) to study interactions between SmHDAC8 and SmRho1 isoforms.

To confirm SmHDAC8 and SmRho interaction in adult worms and schistosomula, we performed co-immunoprecipitation (Co-IP) experiments and additionally demonstrated SmRho1 acetylation using a Nano LC-MS/MS approach. A major impact of SmHDAC8 in cytoskeleton organization was documented by treating adult worms and schistosomula with a selective SmHDAC8 inhibitor or using RNAi followed by confocal microscopy.

CONCLUSIONS/SIGNIFICANCEOur results suggest that SmHDAC8 is involved in cytoskeleton organization via its interaction with the SmRho1.1 isoform. A specific interaction between SmHDAC8 and the C-terminal moiety of this isoform was demonstrated, and we showed that SmRho1 is acetylated on lysine K136. SmHDAC8 inhibition or knockdown using RNAi caused massive disruption of schistosomula actin cytoskeleton. A specific interaction between SmRho1.2 and SmDia suggested the existence of two signaling pathways that could regulate cytoskeleton organization via the two SmRho1 isoforms.

Author summarySchistosoma mansoni is the major parasitic platyhelminth species causing intestinal schistosomiasis, for which around 200 million people are in need of treatment. Currently one drug, praziquantel, is the treatment of choice and its use in mass treatment programs, rendered imperative the development of new therapeutic agents. As new potential targets, we have focused on lysine deacetylases, and in particular Schistosoma mansoni histone deacetylase 8 (SmHDAC8). Previous studies showed that invalidation of the transcription of SmHDAC8 by RNAi led to the impaired survival of the worms after the infection of mice. The analysis of the 3D structure of SmHDAC8 by X-ray crystallography showed that the catalytic domain structure diverges significantly from that of human HDAC8 and this was exploited to develop novel anti-schistosomal drugs. Biological roles of SmHDAC8 are unknown. For this reason, we previously characterized its protein partners and identified the schistosome orthologue of the human RhoA GTPase, suggesting the involvement of SmHDAC8 in the modulation of cytoskeleton organization. Here, we investigated the interaction between SmHDAC8 and SmRho1 and identified two SmRho1 isoforms (SmRho1.1 and SmRho1.2). Our study showed that SmHDAC8 is indeed involved in schistosome cytoskeleton organization.


title: Knocking-out the human face genes TBX15 and PAX1 in mice alters facial and other physical morphology

abstract:
DNA variants in or closed to the human TBX15 and PAX1 genes have been repeatedly associated with facial morphology in independent genome-wide association studies, while their functional roles in determining facial morphology remains to be understood. We generated Tbx15 knockout (Tbx15-/-) and Pax1 knockout (Pax1-/-) mice by applying the one-step CRISPR/Cas9 method. A total of 75 adult mice were used for subsequent phenotype analysis, including 38 Tbx15 mice (10 homozygous Tbx15-/-, 18 heterozygous Tbx15+/-, 10 wild-type WT) and 37 Pax1 mice (12 homozygous Pax1-/-, 15 heterozygous Pax1+/-, 10 WT mice). Facial and other physical morphological phenotypes were obtained from three-dimensional (3D) images acquired with the HandySCAN BLACK scanner. Compared to WT mice, the Tbx15-/- mutant mice had significantly shorter faces (P=1.08E-8, R2=0.61) and their ears were in a significantly lower position (P=3.54E-8, R2=0.62) manifesting an "ear dropping" characteristic. Besides these face alternations, Tbx15-/- mutant mice displayed significantly lower weight as well as shorter body and limb length. Pax1-/- mutant mice showed significantly longer noses (P=1.14E-5, R2=0.46) relative to WT mice, but otherwise displayed less obvious morphological alterations than Tbx15-/- mutant mice did. Because the Tbx15 and Pax1 effects on facial morphology we revealed here in mice are largely consistent with previously reported TBX15 and PAX1 face associations in humans, we suggest that the functional role these two genes play on determining the face of mice is similar to the functional impact their human homologues have on the face of humans.

Author SummarySeveral independent genome-wide association studies (GWASs) on human facial morphology highlighted DNA variants in or closed to TBX15 and PAX1 with genome-wide significant association with human facial phenotypes. However, direct evidence on the functional involvement if these genes in the development and determination of facial morphology has not been established as of yet. In the current study, our in vivo gene editing experiments in mice for two well-replicated human face TBX15 and PAX1 genes provide novel evidence on the functional involvement of these two genes in facial and other physical morphology in mice, at least. Tbx15-/- mice showed a shortened facial length and manifesting an ear dropping characteristic, Pax1-/- mice showed an increased nose length. Our geometric morphometrics analysis further indicate that there are significant facial morphology differences between groups (Tbx15-/-and Tbx15+/-, Tbx15-/- and Tbx15+/+, Tbx15+/- and Tbx15+/+, Pax1-/- and Pax1+/+). We provide the first direct functional evidence that two well-known and replicated human face genes, Tbx15 and Pax1, impact facial and other body morphology in mice. The general agreement between our findings in knock-out mice with those from previous GWASs suggests that the functional evidence we established here in mice may also be relevant in humans.

category: genetics


title: TnCentral: A Prokaryotic Transposable Element Database and Web Portal for Transposon Analysis

abstract:
We describe here the structure and organization of TnCentral (https://tncentral.proteininformationresource.org/). a web resource for prokaryotic transposable elements (TE). TnCentral currently contains ~400 carefully annotated TE, including transposons from the Tn3, Tn7, Tn402 and Tn554 families, compound transposons, integrons and associated insertion sequences (IS). These TE carry passenger genes, including genes conferring resistance to over 25 classes of antibiotics and nine types of heavy metal as well as genes responsible for pathogenesis in plants, toxin/antitoxin gene pairs, transcription factors and genes involved in metabolism. Each TE has its own entry page providing details about its transposition genes, passenger genes, and other sequence features required for transposition as well as a graphical map of all features. TnCentral content can be browsed and queried through text and sequence-based searches with a graphic output. We describe three use cases, which illustrate how the search interface, results tables, and entry pages can be used to explore and compare TEs.

TnCentral also includes downloadable software to facilitate user-driven identification, with manual annotation, of certain types of TE in genomic sequences. Through the TnCentral homepage, users can also access TnPedia which provides comprehensive reviews of the major TE families including an extensive general section, and specialised sections with descriptions of insertion sequence and transposon families. TnCentral and TnPedia are intuitive resources that can be used by clinicians and scientists to assess TE diversity in clinical, veterinary and environmental samples.

ImportanceThe ability of bacteria to undergo rapid evolution and adapt to changing environmental circumstances drives the public health crisis of multiple antibiotic resistance as well as outbreaks of disease in economically important agricultural crops and animal husbandry. Prokaryotic transposable elements (TE) play a critical role in this. Many carry "passenger genes" (not required for the transposition process) conferring resistance to antibiotics or heavy metals or causing disease in plants and animals. Passenger genes are spread by the normal TE transposition activities, by insertion into plasmids which then spread via conjugation within and across bacterial populations. Thus, an understanding of TE composition and transposition mechanisms is key to developing strategies to combat bacterial pathogenesis. Toward this end, we have developed TnCentral, a bioinformatics resource dedicated to describing and exploring the structural and functional features of prokaryotic TE and whose use is intuitive and accessible to users with or without bioinformatics expertise.

category: genomics


title: Purification of immunostimulatory glucose homopolymer from broccoli (Brassica oleracea var. italica)

abstract: We prepared broccoli (Brassica oleracea var. italica) neutral polysaccharides (flow-through fractions of anion exchange column chromatography from hot water extracts) from different broccoli cultivars and compared their immunostimulatory effects in the silkworm muscle contraction assay. The cultivars showed a wide range of activity, with the cultivar  Winter dome showing the highest specific activity (more than 100 times higher than curdlan). Furthermore, the active substance was purified by gel filtration column chromatography. The active substance showed heterogeneous molecular weights of more than 270 kDa. Sugar composition analysis of the purified fraction revealed that more than 95% of its sugar component was glucose, suggesting that the immunostimulatory neutral polysaccharide from broccoli cultivar  Winter dome was a homopolymer of glucose. The purified fraction also induced TNF production in cultured mouse macrophage cells. These results suggest that the glucose homopolymer in broccoli has an immunostimulatory effect on both arthropod and mammalian immune system.


title: Construction of redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides

abstract: Many protein expression and purification systems are commercially available to provide a sufficient amount of pure, soluble and active native protein, such as the pMAL system based on E. coli maltose binding protein tag (MBP). Adding specific amino acid tags to the N- or C-terminus of the protein increases solubility and facilitates affinity purification of proteins. However, many of expressed tagged proteins consequently lose functionality, particularly small peptides such as antimicrobial peptides (AMPs). Objective of this study was to redesign the pMAL expression vector in order to increase the efficacy of MBP tag separation from native peptides. Redesign of the pMAL expression vector included introduction of the His6 tag and the enterokinase cleavage site downstream from the original MBP tag and Xa cleavage site enabling purification of native and active peptide (P) following two-step affinity chromatography. In the first step the entire MBP-His6-P fusion protein is purified through binding to Ni-NTA agarose. In the second step, the purification was performed by adding mixture of amylose and Ni-NTA agarose resins following cleavage of the fusion protein with active His6 tagged enterokinase. This removes MBP-His6 and His6-enterokinase leaving pure native protein in solution. The redesigned pMAL vectors were optimized for cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides expression. Two-step purification protocol was successfully applied in purification of active native AMPs, lactococcin A and human {beta}-defensin. Taken together, we established the optimal conditions and pipeline for overexpression and purification of large amount of native peptides, that can be implemented in any laboratory.


title: Expression of zebrafish Brn1.2 (Pou3f2) and two Brn-3a (Pou4f1) POU genes in brain and sensory structures

abstract: POU genes are characterized by a conserved POU DNA-binding domain, and are divided into six subclasses. Class III and IV POU genes are predominantly expressed in the developing nervous system. POU class III genes are critical for several neuronal cell differentiation and class IV POU genes serve important functions in the differentiation and survival of sensory neurons. In this study, we attempted to identify POU genes in the zebrafish and pufferfish genomes by using existing bioinformatics tools. We analysed the expression of zebrafish brn1.2 and brn3a genes (brn3a1 and brn3a2)) using whole-mount in situ hybridisation. Similarly to the mammalian orthologue, zebrafish brn1.2 was widely expressed in the forebrain, midbrain and hindbrain. During the late stages of embryogenesis, brn1.2 expressing cells were located in the preoptic area and in the auditory vesicles. Expression of both zebrafish brn3a genes was detected in trigeminal ganglia, cranial sensory ganglia, sensory neurons along the dorsal spinal cord, in the anterior and posterior lateral line placodes (ALL and PLL), retinal ganglion cell layer, optic tectum and in small cell clusters in the forebrain and hindbrain. Similar to mammalian Brn3a, zebrafish brn3a genes were detected in the retina and sensory structures. However, different domains of expression were also observed, namely in spinal sensory neurons, and lateral line system.


title: The complete sequence of a human genome

abstract: In 2001, Celera Genomics and the International Human Genome Sequencing Consortium published their initial drafts of the human genome, which revolutionized the field of genomics. While these drafts and the updates that followed effectively covered the euchromatic fraction of the genome, the heterochromatin and many other complex regions were left unfinished or erroneous. Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release. The new T2T-CHM13 reference includes gapless assemblies for all 22 autosomes plus Chromosome X, corrects numerous errors, and introduces nearly 200 million bp of novel sequence containing 2,226 paralogous gene copies, 115 of which are predicted to be protein coding. The newly completed regions include all centromeric satellite arrays and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies for the first time.


title: LMO2 is critical for early metastatic events in breast cancer

abstract:
Metastasis is responsible for the majority of breast cancer-related deaths, however identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage- traced cells have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by TNF and IL6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

One sentence summaryLMO2 modulates STAT3 signaling in breast cancer metastasis.

category: cancer biology


title: Regional Differences in Brain Plasticity and Behaviour as a Function of Sex and Enrichment Type: Oxytocin Matters

abstract: The early environment is critical to brain development, but the relative contribution of physical vs. social stimulation is unclear. Here, we investigated in male and female rats the response to early physical and social environmental enrichment in relation to oxytocin (OT) and brain-derived neurotrophic factor (BDNF) expression. The findings show that males and females respond differently to prolonged sensorimotor stimulation from postnatal day 21-110 in terms of functional, structural and molecular changes in the hippocampus vs. medial prefrontal cortex (mPFC). Physical enrichment promoted motor and cognitive functions and hippocampal BDNF mRNA and protein expression in both sexes. Combined physical and social enrichment, however, promoted functional and structural gain predominantly in females. These changes were accompanied by elevated plasma oxytocin (OT) levels and BDNF mRNA expression in the mPFC while the hippocampus was not affected. Administration of an OT antagonist in females blocked the beneficial effects of enrichment and led to reduced cortical BDNF signaling. These findings suggest that an OT-based mechanism selectively stimulates a region-specific BDNF response which is dependent on the type of experience.


title: EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in developing neurons

abstract: Dendritic spines are the postsynaptic compartment of a functional neuronal synapse, and are critical for synaptic connectivity and plasticity. The developmental precursor to dendritic spines, dendritic filopodia, are highly motile protrusions that facilitate synapse formation by sampling the environment for suitable axon partners during development and learning. Despite the significance of the actin cytoskeleton in driving these protrusions, the actin remodeling factors involved in this process are not fully characterized. In this work, we identify a critical function for the Ena/VASP protein EVL in the regulation of dendritic filopodia. Amongst the Ena/VASP proteins, EVL is uniquely required for the characteristic morphology and dynamics of dendritic filopodia. Using a combination of genetic and optogenetic manipulations, we demonstrate that EVL promotes protrusive motility through membrane-direct actin polymerization at dendritic filopodia tips. EVL forms a complex at nascent protrusions and dendritic filopodia tips with MIM/MTSS1, an I-BAR protein recently discovered to be important for initiation of dendritic filopodia. We propose a model in which EVL cooperates with MIM to elongate and coalesce branched actin filaments, establishing the dynamic lamellipodia-like architecture of dendritic filopodia in developing neurons.

category: cell biology


title: Epigenetic Patterns in a Complete Human Genome

abstract: The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, longrange epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.


title: Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

abstract: The microtubule network of the cardiomyocyte exhibits specialized architecture, stability and mechanical behavior that accommodate the demands of working muscle cells. Stable, post-translationally detyrosinated microtubules are physical coupled to the sarcomere, the contractile apparatus of muscle, and resist sarcomere motion to regulate muscle mechanics and mechanosignaling. Control of microtubule growth and shrinkage dynamics represents a potential intermediate in the formation of a stable, physically coupled microtubule network, yet the molecular determinants that govern dynamics are unknown. Here we test the hypothesis that desmin intermediate filaments may stabilize growing microtubules at the sarcomere Z-disk in a detyrosination-dependent manner. Using a combination of biochemical assays and direct observation of microtubule plus-end dynamics in primary adult cardiomyocytes, we determine that: 1) tyrosination increases the frequency of microtubule depolymerization and reduces the pausing of microtubules at the Z-disk, leading to a more dynamic microtubule; and 2) desmin intermediate filaments stabilize both growing and shrinking microtubules specifically at the Z-disk and protect them from depolymerization. This stabilizes iteratively growing, detyrosinated microtubules between adjacent sarcomeres, which promotes the formation of high-energy microtubules that buckle between sarcomeres and elevates myocyte viscoelasticity. Our findings inform on how the tubulin code and intermediate filaments regulate microtubule dynamics, and provide mechanism to the establishment of a spatially organized, physically coupled, and long-lived microtubule network in the cardiomyocyte.


title: Location and function of all Plasmodium kinesins: key roles in parasite proliferation, polarity, and transmission

abstract: Kinesins are microtubule-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens-Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the lifecycle in both mammalian and mosquito hosts. Here we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other eight kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in microtubule dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of microtubule motors and may be exploited to discover new therapeutic interventions against malaria.


title: Location and function of Plasmodium kinesins: key roles in parasite proliferation, polarity, and transmission


title: Key roles for kinesin-13 and kinesin-20 in malaria parasite proliferation, polarity, and transmission revealed by genome-wide functional analysis


title: Genome-wide screening in human kidney organoids identifies novel aspects of nephrogenesis

abstract: Human organoids allow studying proliferation, lineage specification, and three-dimensional tissue development. Due to the inherent multicellular complexity, interrogation by systematic genetic methodologies is challenging. Here, we present the first genome-wide CRISPR screen in iPSC-derived kidney organoids. The combination of genome editing, longitudinal sampling and sorting of specific cell populations enabled us to uncover novel biology from development to tubular morphogenesis. We validated the high quality of our screen by individual hit follow up and comparisons to kidney disease datasets. The discovery of a novel regulatory mechanism which controls epithelial proliferation via a trans-activating but cis-inhibitory effect of the Notch ligand Jagged1 proves how mosaic knockouts generated by pooled CRISPR screening in organoids can identify novel ways of communication between heterogeneous cell populations in complex tissues. Collectively, these data demonstrate the feasibility of using complex iPSC- derived organoids for genome-scale screening and serves as a benchmark for future organoid CRISPR screens.


title: DYNLL1 mis-splicing is associated with replicative genome instability in SF3B1 mutant cells

abstract: Genome instability is a hallmark of cancer that arises through a panoply of mechanisms driven by oncogene and tumour-suppressor gene mutations. Oncogenic mutations in the core splicing factor SF3B1 have been linked to genome instability. Since SF3B1 mutations alter the selection of thousands of 3 splice sites affecting genes across biological pathways, it is not entirely clear how they might drive genome instability. Here we confirm that while R-loop formation and associated replication stress may account for some of the SF3B1-mutant genome instability, a mechanism involving changes in gene expression also contributes. An SF3B1-H662Q mutant cell line mis-splices the 5UTR of the DNA repair regulator DYNLL1, leading to higher DYNLL1 protein levels, mis-regulation of DNA repair pathway choice and PARP inhibitor sensitivity. Reduction of DYNLL1 protein in these cells restores genome stability. Together these data highlight how SF3B1 mutations can alter cancer hallmarks through subtle changes to the transcriptome.


title: Simple methods for quantifying super-resolved cortical actin

abstract: Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or  corrals, in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by both Super Resolved Radial Fluctuations (SRRF) and expansion microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31{micro}m2 {+/-} 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.


title: PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1

abstract: Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow that is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. Here we have identified peroxisome proliferator gamma coactivator-1 (PGC-1) as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared to oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) increased PGC-1 expression and its transcriptional co-activation. PGC-1 was required for laminar FSS-induced expression of telomerase reverse transcriptase (TERT) in vitro and in vivo via its association with ERR and KLF4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status was heme oxygenase-1 (HMOX1), a gene required for endothelial alignment to laminar FSS. Thus, these data suggest an important role for a PGC-1-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.


title: Sir3 Heterochromatin Protein Promotes NHEJ by Direct Inhibition of Sae2

abstract: Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) complex assembles heterochromatin-like chromatin at subtelomeres. SIR-mediated repressive chromatin limits double strand break (DSB) resection protecting damaged chromosome ends during HR. As resection initiation marks the cross-road between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR- mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Sir3 physically interacts with Sae2 and this interaction impairs Sae2-dependent MRX functions. As a consequence, Sir3 limits Mre11-mediated resection, delays MRX removal from DSB ends and promotes NHEJ.


title: A technical pipeline for screening microbial communities as a function of substrate specificity through single cell fluorescent imaging

abstract:
The study of specific glycan uptake and metabolism has been shown to be an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards target. In this study, we successfully used the fluorescent label 2-aminobenzamide (2-AB), most commonly employed for enhancing the detection of protein anchored glycans, to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, were able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it was possible to comprehensively track the metabolism of the labelled glycans in both supernatants and at a single cell level. We envisage this combination of complimentary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

IMPORTANCEInformation on how bacterial consortia utilize polysaccharides at strain level, whilst progressing rapidly in recent years still lacks a suitable way to study the vast range of ornamentations and structural motifs found in the natural glycans we consume in everyday life. As multi-omic approaches commonly require complex and costly analysis, a screening platform, as described in our work, could be seen as both a complementary and essential new tool in the understanding of microbial polysaccharide metabolism. Our study demonstrates a fast and efficient glycan labelling technique composed of several integrated procedures and advanced analytical methodologies. Chromatography and mass spectrometry are applied in the tracking of metabolized labelled glycans whilst microscopy and flow cytometry are used in the visualization of labelled bacteria at a single cell level.


title: Evolutionary systems biology reveals patterns of rice adaptation to drought-prone agro-ecosystems

abstract: Rice was domesticated around 10,000 years ago and has developed into a staple for half of humanity. The crop evolved and is currently grown in stably wet and intermittently dry agro-ecosystems, but patterns of adaptation to differences in water availability remain poorly understood. While previous field studies have evaluated plant developmental adaptations to water deficit, adaptive variation in functional and hydraulic components, particularly in relation to gene expression, has received less attention. Here, we take an evolutionary systems biology approach to characterize adaptive drought resistance traits across roots and shoots. We find that rice harbors heritable variation in molecular, physiological, and morphological traits that is linked to higher fitness under drought. We identify modules of co-expressed genes that are associated with adaptive drought avoidance and tolerance mechanisms. These expression modules showed evidence of polygenic adaptation in rice subgroups harboring accessions that evolved in drought-prone agro-ecosystems. Fitness-linked expression patterns had predictive value and allowed us to identify the drought-adaptive nature of optimizing photosynthesis and interactions with arbuscular mycorrhizal fungi. Taken together, our study provides an unprecedented, integrative view of rice adaptation to water-limited field conditions.

category: plant biology


title: MtExpress, a Comprehensive and Curated RNAseq-based Gene Expression Atlas for the Model Legume Medicago truncatula

abstract:
Although RNA sequencing has been becoming the main transcriptomic approach in the model legume Medicago truncatula, there is currently no genome-wide gene expression atlas covering the whole set of RNAseq data published for this species. Nowadays, such tool is highly valuable to provide a global view of gene expression in a wide range of conditions and tissues/organs.

Here, we present MtExpress, a gene expression atlas that compiles an exhaustive set of published M. truncatula RNAseq data (https://medicago.toulouse.inrae.fr/MtExpress). MtExpress makes use of recent releases of M. truncatula genome sequence and annotation, as well as up-to-date tools to perform mapping, quality control, statistical analysis and normalization of RNAseq data. MtExpress combines semi-automated pipelines with manual re-labelling and organization of samples, to produce an attractive and user-friendly interface, fully integrated with other available Medicago genomic resources. Importantly, MtExpress is highly flexible, in terms of both queries, e.g. allowing searches with gene names and orthologous gene IDs from Arabidopsis and other legume species, and outputs, to customize visualization and redirect gene study to relevant Medicago webservers.

Thanks to its semi-automated pipeline, MtExpress will be frequently updated to follow the rapid pace of M. truncatula RNAseq data publications, as well as the constant improvement of genome annotation.


title: The evolution of the phenylpropanoid pathway entailed pronounced radiations and divergences of enzyme families

abstract: Land plants constantly respond to fluctuations in their environment. Part of their response is the production of a diverse repertoire of specialized metabolites. One of the foremost sources for metabolites relevant to environmental responses is the phenylpropanoid pathway, which was long thought to be a land plant-specific adaptation shaped by selective forces in the terrestrial habitat. Recent data have however revealed that streptophyte algae, the algal relatives of land plants, have candidates for the genetic toolkit for phenylpropanoid biosynthesis and produce phenylpropanoid-derived metabolites. Using phylogenetic and sequence analyses, we here show that the enzyme families that orchestrate pivotal steps in phenylpropanoid biosynthesis have independently undergone pronounced radiations and divergence in multiple lineages of major groups of land plants; sister to many of these radiated gene families are streptophyte algal candidates for these enzymes. These radiations suggest a high evolutionary versatility in the enzyme families involved in the phenylpropanoid-derived metabolism across embryophytes. We suggest that this versatility likely translates into functional divergence and may explain the key to one of the defining traits of embryophytes: a rich specialized metabolism.


title: Microtubules Promote the Non-cell Autonomy of MicroRNAs by Inhibiting their Cytoplasmic Loading into ARGONAUTE1 in Arabidopsis

abstract: Mobile microRNAs (miRNAs) serve as local and long-distance signals in developmental patterning and stress responses in plants. However, mechanisms governing the non-cell autonomous activities of miRNAs remain elusive. Here, we show that mutations that disrupt microtubule dynamics are specifically defective for the non-cell autonomous actions of mobile miRNAs, including miR165/6 that is produced in the endodermis and moves to the vasculature to pattern xylem cell fates in Arabidopsis roots. We show that KTN1, a subunit of a microtubule-severing enzyme, is required in source and intermediary cells to inhibit the loading of miR165/6 into ARGONUATE1 (AGO1), which is cell-autonomous, to enable the miRNA"s cell exit. Microtubule disruption enhances the association of miR165/6 with AGO1 in the cytosol. These findings suggest that, while cell-autonomous miRNAs load into AGO1 in the nucleus, cytoplasmic AGO1 loading of mobile miRNAs is a key step regulated by microtubules to promote the range of miRNA"s cell-to-cell movement.


title: Notch controls the cell cycle to define leader versus follower identities during collective cell migration.

abstract: Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, as cancer. How cell cycle progression affects migration, and vice-versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish Trunk Neural Crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. We propose that migratory behaviours are defined through the interaction of Notch signalling and cell cycle progression.


title: Identifying interpretable gene-biomarker associations with functionally informed kernel-based tests in 190,000 exomes

abstract: Here we present an exome-wide rare genetic variant association study for 30 blood biomarkers in 191,971 individuals in the UK Biobank. We compare gene-based association tests for separate functional variant categories to increase interpretability and identify 193 significant gene-biomarker associations. Genes associated with biomarkers were ~ 4.5-fold enriched for conferring Mendelian disorders. In addition to performing weighted gene-based variant collapsing tests, we design and apply variant-category-specific kernel-based tests that integrate quantitative functional variant effect predictions for missense variants, splicing and the binding of RNA-binding proteins. For these tests, we present a statistically powerful and computationally efficient combination of the likelihood-ratio and score tests that found 36% more associations than the score test alone. Kernel-based tests identified 13% more associations than their gene-based collapsing counterparts and had advantages in the presence of gain of function missense variants. We introduce local collapsing by amino acid position for missense variants and use it to interpret associations and identify potential novel gain of function variants in PIEZO1. Our results show the benefits of investigating different functional mechanisms when performing rare-variant association tests, and demonstrate pervasive rare-variant contribution to biomarker variability.


title: An in vivo approach to characterize novel variants associated with musculoskeletal disorders

abstract: Nemaline Myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as Cap Myopathy, congenital fiber type disproportion, and distal arthrogrypsosis (DA). TPM2-related disorders have not been modeled in vivo, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found two variants, K49Del and E122K, significantly affected muscle morphogenesis and muscle function, in part by disrupting myotube guidance. Transient overexpression of K49Del and E122K also disrupted the morphogenesis of immortalized mouse myoblasts in vitro, and negatively affected zebrafish muscle development and function in vivo. We used our transient overexpression assays in zebrafish to characterize two novel TPM2 variants that we identified in DA patients (V129A and E139K), and found these variants caused musculoskeletal defects similar to those of the known pathogenic variants. In addition, the severity of musculoskeletal phenotypes in zebrafish expressing TPM2 variants correlated with the severity of clinical phenotypes observed in DA patients. Our study establishes transient overexpression in zebrafish as an efficient platform to characterize variants of uncertain significance in TPM2 in vivo, and suggests that this method can be used to predict the clinical severity of variants associated with DA and congenital myopathies.


title: Transcriptional milestones in Dictyostelium development

abstract: Development of the social amoeba Dictyostelium discoideum begins by starvation of single cells and ends in multicellular fruiting bodies 24 hours later. These major morphological changes are accompanied by sweeping gene expression changes, encompassing nearly half of the 13,000 genes in the genome. To explore the relationships between the transcriptome and developmental morphogenesis, we performed time-series RNA-sequencing analysis of the wild type and 20 mutant strains with altered morphogenesis. These strains exhibit arrest at different developmental stages, accelerated development, or terminal morphologies that are not typically seen in the wild type. Considering eight major morphological transitions, we identified 1,371 milestone genes whose expression changes sharply between two consecutive transitions. We also identified 1,099 genes as members of 21 regulons, which are groups of genes that remain coordinately regulated despite the genetic, temporal, and developmental perturbations in the dataset. The gene annotations in these milestones and regulons validate known transitions and reveal several new physiological and functional transitions during development. For example, we found that DNA replication genes are co-regulated with cell division genes, so they are co-expressed in mid-development even though chromosomal DNA is not replicated at that time. Altogether, the dataset includes 486 transcriptional profiles, across developmental and genetic conditions, that can be used to identify new relationships between gene expression and developmental processes and to improve gene annotations. We demonstrate the utility of this resource by showing that the cycles of aggregation and disaggregation observed in allorecognition-defective mutants involve a dedifferentiation process. We also show unexpected variability and sensitivity to genetic background and developmental conditions in two commonly used genes, act6 and act15, and robustness of the coaA gene. Finally, we propose that gpdA should be used as a standard for mRNA quantitation because it is less sensitive to genetic background and developmental conditions than commonly used standards. The dataset is available for democratized exploration without the need for programming skills through the web application dictyExpress and the data mining environment Orange.


title: Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness

abstract: Helicase regulation is vital for replisome progression, where the helicase enzyme functions to unwind duplex DNA and aids in the coordination of replication fork activities. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased DNA damage and chromosome complexity, less stable genomes, and ultimately less viable and fit strains. Notably, while two mutations stabilized fully constricted states, they have distinct effects on genomic stability, suggesting a complex relationship between helicase regulation mechanisms and faithful, efficient DNA replication. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving SEW and conformational changes and relates current mechanistic understanding to functional helicase behavior.


title: Growth hormone transgenesis affects thermal tolerance in zebrafish (Danio rerio)

abstract:
In fish, growth hormone (GH)-transgenesis may modify physiological mechanisms of adaptation when challenged by biotic and abiotic stressors. Thus, we evaluated whether GH overexpression can alter the thermal tolerance of adult and juvenile GH-transgenic zebrafish (Danio rerio). This study compared the thermal tolerance in non-transgenic (NT) and GH-transgenic (T) zebrafish exposed to 13 {degrees}C, 39 {degrees}C, or 28 {degrees}C (control) for 96 h. Mortality rate was checked every 12 h in juvenile (8 week-old) and adult males (6 month-old). Exposure to different temperatures revealed that GH overexpression increases the tolerance of transgenic juveniles exposed to 13 {degrees}C and diminishes the tolerance of juveniles and adults, when exposed to 39 {degrees}C. Additionally, we have analyzed transcriptional expression from the heat shock proteins (HSPs), which are mainly involved in the thermal tolerance mechanism. The mRNA level analysis results revealed that, under controlled conditions (28 {degrees}C), GH-transgenesis upregulates the expression of hsp47, hsp70, hsp90a and heat shock transcription factor (hsf1a) in transgenic juveniles, although the same result was not observed in transgenic adults. Exposure to low temperature did not alter the expression of any analyzed gene, both in adults and in juveniles. Exposure to 39 {degrees}C decreased the expression of all genes analyzed, in GH-transgenic adults. Furthermore, the HSP expression pattern was analyzed via hierarchical clustering. This analysis revealed two major clusters illustrating the dependency of gene changes related to age. These results indicate that the GH overexpression can alter thermal tolerance of fish, depending of age and temperature.

Highlights- GH-transgenesis increased the survival rate of juveniles at low temperature;
- High temperature is more lethal for juvenile ande adult GH-transgenic zebrafish;
- GH-transgenesis increased expression of hsf1a, hsp47, and hsp70 genes in juvenile zebrafish;
- hsf1a, hsp47, hsp70, hsp90a, and hsp90b genes expression is diminished in adult zebrafish GH-transgenesis exposure at high temperature.

O_FIG O_LINKSMALLFIG WIDTH=142 HEIGHT=200 SRC="FIGDIR/small/445844v1_ufig1.gif" ALT="Figure 1">
View larger version (59K):
org.highwire.dtl.DTLVardef@1847289org.highwire.dtl.DTLVardef@7bb056org.highwire.dtl.DTLVardef@75efc9org.highwire.dtl.DTLVardef@d1a9ac_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: GenomicSuperSignature: interpretation of RNA-seq experiments through robust, efficient comparison to public databases

abstract:
Millions of transcriptomic profiles have been deposited in public archives, yet remain underused for the interpretation of new experiments. We present a novel method for interpreting new transcriptomic datasets through near-instantaneous comparison to public archives without high-performance computing requirements. We performed Principal Component Analysis on 536 studies comprising 44,890 RNA sequencing profiles. Sufficiently similar loading vectors were aggregated to form Replicable Axes of Variation (RAV). RAVs were annotated with metadata of originating studies and samples and by gene set enrichment analysis. Functionality to associate new datasets with RAVs, extract interpretable annotations, and provide intuitive visualization are implemented as the GenomicSuperSignature R/Bioconductor package. We demonstrated the efficient and coherent database searching, robustness to batch effects and heterogeneous training data, and transfer learning capacity of our method using TCGA and rare diseases datasets. GenomicSuperSignature will aid analyzing new gene expression data in the context of existing databases using minimal computing resources.

PURPOSEMillions of transcriptomic profiles have been deposited in public archives, yet remain underused for the interpretation of new experiments. Existing methods for leveraging these public resources have focused on the reanalysis of existing data or analysis of new datasets independently. We present a novel approach to interpreting new transcriptomic datasets by near-instantaneous comparison to public archives without high-performance computing requirements.

METHODSTo identify replicable and interpretable axes of variation in any given gene expression dataset, we performed Principal Component Analysis (PCA) on 536 studies comprising 44,890 RNA sequencing profiles. Sufficiently similar loading vectors, when compared across studies, were aggregated to form Replicable Axes of Variation (RAV). RAVs were annotated with metadata of originating studies and samples and by gene set enrichment analysis. Functionality to associate new datasets with RAVs, extract interpretable annotations, and provide intuitive visualization are implemented as the GenomicSuperSignature R/Bioconductor package.

RESULTSRAVs are robust to batch effects and the presence of low-quality or irrelevant studies, and identify signals that can be lost by merging samples across the training datasets. The GenomicSuperSignature package allows instantaneous matching of PCA axes in new datasets to pre-computed RAVs, cutting down the analysis time from days to the order of seconds on an ordinary laptop. We demonstrate that RAVs associated with a phenotype can provide insight into weak or indirectly measured biological attributes in a new study by leveraging accumulated data from published datasets. Benchmarking against complementary previous works demonstrates that the RAV index 1) identifies colorectal carcinoma transcriptome subtypes that are similar to but more correlated with clinicopathological characteristics than previous disease-specific efforts and 2) can estimate neutrophil counts through transfer learning on new data comparably to the previous efforts despite major differences in training datasets and model building processes with the additional benefits of flexibility and scalability of the model application.

CONCLUSIONGenomicSuperSignature establishes an information resource and software tools to interrogate it. Prior knowledge databases are coherently linked, enabling researchers to analyze new gene expression data in the context of existing databases using minimal computing resources. The robustness of GenomicSuperSignature suggests that we can expand this approach beyond human gene expression profiles, such as single-cell RNA-seq, microbiome abundance, and different species transcriptomics datasets.

category: bioinformatics


title: Single-molecule imaging of cholesterol-dependent cytolysin assembly

abstract: We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore-formation of the archetypical cholesterol-dependent cytolysin, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 millisecond temporal resolution and 20 nanometre spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes.

category: biophysics


title: Single molecule tracking the uncoupling of assembly and membrane insertion in Perfringolysin O