title: Possible Implications of AlphaFold2 for Crystallographic Phasing by Molecular Replacement

abstract:
The AlphaFold2 results in the 14th edition of Critical Assessment of Structure Prediction (CASP14) showed that accurate (low root-mean-square deviation) in silico models of protein structure domains are on the horizon, whether or not the protein is related to known structures through high- coverage sequence similarity. As highly accurate models become available, generated by harnessing the power of correlated mutations and deep learning, one of the aspects of structural biology to be impacted will be methods of phasing in crystallography. We here use the data from CASP14 to explore the prospect for changes in phasing methods, and in particular to explore the prospects for molecular replacement phasing using in silico models.

SynopsisWe discuss the implications of the AlphaFold2 protein structure modelling software for crystallographic phasing strategies.


title: Glycine-Rich RNA-Binding Protein 7 interacts with and potentiates effector-induced immunity by Gpa2 and Rx1 based on an intact RNA Recognition Motif

abstract:
O_LIThe activity of intracellular plant Nucleotide-Binding Leucine-Rich Repeat (NB-LRR) immune receptors is fine-tuned by interactions between the receptors and their partners. Identifying NB-LRR interacting proteins is, therefore, crucial to advance our understanding of how these receptors function.
C_LIO_LIA Co-Immunoprecipitation/Mass-Spectrometry screening was performed in Nicotiana benthamiana to identify host proteins associated with the Gpa2 CC-NB-LRR, which confers resistance against the potato cyst nematode Globodera pallida. A combination of biochemical, cellular, and functional assays was used to assess the role of a candidate interactor in defence.
C_LIO_LIA N. benthamiana homolog of the Glycine-Rich RNA-Binding Protein 7 (NbGRP7) protein was prioritized as a novel Gpa2-interacting protein for further investigations. NbGRP7 also associates in planta with the homologous Rx1 receptor, which confers immunity to Potato Virus X. We show that NbGRP7 positively regulates extreme resistance by Rx1 and cell death by Gpa2. Mutating the NbGRP7 RNA recognition motif compromises its role in Rx1-mediated defence. Strikingly, ectopic NbGRP7 expression impacts the steady-state levels of Rx1, which relies on an intact RNA recognition motif.
C_LIO_LICombined, our findings illustrate that NbGRP7 is a novel pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 functioning at a post-transcriptional level.
C_LI

category: plant biology


title: Thermodynamics drives coenzyme redundancy in metabolism

abstract:
Coenzymes distribute a variety of chemical moieties throughout cellular metabolism, participating in group (e.g., phosphate, acyl) and electron transfer. For a variety of reactions requiring acceptors or donors of specific resources, there often exist degenerate sets of molecules (e.g., NAD(H) and NADP(H)) that carry out similar functions. Although the physiological roles of various coenzyme systems are well established, it is unclear what selective pressures may have driven the emergence of coenzyme redundancy. Here we use genome-wide metabolic modeling approaches to decompose the selective pressures driving enzymatic specificity for either NAD(H) or NADP(H) in the metabolic network of Escherichia coli. We found that few enzymes are thermodynamically constrained to using a single coenzyme, and in principle, a metabolic network relying on only NAD(H) is feasible. However, structural and sequence analyses revealed widespread conservation of residues that retain selectivity for either NAD(H) or NADP(H), suggesting that additional forces may shape specificity. Using a model accounting for the cost of oxidoreductase enzyme expression, we found that coenzyme redundancy universally reduces the minimal amount of protein required to catalyze coenzyme-coupled reactions, inducing individual reactions to strongly prefer one coenzyme over another when reactions are near thermodynamic equilibrium. We propose that protein minimization generically promotes coenzyme redundancy, and that coenzymes typically thought to exist in a single pool (e.g., CoA) may exist in more than one form (e.g., dephospho-CoA).

Significance statementMetabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes (e.g., NAD(H) and NADP(H)) associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling, and structural and sequence analysis indicates that coenzyme redundancy is not essential for metabolism, but rather an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes, and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems.

category: systems biology


title: Protein cost minimization promotes the emergence of coenzyme redundancy


title: A systems-guided approach to discover the intracellular target of a novel evolution-drug lead

abstract:
A promising approach to prevent antibiotic resistance is design of multivalent antibiotics that constrain bacterial evolvability by blocking multiple escape routes. Previously we discovered that an "evolution drug" lead designed to broadly neutralize bacterial dihydrofolate reductase (DHFR) and its trimethoprim escape variants is multivalent in E. coli cells. In this work we identified the second target of this new lead. We analysed antibiotic induced global metabolic perturbations and deployed metabolic-network analysis and structural similarity search of the putative targets and narrowed down the search to a few enzymes. Further, combining in vivo and in vitro analyses, we identified HPPK (folK), an essential protein in bacterial folate-metabolism, as the non-DHFR target of the lead. We observed that compounds with interaction specificity against WT do not develop multivalency against related proteins. This multivalent-design strategy is effective in blocking the emergence of drug-resistant phenotypes.

HighlightsO_LIAn evolution drug lead designed to block bacterial evolvability manifested behaviours suggesting an intra-cellular multivalent activity.
C_LIO_LIA multi-layered metabolomic and structure guided approach unravelled the second intra-cellular target of the evolution-drug lead, showing folate-pathway inhibitions at two metabolic points.
C_LIO_LIThe on-pathway anti-folate activity is exhibited by inhibiting two essential proteins viz. DHFR and its upstream protein HPPK.
C_LIO_LIMultivalent drug development schemes present a promising anti-evolution strategy to address the problem of "drug-resistance" by inhibiting more than one metabolic protein.
C_LI


title: On-pathway multivalency of a broadly neutralizing evolution drug candidate


title: Correcting for Body Size Variation in Morphometric Analysis

abstract: Using an allometric growth model to correct for body size variation has been known for many decades to be superior to several other widely used methods such as ratios, analysis of covariance, principal components analysis, and residual analysis. However, this technique remains relatively obscure and rarely applied. We optimize the implementation of this method through a newly developed and easy-to-use R package GroupStruct and use empirical datasets to test its relative efficacy compared to several commonly used methods. Our results demonstrate the superiority of the allometric method and highlights the negative impacts of applying improper body size correction methods.

category: zoology


title: BleTIES: Annotation of natural genome editing in ciliates using long read sequencing

abstract:
SummaryCiliates are single-celled eukaryotes that eliminate specific, interspersed DNA sequences (internally eliminated sequences, IESs) from their genomes during development. These are challenging to annotate and assemble because IES-containing sequences are much less abundant in the cell than those without, and IES sequences themselves often contain repetitive and low-complexity sequences. Long read sequencing technologies from Pacific Biosciences and Oxford Nanopore have the potential to reconstruct longer IESs than has been possible with short reads, and also the ability to detect correlations of neighboring element elimination. Here we present BleTIES, a software toolkit for detecting, assembling, and analyzing IESs using mapped long reads.

Availability and implementationBleTIES is implemented in Python 3. Source code is available at https://github.com/Swart-lab/bleties (MIT license), and also distributed via Bioconda.

ContactContact: kb.seah@tuebingen.mpg.de

Supplementary informationBenchmarking of BleTIES with published sequence data.

category: bioinformatics


title: Customizing Multifunctional Neural Interfaces through Thermal Drawing Process

abstract: Fiber drawing enables scalable fabrication of multifunctional flexible fibers that integrate electrical, optical and microfluidic modalities to record and modulate neural activity. Constraints on thermomechanical properties of materials, however, have prevented integrated drawing of metal electrodes with low-loss polymer waveguides for concurrent electrical recording and optical neuromodulation. Here we introduce two fabrication approaches: (1) an iterative thermal drawing with a soft, low melting temperature (Tm) metal indium, and (2) a metal convergence drawing with traditionally non-drawable high Tm metal tungsten. Both approaches deliver multifunctional flexible neural interfaces with low-impedance metallic electrodes and low-loss waveguides, capable of recording optically-evoked and spontaneous neural activity in mice over several weeks. We couple these fibers with a light-weight mechanical microdrive (1g) that enables depth-specific interrogation of neural circuits in mice following chronic implantation. Finally, we demonstrate the compatibility of these fibers with magnetic resonance imaging (MRI) and apply them to visualize the delivery of chemical payloads through the integrated channels in real time. Together, these advances expand the domains of application of the fiber-based neural probes in neuroscience and neuroengineering.

category: neuroscience


title: The association of human astrovirus with extracellular vesicles facilitates cell infection and protects the virus from neutralizing antibodies

abstract:
Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years because of acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from astrovirus- and mock-infected cells showed that infection with HAstV Yuc8 enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Some of the extracellular virus was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extraintestinal presence of some astrovirus strains.

ImportanceAstroviruses are an important cause of diarrhea in children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles seems to increase astrovirus infectivity in less susceptible cells, and renders virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

category: microbiology


title: Higher concentrations of bacterial enveloped virus Phi6 can protect the virus from environmental decay

abstract:
Phage Phi6 is an enveloped virus considered as a possible non-pathogenic surrogate for SARS-CoV-2 and other viral pathogens in transmission studies. Higher input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phage are dried in plastic tubes, and when they are stored in saline solution at 4{degrees}C. When bacteriophage Phi6 are placed in LB (Luria-Bertani) growth medium prior to placement on the plastic surface, viral recovery is not influenced by the starting concentration. The protection is reflected in longer half-lives of the phage at higher concentrations compared to lower. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon survival of infectious virus following inoculation of various surfaces, high initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at the higher concentrations, but also because the half-lives themselves are extended at the higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus, and to consider the role of the carrier and matrix if the results are to be clinically relevant.

IMPORTANCEDuring the COVID-19 pandemic, a lot of attention has been paid to the environmental decay of SARS-CoV-2 due to proposed transmission of the virus via fomites. However, published experiments have commenced with very high virus titer inoculums, an experimental design not representative of real-life conditions. The study described here evaluated the impact of initial virus titer on environmental decay of an enveloped virus, using a non-pathogenic surrogate for SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.


title: A protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oocyte maturation and wing development

abstract: Metazoans have evolved various stress response mechanisms to cope with cellular stress inflicted by external and physiological conditions. The Integrated Stress Response (ISR) is an evolutionarily conserved pathway that mediates adaptation to cellular stress via the transcription factor, ATF4. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo, and acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders photoreceptors more vulnerable to age-dependent retinal degeneration. crc mutant adult animals also show greater susceptibility to amino acid deprivation and reduced levels of known crc transcriptional targets. Furthermore, this mutant allele shows defects in wing veins and oocyte maturation, uncovering previously unknown roles for crc in the development of these tissues. Together, our data establish physiological and pathological functions of crc-mediated ISR in adult Drosophila tissues.

category: genetics


title: A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

abstract: For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: "HA Frankenbody". This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

category: cell biology


title: Whole-ExM: Expansion microscopy imaging of all anatomical structures of whole larval zebrafish

abstract: Nanoscale resolution imaging of whole vertebrates is required for a systematic understanding of human diseases, but this has yet to be realized. Expansion microscopy (ExM) is an attractive option for achieving this goal, but the expansion of whole vertebrates has not been demonstrated due to the difficulty of expanding hard body components. Here, we demonstrate whole-body ExM, which enables nanoscale resolution imaging of anatomical structures, proteins, and endogenous fluorescent proteins (FPs) of whole zebrafish larvae and mouse embryos by expanding them fourfold. We first show that post-digestion decalcification and digestion kinetics matching are critical steps in the expansion of whole vertebrates. Then, whole-body ExM is combined with the improved pan-protein labeling approach to demonstrate the three-dimensional super-resolution imaging of antibody- or FP-labeled structures and all major anatomical structures surrounding them. We also show that whole-body ExM enables visualization of the nanoscale details of neuronal structures across the entire body.

category: developmental biology


title: Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy


title: Patients with mesenchymal tumours and high Fusobacteriales prevalence have worse prognosis in colorectal cancer (CRC)

abstract:
ObjectiveTranscriptomic-based subtyping, Consensus Molecular Subtyping (CMS) and CRC Intrinsic Subtyping (CRIS), identify a patient subpopulation with mesenchymal traits (CMS4/CRIS-B) and poorer outcome. Here, we investigated the relationship between prevalence of Fusobacterium nucleatum (Fn) and Fusobacteriales, CMS/CRIS subtyping, cell type composition, immune infiltrates and host contexture to refine patients stratification and identify druggable context-specific vulnerabilities.

DesignWe coupled cell culture experiments with characterization of Fn/Fusobacteriales prevalence and host biology/microenviroment in tumours from 2 independent CRC patient cohorts (Taxonomy: n=140; TCGA-COAD-READ: n=605).

ResultsIn vitro, Fn infection induced inflammation via NF{kappa}B/TNF in HCT116 and HT29 cancer cell lines. In patients, high Fn/Fusobacteriales were found in CMS1, MSI tumours, with infiltration of macrophages M1, reduced macrophages M2, and high IL6/IL8/IL1{beta} signaling. Analysis of the Taxonomy cohort suggested that Fn was prognostic for CMS4/CRIS-B patients, despite having lower Fn load than CMS1 patients. In the TCGA-COAD-READ cohort, we likewise identified a differential association between Fusobacteriales relative abundance and outcome when stratifying patients in mesenchymal (either CMS4 and/or CRIS-B) vs. non-mesenchymal (neither CMS4 nor CRIS-B). Patients with mesenchymal tumours and high Fusobacteriales had approximately 2-fold higher risk of worse outcome. These associations were null in non-mesenchymal patients. Modelling the 3-way association between Fusobacteriales prevalence, molecular subtyping, and host contexture with logistic models with an interaction term disentangled the pathogen/host-signaling relationship and identified aberrations (including EMT/WNT/NOTCH) as candidate targets.

ConclusionThis study identifies CMS4/CRIS-B patients with high Fn/Fusobacteriales prevalence as a high-risk subpopulation that may benefit from therapeutics targeting mesenchymal biology.

Significance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIFusobacterium nucleatum (Fn), a commensal Gram-negative anaerobe from the Fusobacteriales order, is an onco-bacterium in CRC as a causal relationship between Fn prevalence and CRC pathogenesis, progression and treatment response has been reported in vivo.
C_LIO_LIBroad spectrum antibiotics has proven moderately successful in reducing tumour growth in preclinical models. However, the use of antibiotics to treat bacterium-positive cases in the clinic is not a viable option as it may further alter the already dysbiotic gut microbiome of CRC patients and may also have limited efficacy against Fn which penetrates and embeds deeply within the tumour.
C_LIO_LIThe highly heterogenous CRC patient population can be classified into distinct molecular subtypes (CMS and CRIS) based on gene expression profiles mirroring the underlying transcriptional programs. Patients classified as CMS4 and CRIS-B exhibit a mesenchymal phenotype and have poorer outcome.
C_LI

What are the new findings?O_LIFn/Fusobacteriales prevalence is associated with immune involvement (decrease in macrophages M1 and increase in macrophages M2) and activation of specific signalling programs (inflammation, DNA damage, WNT, metastasis, proliferation, cell cycle) in the host tumours.
C_LIO_LIThe prevalence of bacteria from the Fusobacteriales order, largely driven by Fn species, play an active or opportunistic role depending on the underlying host tumour biology and microenvironment.
C_LIO_LIFn and other species of the Fusobacteriales order are enriched in CMS1 (immuno, microsatellite unstable) patients compared to CMS2-4 cases.
C_LIO_LIFn/Fusobacteriales prevalence is associated with worse clinical outcome in patients with mesenchymal-rich CMS4/CRIS-B tumours, but not in patients with other molecular subtypes.
C_LI

How might it impact on clinical practice in the foreseeable future?O_LIFn/Fusobacteriales screening and transcriptomic-based molecular subtyping should be considered to identify patients with mesenchymal-rich tumours and high bacterium prevalence and to inform disease management.
C_LIO_LIFn/Fusobacteriales prevalence may need to be addressed exclusively in patients with mesenchymal-rich high-stromal infiltrating tumours rather than a blanket-approach to treat all pathogen-positive patients.
C_LIO_LIClinical management of the disease for this subpopulation of high-risk patients with unfavourable clinical outcome could be attained by administering compounds currently in clinical trials that target aberrations in the host signaling pathways (NOTCH, WNT, EMT) and tumour microenviroment (inflammasome, activated T cells, complement system, and macrophage chemotactism and activation).
C_LI

category: cancer biology


title: PAK1 and PAK2 in the cell metabolism regulation

abstract: P21-activated kinases (PAKs) regulate processes associated with cytoskeletal rearrangements, such as cell division, adhesion, and migration. The possible regulatory role of PAKs in cell metabolism has not been well explored, but increasing evidence suggests that a cell metabolic phenotype is related to cell interactions with the microenvironment. We analyzed the impact of PAK inhibition by small molecule inhibitors, small interfering RNA, or gene knockout on the rates of mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PAK group I by IPA-3 induced a strong decrease in metabolic rates in human adherent cancer cell lines, leukemia/lymphoma cell lines, and primary leukemia cells. The immediate effect of FRAX597, which inhibits PAK kinase activity, was moderate, indicating that PAK nonkinase functions are essential for cell metabolism. Selective downregulation or deletion of PAK2 was associated with a shift toward oxidative phosphorylation. Alternatively, PAK1 knockout resulted in increased glycolysis. However, the overall metabolic capacity was not substantially reduced by PAK1 or PAK2 deletion, possibly due to partial redundancy in PAK1/PAK2 regulatory roles or to activation of other compensatory mechanisms.


title: PAK1 and PAK2 in cell metabolism regulation


title: Multi-Factor Authentication of Potential 5' Splice Sites by the Saccharomyces cerevisiae U1 snRNP

abstract:
In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5 splice site (5 SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5 SS recognition have largely been done in vivo or in cellular extracts where the U1/5 SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5 SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5 SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5 SS and preferentially accumulate at these sequences rather than on close cognates.

IMPACT STATEMENTThe yeast U1 snRNP recognizes multiple features of target RNAs to reversibly identify splicing-competent 5 splice sites.

category: biochemistry


title: In Silico Molecular-Based Rationale for SARS-CoV-2 Spike Circulating Mutations Able to Escape Bamlanivimab and Etesevimab Monoclonal Antibodies

abstract: The purpose of this work was to provide an in silico molecular rationale of the role eventually played by currently circulating S-RBDCoV-2 mutations in evading the immune surveillance effects elicited by the two Eli Lilly LY-CoV555/bamlanivimab and LY-CoV016/etesevimab monoclonal antibodies. The main findings from this study and shows that, compared to the wild-type SARS-CoV-2 spike protein, mutations E484A/G/K/Q/R/V, Q493K/L/R, S494A/P/R, L452R and F490S are predicted to be markedly resistant to neutralization by LY-CoV555, while mutations K417E/N/T, D420A/G/N, N460I/K/S/T, T415P, and Y489C/S are predicted to confer LY-CoV016 escaping advantage to the viral protein. A challenge of our global in silico results against the relevant experimental data resulted in an overall 90% agreement. This achievement not only constitutes a further, robust validation of our computer-based approach but also yields a molecular-based rationale for all relative experimental findings, and leads us to conclude that the current circulating SARS-CoV-2 and all possible emergent variants carrying these mutations in the spike protein can present new challenges for mAb-based therapies and ultimately threaten the fully-protective efficacy of currently available vaccines.

category: biophysics


title: The processing of semantic complexity and co-speech gestures in schizophrenia: a naturalistic, multimodal fMRI study

abstract: Schizophrenia is marked by aberrant processing of complex speech and gesture, which may contribute functionally to its impaired social communication. To date, extant neuroscientific studies of schizophrenia have largely investigated dysfunctional speech and gesture in isolation, and no prior research has examined how the two communicative channels may interact in more natural contexts. Here, we tested if patients with schizophrenia show aberrant neural processing of semantically complex story segments, and if speech-associated gestures (co-speech gestures) might modulate this effect. In a functional MRI study, we presented to 34 participants (16 patients and 18 matched-controls) an ecologically-valid retelling of a continuous story, performed via speech and spontaneous gestures. We split the entire story into ten-word segments, and measured the semantic complexity for each segment with idea density, a linguistic measure that is commonly used clinically to evaluate aberrant language dysfunction at semantic level. Per segment, the presence of numbers of gestures varied (n = 0, 1, +2). Our results suggest that, in comparison to controls, patients showed reduced activation for more complex segments in the bilateral middle frontal and inferior parietal regions. Importantly, this neural aberrance was reduced in segments presented with gestures. Thus, for the first time with a naturalistic multimodal stimulation paradigm, we show that gestures reduced group differences when processing a natural story, probably by facilitating the processing of semantically complex segments of the story in schizophrenia.


title: Evidence for PKD2L1-positive neurons present distant from the central canal in the ventromedial spinal cord and Medulla of the adult mouse

abstract:
Neurons in contact with the cerebrospinal fluid (CSF) are found around the medullo-spinal central canal (CC) in adult mice. These neurons (CSF-cNs), located within or below the ependymal cell layer known as the stem cell niche, present a characteristic morphology with a dendrite projecting to the CC and ending with a protrusion. They are GABAergic, characterized by an immature neuronal phenotype and selectively express PKD2L1, a channel member of the TRP channel superfamily with properties of sensory receptor.

Using immunohistological techniques in mice, we characterize a new population of PKD2L1 positive cells that is observed around embryonic day 16 (E16), is present distant from the CC in a zone enriched with astrocytes and ependymal fibers of the ventro-medial spinal cord and medulla. With development, their number appears stable although smaller than that of CSF-cNs and they progressively become more distant from the CC with the reorganization of the CC region. These neurons share both functional and phenotypical properties with CSF-cNs, but they appear subdivided in two groups. One, present along the midline, has a bipolar morphology and extend a long dendrite along ependymal fibers and towards the CC. The second group, localized in more ventro-lateral regions, has a multipolar morphology and no apparent projection to the CC

Altogether, we describe a novel population of PKD2L1+ neurons distant from the CC but with properties similar to CSF-cNs that might serve to sense modification in the composition of either CSF or interstitial liquid, a function that will need to be confirmed.


title: Distinct Origins of Two-order Hierarchical Cognitive Abilities in Human Adults

abstract: Human cognitive abilities are considerably diverse from basic perceptions to complex social behaviors. All human cognitive functions are principally categorized into a two-order hierarchy. Almost all of the first-order cognitive abilities investigated in behavioral genetics have been found to be dominantly heritable. However, the origins of the human second-order cognitive abilities in metacognition and mentalizing so far remain unclear. We here systematically compared the origins of the first-order and second-order cognitive abilities involved in the metacognition and mentalizing tasks using the classical twin paradigm on human adults. Our results demonstrated a double dissociation of the genetic and environmental contributions to the first-order and second-order cognitive abilities. All the first-order cognitive abilities involved in the metacognition and mentalizing tasks were dominantly heritable. In contrast, the shared environmental effects, rather than the genetic effects, had dominant contributions to the second-order cognitive abilities of metacognition and mentalizing in human adults. Hence, our findings suggest that human adults monitoring sensitivities in metacognition and mentalizing are profoundly sculpted by their social or cultural experiences, but less preconditioned by their biological nature.


title: Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

abstract: The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the worlds population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

category: immunology


title: SARS-like coronaviruses in horseshoe bats (Rhinolophus spp.) in Russia, 2020.

abstract: We found and genetically described two novel SARS-like coronaviruses in feces and oral swabs of the great (R. ferrumequinum) and the lesser (R. hipposideros) horseshoe bats in southern region of Russia. The viruses, named Khosta-1 and Khosta-2, together with related viruses from Bulgaria and Kenya, form a separate phylogenetic lineage. We found an evidence of recombination events in evolutionary history of Khosta-1, which involved the acquisition of structural proteins S, E, and M as well as nonstructural genes ORF3, ORF6, ORF7a, and ORF7b from a virus that is closely related to Kenyan isolate BtKY72. Examination of bats by RT-PCR revealed that 62,5% of great horseshoe bats in one of the caves were positive for Khosta-1 virus while its overall prevalence was 14%. The prevalence of Khosta-2 was 1,75%. Our results show that SARS-like coronaviruses circulate in horseshoe bats in the region and provide a new data on their genetic diversity.


title: Effects of common mutations in the SARS-CoV-2 Spike RBD domain and its ligand the human ACE2 receptor on binding affinity and kinetics

abstract: The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD domain are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the more transmissible B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD domain that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations, and combinations found in new SARS-CoV-2 variants first identified in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P1). Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations primarily enhance transmission, the K417N/T mutations facilitate immune escape, and the E484K mutation facilitates both transmission and immune escape.


title: An intranasal ASO therapeutic targeting SARS-CoV-2

abstract: The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity1. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic2. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identified an LNA ASO binding to the 5 leader sequence of SARS-CoV-2 ORF1a/b that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the K18-hACE2 humanized COVID-19 mouse model potently (98-99%) suppressed viral replication in the lungs of infected mice, revealing strong prophylactic and treatment effects. We found that the LNA ASO also represses viral infection in golden Syrian hamsters, and is highly efficacious in countering all SARS-CoV-2 "variants of concern" tested in vitro and in vivo, including B.1.427, B.1.1.7, and B.1.351 variants3. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce transmission of variants partially resistant to vaccines and monoclonal antibodies, and could be deployed intranasally for prophylaxis or via lung delivery by nebulizer to decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences4, and they may have particular impact in areas where vaccine distribution is a challenge, and could be stockpiled for future coronavirus pandemics.


title: Genomic evidence for inbreeding depression and purging of deleterious genetic variation in Indian tigers

abstract:
Increasing habitat fragmentation leads to wild populations becoming small, isolated, and threatened by inbreeding depression. However, small populations may be able to purge recessive deleterious alleles as they become expressed in homozygotes, thus reducing inbreeding depression and increasing population viability. We used whole genomes sequencing from 57 tigers to estimate individual inbreeding and mutation loads in a small-isolated, and two large-connected populations in India. As expected, the small-isolated population had substantially higher average genomic inbreeding (FROH=0.57) than the large-connected (FROH=0.35 and FROH=0.46) populations. The small-isolated population had the lowest loss-of-function mutation load, likely due to purging of highly deleterious recessive mutations. The large populations had lower missense mutation loads than the small-isolated population, but were not identical, possibly due to different demographic histories. While the number of the loss-of-function alleles in the small-isolated population was lower, these alleles were at high frequencies and homozygosity than in the large populations. Together, our data and analyses provide evidence of (a) high mutation load; (b) purging and (c) the highest predicted inbreeding depression, despite purging, in the small-isolated population. Frequency distributions of damaging and neutral alleles uncover genomic evidence that purifying selection has removed part of the mutation load across Indian tiger populations. These results provide genomic evidence for purifying selection in both small and large populations, but also suggest that the remaining deleterious alleles may have inbreeding associated fitness costs. We suggest that genetic rescue from sources selected based on genome-wide differentiation should offset any possible impacts of inbreeding depression.

Significance statementHabitat fragmentation is sequestering species into small and isolated populations with high chances of extinction. But are small and isolated populations at risk of extinction from inbreeding depression, or does inbreeding and purging of deleterious alleles reduce such threat? Using whole genomes from several wild Indian tiger populations, we provide evidence supporting purging of highly deleterious variants in a small isolated population. However, our analyses also indicate that the remaining highly deleterious alleles are at high frequencies, suggesting continued inbreeding depression despite some successful purging. We discuss the implications of our results for conservation, including possible genomics-informed genetic rescue strategies.

category: evolutionary biology


title: Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells

abstract: Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results defined chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identified both promoter selective as well as shared cis-regulatory interactions with the Paupar and Pax6 promoters involved in regulating Paupar-Pax6 co-expression in neuronal cells. The TCF7L2 transcription factor, a major regulator of chromatin architecture and effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We identify a functional TCF7L2 bound cis-regulatory element within the Paupar gene, suggesting that the Paupar DNA locus itself regulates Pax6 expression in addition to its previously described transcriptdependent modes of action. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

category: genomics


title: Automated Assignment of Proteoform Classification Levels

abstract: Proteoform identification is required to fully understand the biological diversity present in a sample. However, these identifications are often ambiguous because of the challenges in analyzing full length proteins by mass spectrometry. A five-level proteoform classification system was recently developed to delineate the ambiguity of proteoform identifications and to allow for comparisons across software platforms and acquisition methods. Widespread adoption of this system requires software tools to provide classification of the proteoform identifications. We describe here implementation of the five-level classification system in the software program MetaMorpheus, which provides both bottom-up and top-down identifications. Additionally, we developed a stand-alone program called ProteoformClassifier that allows users to classify proteoform results from any search program, provided that the program writes output that includes the information necessary to evaluate proteoform ambiguity. This stand-alone program includes a small test file and database to evaluate if a given program provides sufficient information to evaluate ambiguity. If the program does not, then ProteoformClassifier provides meaningful feedback to assist developers with implementing the classification system. We tested currently available top-down software programs and found that none of them other than MetaMorpheus provided sufficient information regarding identification ambiguity to permit classification.


title: Incorporating structural knowledge into unsupervised deep learning for two-photon imaging data

abstract:
Live imaging techniques, such as two-photon imaging, promise novel insights into cellular activity patterns at a high spatio-temporal resolution. While current deep learning approaches typically focus on specific supervised tasks in the analysis of such data, we investigate how structural knowledge can be incorporated into an unsupervised generative deep learning model directly at the level of the video frames. We exemplify the proposed approach with two-photon imaging data from hippocampal CA1 neurons in mice, where we account for spatial structure with convolutional neural network components, disentangle the neural activity of interest from the neuropil background signal with separate foreground and background encoders and model gradual temporal changes by imposing smoothness constraints. Taken together, our results illustrate how such architecture choices facilitate a modeling approach that combines the flexibility of deep learning with the benefits of domain knowledge, providing an interpretable, purely image-based model of activity signals from live imaging data.

Teaser sentenceUsing a neural network architecture that reflects domain knowledge provides an interpretable model of live cell imaging data.


title: Permutation-based significance analysis reduces the type 1 error rate in bisulfite sequencing data analysis of human umbilical cord blood samples

abstract:
BackgroundDNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2 % of human CpG sites. To detect such associations outside these regions, we chose the bisulfite sequencing approach.

MethodsWe collected and curated all available clinical data on 200 newborn infants; whose umbilical cord blood samples were analyzed with the reduced representation bisulfite sequencing (RRBS) method. A generalized linear mixed effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis.

ResultsWe discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.

ConclusionsThe inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. With standard significance thresholds, type 1 error rates were high with both these implementations, across alternative parameter settings and analysis strategies. We conclude that comb-p and RADMeth are convenient methods for the detection of differentially methylated regions, but the statistical significance should either be determined empirically or before the spatial adjustment. Our RRBS data analysis workflow is available in https://github.com/EssiLaajala/RRBS_workflow.


title: Implications Derived from S-Protein Variants of SARS-CoV-2 from Six Continents

abstract: Spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical determinants of the infectivity and antigenicity of the virus. Several mutations in the spike protein of SARS-CoV-2 have already been detected, and their effect in immune system evasion and enhanced transmission as a cause of increased morbidity and mortality are being investigated. From pathogenic and epidemiological perspectives, spike proteins are of prime interest to researchers. This study focused on the unique variants of S proteins from six continents Asia, Africa, Europe, Oceania, South America, and North America. In comparison to the other five continents, Africa (29.065%) had the highest percentage of unique S proteins. Notably, only North America had 87% (14046) of the total (16143) specific S proteins available in the NCBI database(across all continents). Based on the amino acid frequency distributions in the S protein variants from all the continents, the phylogenetic relationship implies that unique S proteins from North America were significantly different from those of the other five continents. Overtime, the unique variants originating from North America are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. Hence it is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of COVID-19 pandemic. It is also further suggested that the efficacy of existing vaccines and future vaccine development must be reviewed with careful scrutiny, and if needed, further re-engineered based on requirements dictated by new emerging S protein variants.


title: Mechanism of the Switch from NO to H2O2 in Endothelium-Dependent Vasodilation in Diabetes

abstract: Coronary microvascular dysfunction is prevalent among diabetics and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but the mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a major risk factor for CAD we hypothesized that a similar switch from NO-to-H2O2 occurs in diabetes. Methods: Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice fed chow or high fat and sugar diet, and LepR null (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells and hearts were analyzed via qPCR for gene expression and protein expression was assessed via Western-Blot analyses. Superoxide was detected via electron paramagnetic resonance (EPR). Results: 1) Ex vivo coronary EDD and in vivo MBF was impaired in diabetes. 2) L-NAME (NO-synthase inhibitor) inhibited ex vivo coronary EDD and in vivo MBF in WT, while PEG-catalase (H2O2 scavenger) inhibited diabetic EDD ex vivo and MBF in vivo. 5) miR-21 deficiency blocked the NO-to-H2O2 switch and prevented diabetic vasodilation impairments. 6) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav-1, and downregulated Pgc-1. Deficiency of miR-21 reversed these changes. 7) miR-21 deficiency increased PGC1, PPAR and eNOS protein and reduced detection of endothelial superoxide. Conclusions: Diabetics exhibit an NO-to-H2O2 switch in the mediator of EDD coronary dilation, which contributed to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients.

category: physiology


title: Somatosensory signals from the controllers of an extra robotic finger support motor learning

abstract: Considerable resources are being invested to provide bidirectional control of substitutive and augmentative motor interfaces through artificial somatosensory feedback. Here, we investigated whether intrinsic somatosensory information, from body part(s) proportionally controlling an augmentation device, can be utilised to infer the devices state and position, to better support motor control and learning. In a placebo-controlled design, we used local anaesthetic to attenuate somatosensory inputs to the big toes while participants learned to operate a toe-controlled robotic extra finger (Third Thumb) using pressure sensors. Motor learning outcomes were compared against a control group who received sham anaesthetic. The availability of somatosensory cues about the amount of exerted pressure generally facilitated acquisition, retention and transfer of motor skills, and performance under cognitive load. Motor performance was not impaired by anaesthesia when tasks involved close collaboration with the biological fingers, indicating that the brain could  close the gap of the missing pressure signals by alternative means, including feedback from other body parts involved in the motor task. Together, our findings demonstrate that there are intrinsic natural avenues to provide surrogate position information to support motor control of an artificial body part, beyond artificial extrinsic signalling.


title: Dissociation in basolateral and central amygdala effective connectivity predicts the stability of emotion-related impulsivity in adolescents with borderline personality symptoms: a resting-state fMRI study

abstract:
BackgroundBorderline personality disorder (BPD) is associated with altered activity in the prefrontal cortex (PFC) and amygdala, yet no studies have examined fronto-limbic circuitry in borderline adolescents. Here, we examined the contribution of fronto-limbic connectivity to the longitudinal stability of emotion-related impulsivity (ERI), a key feature of BPD, in symptomatic adolescents and young adults.

MethodsWe compared resting-state effective connectivity (EC) in 82 adolescents and emerging adults with and without clinically significant borderline symptoms (n BPD = 40, ages 13-30). Group-specific directed networks were estimated amongst fronto-limbic nodes including PFC, ventral striatum (VS), central amygdala (CeN), and basolateral amygdala (BLA). We calculated directed centrality metrics and examined if these values were associated with initial levels and rates of change in ERI symptoms over a one-year follow-up using latent growth curve models (LGCMs).

ResultsIn the healthy group, ventromedial prefrontal cortex (vmPFC) and dorsal ACC had a directed influence on CeN and VS respectively. In the borderline group bilateral BLA had a directed influence on CeN, whereas in the healthy group CeN influenced BLA. LGCMs revealed that in borderline adolescents, ERI remained stable across follow-ups. Further, higher output of R CeN in controls was associated with stronger within-person decreases in ERI.

ConclusionsFunctional inputs from BLA and vmPFC appear to play competing roles in influencing CeN activity. In borderline adolescents BLA may predominate over CeN activity, while in controls the ability of CeN to conversely influence BLA activity is associated with more rapid reductions in ERI.


title: Contactless recordings of retinal activity using optically pumped magnetometers

abstract: Optically pumped magnetometers (OPMs) have been adopted for the recording of brain activity. Without the need to be cooled to cryogenic temperatures, an array of these sensors can be placed more flexibly, which allows for the recording of neuronal structures other than neocortex. Here we use eight OPM sensors to record human retinal activity following flash stimulation. We compare this magnetoretinographic (MRG) activity to the simultaneously recorded electroretinogram of the eight participants. The MRG shows the familiar flash-evoked potentials (a-wave and b-wave) and shares a highly significant amount of information with the electroretinogram recording (both in a simultaneous and separate recording). We conclude that OPM sensors have the potential to become a contactless alternative to fiber electrodes for the recording of retinal activity. Such a contactless solution can benefit both clinical and neuroscientific settings.


title: Encoding of inflammatory hyperalgesia in mice spinal cord

abstract: Inflammation modifies the input-output properties of peripheral nociceptive neurons, thus leading to hyperalgesia, a condition in which the perception of noxious heat stimuli is altered such that the same stimulus produces enhanced pain. The increased nociceptive output enters the superficial dorsal spinal cord (SDH), which comprises the first CNS network integrating the noxious information. Here we used in vivo calcium imaging and a computational approach to investigate how the SDH network in mice encodes the injury-mediated abnormal input from peripheral nociceptive neurons. We show that the application of noxious heat stimuli to the hind paw in naive mice before induction of injury affects the activity of 70% of recorded neurons by either increasing or suppressing it. Application of the same noxious heat stimuli to hyperalgesic skin following injury leads to activation of previously non-responded cells and de-suppression of the "suppressed" neurons. We further demonstrate that reduction in synaptic inhibition mimics the response to the noxious stimuli in hyperalgesic conditions. Using a computational model of the SDH network, we predict that the "disinhibitory" effect of hyperalgesic stimuli results from the inflammation-mediated increased afferent input to the SDH network and a decrease in SDH inhibition. Both of these processes synergistically contribute to the injury-mediated increase in SDH output towards higher brain centers.


title: Standard PBMC cryopreservation selectively decreases detection of nine clinically-relevant T-cell markers

abstract: Biobanking is an operational component of various epidemiological studies and clinical trials. Although peripheral blood is routinely acquired and stored in biobanks, the effects of specimen processing on cell composition and clinically-relevant functional markers of T cells still require a systematic evaluation. Here we assessed 25 relevant T-cell markers and showed that the detection of nine membrane markers, e.g., PD-1, CTLA4, KLRG1, CD25, CD122, CD127 and others reflecting exhaustion, senescence and other functions was reduced among at least one T-cell subset following standard processing, although the frequency of CD4, CD8 and regulatory T cells was unaffected. Nevertheless, a six-month-long cryopreservation did not impair the expression levels of many other membrane and all the eight tested intracellular lineage or functional T-cell markers. Our findings uncover that several clinically-relevant markers are particularly affected by processing and the interpretation of those results in clinical trials and translational research should be done with caution.


title: Somatic structural variant formation is guided by and influences genome architecture

abstract: The occurrence and formation of genomic structural variants (SV) is known to be influenced by the 3D chromatin architecture, but the extent and magnitude has been challenging to study. Here, we apply Hi-C to study chromatin organization before and after induction of chromothripsis in human cells. We use Hi-C to manually assemble the derivative chromosomes following the massive complex rearrangements, which allowed us to study the sources of SV formation and their consequences on gene regulation. We observe an action-reaction interplay whereby the 3D chromatin architecture directly impacts on the location and formation of SVs. In turn, the SVs reshape the chromatin organization to alter the local topologies, replication timing and gene regulation in cis. We show that genomic compartments and replication timing are important determinants for juxtaposing distant loci to form SVs across 30 different cancer types with a pronounced abundance of SVs between early replicating regions in uterine cancer. We find that SVs frequently occur at 3D loop-anchors, cause compartment switching and changes in replication timing, and that this is a major source of SV-mediated effects on nearby gene expression changes.


title: Large-scale genomic study reveals robust activation of the immune system following advanced Inner Engineering meditation retreat.

abstract: The positive impact of meditation on human wellbeing is well documented, yet its molecular mechanisms are incompletely understood. We applied a comprehensive systems biology approach starting with whole blood gene expression profiling combined with multi-level bioinformatic analyses to characterize the co-expression, transcriptional, and protein-protein interaction networks to identify meditation-specific core network after an advanced 8-day Inner Engineering retreat program. We found the response to oxidative stress, detoxification, and cell cycle regulation pathways were downregulated after meditation. Strikingly, 220 genes directly associated with immune response, including 68 genes related to interferon (IFN) signaling were upregulated, with no significant expression changes in the inflammatory genes. This robust meditation-specific immune response network is significantly dysregulated in multiple sclerosis and severe COVID-19 patients. The work provides a foundation for understanding the effect of meditation and potential implications to voluntarily and non-pharmacologically improve the immune response before immunotherapy for many conditions, including multiple sclerosis and COVID-19 vaccination.


title: The dilution effect limits plasmid horizontal transmission in multispecies bacterial communities

abstract: By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multispecies communities are poorly understood. Here, we show, using experimental multispecies communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to  the dilution effect; an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from coinfected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid-plasmid interactions reducing the rate of horizontal transmission.


title: Asymptomatic gut colonization by extended-spectrum beta-lactamase-producing Escherichia coli is not associated with an altered gut microbiome or metabolome in Dutch adults

abstract:
BackgroundGut colonization by antibiotic resistant E. coli strains, including extended-spectrum beta-lactamase (ESBL)-producing E. coli is a risk factor for developing overt infection. The gut microbiome can provide colonization resistance against enteropathogens, but it remains unclear whether it confers resistance against potentially pathogenic ESBL-producing E. coli.

MaterialsFrom a Dutch cross-sectional population study (PIENTER-3), feces from 2751 individuals were used to culture ESBL-producing bacteria. Of these, we selected 49 samples which were positive for an ESBL-producing Escherichia coli (ESBL+), and negative for a variety of variables known to affect microbiome composition. These were matched in a 1:1 ratio to ESBL- samples based on age, sex, having been abroad in the past six months and ethnicity. Shotgun metagenomic sequencing was performed and taxonomic species composition and functional annotations (microbial metabolism and carbohydrate-active enzymes) were determined. Targeted quantitative metabolic profiling (1H NMR-spectroscopy) was performed to investigate metabolomic profiles.

ResultsNo differences in alpha or beta diversity were observed, nor in relative abundance, between ESBL+ and ESBL- individuals based on bacterial species level composition. Machine learning approaches based on microbiota composition did not accurately predict ESBL status (area under the receiver operating characteristic curve (AUROC)=0.53), neither when based on functional profiles. The metabolome did also not convincingly differ between ESBL groups as assessed by a variety of approaches, including machine learning through random forest (AUROC=0.61).

ConclusionUsing a combination of multi-omics and machine learning approaches, we conclude that asymptomatic gut carriage of ESBL-producing E. coli is not associated with an altered microbiome composition or function. This may suggest that microbiome-mediated colonization resistance against ESBL-producing E. coli is not as relevant as it is against other enteropathogens.


title: Rad52 oligomeric N-terminal domain stabilizes Rad51 nucleoprotein filaments and contributes to their protection against Srs2

abstract: Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

category: molecular biology


title: Changes in the urinary proteome in rats with regular swimming exercise

abstract:
PurposeUrine can sensitively reflect early pathophysiological changes in the body. The purpose of this study was to determine whether the urine proteome could reflect changes in regular swimming exercise.

MethodsIn this study, experimental rats were subjected to daily moderate-intensity swimming exercise for 7 weeks. Urine samples were collected at weeks 2, 5, and 7 and were analyzed by using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

ResultsUnsupervised clustering analysis of all urinary proteins identified at week 2 showed that the swimming group was distinctively different from the control group. Compared to the control group, a total of 112, 61 and 44 differential proteins were identified in the swimming group at weeks 2, 5 and 7, respectively. Randomized grouping statistical analysis showed that more than 85% of the differential proteins identified in this study were caused by swimming exercise rather than random allocation. According to the Human Protein Atlas, the differential proteins that have human orthologs were strongly expressed in the liver, kidney and intestine. Functional annotation analysis revealed that these differential proteins were involved in glucose metabolism and immunity-related pathways.

ConclusionOur results revealed that the urinary proteome could reflect significant changes following regular swimming exercise. These findings may suggest an approach to monitoring whether the amount of exercise is appropriate.


title: Identifiability analysis for models of the translation kinetics after mRNA transfection

abstract: Mechanistic models are a powerful tool to gain insights into biological processes. The parameters of such models, e.g. kinetic rate constants, usually cannot be measured directly but need to be inferred from experimental data. In this article, we study dynamical models of the translation kinetics after mRNA transfection and analyze their parameter identifiability. Previous studies have considered ordinary differential equation (ODE) models of the process, and here we formulate a stochastic differential equation (SDE) model. For both model types, we consider structural identifiability based on the model equations and practical identifiability based on simulated as well as experimental data and find that the SDE model provides better parameter identifiability than the ODE model. Moreover, our analysis shows that even for those parameters of the ODE model that are considered to be identifiable, the obtained estimates are sometimes unreliable. Overall, our study clearly demonstrates the relevance of considering different modeling approaches and that stochastic models can provide more reliable and informative results.


title: A combined lifestyle intervention induces a sensitization of the blood transcriptomic response to a nutrient challenge

abstract:
The global population is growing older. As age is a primary risk factor of (multi)morbidity, there is a need for novel indicators to predict, track, treat and prevent the development of disease. Lifestyle interventions have shown promising results in improving the health of participants and reducing the risk for disease, but in the elderly population, such interventions often show less reliable or subtle effects on health outcomes. This is further complicated by a poor understanding of the homeodynamics and the molecular effects of lifestyle interventions, by which their effects of a lifestyle intervention remain obscured. In the Growing Old Together (GOTO) study, we examined the responses of 164 healthy, elderly men and women to a 13-week combined physical and dietary lifestyle intervention. In addition to collecting blood samples at a fasted state, we sampled blood also 30 minutes following a standardized meal. This allows us to investigate an intervention response not only in the traditional fasted state, but also in the blood metabolic and cellular responses to a nutrient challenge. We investigated the transcriptomic and metabolomic responses to this nutrient challenge, how these responses relate to each other, and how this response is affected by the lifestyle intervention.

We find that the intervention has very little effect on the fasted blood transcriptome, but that the nutrient challenge induces a large translational inhibition, and an innate immune activation, which together comprise a cellular stress response that is stimulated by the intervention. A sex-specific analysis reveals that although the same set of genes respond in the same direction in both males and females, the magnitude of these effects differ, and are modulated differently by the intervention. On the other hand, the metabolomic response to the nutrient challenge is largely unaffected by the intervention, and the correlation between the metabolomic nutrient response and transcriptomic modules indicates that the change in transcriptomic response to the nutrient challenge is independent from a change in cellular metabolomic environment.

This work constitutes a glance at the acute transcriptomic stress response to nutrient intake in blood, and how a lifestyle intervention affects this response in healthy elderly, and may lead to the development of novel biomarkers to capture the phenotypic flexibility of health.


title: Precise estimation of genome size from NGS data

abstract: Precise estimates of genome sizes are important parameters for both theoretical and practical biodiversity genomics. We present here a fast, easy-to-implement and precise method to estimate genome size from the number of bases sequenced and the mean sequence coverage. To estimate the latter, we take advantage of the fact that a precise estimation of the Poisson distribution parameter lambda is possible from truncated data, restricted to the part of the coverage distribution representing the true underlying distribution. With simulations we could show that reasonable genome size estimates can be gained even from low-coverage (10X), highly discontinuous genome drafts. Comparison of estimates from a wide range of taxa and sequencing strategies with flow-cytometry estimates of the same individuals showed a very good fit and suggested that both methods yield comparable, interchangeable results.


title: ModEst - Precise estimation of genome size from NGS data


title: The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates metabolism and development via translation

abstract: Ribosomal RNAs (rRNAs) have long been known to carry modifications, including numerous sites of 2O-methylation and pseudouridylation, as well as N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry m6A, and while ZCCHC4 has been identified as the methyltransferase responsible for the 28S rRNA m6A site, the methyltransferase responsible for the 18S rRNA m6A site has remained uncharacterized until recently. Here, we show that the METTL5-TRMT112 complex is the methyltransferase responsible for installing m6A at position 1832 of human 18S rRNA. TRMT112 is required for the metabolic stability of METTL5, and human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. Loss of METTL5 in human cancer lines alters the translation of transcripts associated with mitochondrial biogenesis and function. Mettl5 knockout mice display reduced body size and evidence of metabolic defects. This m6A site is located on the 3 end of 18S rRNA, which may become surface-exposed under some circumstances and thus may play a regulatory role in translation of specific transcripts. While recent work has focused heavily on m6A modifications in mRNA and its roles in mRNA processing and translation, deorphanizing putative methyltransferase enzymes is revealing previously unappreciated regulatory roles for m6A in noncoding RNAs.


title: A transcriptomics-guided drug target discovery strategy identifies novel receptor ligands for lung regeneration

abstract: Currently, there is no pharmacological treatment targeting defective tissue repair in chronic disease. Here we utilized a transcriptomics-guided drug target discovery strategy using gene signatures of smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke, identifying druggable targets expressed in alveolar epithelial progenitors of which we screened the function in lung organoids. We found several drug targets with regenerative potential of which EP and IP prostanoid receptor ligands had the most significant therapeutic potential in restoring cigarette smoke-induced defects in alveolar epithelial progenitors in vitro and in vivo. Mechanistically, we discovered by using scRNA-sequencing analysis that circadian clock and cell cycle/apoptosis signaling pathways were enriched in alveolar epithelial progenitor cells in COPD patients and in a relevant model of COPD, which was prevented by PGE2 or PGI2 mimetics. Conclusively, specific targeting of EP and IP receptors offers therapeutic potential for injury to repair in COPD.

category: pharmacology and toxicology


title: Peripheral neuropathy linked mRNA export factor GANP reshapes gene regulation in human motor neurons

abstract: Loss-of-function of the mRNA export protein GANP (MCM3AP gene) cause early-onset sensorimotor neuropathy, characterised by axonal degeneration in long peripheral nerves. GANP functions as a scaffold at nuclear pore complexes, contributing to selective nuclear export of mRNAs. Here, we aimed to identify motor neuron specific transcripts that are regulated by GANP and may be limiting for local protein synthesis in motor neuron axons. We compared motor neurons with a gene edited mutation in the Sac3 mRNA binding domain of GANP to isogenic controls. We also examined patient-derived motor neurons. RNA sequencing of motor neurons as well as nuclear and axonal subcompartments showed that mutant GANP had a profound effect on motor neuron transcriptomes, with alterations in nearly 40 percent of all expressed genes and broad changes in splicing. Expression changes in multiple genes critical for neuronal functions, combined with compensatory upregulation of protein synthesis and early-stage metabolic stress genes, indicated that RNA metabolism was abnormal in GANP-deficient motor neurons. Surprisingly, limited evidence was found for large-scale nuclear retention of mRNA. This first study of neuropathy-linked GANP defects in human motor neurons shows that GANP has a wide gene regulatory role in a disease-relevant cell type that requires long-distance mRNA transport.


title: Functional and structural modifications of influenza antibodies during pregnancy

abstract: Pregnancy represents a unique tolerogenic immune state which may alter susceptibility to infection and vaccine-response. Here we characterized humoral immunity to seasonal influenza vaccine strains in pregnant and non-pregnant women. Pregnant women had reduced hemagglutinin subtype-1 (H1)-IgG, IgG1, and IgG2, hemagglutination inhibition and group 1 and 2 stem IgG. However, H1-specific avidity and Fc{gamma}R1 binding increased. Influenza-antibodies in pregnancy had distinct Fc and Fab glycans characterized by di-galactosylation and di-sialylation. In contrast, agalactosylation and bisection were prominent outside of pregnancy. H1-specific Fc-functionality was moderately reduced in pregnancy, although likely compensated by stronger binding to cognate antigen and FcR. Multivariate analysis revealed distinct populations characterized by Fc{gamma}R1 binding, H1-IgG levels, and glycosylation. Pooled sera from pregnant women exhibited longer retention in vivo. Our results demonstrate structural and functional modulation of humoral immunity during pregnancy in an antigen-specific manner towards reduced inflammation, increased retention in circulation, and efficient placental transport.


title: Phenotypic and Kinetic Changes of Myeloid Lineage Cells in Innate Response to Chikungunya Infection in Cynomolgus Macaques

abstract: Chikungunya is an emerging threat, spreading across multiple continents. The immune response to infection can lead to protection and convalescence, or result in long term sequelae such as arthritis. Early innate immune events during acute infection have been characterized for some cell types, but more must be elucidated with respect to cellular responses of monocytes and other myeloid lineage cells. In addition to their roles in protection and inflammation resolution, monocytes and macrophages are sites for viral replication and reservoirs for the virus. They are also found in joints post-infection, possibly playing a role in long term CHIKV-induced pathology. We examined kinetic and phenotypic changes in myeloid lineage cells, including monocytes, in cynomolgus macaques early after infection with CHIKV. We found increased turnover of monocytes and mDCs early during infection, with an accompanying decreases in both cell types, as well as a simultaneous increase in pDC number. An increase in CD16 and CD14 was seen along with a decrease in monocyte HLA-DR expression within 3 days of infection, potentially indicating monocyte deactivation. A transient decrease in T cells, B cells and NK cells correlates with lymphocytopenia observed during human infections with CHIKV. CD4+ T cell turnover decreased in blood, indicating relocation of cells to effector sites. This data indicates CHIKV influences turnover rates and kinetics of myeloid lineage cells early during infection, and may prove useful in development of therapeutics and evaluation of infection-induced pathogenesis.


title: Evolutionary coexistence in a fluctuating environment by specialization on resource level

abstract:
Microbial communities in fluctuating environments can contain many species and diversity within species, both in natural environments such as the human gut, and in laboratory settings when communities are propagated for a long time. Whether this diversity is at the species level, within the species level, or a combination of both, the question remains: what processes lead to the origination and maintenance of this diversity?

When nutrient levels fluctuate over time, one possibly relevant process is that different types specialize on low and high nutrient levels. The relevance of this process is supported by observations of types co-existing through this mechanism when put together in the laboratory, and by simple models, that show that negative frequency dependence of two types, specialized on low and high resource level, can stabilize coexistence. However, when microbial populations are in an environment for a long time, they evolve. In this article we determine what happens when species can evolve; whether branching can occur to create diversity and whether evolution will destabilize coexistence.

We find that for the trade-off data between growth at low and high substrate concentrations, available for the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae on glucose, there is only a small portion of the trait-space that allows for coexistence. Moreover, this coexistence is destabilized by evolution, and the only evolutionary stable outcome is a single type. When we combine two species that are well-adapted on their own, we do find that they can form an evolutionary singular coalition, i.e. a coexistence that is evolutionarily stable. We conclude that although specialization on resource level can support diversity within a species, it is likely not a cause by itself. In contrast, for species consortia this specialization can lead to evolutionarily stable coexistence.


title: The Global Protein-RNA Interaction map of Epithelial Splicing Regulatory Protein 1 defines a post-transcriptional program that is essential for epithelial cell function

abstract: The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3 and 5 untranslated regions (UTRs) of genes involved in epithelial cell function suggesting that its post-transcriptional functions extend beyond splicing regulation.


title: Renal Angiotensinogen in Nonhuman Primates is Predominantly Liver-Derived

abstract:
AGT (Angiotensinogen) is the unique substrate of the renin-angiotensin system. Liver is the primary source of circulating AGT. The present study determined whether hepatocyte-derived AGT regulates renal AGT accumulation by injecting ASO (antisense oligonucleotides) targeting hepatocyte-derived AGT (GalNAc AGT ASO) into female cynomolgus monkeys. Hepatocyte-specific inhibition of AGT led to profound reductions of plasma AGT concentrations. AGT protein in S1 and S2 of renal proximal tubules was greatly diminished by GalNAc AGT ASO. Given the similarity between nonhuman primates and human, our findings support the notion that renal AGT is predominantly derived from liver, and liver regulates renal angiotensin II production in humans.

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title: Mechanosensitive calcium signaling promotes epithelial tight junction remodeling by activating RhoA

abstract: Epithelia maintain an effective barrier by actively remodeling cell-cell junctions in response to mechanical stimuli. Cells often respond to mechanical stress through activation of RhoA and dynamic remodeling of actomyosin. Previously, we found that local leaks in the epithelial tight junction barrier are rapidly repaired by localized, transient activation of RhoA, a process we termed "Rho flares", but how Rho flares are initiated remains unknown. Here, we discovered that intracellular calcium flashes occur in Xenopus laevis embryonic epithelial cells undergoing rapid remodeling of tight junctions via activation of Rho flares. Calcium flashes originate at the site of leaks and propagate into the cell. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSC) reduced the amplitude of calcium flashes and diminished the activation of Rho flares. Furthermore, MSC-dependent calcium influx was necessary to maintain global barrier function by regulating repair of local tight junction proteins through efficient contraction of junctions. Collectively, we propose that MSC-dependent calcium flashes are an important mechanism allowing epithelial cells to sense and respond to local leaks induced by mechanical stimuli.


title: Hypertrophic Chondrocytes Serve as a Reservoir for Unique Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development

abstract: Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.


title: Hypertrophic Chondrocytes Serve as a Reservoir for Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development


title: Hybridization and range expansion in tamarisk beetles (Diorhabda spp.) introduced to North America for classical biological control

abstract: With the global rise of human-mediated translocations and invasions, it is critical to understand the genomic consequences of hybridization and mechanisms of range expansion. Conventional wisdom is that high genetic drift and loss of genetic diversity due to repeated founder effects will constrain introduced species. However, reduced genetic variation can be countered by behavioral aspects and admixture with other distinct populations. As planned invasions, classical biological control (biocontrol) agents present important opportunities to understand the mechanisms of establishment and spread in a novel environment. The ability of biocontrol agents to spread and adapt, and their effects on local ecosystems, depends on genomic variation and the consequences of admixture in novel environments. Here we use a biocontrol system to examine the genome-wide outcomes of introduction, spread, and hybridization in four cryptic species of a biocontrol agent, the tamarisk beetle (Diorhabda carinata, D. carinulata, D. elongata, and D. sublineata), introduced from six localities across Eurasia to control the invasive shrub tamarisk (Tamarix spp.) in western North America. We assembled a de novo draft reference genome and applied RADseq to over 500 individuals from laboratory cultures, the native ranges, and across the introduced range. Despite evidence of a substantial genetic bottleneck among D. carinulata in N. America, populations continue to establish and spread, possibly due to aggregation behavior. We found that D. carinata, D. elongata, and D. sublineata hybridize in the field to varying extents, with D. carinata x D. sublineata hybrids being the most abundant. Genetic diversity was greater at sites with hybrids, highlighting potential for increased ability to adapt and expand. Our results demonstrate the complex patterns of genomic variation that can result from introduction of multiple ecotypes or species for biocontrol, and the importance of understanding them to predict and manage the effects of biocontrol agents in novel ecosystems.


title: Genetic signatures of divergent selection in European beech (Fagus sylvatica L.) are associated with the variation in temperature and precipitation across its distribution range

abstract:
High genetic variation and extensive gene flow may help forest trees with adapting to ongoing climate change, yet the genetic bases underlying their adaptive potential remain largely unknown. We investigated range-wide patterns of potentially adaptive genetic variation in 64 populations of European beech (Fagus sylvatica L.) using 270 SNPs from 139 candidate genes involved either in phenology or in stress responses. We inferred neutral genetic structure and processes (drift and gene flow) and performed differentiation outlier analyses and gene-environment association (GEA) analyses to detect signatures of divergent selection.

Beech range-wide genetic structure was consistent with the species previously identified postglacial expansion scenario and recolonization routes. Populations showed high diversity and low differentiation along the major expansion routes. A total of 52 loci were found to be putatively under selection and 15 of them turned up in multiple GEA analyses. Temperature and precipitation related variables were equally represented in significant genotype-climate associations. Signatures of divergent selection were detected in the same proportion for stress response and phenology-related genes. The range-wide adaptive genetic structure of beech appears highly integrated, suggesting a balanced contribution of phenology and stress-related genes to local adaptation, and of temperature and precipitation regimes to genetic clines. Our results imply a best-case scenario for the maintenance of high genetic diversity during range shifts in beech (and putatively other forest trees) with a combination of gene flow maintaining within-population neutral diversity and selection maintaining between-population adaptive differentiation.


title: The draft genome sequence of Eucalyptus polybractea based on hybrid assembly with short- and long-reads reads

abstract: Eucalyptus polybractea is a small, multi-stemmed tree, which is widely cultivated in Australia for the production of Eucalyptus oil. We report the hybrid assembly of the E. polybractea genome utilizing both short- and long-read technology. We generated 44 Gb of Illumina HiSeq short reads and 8 Gb of Nanopore long reads, representing approximately 83x and 15x genome coverage, respectively. The hybrid-assembled genome, after polishing, contained 24,864 scaffolds with an accumulated length of 523 Mb (N50 = 40.3 kb; BUSCO-calculated genome completeness of 94.3%). The genome contained 35,385 predicted protein-coding genes detected by combining homology-based and de novo approaches. We have provided the first assembled genome based on hybrid sequences from the highly diverse Eucalyptus subgenus Symphyomyrtus, and revealed the value of including long-reads from Nanopore technology for enhancing the contiguity of the assembled genome, as well as for improving its completeness. We anticipate that the E. polybractea genome will be an invaluable resource supporting a range of studies in genetics, population genomics and evolution of related species in Eucalyptus.


title: 3'HS1 CTCF binding site in human β-globin locus regulates fetal hemoglobin expression

abstract: Mutations in the adult {beta}-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and {beta}-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named Hereditary Persistence of Fetal Hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3 end of the {beta}-globin locus, including a CTCF binding site termed 3HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.


title: Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III

abstract: During cellular respiration, electron transfer between the integral membrane protein complexes of the electron transport chain is coupled to proton translocation across the inner mitochondrial membrane, which in turn powers synthesis of ATP and transmembrane transport processes. The homodimeric electron transport chain Complex III (CIII2) oxidizes ubiquinol (UQH2) to ubiquinone (UQ), transferring electrons to cytochrome c, and translocating protons through a mechanism known as the Q cycle. The Q cycle involves UQH2 oxidation and UQ reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We used cryoEM to determine the structure of CIII2 from Candida albicans, revealing density for endogenous UQ in the structure and allowing us to directly visualize the continuum of conformations of the Rieske head domain. Analysis of these conformations does not indicate cooperativity in the position of the Rieske head domains or binding of ligands in the two CIIIs of the CIII2 dimer. CryoEM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that inhibition by Inz-5 alters the equilibrium of the Rieske head domain positions.


title: Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC

abstract: KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We unambiguously show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-{pi} stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.