title: Multi-omics profiling of collagen-induced arthritis mouse model reveals early metabolic dysregulation via SIRT1 axis

abstract: Rheumatoid arthritis (RA) is characterized by joint infiltration of immune cells and synovial inflammation which leads to progressive disability. Current treatments improve the disease outcome, but the unmet medical need is still high. New discoveries over the last decade have revealed the major impact of cellular metabolism on immune cell functions. So far, a comprehensive understanding of metabolic changes during disease development, especially in the diseased microenvironment, is still limited. Therefore, we studied the longitudinal metabolic changes during the development of murine arthritis integrating metabolomics and bulk RNA-seq data. We identified an early change in macrophage pathways which was accompanied by oxidative stress, a drop in NAD+ level and induction of glucose transporters. We discovered inhibition of SIRT1, a NAD-dependent histone deacetylase and confirmed its dysregulation in human macrophages and synovial tissue of RA patients. Mining this database should enable the discovery of novel metabolic targets and therapy opportunities in RA.


title: Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa)

abstract: The jellyfish, or medusa, is a life cycle stage characteristic of the cnidarian subphylum Medusozoa. By contrast, the other cnidarian subphyla Anthozoa and Endocnidozoa lack a medusa stage. Of the medusozoan classes, Hydrozoa is the most diverse in terms of species number and life cycle variation. A notable pattern in hydrozoan evolution is that the medusa stage has been lost or reduced several times independently. Although this loss of the jellyfish stage is thought to be due to heterochrony, the precise developmental mechanisms underlying this complex pattern of medusa evolution are unknown. We found that the presence of the homeobox gene Tlx in cnidarian genomes is correlated with those medusozoans that have a medusa stage as part of their life cycle. Although Tlx is conserved in Bilateria and Cnidaria, it is missing in the genomes of anthozoans, endocnidozoans, and those hydrozoans that have lost the medusa stage. Selection analyses of Tlx across medusozoans revealed that hydrozoans undergo relatively relaxed selection compared to the other medusozoan classes, which may in part explain the pattern of multiple medusa losses. Differential expression analyses on three distantly related medusozoan representatives indicate an upregulation of Tlx during medusa development. In addition, Tlx expression is spatially restricted to regions of active development in medusae of the hydrozoan Podocoryna carnea. Our results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage.

category: evolutionary biology


title: Remodeled Connexin 43 hemichannels alter cardiac excitability and promote arrhythmias

abstract: Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of a dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the {beta}-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pre-treatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability and greater plasma membrane depolarization, which subsequently leads to triggered activity. These cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac stress-induced arrhythmogenicity.

category: physiology


title: Cohesin is required for meiotic spindle assembly independent of its role in cohesion in C. elegans

abstract: Accurate chromosome segregation requires a cohesin-mediated physical attachment between chromosomes that are to be segregated apart, and a bipolar spindle with microtubule plus ends emanating from exactly two poles toward the paired chromosomes. We asked whether the striking bipolar structure of C. elegans meiotic chromosomes is required for bipolarity of acentriolar female meiotic spindles by analyzing mutants that lack cohesion between chromosomes. Both a spo-11, rec-8, coh-3, coh-4 quadruple mutant and a spo-11, rec-8 double mutant entered M phase with single chromatids lacking any cohesion. However, the quadruple mutant formed an apolar spindle whereas the double mutant formed a bipolar spindle that segregated chromatids into two roughly equal masses. Residual non-cohesive COH-3/4-dependent cohesin on single chromatids of the double mutant was sufficient to recruit haspin- dependent Aurora B kinase, which regulated the localization of the spindle-assembly factors CLASP-2 and kinesin-13 to mediate bipolar spindle assembly in the apparent absence of chromosomal bipolarity. These results demonstrate that cohesin is essential for spindle assembly and chromosome segregation independent of its role in sister chromatid cohesion.

category: cell biology


title: Altered excitatory and inhibitory neuronal subpopulation parameters are distinctly associated with tau and amyloid in Alzheimer's disease

abstract:
BackgroundNeuronal and circuit level abnormalities of excitation and inhibition are shown to be associated with tau and amyloid-beta (A{beta}) in preclinical models of Alzheimers disease (AD). These relationships remain poorly understood in patients with AD.

MethodsUsing empirical spectra from magnetoencephalography (MEG) and computational modeling (neural mass model; NMM) we examined excitatory and inhibitory parameters of neuronal subpopulations and investigated their specific associations to regional tau and A{beta}, measured by positron emission tomography (PET), in patients with AD.

ResultsPatients with AD showed abnormal excitatory and inhibitory time-constants and neural gains compared to age-matched controls. Increased excitatory time-constants distinctly correlated with higher tau depositions while increased inhibitory time-constants distinctly correlated with higher A{beta} depositions.

ConclusionsOur results provide critical insights about potential mechanistic links between abnormal neural oscillations and cellular correlates of impaired excitatory and inhibitory synaptic functions associated with tau and A{beta} in patients with AD.

FundingThis study was supported by the National Institutes of Health grants: K08AG058749 (KGR), F32AG050434-01A1 (KGR), K23 AG038357 (KAV), P50 AG023501, P01 AG19724 (BLM), P50-AG023501 (BLM & GDR), R01 AG045611 (GDR); AG034570, AG062542 (WJ); NS100440 (SSN), DC176960 (SSN), DC017091 (SSN), AG062196 (SSN); a grant from John Douglas French Alzheimers Foundation (KAV); grants from Larry L. Hillblom Foundation: 2015-A-034-FEL and (KGR); 2019-A-013-SUP (KGR); a grant from the Alzheimers Association: (PCTRB-13-288476) (KAV), and made possible by Part the CloudTM, (ETAC-09-133596); a grant from Tau Consortium (GDR & WJJ), and a gift from the S. D. Bechtel Jr. Foundation.

category: neuroscience


title: Rickettsia typhi peptidoglycan mapping with data-dependent tandem mass spectrometry

abstract: Rickettsia species are diverse Gram-negative obligate intracellular bacteria often pervasive in numerous invertebrates, as well as fungal, nematode and microeukaryotic hosts. Certain species are etiological agents for well-known arthropod-borne illnesses; e.g., R. rickettsii (Rocky Mountain Spotted Fever), R. prowazekii (Epidemic Typhus), and R. typhi, (Endemic Typhus). Living freely in eukaryotic cytosol presumably exposes rickettsiae to host cell immune receptors, particularly those recognizing bacterial cell envelope glycoconjugates. However, the mechanics of host recognition of rickettsiae remain poorly defined. As rickettsiae synthesize a canonical Gram-negative cell envelope that includes peptidoglycan (PGN) and lipopolysaccharide (LPS), structural insight on these macromolecules is important for deciphering host responses to these pathogens. In this work, PGN from R. typhi was digested and the resultant subunits were analyzed by two different, albeit complementary, sample preparation methods. Both approaches were subsequently subjected to liquid chromatography/mass spectrometry analysis to infer PGN structure. R. typhi PGN was determined to be similar to most other Gram-negative bacteria, with mDAP-type muropeptide subunits. However, additional alanine residues were observed elongating the muropeptide stems, rather than the glycine residues usually observed in Gram-negative bacterial PGN. Despite this deviation, R. typhi contains a murein layer that is predicted to agonize host cellular PGN receptors and be susceptible to PGN-targeting antimicrobials. This same structure is likely synthesized by all Rickettsia species, as bioinformatics and comparative genomics analyses indicate the biosynthesis of PGN is highly conserved. Determining how host cells process this canonical glycoconjugate during infection is crucial for identifying factors behind rickettsial pathogenesis, including immunoavoidance or proinflammatory mechanisms possibly employed by rickettsiae with varying pathogenic potential.

category: microbiology


title: CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

abstract: CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities are in vitro irregularities.


title: Development of a Novel Bruton's Tyrosine Kinase Inhibitor that exerts Anti-Cancer Activities Potentiates Response of Chemotherapeutic Agents In Multiple Myeloma Stem Cell-Like Cells

abstract: Despite recent improvements in multiple myeloma (MM) treatment, MM remains an incurable disease and most patients experience a relapse. The major reason for myeloma recurrence is the persistent stem cell-like population. It has been demonstrated that overexpression of Brutons tyrosine kinase (BTK) in MM stem cell-like cells is correlated with drug resistance and poor prognosis. We have developed a novel small BTK inhibitor, KS151, which is unique compared to other BTK inhibitors. Unlike ibrutinib, and the other BTK inhibitors such as acalabrutinib, orelabrutinib, and zanubrutinib that covalently bind to the C481 residue in the BTK kinase domain, KS151 can inhibit BTK activities without binding to C481. This feature of KS151 is important because C481 becomes mutated in many patients and causes drug resistance. We demonstrated that KS151 inhibits in vitro BTK kinase activities and is more potent than ibrutinib. Furthermore, by performing a semi-quantitative, sandwich-based array for 71-tyrosine kinase phosphorylation, we found that KS151 specifically inhibits BTK. Our western blotting data showed that KS151 inhibits BTK signaling pathways and is effective against bortezomib-resistant cells as well as MM stem cell-like cells. Moreover, KS151 potentiates the apoptotic response of bortezomib, lenalidomide, and panobinostat in both MM and stem cell-like cells. Interestingly, KS151 inhibits stemness markers and is efficient in inhibiting Nanog and Gli1 stemness markers even when MM cells were co-cultured with bone marrow stromal cells (BMSCs). Overall, our results show that we have developed a novel BTK inhibitor effective against the stem cell-like population, and potentiates the response of chemotherapeutic agents.

category: molecular biology


title: A novel Vps34 complex constrains loop extrusion-mediated P-H2A spreading

abstract: The establishment of appropriate phosphorylated histone H2AX ({gamma}H2AX) foci is required for the precise initiation of DNA damage response (DDR) signaling, which contributes to the efficient repair of DNA double-strand breaks (DSB)1. Therefore, it is important to understand exactly how {gamma}H2AX foci are established in response to DSB. Recently, an attractive mechanism has been proposed to explain this process2. DSB-anchored cohesin-mediated loop extrusion promotes H2AX-containing chromatin to pass through DSB-recruited ATM, by which H2AX is phosphorylated to form {gamma}H2AX foci on each side of the DSB. The loop extrusion-promoted enlargement of {gamma}H2AX-modified chromatin is halted upon cohesin encountering boundary factors such as CTCF. This cohesin-dependent mechanism was shown to be conserved among eukaryotic cells, because deletion of the cohesin regulator Pds5 in budding yeast also results in extended spreading of phosphorylated H2A at Ser129 (P-H2A)2. However, yeast lacks CTCF or CTCF-like boundary factors that restrict cohesion-mediated loop extrusion. Here, we report a novel vacuolar sorting protein 34 (Vps34) complex, which might function as a boundary factor that constrains cohesin-dependent loop extrusion-mediated P-H2A spreading.


title: Cryo-EM reconstructions of LiCl core particles show strong similarities between disassembly and assembly of the ribosomal 50S subunit

abstract: Bacterial ribosome assembly is a complex process. Purified 50S ribosomes subjected to high-salt wash removing a subset of ribosomal proteins (r-proteins), were early shown competent for in vitro assembly into functional 50S subunits. We here used cryo-EM to determine the structure of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variation in extent of ordered 23S rRNA and occupancy of r-proteins could be identified, and resolved to between 2.8 [A] and 9 [A] resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed ordered density for 23S rRNA domains 0, I, II, III, VI and the 5-half of domain IV. In addition, smaller core particles were discovered, which may mimic earlier stages of 50S biogenesis. Our data support a multi-pathway disassembly process based on independent folding blocks, similar but reverse to the assembly process. The study provides examples of dependencies between complex tertiary RNA structure and RNA-protein interactions, exemplifies the need for proteins to stabilize native RNA structure also after folding, and supports the relevance of salt-washed core particles as a mimics of early ribosome assembly intermediates.


title: The mitochondrial genome segregation system of T. brucei: Single p197 molecules connect the basal body with the outer membrane

abstract:
The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body of the flagellum. Here we studied the architecture of the exclusion zone filament of the TAC that connects the basal body with the mitochondrial outer membrane. The only known component of the exclusion zone filaments is p197. Using genetical, biochemical and microscopical methods we show that p197 has three domains all of which are essential for mitochondrial DNA inheritance. The C-terminus of p197 interacts with the mature and pro-basal body whereas its N-terminus binds to the peripheral outer membrane protein TAC65. The large central region of p197 has a high -helical content and likely acts as a flexible spacer. Replacement of endogenous p197 with a functional version containing N- and C-terminal epitope tags together with expansion microscopy demonstrates that p197 alone can bridge the approximately 170 nm gap between the basal body and the periphery of the outer membrane. This demonstrates the power of expansion microscopy which allows to localize distinct regions within the same molecule and suggests that p197 is the TAC subunit most proximal to the basal body.

Significance statementSegregation of the replicated single unit mitochondrial genome of Trypanosoma brucei requires a large hardwired structure that connects the organellar DNA with the flagellar basal body. The cytosolic part of this structure consists of filaments made of single p197 molecules, a protein larger than 600 kDa. p197 has three domains all of which are essential for its function. The N-terminus of p197 is anchored to the peripheral outer membrane protein TAC65 whereas its C-terminus connects to the base of the basal body. The large central domain forms an -helix and consists of at least 26 repeats of 175 aa in length. It provides a flexible linker bridging the approximately 170 nm between the outer membrane and the basal body


title: DNA Double Strand Breaks cause chromosome loss through sister chromatid tethering in human embryos

abstract: Summary paragraphGenome editing by DNA double-strand breaks (DSB) is currently being investigated as a tool to treat or even prevent heritable diseases1. However, DNA repair mechanisms in the human embryo remain poorly understood and DSBs may result in chromosome loss 2,3. Here we provide evidence of whole and segmental chromosome loss in over one third of chromosomes 16, 17 and X targeted by CRISPR/Cas9-induced DNA DSB, including pericentromeric and mid-arm sites. Chromosomal changes were asymmetric relative to the Cas9 cut site: segmental losses occurred on both centric as well as acentric chromosome arms, while gains were exclusively found on acentric arms, suggesting that centromeres in broken chromosomes continued to mediate sister chromatid separation. Using this pattern of chromosomal errors, we were able to define new genomic coordinates of the active centromere on chromosome 16. Asymmetry was also found in the attrition of gDNA at the break site: attrition occurred centromeric of the DSB, while telomeric to the break, chromosomal ends were protected. Thus, spindle forces at centromeres and end tethering and protection at DSBs are antagonistic forces that interfere with accurate segregation of sister chromatids. Thereby, a single DSB is sufficient to result in the loss of a chromosome from the embryo. These results highlight the risks of aneuploidy in CRISPR/Cas9 genome editing, while also providing a mechanism for mitotically acquired aneuploidy caused by DNA breaks in human embryos.

category: developmental biology


title: Single-cell multiome of the human retina and deep learning nominate causal variants in complex eye diseases

abstract: Genome-wide association studies (GWAS) of eye disorders have identified hundreds of genetic variants associated with ocular disease. However, the vast majority of these variants are noncoding, making it challenging to interpret their function. Here, we present a joint single-cell atlas of gene expression and chromatin accessibility of the adult human retina with >50,000 cells, which we used to analyze noncoding single-nucleotide polymorphisms (SNPs) implicated by GWAS of age-related macular degeneration, glaucoma, diabetic retinopathy, myopia, and type 2 macular telangiectasia. We integrate this atlas with a HiChIP enhancer connectome, expression quantitative trait loci (eQTL) data, and base-resolution deep learning models to predict noncoding SNPs with causal roles in eye disease, assess SNP impact on transcription factor binding, and define their known and novel target genes. Our efforts nominate pathogenic SNP-target gene interactions for multiple vision disorders and provide a potentially powerful resource for interpreting noncoding variation in the eye.

category: genomics


title: Structural variation turnovers and defective genomes: key drivers for the in vitro evolution of the large double-stranded DNA koi herpesvirus (KHV)

abstract: Structural variations (SVs) constitute a significant source of genetic variability in virus genomes. Yet knowledge about SV variability and contribution to the evolutionary process in large double-stranded (ds)DNA viruses is limited. Cyprinid herpesvirus 3 (CyHV-3), also commonly known as koi herpesvirus (KHV), has the largest dsDNA genome within herpesviruses. This virus has become one of the biggest threats to common carp and koi farming, resulting in high morbidity and mortalities of fishes, serious environmental damage, and severe economic losses. A previous study analyzing CyHV-3 virulence evolution during serial passages onto carp cell cultures suggested that CyHV-3 evolves, at least in vitro, through an assembly of haplotypes that alternatively become dominant or under-represented. The present study investigates the SV diversity and dynamics in CyHV-3 genome during 99 serial passages in cell culture using, for the first time, ultra-deep whole-genome and amplicon-based sequencing. The results indicate that KHV polymorphism mostly involves SVs. These SVs display a wide distribution along the genome and exhibit high turnover dynamics with a clear bias towards inversion and deletion events. Analysis of the pathogenesis-associated ORF150 region in ten intermediate cell passages highlighted mainly deletion, inversion and insertion variations that deeply altered the structure of ORF150. Our findings indicate that SV turnovers and defective genomes represent key drivers in the viral population dynamics and in vitro evolution of KHV. Thus, the present study can contribute to the basic research needed to design safe live-attenuated vaccines, classically obtained by viral attenuation after serial passages in cell culture.


title: Structural basis for APE1 processing DNA damage in the nucleosome

abstract: Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determined that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site identified that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identified contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

category: biochemistry


title: Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases

abstract: CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations 1,2. The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes 3,4. Here we report engineered Cas9 nucleases that engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that suppresses otherwise dominant small indels produced by nonhomologous end-joining (NHEJ) repair. Instead, vCas9 makes repair pathways utilizing homologous sequences dominant, specifically indel-producing microhomology-mediated end-joining (MMEJ) and precise templated mutation-producing homology-directed repair (HDR). Consequently, vCas9 enables efficient precise templated mutations in dividing and non-dividing cells - establishing a new paradigm of targeted nucleases custom-designed for specific mutational applications.

category: bioengineering


title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat generated by CRISPR/Cas9

abstract: Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3- puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-{kappa}B signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-{kappa}B-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

category: immunology


title: Genetic diversity and characterization of circular replication(Rep)-encoding single-stranded (CRESS) DNA viruses

abstract:
The CRESS-DNA viruses are the ubiquitous virus detected in almost all eukaryotic life trees and play an essential role in the maintaining ecosystem of the globe. Still, their genetic diversity is not fully understood. Here we bring to light the genetic diversity of Replication (Rep) and Capsid (Cap) proteins of CRESS-DNA viruses. We divided the Rep protein of the CRESS-DNA virus into ten clusters using CLANS and phylogenetic analyzes. Also, most of the Rep protein in Rep cluster 1 (R1) and R2 (Circoviridae, Smacoviridae, Nanoviridae, and CRESSV1-5) contain the Viral_Rep superfamily and P-loop_NTPase superfamily domains, while the Rep protein of viruses in other clusters has no such characterized functional domain. The Circoviridae, Nanoviridae, and CRESSV1-3 viruses contain two domains, such as Viral_Rep and P-loop_NTPase; the CRESSV4 and CRESSV5 viruses have only the Viral_Rep domain, and most of the sequences in the pCRESS-related group have only P-loop_NTPase, and Smacoviridae do not have these two domains. Further, we divided the Cap protein of the CRESS-DNA virus into 20 clusters using CLANS and phylogenetic analyzes. The Rep and Cap proteins of Circoviridae and Smacoviridae are grouped into a specific cluster. Cap protein of CRESS-DNA viruses grouped with one cluster and Rep protein with another cluster. Further, our study reveals that selection pressure plays a significant role in the evolution of CRESS-DNA viruses Rep and Cap genes rather than mutational pressure. We hope this study will help determine the genetic diversity of CRESS-DNA viruses as more sequences are discovered in the future.

ImportanceThe genetic diversity of CRESS-DNA viruses is not fully understood. CRESS-DNA viruses are classified as CRESSV1 to CRESSV6 using only Rep protein. This study revealed that the Rep protein of the CRESS-DNA viruses is classified as CRESSV1 to CRESSV6 groups and the new Smacoviridae-related, CRESSV2-related pCRESS-related, Circoviridae-related, and 1 to 4 outgroups, according to the Viral_Rep and P-loop_NTPase domain organization, CLANS, and phylogenetic analysis. Furthermore, for the first time in this study, the Cap protein of CRESS-DNA viruses was classified into 20 distinct clusters by CLANS and phylogenetic analysis. Through this classification, the genetic diversity of CRESS-DNA viruses clarifies the possibility of recombinations in Cap and Rep proteins. Finally, it has been shown that selection pressure plays a significant role in the evolution and genetic diversity of Cap and Rep proteins. This study explains the genetic diversity of CRESS-DNA viruses and hopes that it will help classify future detected viruses.


title: The characterization of multiple novel paramyxovirus species highlights the diverse nature of the subfamily Orthoparamyxovirinae

abstract:
The subfamily Orthoparamyxovirinae is a group of single-stranded, negative-sense RNA viruses that contains many human, animal and zoonotic pathogens. While there are currently only 34 recognized member species in this subfamily, recent research has revealed that much of its diversity remains to be characterized. Using a newly developed nested PCR-based screening assay, we report here the discovery of fifteen orthoparamyxoviruses in rodents and shrews from Belgium and Guinea, thirteen of which are believed to represent new species. Using nanopore sequencing, complete genomes could be determined for almost all of these viruses, enabling a detailed evaluation of their genome characteristics. While most viruses are thought to belong to the rapidly expanding genus Jeilongvirus, we also identify novel members of the genera Narmovirus, Henipavirus and Morbillivirus. Together with other recently discovered orthoparamyxoviruses, both the henipaviruses and the morbillivirus discovered here appear to form distinct rodent-/shrew-borne clades within their respective genera, clustering separately from all currently classified member species. In the case of the henipaviruses, a comparison of the different members of this clade revealed the presence of a secondary conserved open reading frame, encoding for a transmembrane protein, within the F gene, the biological relevance of which remains to be established. While the characteristics of the viruses described here shed further light on the complex evolutionary origin of paramyxoviruses, they also illustrate that the diversity of this group of viruses in terms of genome organization appears to be much larger than previously assumed.

Data availabilityThe genome sequences generated in this study have been submitted to GenBank (accession numbers OK623353-OK623368).


title: IPMK modulates insulin-mediated suppression of hepatic glucose production

abstract:
Hepatic glucose production is crucial for the maintenance of normal glucose homeostasis. Although hepatic insulin resistance contributes to excessive glucose production, its mechanism is not well understood. Here, we show that inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate biosynthesis, plays a role in regulating hepatic insulin signaling and gluconeogenesis both in vitro and in vivo.IPMK-deficient hepatocytes exhibit decreased insulin-induced activation of Akt-FoxO1 signaling. The expression of mRNA levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose 6-phosphatase (G6pc), key enzymes mediating gluconeogenesis, are increased in IPMK-deficient hepatocytes compared to wild type (WT) hepatocytes. Importantly, re-expressing IPMK restores insulin sensitivity and alleviates glucose production in IPMK-deficient hepatocytes. Moreover, hepatocyte-specific IPMK deletion exacerbates hyperglycemia and insulin sensitivity in mice fed a high-fat diet (HFD), accompanied by an increase in hepatic glucose production during pyruvate tolerance test and reduction in Akt phosphorylation in IPMK deficient liver. Our results demonstrate that IPMK mediates insulin signaling and gluconeogenesis and may be potentially targeted for treatment of diabetes.

HighlightsIPMK expression is reduced in livers of HFD-fed mice.

Hepatocyte-specific deletion of IPMK in mice aggravated HFD-induced insulin resistance.

Loss of IPMK decreased insulin-induced activation of Akt-FoxO1 signaling, leading to the increase of glucose production in hepatocytes.


title: Structure and mechanism of a mitochondrial AAA+ disaggregase CLBP

abstract: The human AAA+ ATPase CLPB (aka, HSP78 and Skd3) is a protein disaggregase and functions to promote the solubilization of proteins in the mitochondrial intermembrane space. Unlike other AAA+ protein unfoldases or disaggregases, CLPB contains an ankyrin repeat containing domain (ANK) at its N-terminus. Mutations of CLPB are closely associated with a few human diseases, such as 3-methylglutaconic aciduria (3-MGA) and severe congenital neutropenia (SCN). The mechanism of CLPB functions as a disaggregase and the role of its unique ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. Our data show that CLPB assembles into homo-tetradecamers in apo-state and dodecamers in the presence of a substrate, mediated by ANK domains via a "head-to-head" organization. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grasp and translocate the substrate in a step-size of two amino acid residues. We also show that the ANK domain is functionally essential, and more important, responsible for direct binding to various mitocondiral proteins in the inner membrane or intermembrane space. These results suggest that CLPB functions as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.


title: Small non-coding RNA landscape of extracellular vesicles from a post-traumatic model of equine osteoarthritis

abstract: Extracellular vesicles comprise an as yet inadequately investigated intercellular communication pathway in the field of early osteoarthritis. We hypothesised that small non-coding RNA expression pattern in synovial fluid and plasma would change during progression of experimental osteoarthritis. In this study, we used small RNA sequencing to provide a comprehensive overview of the temporal expression profiles of small non-coding transcripts carried by EVs derived from plasma and synovial fluid for the first time in a post-traumatic model of equine osteoarthritis. Additionally, we characterised synovial fluid and plasma-derived extracellular vesicles with respect to quantity, size, and surface markers. The differential expression of seven microRNAs in plasma and synovial fluid-derived extracellular vesicles; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a and four snoRNAs; U3, snord15, snord46, snord58 represent potential biomarkers for early OA. Bioinformatics analysis of the differentially expressed microRNAs in synovial fluid highlighted that in early OA these related to the inhibition of cell cycle, cell cycle progression, DNA damage and cell proliferation but increased cell viability, and differentiation of stem cells. Plasma and synovial fluid-derived extracellular vesicle small non-coding signatures have been established for the first time in a temporal model of osteoarthritis. These could serve as novel biomarkers for the evaluation of osteoarthritis progression or act as potential therapeutic targets.


title: S. pombe Rtf2 is Important for Replication Fork Barrier Activity of RTS1 via Splicing of Rtf1

abstract: Arrested replication forks, when restarted by homologous recombination, result in error-prone DNA syntheses and non-allelic homologous recombination. Fission yeast RTS1 is a model fork barrier used to probe mechanisms of recombination-dependent restart. RTS1 barrier activity is entirely dependent on the DNA binding protein Rtf1 and partially dependent on a second protein, Rtf2. Human RTF2 was recently implicated in fork restart, leading us to examine fission yeast Rtf2s role in more detail. In agreement with previous studies, we observe reduced barrier activity upon rtf2 deletion. However, we identified Rtf2 to be physically associated with mRNA processing and splicing factors and rtf2 deletion to cause increased intron retention. One of the most affected introns resided in the rtf1 transcript. Using an intronless rtf1 we observed no reduction in RFB activity in the absence of Rtf2. Thus, Rtf2 is essential for correct rtf1 splicing to allow optimal RTS1 barrier activity.

category: genetics


title: AlphaFold Models of Small Proteins Rival the Accuracy of Solution NMR Structures

abstract: Recent advances in molecular modeling using deep learning have the potential to revolutionize the field of structural biology. In particular, AlphaFold has been observed to provide models of protein structures with accuracy rivaling medium-resolution X-ray crystal structures, and with excellent atomic coordinate matches to experimental protein NMR and cryo-electron microscopy structures. Here we assess the hypothesis that AlphaFold models of small, relatively rigid proteins have accuracies (based on comparison against experimental data) similar to experimental solution NMR structures. We selected six representative small proteins with structures determined by both NMR and X-ray crystallography, and modeled each of them using AlphaFold. Using several structure validation tools integrated under the Protein Structure Validation Software suite (PSVS), we then assessed how well these models fit to experimental NMR data, including NOESY peak lists (RPF-DP scores), comparisons between predicted rigidity and chemical shift data (ANSURR scores), and 15N-1H residual dipolar coupling data (RDC Q factors) analyzed by software tools integrated in the PSVS suite. Remarkably, the fits to NMR data for the protein structure models predicted with AlphaFold are generally similar, or better, than for the corresponding experimental NMR or X-ray crystal structures. Similar conclusions were reached in comparing AlphaFold2 predictions and NMR structures for three targets from the Critical Assessment of Protein Structure Prediction (CASP). These results contradict the widely held misperception that AlphaFold cannot accurately model solution NMR structures. They also document the value of PSVS for model vs. data assessment of protein NMR structures, and the potential for using AlphaFold models for guiding analysis of experimental NMR data and more generally in structural biology.


title: Alterations to the broad-spectrum formin inhibitor SMIFH2 improve potency

abstract: SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.


title: Comparison of sampling methods for small oxbow wetland fish communities

abstract: Throughout the world, wetlands have experienced degradation and declines in areal coverage. Fortunately, recognition of the value of wetlands has generated interest in preserving and restoring them. Post-restoration monitoring is necessary to analyze success or failure, thereby informing subsequent management decisions. Restoration of oxbow wetlands has become the focus of targeted restoration efforts to promote recovery of biodiversity and sensitive species, and to enhance ecosystem services. The fish communities of oxbows have been the subject of many monitoring studies. However, an optimal method for monitoring the fish communities of oxbows has not been described, thereby limiting our capacity to effectively manage these ecosystems. We compared four sampling methodologies (backpack electrofishing, fyke netting, minnow trapping, and seining) for fish community data collection with a primary objective of determining an optimal method for sampling fish communities in small oxbow wetlands. Seining and fyke netting were determined to be optimal methods for sampling oxbow fish communities. Backpack electrofishing and minnow trapping produced lower total catch and taxonomic richness values than seining and fyke netting. Although seining and fyke netting produced similar taxonomic diversity and abundance values, qualitative analysis revealed that seining caused greater habitat disturbance and potential stress to fish. Therefore, consideration must be given to how species present (especially sensitive species) within the wetland could be impacted by sampling disturbance when choosing between seining and fyke netting.

category: ecology


title: Towards a bionic IoT: environmental monitoring using smartphone interrogated plant sensors

abstract: The utilisation of plants directly as quantifiable natural sensors is proposed. A case study measuring surface wettability of Aucuba japonica, or Japanese Laurel, plants using a novel smartphone field interrogator is demonstrated. This plant has been naturalised globally from Asia. Top-down contact angle measurements map wettability on-site and characterise a range of properties impacting plant health, such as aging, solar and UV exposure, and pollution. Leaves at an early age or in the shadow of trees are found to be hydrophobic with contact angle{theta} ~ 99{degrees}, while more mature leaves under sunlight are hydrophilic with{theta} ~ 79{degrees}. Direct UVA irradiation at{lambda} = 365 nm is shown to accelerate aging, changing contact angle of one leaf from slightly hydrophobic at{theta} ~ 91{degrees} to be hydrophilic with{theta} ~ 87 {degrees} after 30 min. Leaves growing beside a road with heavy traffic are observed to be substantially hydrophilic, as low as{theta} ~ 47{degrees}, arising from increased wettability with particulate accumulation on the leaf surface. Away from the road, the contact angle increases as high as{theta} ~ 96{degrees}. The results demonstrate that contact angle measurements using a portable diagnostic IoT edge device can be taken into the field for environmental detection, pollution assessment and more. Using an internet connected smartphone combined with a plant sensor allows multiple measurements at multiple locations together in real-time, potentially enabling tracking of parameter change anywhere where plants are present or introduced. This hybrid integration of widely distributed living organic systems with the internet marks the beginning of a new bionic internet-of-things (b-IoT).

category: plant biology


title: Diet suppresses tumour initiation by maintaining quiescence of mutation-bearing neural stem cells

abstract: Glioblastoma is thought to originate from neural stem cells (NSCs) of the subventricular zone that acquire genetic alterations. In the adult brain, NSCs are largely quiescent, suggesting that deregulation of quiescence maintenance may be a pre-requisite for tumour initiation. Although inactivation of the tumour suppressor p53 is a frequent event in gliomagenesis, whether, or how, it affects quiescent NSCs (qNSCs) remains unclear. Here we show that p53 maintains quiescence by inducing fatty acid oxidation (FAO) and that acute p53 deletion in qNSCs results in their premature activation to a proliferative state. Mechanistically, this occurs through direct transcriptional induction of PPARGC1a, which in turn activates PPAR to upregulate FAO genes. Strikingly, dietary supplementation with fish oil containing omega-3 fatty acids, natural PPAR ligands, fully restores quiescence of p53-deficient NSCs and delays tumour initiation in a glioblastoma mouse model. Thus, diet can silence glioblastoma driver mutations, with important implications for cancer prevention.

category: cancer biology


title: On optimal temozolomide scheduling for slowly growing gliomas

abstract:
BackgroundTemozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma, and is commonly used in low-grade gliomas. In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes.

MethodsAgent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR wt or EGFR vIII in primary progenitors from p16/p19 ko mice and grown in vitro and in vivo in orthotopic allografts, and human glioblastoma U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study.

ResultsSlow growth  virtual murine gliomas benefited from increasing TMZ dose separation in silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly-growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically-driven changes in tumor phenotype, which could explain the reduction in glioma growth speed. In-silico trials provided support for methods of implementation in human patients.

ConclusionsIn-silico simulations, and in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in gliomas.

IMPORTANCE OF THE STUDYIn-vivo evidence is provided of improvements in survival, resistance, and toxicity from TMZ schemes with long rest periods between doses in slowly-growing GBM mouse models. The results match hypotheses generated in silico using a mathematical model incorporating the main biological features and fed with real patient data. An epigenetically-driven change in tumor phenotype was also revealed experimentally, which could explain the reduction in glioma growth speed under the  long cycle scheme. To determine the extent to which our results hold for human patients, large sets of simulations were performed on virtual patients. These in-silico trials suggest different ways to bring the benefits observed in experimental models into clinical practice.


title: Actin-dependent recruitment of Ago2 to the zonula adherens

abstract:
Adherens junctions are cadherin-based structures critical for cellular architecture. E-cadherin junctions in mature epithelial cell monolayers tether to an apical actomyosin ring to form the zonula adherens (ZA). We have previously shown that the adherens junction protein PLEKHA7 associates with and regulates the function of the core RNA interference (RNAi) component Ago2, specifically at the ZA. However, the mechanism mediating Ago2 recruitment to the ZA remained unexplored. Here, we reveal that this ZA-specific recruitment of Ago2 depends on both the structural and tensile integrity of the actomyosin cytoskeleton. We found that depletion of not only PLEKHA7, but also either of three PLEKHA7-interacting, LIM-domain family proteins, namely LMO7, LIMCH1, and PDLIM1, results in disruption of actomyosin organization and tension, as well as loss of Ago2 junctional localization. These results demonstrate that recruitment of Ago2 to the ZA is sensitive to actomyosin perturbations, introducing the idea of a mechanosensitive RNAi machinery, with potential implications in tissue remodeling and in disease.

SummaryE-cadherin junctions at the zonula adherens recruit the key RNAi component Ago2 through a still unexplored mechanism. The present work shows that junctional recruitment of Ago2 depends on the integrity and tension of the actin cytoskeleton, revealing a mechanosensitive aspect of RNAi.


title: Lamin A/C mediated invaginations in the nuclear surface allow the nucleus to pass unimpeded through a dense array of fiber-like obstacles

abstract: Migrating cells must deform their stiff cell nucleus to move through pores and fibers in tissue. Lamin A/C is known to hinder cell migration by limiting nuclear deformation and passage through confining channels, but its role in nuclear deformation and passage through fibrous environments is less clear. We studied cell and nuclear migration through discrete, closely spaced, slender obstacles which mimic the mechanical properties of collagen fibers. Nuclei bypassed slender obstacles while preserving their overall morphology by deforming around them with deep local invaginations of little resisting force. The obstacles did not impede the nuclear trajectory or cause a rupture of the nuclear envelope. Nuclei likewise deformed around single collagen fibers in cells migrating in 3D collagen gels. In contrast to its limiting role in nuclear passage through confining channels, lamin A/C facilitated nuclear deformation and passage through fibrous environments; nuclei in lamin-null (Lmna-/-) cells lost their overall morphology and became entangled on the obstacles. Analogous to surface tension-mediated deformation of a liquid drop, lamin A/C imparts a surface tension on the nucleus that allows nuclear invaginations with little mechanical resistance, preventing nuclear entanglement and allowing nuclear passage through fibrous environments.


title: The nucleus bypasses obstacles by deforming like a drop with surface tension mediated by lamin A/C


title: Constitutive 5-HT2C receptor knock-out facilitates fear extinction through altered activity of a dorsal raphe-bed nucleus of the stria terminalis pathway

abstract: Serotonin 2C receptors (5-HT2CRs) are widely distributed throughout the brain and are strongly implicated in the pathophysiology of anxiety disorders such as post-traumatic stress disorder (PTSD). Although in recent years, a considerable amount of evidence supports 5-HT2CRs facilitating effect on anxiety behavior, the involvement in learned fear responses and fear extinction is rather unexplored. Here we used a 5-HT2CR knock-out mouse line (2CKO) to gain new insights into the involvement of 5-HT2CRs in the neuronal fear circuitry. Using a cued fear conditioning paradigm, our results revealed that global loss of 5-HT2CRs exclusively accelerates fear extinction, without affecting fear acquisition and fear expression. To investigate the neuronal substrates underlying the extinction enhancing effect, we mapped the immediate-early gene product cFos, a marker for neuronal activity, in the dorsal raphe nucleus (DRN), amygdala and bed nucleus of the stria terminalis (BNST). Surprisingly, besides extinction-associated changes, our results revealed alterations in neuronal activity even under basal home cage conditions in specific subregions of the DRN and the BNST in 2CKO mice. Neuronal activity in the dorsal BNST was shifted in an extinction-supporting direction due to 5-HT2CR knock-out. Finally, the assessment of DRN-BNST connectivity using antero- and retrograde tracing techniques uncovered a discrete serotonergic DRC-BNSTad pathway showing increased activity in 2CKO mice. Thus, our results provide new insights for the fear extinction network by revealing a specific serotonergic DRC-BNSTad pathway underlying a 5-HT2CR-sensitive mechanism with high significance in the treatment of PTSD.


title: Type I interferons govern Zika virus resistance of human macrophages and microglia

abstract: The role of the human type I interferon (IFN-I) system in restricting Zika virus (ZIKV) is uncertain. Here, genetic and pharmacological ablation of IFN-I signalling enhanced ZIKV replication and cytopathicity in macrophages and microglia, key cells in ZIKV transmission and pathogenesis. Thus, despite the extensive IFN-I countermeasures employed by ZIKV, IFN-I dictates the outcome of infection in macrophages. Therapeutic manipulation of the IFN-I system may bring clinical benefit in ZIKV.


title: Injury-induced pulmonary tuft cells are heterogenous, arise independent of key Type 2 cytokines, and are dispensable for dysplastic repair

abstract: While the lung bears significant regenerative capacity, severe viral pneumonia can chronically impair lung function by triggering dysplastic remodeling. The connection between these enduring changes and chronic disease remains poorly understood. We recently described the emergence of tuft cells within Krt5+ dysplastic regions after influenza injury. Using bulk and single cell transcriptomics, we characterized and delineated multiple distinct tuft cell populations that arise following influenza clearance. Distinct from intestinal tuft cells which rely on Type 2 immune signals for their expansion, neither IL-25 nor IL-4R signaling are required to drive tuft cell development in dysplastic/injured lungs. Furthermore, tuft cells were also observed upon bleomycin injury, suggesting that their development may be a general response to severe lung injury. While intestinal tuft cells promote growth and differentiation of surrounding epithelial cells, in the lungs of tuft cell deficient mice, Krt5+ dysplasia still occurs, goblet cell production is unchanged, and there remains no appreciable contribution of Krt5+ cells into more regionally appropriate alveolar Type 2 cells. Together, these findings highlight unexpected differences in signals necessary for lung tuft cell amplification and establish a framework for future elucidation of tuft cell functions in pulmonary health and disease.


title: Extracellular prostaglandins E1 and E2 and inflammatory cytokines are regulated by the senescence program in potentially premalignant oral keratinocytes

abstract:
Potentially pre-malignant lesions (PPOLs) are composed of keratinocytes that are either mortal (MPPOL) or immortal (IPPOL) in vitro. We report here that MPPOL, but not generally IPPOL keratinocytes upregulate various extracellular tumor promoting cytokines (interleukins 6 and 8) and prostaglandins E1 (ePGE1) and E2 (ePGE2) relative to normal oral keratinocytes (NOKs). ePGE upregulation in MPPOL was independent of PGE receptor status, associated with some but not all markers of cellular senescence. Nevertheless, ePGE upregulation was dependent on the senescence program, cyclo-oxygenase 2 (COX2) and p38 mitogen-activated protein kinase and partially regulated by hydrocortisone. Following senescence in the absence of p16INK4A ePGEs accumulated in parallel with a subset of tumor promoting cytokine and metalloproteinase (MMP) transcripts all of which were ablated by ectopic telomerase. Surprisingly, ataxia telangiectasia mutated (ATM) function was not required for ePGE upregulation and actually increased in expression in IPPOL keratinocytes in line with its recently reported role in telomerase function. Only ePGE1 was dependent on p53 function suggesting that ePGEs 1 and 2 are regulated differently in oral keratinocytes. We show here that ePGE2 stimulates IPPOL keratinocyte proliferation in vitro. Therefore, we propose that MPPOL keratinocytes promote the progression of IPPOL to oral SCC in a pre-cancerous field by supplying PGEs, interleukins and MMPs in a paracrine manner. Our results suggest that the therapeutic targeting of COX-2 might be enhanced by strategies that target keratinocyte senescence.

Simple SummaryThe early treatment of oral cancer is a high priority, as improvements in this area could lead to greater cure rates and reduced disability due to extensive surgery. Oral cancer is very difficult to detect in over 70% of cases as it develops unseen until quite advanced, sometimes rapidly. It has become apparent that there are at least two types of epithelial cells (keratinocytes) found in oral tissue on the road to cancer (premalignant). One type secretes molecules called prostaglandins but the other does not and the former may stimulate the latter to progress to malignancy, either by stimulating their proliferation or encouraging the influx of blood vessels to feed them. Additionally, we have identified regulators of prostaglandin secretion in premalignant oral cells that could be targeted in future therapies such as inducers of cellular senescence, drugs which kill senescent cells (senolytics), steroid metabolism, cyclo-oxygenase 2 (COX2) and p38 mitogen-activated protein kinase.


title: Minimizing cell number fluctuations in self-renewing tissues with a stem cell niche

abstract: Self-renewing tissues require that a constant number of proliferating cells is maintained over time. This maintenance can be ensured at the single-cell level or the population level. Maintenance at the population level leads to fluctuations in the number of proliferating cells over time. Often, it is assumed that those fluctuations can be reduced by increasing the number of asymmetric divisions, i.e. divisions where only one of the daughter cells remains proliferative. Here, we study a model of cell proliferation that incorporates a stem cell niche of fixed size, and explicitly model the cells inside and outside the niche. We find that in this model fluctuations are minimized when the difference in growth rate between the niche and the rest of the tissue is maximized and all divisions are symmetric divisions, producing either two proliferating or two non-proliferating daughters. We show that this optimal state leaves visible signatures in clone size distributions and could thus be detected experimentally.


title: Recombinant annexin A6 promotes membrane repair in a stem cell derived-cardiomyocyte model of dystrophic cardiomyopathy

abstract: Heart failure is a major source of mortality in Duchenne muscular dystrophy (DMD). DMD arises from mutations that ablate expression of the protein dystrophin, which render the plasma membrane unusually fragile and prone to disruption. In DMD patients, repeated mechanical stress leads to membrane damage and cardiomyocyte loss. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study specific mutations in the context of a human cell, but these models can be improved by adding physiologic stressors. We modeled the primary defect underlying DMD by applying equibiaxial mechanical strain to DMD iPSC-CMs. DMD iPSC-CMs demonstrated an increased susceptibility to equibiaxial strain after 2 hours at 10% strain relative to healthy control cells, measured as increased lactate dehydrogenase (LDH) release. After 24 hours, both DMD and healthy control iPSC-CMs showed evidence of injury with release of LDH and cardiac troponin T. We exposed iPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced LDH and troponin release in DMD and control iPSC-CMs that had been subjected to 24 hour strain at 10%. We used aptamer protein profiling of media collected from DMD and control iPSC-CMs and compared these results to serum protein profiling from DMD patients. We found a strong correlation between the proteins in DMD patient serum and media from DMD iPSC-CMs subjected to mechanical stress. By developing an injury assay that specifically targets an underlying mechanism of injury seen in DMD-related cardiomyopathy, we demonstrated the potential therapeutic efficacy of the protein membrane resealer, recombinant annexin A6, for the treatment of DMD-related cardiomyopathy and general cardiac injury.


title: {Pi}-{Pi} Interactions Stabilize PeptoMicelle-Based Formulations of Pretomanid Derivatives Leading to Promising Therapy Against Tuberculosis in Zebrafish and Mouse Models

abstract: Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic, we expected that such micellar formulations would increase drug bioavailability, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by {pi}-{pi} interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these {pi}-{pi}-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The pretomanid derivative with the best in vitro potency against Mycobacterium marinum ("drug D") was also the most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model, almost completely eradicating the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug D micellar formulation significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.


title: Efficacy and safety of a patch containing adipose-derived stem cells for skin wound healing. Results form a comprehensive pre-clinical evaluation program

abstract: Mesenchymal stem cell-based therapies are emerging as innovative approaches to treat chronic wounds. A common administration route used in clinical trials consists of local injections leading to uncontrolled/sub-optimal delivery. This study reports a comprehensive pre-clinical evaluation program on the mechanism of action, efficacy and safety of an easy-to-use patch that concentrates Adipose-derived Stem Cells (ASCs) in a clinical-grade sponge of porcine crosslinked-gelatin. ASCs were prepared from the fat of ischemic patients. Transcriptome and proteome of ASC-patches and ASC monolayers were assessed by microarrays, bio-arrays and mass spectrometry. Tumorigenesis was investigated in immunosuppressed mice according to the European Pharmacopeia. Angiogenesis was assessed in vivo in the chick chorioallantoic membrane model. Efficacy of the ASC-patch was tested in a rat model of ischemic full-thickness skin defect. Cell stability was assessed by luminescence using ASC-patches generated from ASCs stably transduced with firefly luciferase. ASCs from ischemic patients upregulated the transcription of multiple genes involved in skin wound healing when cultured within the ASC-patch formulation. The patch was not only a concentrator, but also a reservoir of both ASC-derived regenerative factors and sponge-derived soluble fragments having healing capacity. The secretome of the ASC-patch promoted dermal fibroblast survival and epidermal epithelialization. No tumor formation was observed in immunodeficient Nude mice subcutaneously transplanted with the ASC-patch. Transplanted patches were early invaded by new vessels in vivo, and a marked angiogenesis was confirmed in two independent animal models. Finally, ASC-patches prepared from syngeneic rats promoted faster healing and re-vascularization of full-thickness skin defects in a rat animal model. Of note, ASCs were viable and locally stable for at least two weeks in vivo. We provide here compelling pre-clinical evidence that patches concentrating ASCs within crosslinked gelatin may represent a convenient and effective tool for the management of chronic wounds.


title: The chromatin associated Sin3B is a critical regulator of DNA damage repair through the engagement of the Non-Homologous End Joining.

abstract: Transcription and DNA damage repair act in a coordinated manner. The scaffolding protein Sin3B serves as a transcriptional corepressor of hundreds of cell-cycle-related genes. However, the role of Sin3B during the DNA damage response still remains unknown. Here, we show that Sin3B depletion delays the resolution of DNA double-strand breaks (DSBs) and sensitizes cells to diverse DNA-damaging agents, including cisplatin and doxorubicin. Furthermore, Sin3B is rapidly recruited to DNA damage sites and interacts with other DDR proteins. Mechanistically, Sin3B promotes non-homologous end joining DNA repair by directing the accumulation of MDC1 at DBSs. Altogether, our findings impute an unexpected function for the transcriptional co-repressor Sin3B as a gatekeeper of genomic integrity, and point to the inhibition of the Sin3B chromatin modifying complex as a novel therapeutic vulnerability in cancer cells.


title: Tailoring CRISPR-Cas Immunity for the Degradation of Antibiotic Resistance Genes

abstract:
The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes are key barriers preventing the spread of ARGs, they are often sources of ARGs at the same time, as ARGs may be required and accumulate in the biological treatment units. An upgrading of environmental biotechnology is imperative and urgent. ARGs confer antibiotic resistance based on the DNA sequences rather than the chemistry of DNA molecules. An ARG can be considered degraded if its sequence was disrupted. Therefore, we present here that CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, can be repurposed and tailored for the degradation of ARGs. By deploying an artificial IncP machinery, the designed system, namely VADER, can be successfully delivered via bacterial conjugation. Then, we propose a new sector for ARG degradation to be implemented as a complement to the biological units in the framework of environmental processes. In this endeavor, a prototype conjugation reactor at a 10-mL-scale was devised, and 100% of the target ARG were eliminated in the transconjugated microbes receiving VADER in the reactor. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future.

ImportanceAntibiotic resistance has been causing severe health problems and leading to millions of deaths in recent years. Environmental processes, especially the wastewater treatment sector, are important to barrier the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as the source of antibiotic resistance at the same time, as antibiotic resistance with its main cause antibiotic resistance genes (ARGs) may be required and accumulate in the biological treatment units, leading to the dissemination of ARGs. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to environmental biotechnology for tackling the antibiotic resistance dilemma thereof, and we propose a new sector in environmental processes specialized in ARG removal with a reactor inhabiting the CRISPR-Cas system per se. Our study provides a new angle to resolve public health issues via the implementation of synthetic biology at the process level.

category: synthetic biology


title: Large-Scale Circuit Configuration for Flexible Sensory-Motor Decisions

abstract: Humans and non-human primates can acquire, and rapidly switch between, arbitrary rules that govern the mapping from sensation to action. It has remained unknown if and how the brain configures large-scale sensory-motor circuits to establish such flexible information flow. Here, we developed an approach that elucidates the dynamic configuration of task-specific sensory-motor circuits in humans. Participants switched between arbitrary mapping rules for reporting visual orientation judgments during fMRI. Rule switches were either signaled explicitly or inferred by the participants from ambiguous cues, and we used behavioral modeling to reconstruct the time course of their belief about the active rule. In both contexts, patterns of correlations between ongoing fluctuations in feature-specific activity across visual and action-related brain regions tracked participants belief about the active rule. These rule-specific, intrinsic correlation patterns broke down on error trials and predicted individuals model-inferred internal noise. Our findings indicate that internal beliefs about task state are instantiated in specific large-scale patterns of selective, correlated neural variability.


title: An integrated cell atlas of the human lung in health and disease

abstract: Organ- and body-scale cell atlases have the potential to transform our understanding of human biology. To capture the variability present in the population, these atlases must include diverse demographics such as age and ethnicity from both healthy and diseased individuals. The growth in both size and number of single-cell datasets, combined with recent advances in computational techniques, for the first time makes it possible to generate such comprehensive large-scale atlases through integration of multiple datasets. Here, we present the integrated Human Lung Cell Atlas (HLCA) combining 46 datasets of the human respiratory system into a single atlas spanning over 2.2 million cells from 444 individuals across health and disease. The HLCA contains a consensus re-annotation of published and newly generated datasets, resolving under- or misannotation of 59% of cells in the original datasets. The HLCA enables recovery of rare cell types, provides consensus marker genes for each cell type, and uncovers gene modules associated with demographic covariates and anatomical location within the respiratory system. To facilitate the use of the HLCA as a reference for single-cell lung research and allow rapid analysis of new data, we provide an interactive web portal to project datasets onto the HLCA. Finally, we demonstrate the value of the HLCA reference for interpreting disease-associated changes. Thus, the HLCA outlines a roadmap for the development and use of organ-scale cell atlases within the Human Cell Atlas.


title: A chemical molecule promotes Trop2+ biliary duct organoids differentiation into insulin-secreting cells

abstract:
Cell replacement therapy is a hopeful strategy for diabetes treatment, whereas lack of pancreas donors leads this strategy to a dilemma. Here, we demonstrate that Trop2 is a useful marker to enrich the progenitors with a long-term organoid formation capability from extrahepatic bile ducts in both mouse and human. Based on BMP7 initiation, the Trop2-positive mouse extrahepatic bile duct organoids (mBDOs) could be induced to produce 8.49 {+/-} 0.45% of insulin-secreting cells. We further screened out a chemical small molecule TLY142, which significantly increased the proportion of insulin-secreting cells to 19.9 {+/-} 0.62% and 25.50 {+/-} 4.82% in induced mouse and human extrahepatic bile duct organoids (hBDOs) in vitro, and transplantation of these differentiated organoids into STZ-induced diabetic nude mice remarkably improved their blood glucose and enhanced glucose sensitivity. This study provides a novel strategy that using small molecules promotes biliary duct organoids differentiation into {beta} cells to cure diabetes.

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title: Epithelial monolayer development and tight junction assembly on nanopillar arrays

abstract: Nanostructured materials provide an outstanding opportunity to both stimulate and measure cellular processes. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO-1, claudins, and F-actin that increases paracellular macromolecular flux. In excitable tissues, nanomaterials have been used to apply and measure electrical signals, such action potentials. As a first step towards translating these technologies for use in analysis of epithelial function, we sought to culture monolayers composed of transporting epithelia over nanopillar arrays without perturbing cellular structure or function. Madin-Darby Canine kidney I (MDCK I) cells were cultured on collagen-coated silicon chips with [~]1 m diameter nanopillar arrays. Fluorescence and scanning electron microscopy were used to assess the impact of height on nanopillar-epithelial interactions. Monolayers formed over and were largely unaffected by short nanopillars. These nanopillars were located beneath basal epithelial surfaces and were not preferentially located within lateral intercellular spaces or beneath ZO-1-containing junctions. In contrast, tall nanopillars that exceeded cell height disrupted MDCK I monolayer growth. Cells interacted with, encircled, and extended cytoplasm over the top of tall nanopillars, and dense ZO-1 and F-actin accumulations occasionally surrounded apical membranes adjacent to nanopillars. Finally, when grown over arrays composed of nanopillars 1 - 2 m shorter than cells, MDCK I frequently grew between nanopillars. As a result, nanopillars were more commonly present within lateral intercellular spaces beneath junctions. Apical complex structure was intact, as assessed by fluorescence microscopy of ZO-1, occludin, claudin-2, F-actin, and E-cadherin. Apical microvilli were also unaffected. We therefore show that conditions can be defined to allow growth of mature, correctly assembled epithelial monolayers with nanopillars localized to lateral intercellular spaces. This sets the stage for application of nanotechnologies for perturbation and analysis of epithelial biology.


title: A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development.

abstract: Defects in embryonic patterning resulting in craniofacial abnormalities account for approximately 1/3 of birth defects. The regulatory programs that build and shape the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Large regions with coactivation of enhancer elements and co-regulation of multiple genes, referred to as superenhancers, are important in determining cell identity and perturbation could result in developmental defects. Building upon a previously published epigenomic atlas of human embryonic craniofacial tissue in which we identified over 75,000 putative embryonic craniofacial enhancer regions, we have identified 531 superenhancer regions unique to embryonic craniofacial tissue, including 37 which fall in completely noncoding regions. To demonstrate the utility of this data for the understanding of craniofacial development and the etiology of craniofacial abnormalities, we focused on a craniofacial-specific superenhancer in a [~]600kb noncoding region located between NPVF and NFE2L3. This region harbors over 100 individual putative craniofacial enhancer segments and 7 in vivo validated craniofacial enhancers from primary craniofacial tissue as well as strong enhancer activation signatures in a culture model of cranial neural crest cell (CNCC) development. However, none of the directly adjacent genes have been implicated in neural crest specification, craniofacial development, or abnormalities. To identify potential regulatory targets of this superenhancer region, we characterized three-dimensional chromatin structure of this region in CNCCs and mouse embryonic craniofacial tissues using multiple techniques (4C-Seq, HiC). We identified long range interactions that exclude most intervening genes and specifically target the anterior portion of the HOXA gene cluster located 1.2 to 1.8 Mb away. We demonstrate the specificity of the enhancer region for regulation of anterior HOXA genes through CRISPR/Cas9 editing of human embryonic stem cells. Mice homozygous for deletion of the superenhancer confirm the specificity of the enhancer region and demonstrate that the region is essential for viability. At fetal stages homozygotes develop at the same rate as heterozygous and wild type littermates but die at P0-P1 and have high penetrance of orofacial clefts that phenocopy previously described Hoxa2-/- mice. Moreover, we identified a de novo deletion partially overlapping the superenhancer in a human fetus with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and whose deletion is likely pathogenic in human patients.


title: Cohesin erases genomic-proximity biases to drive stochastic Protocadherin expression for proper neural wiring

abstract: Clustered Protocadherin (Pcdh) proteins act as cell-surface recognition barcodes for neural circuit formation. Neurites expressing the same barcode repel each other, but this mechanism is deployed in two different ways. For instance, convergence of olfactory sensory neuron (OSN) projections requires stochastic expression of distinct Pcdh isoforms in individual cells, while tiling of neural arbors of serotonergic neurons (5-HTs) requires expression of the same isoform, Pcdhc2. Despite their essential role, however, the molecular mechanisms of cell-type specific Pcdh barcoding remain a mystery. Here, we uncover a new role of cohesin: that of regulating distance-independent enhancer-promoter interactions to enable random Pcdh isoform choice via DNA loop extrusion in OSNs. Remarkably, this step mediates DNA demethylation of Pcdh promoters and their CTCF binding sites, thus directing CTCF to the chosen promoter. In contrast, the uniform pattern of Pcdh expression in 5-HTs is achieved through conventional cohesin-independent, distance-dependent enhancer/promoter interactions, that favor choice of the nearest isoform. Thus, cell-type specific cohesin deployment converts a distance-dependent and deterministic regulatory logic into a distance-independent and stochastic one. We propose that this mechanism provides an elegant strategy to achieve distinct patterns of Pcdh expression that generate wiring instructions to meet the connectivity requirements of different neural classes.


title: NudF-boosted strategy to improve the yield of DXS pathway

abstract:
BackgroundTerpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and pharmacological properties. The DXP pathways end enzyme, nuclear distribution protein (NudF), interacting with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), is critical for the synthesis of isoprenol/prenol/downstream compounds. The enzyme is yet to be thoroughly investigated to increase the overall yield of terpenoids in the Bacillus subtilis, which is widely used in industry and is generally regarded as safe (GRAS) bacterium. The study aims to analyze the evolutionary conservation across the active site, and map the key residues for mutagenesis studies. The study would allow us customize the metabolic load towards the synthesis of prenol or isoprenol or any of the downstream molecules.

ResultsThe 37-sequence dataset, extracted from 103 Bacillus subtilis entries, show a high phylogenetic divergence, and only six one-motif sequences ASB92783.1, ASB69297.1, ASB56714.1, AOR97677.1, AOL97023.1, and OAZ71765.1 show monophyly relationship, unlike a complete polyphyly relationship between the other 31 three-motif sequences. Further, only 47 of 179 residues of the representative sequence CUB50584.1 are observed to be significantly conserved. Docking analysis shows a preferential bias of ADP-ribose pyrophosphatase towards IPP, and a nearly 3-fold energetic difference is observed between IPP and DMAPP. Computational saturation mutagenesis of the seven hotspot residues identifies two key positions LYS78 and PHE116, encoded within loop1 and loop7, majorly interact with the ligands DMAPP and IPP, and their mutants K78I/K78L and PHE116D/PHE116E are found to stabilize the overall conformation. The loops are hereby shown to play a regulatory role in guiding the promiscuity of NudF towards a specific ligand.

ConclusionThe study map the phylogenetic relationship between the 37 representative B.subtiis NudF sequences, and through sequence conservation, structural contact map, topological flexibility, and saturation mutagenesis of the active site residues, the essential residues regulating the interaction of NudF with IPP/DMAPP are deciphered. The study robustly screens its mutational landscape and localizes the two crucial residues LYS78 and PHE116 for directing the mutagenesis studies. The preliminary docking and simulation results also suggest a preferential bias of ADP-ribose pyrophosphatase towards IPP over DMAPP. The findings would pave the way for the development of novel enzyme variants with highly improved catalytic ability for the large-scale bioproduction of specific terpenoids with significant neutraceutical or commercial value.


title: Stretching of long double-stranded DNA and RNA described by the same model.

abstract: We propose a bead-spring model that accurately reproduces a variety of experimental force-extension curves of long double-stranded DNA and RNA, including torsionally constrained and unconstrained DNA, and negatively supercoiled DNA. A key feature of the model is a specific non-convex energy function of the spring. We provide an algorithm for obtaining five required parameters of the model from experimental force-extension curves. In the plateau region of the force-extension curves, our molecular dynamics simulations show that the polymer separates into a mix of weakly and strongly stretched states without forming macroscopically distinct phases.

category: biophysics


title: Longitudinal dynamics of microvascular recovery after acquired cortical injury

abstract: Acquired brain injuries due to trauma damage cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury (TBI), but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14-21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (>200 m), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.


title: Polyploidy and plant-fungus symbiosis: evidence of cytotype-specific microbiomes in the halophyte Salicornia (Amaranthaceae)

abstract: Polyploidy is recognized as a mechanism of speciation in plants with cascading effects on biotic interactions. However, a limited number of studies have investigated the effects of polyploidy on the association of plants and microorganisms. Herein, we investigated whether two Salicornia cytotypes (S. europaea - 2x and S. procumbens - 4x) show different root-associated fungal communities. Additionally, we explored the existence of cytotype-specific root anatomical traits, which could influence fungal recruitment and establishment. Salicornia spp. were identified based on their ploidy level. The root-associated fungal microbiome of Salicornia was analyzed using high throughput amplicon sequencing (ITS1 of rDNA) in spring and summer. The following root anatomical traits were investigated: maximum root diameter, periderma thickness, parenchyma thickness, diameter of the vascular cylinder and maximum diameter of parenchyma cells. Our results showed that Shannon diversity and evenness indices were higher in samples of Salicornia procumbens (4x) compared to those of S. europaea (2x), and in summer the root-associated fungal community of S. procumbens (4x) was significantly different from that of S. europaea (2x). The orders Xylariales, Malasseziales and Pleosporales were the most frequent root colonizers in both cytotypes and most of the taxa associated with Salicornia were functionally classified as saprophytes or plant pathogens. Finally, we observed larger periderma and parenchyma layers in S. procumbens (4x) than S. europaea (2x) that may contribute to the observed differences in community composition between the two cytotypes. Our results suggest that differences in ploidy may modulate plant interaction with fungi by affecting species recruitment and microbiome structure. In addition, cytotype-specific root traits may also have the potential to affect differently community assembly in the two cytotypes.


title: Analysis of the persistence and particle size distributional shift of sperm-derived environmental DNA to monitor Jack Mackerel spawning activity

abstract:
Environmental DNA (eDNA) analysis holds great promises as an efficient and noninvasive method to monitor not only the presence and biomass of organisms but also their spawning activity. In eDNA analysis-based monitoring of spawning activity, the detection of sperm-derived eDNA is a key point; however, its characteristics and dynamics are completely unknown. The present study focuses on the persistence and particle size distribution (PSD) of eDNA derived from the sperm of Japanese jack mackerel. First, we investigated the time-dependent degradation and the PSD of sperm-derived eDNA by artificially adding sperm to fish eDNA-free seawater. Next, we kept fish in tanks and examined the changes in DNA concentration and PSD before and after spawning. The data obtained from the two experiments showed that the degradation of sperm-derived eDNA proceeded rapidly, with PSD shifting to a smaller size, regardless of the DNA region (Cyt b or ITS1). Additionally, it was shown that the nuclei and mitochondria released from sperm through degradation had a size distribution that was not simply dependent on each organelle size. The results of this study will contribute to elucidating the characteristics and dynamics of eDNA specifically during the spawning season and to further developing eDNA analysis as a powerful tool for the monitoring of spawning activity.

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title: GENESPACE: syntenic pan-genome annotations for eukaryotes

abstract: The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes.


title: Emergent periodicity in the collective synchronous flashing of fireflies

abstract: Left alone, Photinus carolinus fireflies flash without an intrinsic period, making it uncertain when they may flash next. Yet when gathering at the mating lek in large swarms, these fireflies transition into predictability, synchronizing with their neighbors with a rhythmic periodicity. Here we propose a mechanism for emergence of synchrony and periodicity, and formulate the principle in a mathematical framework. Remarkably, with no fitting parameters, analytic predictions from this simple principle and framework agree strikingly well with data. Next, we add further sophistication to the framework using a computational model featuring groups of random oscillators via integrate-and-fire interactions controlled by a tunable parameter. This agent-based model of P. carolinus fireflies interacting in swarms of increasing density also shows quantitatively similar phenomenology and reduces to the analytic framework in the appropriate limit of the tunable parameter. We discuss our findings and note that the resulting dynamics follow the style of a decentralized follow-the-leader synchronization, where any of the randomly flashing individuals may take the role of the leader of any subsequent synchronized flash burst.

category: animal behavior and cognition


title: Structure and regulation of the Myotonic dystrophy kinase-Related Cdc42-binding Kinase

abstract: Remodeling of the cytoskeleton underlies myriad processes essential for life. Protein kinases of the DMPK family are critical regulators of actomyosin contractility in cells. In the nematode worm, Caenorhabditis elegans, MRCK1 is required for the activation of myosin, leading to the development of cortical tension, apical constriction and early gastrulation. Here, we present the structure, conformation, and membrane-binding properties of C. elegans MRCK1. MRCK1 forms an obligate homodimer with N-terminal kinase domains, a parallel coiled-coil of 55 nm, and a C-terminal tripartite module of C1, PH and CNH domains. High-throughput liposome binding assays indicate binding to specific phosphoinositides, while the C-terminal Cdc42-binding (CRIB) motif binds specifically to activated Cdc42. The length of the coiled-coil domain of MRCK, as well as those of the related DMPK kinases ROCK, CRIK and DMPK, is remarkably conserved over millions of years of evolution, suggesting that they may function as molecular rulers to precisely position kinase activity at a fixed distance from the membrane.


title: A Strategy to Quantify Myofibroblast Activation on a Continuous Spectrum

abstract: Myofibroblasts are a highly secretory and contractile phenotype most commonly identified by the de novo expression and assembly of alpha-smooth muscle actin stress fibers. Traditionally, this activation process has been thought of as a binary process, with cells being labeled as "activated" or "quiescent (non-activated)". More recently, this view has been expanded to consider activation on a continuous spectrum. However, there is no established method to quantify a cells position on this spectrum, and as a result, the binary labeling system is still widely used. While transcriptomic analyses provide a continuous measure of myofibroblast markers, a faster and more facile screening method is needed. To this end, we utilized optical microscopy and machine learning methods to quantify myofibroblast activation on a spectrum. We first measured size and shape features of over 1,000 individual cardiac fibroblasts and found that these features provide enough information to predict activation state, on the binary scale, with 94% accuracy as compared to manual classification. We next performed dimensionality reduction techniques on these features to create a continuous scale of activation. Importantly, this new classification system captures a range of fibroblast activation states, but still possesses inherent bias due to choice of morphological features. Thus, we next used self-supervised machine learning to create a second continuous labeling system free from biases associated with the manually measured features. Lastly, we compared our findings for mechanically activated cardiac fibroblasts to a distribution of cell phenotypes generated from transcriptomic data using single-cell RNA sequencing. Altogether, these results demonstrate a continuous spectrum of activation from fibroblast to myofibroblast and provide a strategy to quantify a cells position on that spectrum.


title: Whole brain network analysis of neural synchrony and information flow during transition from wakefulness to light non-rapid eye movement sleep

abstract: Sleep is a highly stereotyped phenomenon, requiring robust spatial and temporal coordination of neural activity. How the brain coordinates neural activity with sleep onset can provide insight into the physiological functions subserved by sleep and pathologic phenomena associated with sleep onset. Here, we quantified whole-brain network changes during the transition from wake to non-rapid eye movement (NREM) sleep using magnetoencephalography imaging in healthy subjects. The sleep transition was identified to be encoded in spatially and temporally specific patterns of local and long-range neural synchrony. Patterns of information flow revealed that mesial frontal regions receive hierarchically organized inputs from broad cortical regions upon sleep onset. Finally, biophysical neural mass modeling demonstrated spatially heterogeneous properties of cortical excitation-to-inhibition from wake to NREM. Together, these findings provide evidence for the structured corticocortical modulation in sleep-wake behavior and demonstrate the orchestration of local and long-range, frequency-specific cortical interactions that are fundamental to sleep onset.