title: Eliminating fibroblast activation protein-α expressing cells by photoimmunotheranostics

abstract: Photoimmunotherapy (PIT) using an antibody conjugated to a near infrared dye IR700 is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-) is an important target in cancer because of its expression by cancer associated fibroblasts (CAFs) as well as by some cancer cells. CAFs that express FAP- have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-+ CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP- expression increased in fibroblasts co-cultured with cancer cells. In proof-of-principle studies, we engineered human FAP- overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP- overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP- antibodies and selected AF3715 based on its high binding-affinity with both human and mouse FAP-. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant antibody conjugate, FAP--IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP- expressing cell populations with PIT. FAP--IR700-PIT resulted in effective FAP--specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP--IR700 retention was three-fold higher than IgG-IR700 in FAP- overexpressing tumors, and two-fold higher compared to wild-type tumors. FAP--IR700-PIT resulted in significant growth inhibition of tumors derived from FAP- overexpressing human cancer cells. A reduction of endogenous FAP-+ murine CAFs was identified at 7 days after FAP--IR700-PIT. FAP--targeted NIR-PIT presents a promising strategy to eliminate FAP-+ CAFs.


title: From sequence to yield: deep learning for protein production systems

abstract: Recent progress in laboratory automation has enabled rapid and large-scale characterization of strains engineered to express heterologous proteins, paving the way for the use of machine learning to optimize production phenotypes. The ability to predict protein expression from DNA sequence promises to deliver large efficiency gains and reduced costs for strain design. Yet it remains unclear which models are best suited for this task or what is the size of training data required for accurate prediction. Here we trained and compared thousands of predictive models of protein expression from sequence, using a large screen of Escherichia coli strains with varying levels of GFP expression. We consider models of increasing complexity, from linear regressors to convolutional neural networks, trained on datasets of variable size and sequence diversity. Our results highlight trade-offs between prediction accuracy, data diversity, and DNA encoding methods. We provide robust evidence that deep neural networks can outperform classic models with the same amount of training data, achieving prediction accuracy over 80% when trained on approximately 2,000 sequences. Using techniques from Explainable AI, we show that deep learning models capture sequence elements that are known to correlate with expression, such as the stability of mRNA secondary structure. Our results lay the groundwork for the more widespread adoption of deep learning for strain engineering across the biotechnology sector.

category: synthetic biology


title: The proton motive force determines Escherichia coli's robustness to extracellular pH

abstract: Maintaining intracellular homeostases is a hallmark of life, and key physiological variables, such as cytoplasmic pH, osmotic pressure, and proton motive force (PMF), are typically interdependent. Developing a mathematical model focused on these links, we predict that Escherichia coli uses proton-ion antiporters to generate an out-of-equilibrium plasma membrane potential and so maintain the PMF at the constant levels observed. The strength of the PMF consequently determines the range of extracellular pH over which the cell is able to preserve its near neutral cytoplasmic pH. In support, we concurrently measure the PMF and cytoplasmic pH in single cells and demonstrate both that decreasing the PMFs strength impairs E. colis ability to maintain its pH and that artificially collapsing the PMF destroys the out-of-equilibrium plasma membrane potential. We further predict the observed ranges of extracellular pH for which three of E. colis antiporters are expressed, through defining their cost by the rate at which they divert imported protons from generating ATP. Taken together, our results suggest a new perspective on bacterial electrophysiology, where cells regulate the plasma membrane potential by changing the activities of antiporters to maintain both the PMF and cytoplasmic pH.

category: microbiology


title: Three orphan histidine kinases inhibit Clostridioides difficile sporulation

abstract:
The ability of the anaerobic gastrointestinal pathogen, Clostridioides difficile, to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a conserved phosphotransfer protein, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional conserved orphan phosphotransfer proteins, CD2492, CD1579, and CD1949 which are hypothesized to regulate Spo0A phosphorylation. Disruption of the conserved phosphotransfer protein, CD2492, also increased sporulation frequency, similarly to the CD1492 mutant, and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of the conserved CD1579 phosphotransfer protein also variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492 and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant negative hypersporulation phenotype. Altogether, at least three phosphotransfer proteins, CD1492, CD2492 and CD1579 (herein, PtpA, PtpB and PtpC) repress C. difficile sporulation initiation by regulating activity of Spo0A.

IMPORTANCEThe formation of inactive spores is critical for the long-term survival of the gastrointestinal pathogen Clostridioides difficile. The onset of sporulation is controlled by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple kinases and phosphatases control Spo0A phosphorylation; however, this regulatory pathway is not defined in C. difficile. We show that two conserved phosphotransfer proteins, CD1492 (PtpA) and CD2492 (PtpB), function in the same regulatory pathway to repress sporulation by preventing Spo0A phosphorylation. We show that another conserved phosphotransfer protein, CD1579 (PtpC), also represses sporulation, and we eliminate the possibility that a fourth orphan histidine kinase protein, CD1949, impacts C. difficile sporulation. These results support the idea that C. difficile inhibits sporulation initiation through multiple phosphatases.


title: Progressive sub-MIC Exposure of Klebsiella pneumoniae 43816 to Cephalothin Induces the Evolution of beta-lactam Resistance without Acquisition of beta-lactamase Genes

abstract:
Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in the greater environment. This study aimed to evaluate the adaptive genetic changes in Klebsiella pneumoniae 43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 g/mL to 7.5 g/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 g/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in the rpoB subunit of RNA Polymerase, the tetR/acrR regulator, and the wcaJ sugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increases. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS, may contribute to the resistant phenotype. These data demonstrate that very low, sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.

ImportanceBacteria are constantly exposed to low levels of antibiotics in the environment. The impact of this low-level exposure on bacterial evolution is not well understood. In this work, we developed a model to expose Klebsiella pneumoniae to progressive, low doses of the antibiotic cephalothin. After a fourteen-day exposure regimen, our culture exhibited full clinical resistance to this antibiotic without the traditional acquisition of inactivating genes. This culture also exhibited resistance to tetracycline, had a highly mucoid appearance, and exhibited altered, elongated cellular morphology. Whole genome sequencing identified a collection of mutations to the bacterial genome that could be mapped to the emergence of the resistant phenotype. This study demonstrates that antibiotic resistance can be achieved in response to low level antibiotic exposure and without the traditional acquisition of resistance genes. Further, this study identifies new genes that may play a role in the evolution of antibiotic resistant bacteria.


title: Human macrophage polarization determines bacterial persistence of Staphylococcus aureus in a liver-on-chip-based infection model

abstract: Infections with Staphylococcus aureus (S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis associated with multiple organ dysfunction. Although considered an extracellular pathogen, S. aureus can invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate phagocytes is considered a critical step to evade host antimicrobial reactions. For the first time we leveraged a human liver-on-chip model and tailored image analysis algorithms to demonstrate that S. aureus (USA300) specifically targets macrophages in the liver models as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs). In vitro M2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about the role of liver macrophages in the course of systemic infection. Further, the results might be relevant for understanding infection mechanisms in patients with chronic liver disease such as fibrosis that display increased frequencies of M2 polarized liver macrophages and have a higher risk for developing chronic infections and relapsing bacteremia.


title: A scalable analytical approach from bacterial genomes to epidemiology

abstract: Recent years have seen a remarkable increase in the practicality of sequencing whole genomes from large numbers of bacterial isolates. The availability of this data has huge potential to deliver new insights into the evolution and epidemiology of bacterial pathogens, but the scalability of the analytical methodology has been lagging behind that of the sequencing technology. Here we present a step-by-step approach for such large-scale genomic epidemiology analyses, from bacterial genomes to epidemiological interpretations. A central component of this approach is the dated phylogeny, which is a phylogenetic tree with branch lengths measured in units of time. The construction of dated phylogenies from bacterial genomic data needs to account for the disruptive effect of recombination on phylogenetic relationships, and we describe how this can be achieved. Dated phylogenies can then be used to perform fine-scale or large-scale epidemiological analyses, depending on the proportion of cases for which genomes are available. A key feature of this approach is computational scalability, and in particular the ability to process hundreds or thousands of genomes within a matter of hours. This is a clear advantage of the step-by-step approach described here. We discuss other advantages and disadvantages of the approach, as well as potential improvements and avenues for future research.


title: Replication protein Rep provides selective advantage to viruses in the presence of CRISPR-Cas immunity

abstract:
Prokaryotic viruses express anti-CRISPR (Acr) proteins to inhibit the host adaptive immune system, CRISPR-Cas. While the virus infection biology was shown to be strongly dependent on the relative strengths of the host CRISPR-Cas and viral Acrs, little is known about the role of the core processes of viral life cycle (replication, packaging etc) in defence/anti-defence arms race. Here, we demonstrate the selective advantage provided by a replication initiator, Rep, in the context of CRISPR-Acr interactions. First, we developed a two-host based CRISPR-Cas genome editing tool for the deletion of highly conserved and thus potentially important viral genes. Using this strategy, we deleted a highly conserved Rep-coding gene, gp16, from the genome of Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The knockout mutant ({Delta}gp16) produced around 4 fold less virus in a CRISPR-null host, suggesting that Rep is the major contributor to replication initiation in Rudiviridae. Indeed, DNA sequencing revealed Rep-dependent replication initiation from the viral genome termini, in addition to Rep-independent replication initiation from non-terminal sites. Intriguingly, the lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the {Delta}gp16 mutant much more quickly in CRISPR-containing host than in CRISPR-null host, demonstrating a selective advantage provided by Rep in the presence of host CRISPR-Cas immunity. Despite the non-essentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.

ImportanceCRISPR-Cas and anti-CRISPR proteins are accessary to prokaryotes and their viruses respectively. To date, research has been focused on their diversity, molecular mechanisms and application in genome editing. How CRISPR-Acr arms race influence the evolution of viral core genes involved in the basic virus life cycle remained a gap of knowledge so far. This study provides the first evidence that CRISPR-Acr arms race poses a selection pressure on the efficiency of viral genome replication, forcing viruses to evolve highly productive replication machineries..


title: A marine fungus efficiently degrades polyethylene

abstract: Plastics pollution has been a global concern. Huge quantities of polyethylene (PE), the most abundant and refractory plastic in the world, have been accumulating in the environment causing serious ecological problems. However, the paucity of microorganisms and enzymes that efficiently degrading PE seriously impedes the development of bio-products to eliminate this environmental pollution. Here, by screening hundreds of plastic waste-associated samples, we isolated a fungus (named Alternaria sp. FB1) that possessing a prominent capability of colonizing, degrading and utilizing PE. Strikingly, the molecular weight of PE film decreased 95% after the fungal treatment. Using GC-MS, we further clarified that a four-carbon product (named Diglycolamine) accounted for 93.28% of all degradation products after the treatment by strain FB1. We defined potential enzymes that involved in the degradation of PE through a transcriptomic method. The degradation capabilities of two representative enzymes including a laccase and a peroxidase were verified. Lastly, a complete biodegradation process of PE is proposed. Our study provides a compelling candidate for further investigation of degradation mechanisms and development of biodegradation products of PE.


title: Metformin Suppresses SARS-CoV-2 in Cell Culture

abstract: People with diabetes are reported to have a higher risk of experiencing severe COVID-19 complications. Metformin, a first-line medication for type 2 diabetes, has antiviral properties. Some studies have indicated its prognostic potential in COVID-19. Here, we report that metformin significantly inhibits SARS-CoV-2 growth in cell culture models. SARS-CoV-2 infection of gut epithelial cell line, Caco2, resulted in higher phosphorylation of AMPK. Metformin reduced viral titers in the infected cells by nearly 99%, and by about 90% when cells were treated prior to infection. Metformin pre-treatment resulted in further phosphorylation of AMPK and caused a ten-fold reduction of viral titers indicating its potential in preventing naive infections. Confirming the positive impact of AMPK activation, another AMPK activator AICAR substantially inhibited of viral titers and, AMPK inhibitor Compound C, augmented it considerably. Metformin treatment post-SARS-CoV-2 infection resulted in nearly hundred-fold reduction of viral titers, indicating that the antiviral potency of the drug is far higher in infected cells, while still being able to reduce fresh infection. Metformin displayed SARS-CoV-2 TCID50 and TCID90 at 3.5 and 8.9 mM, respectively. In conclusion, our study demonstrates that metformin is very effective in limiting the replication of SARS-CoV-2 in cell culture and thus possibly could offer double benefits to diabetic COVID-19 patients by lowering both blood glucose levels and viral load.


title: RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity

abstract: The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1- deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

category: molecular biology


title: TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

abstract:
Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease (CAD). ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating CAD; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EMEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

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title: A Comprehensive Roadmap Towards Generation of Influenza B Reporter Assay Using a Single DNA Polymerase Based Cloning of Reporter RNA Construct

abstract: Mini-genome reporter assay is a key tool for conducting RNA virus research. But, procedural complications and lack of adequate literature pose major challenge towards developing these assay systems. Here we present a novel yet generic and simple cloning strategy for construction of influenza B virus reporter RNA template and describe extensive standardization of the reporter RNP/ polymerase activity assay for monitoring viral RNA synthesis in infection free setting. Using this assay system, we, for the first time showed the effect of viral protein NS1 and host protein PKC-Delta upon influenza B virus RNA synthesis. Additionally, the assay system showed promising results in evaluating efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for standardization of influenza virus mini-genome assay and an excellent tool for screening of host factors and antivirals in a fast, user friendly and high throughput manner.


title: Enhancer and super-enhancer landscape in polycystic kidney disease

abstract: Widespread aberrant gene expression is pathological hallmark of polycystic kidney disease (PKD). Numerous pathogenic signaling cascades, including c-Myc, Fos, and Jun are transactivated. However, the underlying epigenetic regulators are poorly defined. Here we show that H3K27ac, a histone modification that marks active enhancers, is elevated in mouse and human ADPKD samples. Using comparative H3K27ac ChIP-Seq analysis, we mapped >16000 active intronic and intergenic enhancer elements in Pkd1-mutant mouse kidneys. We find that the cystic kidney epigenetic landscape resembles that of a developing kidney, and >90% of upregulated genes in Pkd1-mutant kidneys are co-housed with activated enhancers in the same topologically associated domains. Furthermore, we identify an evolutionarily-conserved enhancer cluster downstream of the c-Myc gene and super-enhancers flanking both Jun and Fos loci in mouse and human ADPKD models. Deleting these regulatory elements reduces c-Myc, Jun, or Fos abundance and suppresses proliferation and 3D cyst growth of Pkd1-mutant cells. Finally, inhibiting glycolysis and glutaminolysis or activating Ppara in Pkd1-mutant cells lowers global H3K27ac levels and on c-Myc enhancers. Thus, our work suggests that epigenetic rewiring mediates the transcriptomic dysregulation in PKD, and the regulatory elements can be targeted to slow cyst growth.


title: The sperm protein Spaca6 is essential for fertilization in zebrafish

abstract: Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine SPACA6, which was reported not to be required for sperm-egg membrane binding but necessary for fusion. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.

category: developmental biology


title: Improved resolution of recalcitrant nodes in the animal phylogeny through the analysis of genome gene content and morphology

abstract: An accurate phylogeny of animals is needed to clarify their evolution, ecology, and impact on shaping the biosphere. Although datasets of several hundred thousand amino acids are nowadays routinely used to test phylogenetic hypotheses, key deep nodes in the metazoan tree remain unresolved: the root of animals, the root of Bilateria, and the root of Deuterostomia. To assess patterns of congruence with established amino-acid derived phylogenetic hypotheses for these problematic nodes, we independently and extensively analysed newly assembled genome gene content and morphological datasets. Our datasets strongly support sponges as the sister group of all the other animals, the worm-like bilaterian lineage Xenacoelomorpha as the sister group of the other Bilateria, and largely support monophyletic Deuterostomia. We conclude that the last common animal ancestor may have been a simple, microphagous organism without a nervous system and muscles, while the last common ancestor of Bilateria might have been a small, acoelomate-like worm without a through gut.

category: evolutionary biology


title: Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo

abstract: Zinc (Zn2+) is considered as an important mediator for thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these well-known Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

category: biochemistry


title: Polygalacturonase production enhancement by Piriformospora indica from sugar beet pulp under submerged fermentation using surface methodology

abstract: This study proposed a novel and cost-effective approach to enhance and optimize the polygalacturonase from P. indica. In current investigation, the impact of ammonium sulfate, sugar beet pulp (SBP) and glucose as variables on induction of polygalacturonase from P. indica was optimized using the central composite design (CCD) of response surface methodology (RSM) under SmF. Additionally, partial polygalacturonase purification and in situ analysis were performed. The optimal reaction conditions, which resulted in the highest enzyme activity were observed as the following conditions: ammonium sulfate (4 g/L), SBP (20 g/L), glucose (60 g/L). Under the optimized condition, the maximum enzyme activity reached to 19.4 U/ml (127 U/mg) which increased by 5.84 times compared to non-optimized conditions. The partial purified polygalacturonase molecular weight was estimated 60 KDa. In line with the bioinformatic analysis, exo-polygalacturonase sequence of P. indica showed similarity with Rhizoctonia solanis and Thanateporus cucumeris. These results indicated that SBP act as a cheap and suitable inducer of polygalacturonase production by P. indica in a submerged cultivation. The outcome of this study will be useful for industries to decrease environmental pollution with cost-effective approaches.


title: Chemical Reversible Crosslinking Enables Measurement of RNA 3D Distances and Alternative Conformations in Cells

abstract: Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a new method, Spatial 2-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a new strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.


title: evSeq: Cost-Effective Amplicon Sequencing of Every Variant in a Protein Library

abstract: Widespread availability of protein sequence-fitness data would revolutionize both our biochemical understanding of proteins and our ability to engineer them. Unfortunately, even though thousands of protein variants are generated and evaluated for fitness during a typical protein engineering campaign, most are never sequenced, leaving a wealth of potential sequence-fitness information untapped. This largely stems from the fact that sequencing is unnecessary for many protein engineering strategies; the added cost and effort of sequencing is thus unjustified. Here, we present every variant sequencing (evSeq), an efficient protocol for sequencing a variable region within every variant gene produced during a protein engineering campaign at a cost of cents per variant. Execution of evSeq is simple, requires no sequencing experience to perform, relies only on resources and services typically available to biology labs, and slots neatly into existing protein engineering workflows. Analysis of evSeq data is likewise made simple by its accompanying software (found at github.com/fhalab/evSeq, documentation at fhalab.github.io/evSeq), which can be run on a personal laptop and was designed to be accessible to users with no computational experience. Low-cost and easy to use, evSeq makes collection of extensive protein variant sequence-fitness data practical.

category: bioengineering


title: Inactivation effect and damage of multi-irradiance by UVCLED on Acinetobacter baumannii

abstract:
It is acknowledged that the inactivation of ultraviolet has been widely used in various fields. Much literature has been reported that ultraviolet C caused DNA damage to achieve inactivation of microorganisms. There is a lack of unified dose calibration and related parameters in this field. In this study, we used a device consisted of the LED of 272 nm to conduct sterilization experiments against A. baumanii. We confirmed the effectiveness of ultraviolet C sterilization for both sensitive and drug resistance strains and explored the relationship between bactericidal rate and ultraviolet doses under various irradiance. Dose requirements of various irradiance were clarified. High irradiance improved sterilization efficiency greatly. The overall damage to the total genome was observed though gel electrophoresis. Ultrastructure of damaged bacteria were investigated by transmission electron microscope in detail. The study revealed that damage to DNA and to the cytoplasm matrix and ribosomes. The study has yielded the possible effects of ultraviolet light on cells by amplifying the energy. The radiation significantly promoted the production of cell wall and cellular membrane.

Significance StatementThe statistical results of bactericidal efficiency are influenced by the quantity of bacteria on the medium. Irradiance on the target surface affects the sterilization efficiency directly. And doses required in low irradiance are much more than high. A high irradiance reduced dosage efficiently to achieve the sterilization which improves the sterilization efficiency. There is a difference between low and high UVC dosage damage to the structure of bacteria. Less energy can make DNA coagulation solidified or be dispersed to the edge. Meanwhile the cytoplasm matrix is ruined. When the energy was enough, there is a boost of cell wall and cellular membrane production. The invisible light causes comprehensive damage to bacteria.


title: TK216 targets microtubules in Ewing sarcoma cells

abstract: Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11;22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for EWS patients. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding -tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

category: cancer biology


title: Fusobacterium nucleatum infection induces pancreatic cancer cell proliferation and migration through regulation of host cytokine signaling

abstract: Pancreatic ductal adenocarcinoma (PDAC) harbors a complex tumor microbiome that has been implicated in cancer progression and resistance to chemotherapy. Recent clinical investigations uncovered a correlation between high loads of intratumor Fusobacterium nucleatum and decreased patient survival. Here we show that healthy and cancerous pancreatic cell lines harboring intracellular F. nucleatum secrete increased levels of cancer-associated cytokines including GM-CSF, CXCL1, IL-8, and MIP-3. We report that GM-CSF (granulocyte-macrophage colony stimulating factor) secretion directly increases the proliferation and migration of pancreatic cancer cells via an autocrine mechanism, notably in the absence of immune cell participation. Furthermore, we show that non-cancerous pancreatic epithelial cells do not exhibit increased proliferation or migration in response to these cytokines, but nevertheless, their secreted cytokines stimulate these responses in cancerous cell lines through paracrine signaling. Our results provide evidence that intratumor F. nucleatum in the pancreas elicits an infection-specific cytokine secretion profile from both normal and cancerous cells that adversely contributes to cancer progression through autocrine and paracrine mechanisms. Therefore, these results support the importance of investigating the contributions of both microbiome and host driven processes in pancreatic cancer to guide future therapeutic interventions.


title: Fusobacterium nucleatum induces pancreatic cancer cell proliferation and migration by regulating host autocrine and paracrine signaling


title: Nonenzymatic loop-closing ligation generates RNA hairpins and is a template-free way to assemble functional RNAs

abstract: RNA hairpin loops are the predominant element of secondary structure in functional RNAs. The emergence of primordial functional RNAs, such as ribozymes that fold into complex structures that contain multiple hairpin loops, is generally thought to have been supported by template-directed ligation. However, template inhibition and RNA misfolding problems impede the emergence of function. Here we demonstrate that RNA hairpin loops can be synthesized directly from short RNA duplexes with single-stranded overhangs by nonenzymatic loop-closing ligation chemistry. We show that loop-closing ligation allows full-length functional ribozymes containing a hairpin loop to be assembled free of inhibitory template strands. This approach to the assembly of structurally complex RNAs suggests a plausible pathway for the emergence of functional RNAs before a full-length RNA copying process became available.


title: Mitochondrial dysfunction promotes alternative gasdermin D-mediated inflammatory cell death and susceptibility to infection

abstract:
Human mutations in mitochondrial-associated genes are associated with inflammatory diseases and susceptibility to infection. However, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) promotes mitochondrial hyper-fission, depolarization, and oxidative stress in macrophages. In the presence of Lrrk2G2019S-dependent mitochondrial perturbations, AIM2 inflammasome activation promotes more cell death but not more pyroptotic IL-1b release. Instead, inflammasome activation in Lrrk2G2019S macrophages triggers gasdermin D (GSDMD)-mediated mitochondrial pores, driving up ROS-mediated RIPK1/RIPK3/MLKL dependent necroptosis. Consequently, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and immunopathology via enhanced neutrophil infiltration. By uncovering that GSDMD promotes non-pyroptotic cell death in Lrrk2G2019S macrophages, our findings demonstrate that altered mitochondrial function can reprogram cell death modalities to elicit distinct immune outcomes. This provides mechanistic insights into why mutations in LRRK2 are associated with susceptibility to chronic inflammatory and infectious diseases.

HIGHLIGHTSO_LIAltered mitochondrial homeostasis reprograms cell death modalities
C_LIO_LIGSDMD associates with and depolarizes mitochondrial membranes following AIM2 activation
C_LIO_LIGSDMD initiates a shift from pyroptotic to necroptotic cell death in Lrrk2G2019S macrophages
C_LIO_LILrrk2G2019S elicits hyperinflammation and susceptibility to infection in flies and mice
C_LI

category: immunology


title: Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

abstract: It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.


title: Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state

abstract: The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL- catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential- dependent oligomeric switch.

category: cell biology


title: ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

abstract: Ciliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase in Chlamydomonas. Under physiological condition, ARL3GDP binds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTP dissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis of Chlamydomonas cells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.


title: IGF1 Signaling in Temporomandibular Joint Fibrocartilage Stem Cells Regulates Cartilage Growth and Homeostasis in Mice

abstract:
ObjectiveInvestigate functional roles of Igf1 in fibrocartilage stem cell (FCSC) for temporomandibular joint (TMJ) cartilage growth and homeostasis.

MethodsGli1-CreER+; RosaTdTomato mice were used for validating FCSCs lineage labeling efficiency. In Gli1-/Col2-CreER+; Igf1fl/fl mice, TMJ cartilage morphological and functional changes were characterized at 4 weeks and 5 months after Igf1 deletion. H&E, Safranine O and immuno-histochemistry staining were performed. FCSCs specificity were characterized using EdU and TUNEL staining. A unilateral anterior crossbite (UAC) mouse model was generated for mimicking TMJ osteoarthritis status.

ResultsIn Gli1-CreER+; RosaTdTomato mice, RFP labeled FCSCs showed favorable proliferative capacity. 4 weeks after Igf1 deletion, Gli1+ and Col2+ cell lineages led to distinct pathological changes of TMJ cartilage morphology. A more serious reduction of cartilage thickness and cell density were found in the superficial layers in Gli1-CreER+; Igf1fl/fl mice. 5 months after Igf1 deletion, more severe disordered cell arrangement in TMJ cartilage were found in both groups with Gli1+ and Col2+ specific deletion of Igf1. Immunostaining showed that PI3K/Akt signaling pathway was blocked in the superficial layers of TMJ in Gli1-CreER+; RosaTdTomato mice. Finally, deletion of Igf1 in FCSCs significantly aggravated osteoarthritis (OA) phenotypic changes in TMJ in UAC mice model, characterized in decreased cartilage thickness, cell numbers and loss of extracellular matrix secretion.

ConclusionIgf1 deletion disrupted stem cell functions of FCSCs, leading to disordered cell distribution during TMJ growth, as well as exaggerated the OA process in TMJ under pathological condition. In TMJ cartilage, Igf1 expression in FCSCs is critical for PI3K/Akt activation, which may be involved in regulating FCSCs self-renewal and differentiation.


title: Pre-implantation mouse embryo movement under hormonally altered conditions

abstract: Precise regulation of embryo movement is crucial to successful implantation, but the role of ovarian hormones in this process is not understood. We ascertain the effects of altered hormonal environment on embryo movement using two delayed implantation models: Natural lactational Diapause (ND), a naturally occurring alternate model of pregnancy, and Artificially induced Diapause (AD), a laboratory version of ND. Our previous work suggests that embryos in a natural pregnancy (NP) first display unidirectional clustered embryo movement, followed by bidirectional scattering and spacing movement. In contrast, in the ND model, embryos are present as clusters near the oviductal-uterine junction for ~24-hours longer than NP, followed by locations consistent with a unidirectional scattering and spacing movement. Intriguingly, the AD model closely resembles embryo location in NP and not ND. Further, unlike the popular paradigm of reduced estrogen (E2) levels in diapause E2 levels are comparable across NP, ND, and AD, while progesterone (P4) levels are reduced in ND and highly increased in AD when compared to NP. Exogenous administration of E2 or P4 modifies the unidirectional clustered embryo movement, while E2 treatment causes a reduction in P4 and affects the bidirectional phase of embryo movement. Taken together, our data suggest embryo movement can be modulated by both P4 and E2. Understanding natural hormonal adaptation in diapause provides an opportunity to determine key players regulating embryo movement and implantation success. This knowledge can be leveraged to understand pregnancy survival and implantation success in hormonally altered conditions in the clinic.


title: Monocarboxylate Transporter 1 (MCT1) mediates succinate export in the retina

abstract:
PurposeSuccinate is exported by the retina and imported by eyecup tissue. The transporter(s) mediating this process have not yet been identified. Recent studies showed that Monocarboxylate Transporter 1 (MCT1) can transport succinate across plasma membranes in cardiac and skeletal muscle. Retina and retinal pigment epithelium (RPE) both express multiple MCT isoforms including MCT1. We tested the hypothesis that MCTs facilitate retinal succinate export and RPE succinate import.

MethodsWe assessed retinal succinate export and eyecup succinate import in short term ex vivo culture using gas chromatography-mass spectrometry. We test the dependence of succinate export and import on pH, proton ionophores, conventional MCT substrates, and the MCT inhibitors AZD3965, AR-C155858, and diclofenac.

ResultsSuccinate exits retinal tissue through MCT1 but does not enter RPE through MCT1 or any other MCT. Intracellular succinate levels are a contributing factor that determines if an MCT1-expressing tissue will export succinate.

ConclusionsMCT1 facilitates export of succinate from retinas. An unidentified, non-MCT transporter facilitates import of succinate into RPE.


title: What is the best fitness measure in wild populations? A case study on the power of short-term fitness proxies to predict reproductive value

abstract: Fitness is at the core of evolutionary theory, but it is difficult to measure accurately. One way to measure long-term fitness is by calculating the individuals reproductive value, which represents the expected number of allele copies an individual passes on to distant future generations. However, this metric of fitness is scarcely used because the estimation of individuals reproductive value requires long-term pedigree data, which is rarely available in wild populations where following individuals from birth to death is often impossible. Wild study systems therefore use short-term fitness metrics as proxies, such as the number of offspring produced. This study obtained three frequently used short-term proxies for fitness obtained at different offspring life stages (eggs, hatchlings, fledglings and recruits), and compared their ability to predict reproductive values derived from the genetic pedigree of a wild passerine bird population. We used twenty years of precise field observations and a near-complete genetic pedigree to calculate reproductive success, individual growth rate and de-lifed fitness as lifetime fitness measures, and as annual de-lifed fitness. We compared the power of these metrics to predict reproductive values and lineage survival to the end of the study period. The three short-term fitness proxies predict the reproductive values and lineage survival only when measured at the recruit stage. There were no significant differences between the different fitness proxies at the same offspring stages in predicting the reproductive values and lineage survival. Annual fitness at one year old predicted reproductive values equally well as lifetime de-lifed fitness. However, none of the short-term fitness proxies was strongly associated with the reproductive values. In summary, the commonly short-term fitness proxies capture long-term fitness with intermediate accuracy at best, if measured at recruitment stage. As lifetime fitness measured at recruit stage and annual fitness in the first year of life were the best proxies of long-term fitness, we encourage their future use.


title: Cultured deep-sea PVC bacteria shed light on eukaryogenesis

abstract: Evolutionary relationship between prokaryotes and eukaryotes continues to fascinate biologists. Accumulated studies suggest a eukaryogenesis model based on the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) bacteria. However, a decisive PVC-based eukaryogenesis scenario has not yet been reported. Here, we isolated PVC bacteria, unique for dividing by budding and for possessing developed endomembrane systems, from the deep sea. In cultured PVC bacterial strains, we detected typical eukaryotic organelle-like structures including endoplasmic reticulum, Golgi apparatus, vesicles, vacuoles and actin/tubulin-based microfilaments. Strikingly, we observed a nucleolus-containing nucleus in a Verrucomicrobia strain, which divides by mitosis. Transcriptomic results further demonstrated abundant presence of genes associated with eukaryotic cellular processes including membrane fusion. We propose that the prominent capability of membrane fusion drives eukaryogenesis by enabling PVC bacteria to evolve eukaryotic cellular features.


title: Human-Plant Coevolution: A modelling framework for theory-building on the origins of agriculture

abstract: The domestication of plants and the origin of agricultural societies has been the focus of much theoretical discussion on why, how, when, and where these happened. The  when and  where have been substantially addressed by bioarchaeology, thanks to advances in methodology and the broadening of the geographical and chronological scope of evidence. However, the  why and  how have lagged behind, holding on to relatively old models with limited explanatory power. Armed with the evidence now available, we can return to theory by revisiting the mechanisms allegedly involved, disentangling their connection to the diversity of trajectories, and identifying the weight and role of the parameters involved. We present the Human-Plant Coevolution (HPC) model, which represents the dynamics of coevolution between a human and a plant population. The model consists of an ecological positive feedback system (mutualism), which can be reinforced by positive evolutionary feedback (coevolution). The model formulation is the result of wiring together relatively simple simulation models of population ecology and evolution, through a computational implementation in R. The HPC model captures a variety of potential scenarios, though which conditions are linked to the degree and timing of population change and the intensity of selective pressures. Our results confirm that the possible trajectories leading to neolithisation are diverse and involve multiple factors. However, simulations also show how some of those factors are entangled, what are their effects on human and plant populations under different conditions, and what might be the main causes fostering agriculture and domestication.

category: systems biology


title: STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia

abstract: STAT3 and HIF1 are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. Here we report that STAT3 is required for the HIF1-dependent response to hypoxia. In Stat3 knock-out pluripotent embryonic stem cells (ESCs), a large fraction of HIF1 target genes is not induced by hypoxia. Mechanistically, STAT3 does not regulate neither HIF1 expression nor stability, rather, it physically interacts with it in the nucleus. In vivo, we observed that both genetic and chemical inactivation of Stat3 blunted physiological responses to hypoxia, such as angiogenesis, erythropoiesis, and immune cell mobilization. Such defects were accompanied with faulty transcriptional activity of HIF1. In sum, our data reveal that STAT3 and HIF1 cooperatively mediate the physiological response to hypoxia.


title: Scoping the Line Up: A Comparison of Biomonitoring Methodologies for Surf Zone Fish Communities

abstract: Surf zones are highly dynamic marine ecosystems that are subject to increasing anthropogenic and climatic pressures, posing multiple challenges for biomonitoring. Traditional methods such as seines and hook and line surveys are often labor intensive, taxonomically biased, and can be physically hazardous. Emerging techniques, such as baited remote underwater video (BRUV) and environmental DNA (eDNA) are promising nondestructive tools for assessing marine biodiversity in surf zones of sandy beaches. Here we compare the relative performance of beach seines, BRUV, and eDNA in characterizing community composition of bony (teleost) and cartilaginous (elasmobranch) fishes of surf zones at 18 open coast sandy beaches in southern California. Seine and BRUV surveys captured overlapping, but distinct fish communities with 50% (18/36) of detected species shared. BRUV surveys more frequently detected larger species (e.g. sharks and rays) while seines more frequently detected one of the most abundant species, barred surfperch (Amphistichus argenteus). In contrast, eDNA metabarcoding captured 83.3% (30/36) of all fishes observed in seine and BRUV surveys plus 59 additional species, including 13 that frequent surf zone habitats. eDNA approaches showed significantly higher sensitivity than seine and BRUV methods and more consistently detected 29 of the 30 (96.7%) jointly observed species across beaches. The six species detected by BRUV/seines, but not eDNA either lacked reference sequences, were only resolved at higher taxonomic ranks (e.g. Embiotocidae surfperches), or were detected below occupancy thresholds. Low site-species overlap between methods limited comparisons of richness and abundance estimates, highlighting the challenge of comparing biomonitoring approaches. Despite potential for improvement, results overall demonstrate that eDNA can provide a cost-effective tool for long-term surf zone monitoring that complements data from seine and BRUV surveys, allowing more comprehensive surveys of vertebrate diversity in surf zone habitats.

category: ecology


title: Joint estimation of selection intensity and mutation rate under balancing selection withapplications to HLA

abstract: A composite likelihood method is introduced for jointly estimating the intensity of selection and the rate of mutation, both scaled by the effective population size, when there is balancing selection at a single multi-allelic locus in an isolated population at demographic equilibrium. The performance of the method is tested using simulated data. Average estimated mutation rates and selection intensities are close to the true values but there is considerable variation about the averages. Allowing for both population growth and population subdivision do not result in qualitative differences but the estimated mutation rates and selection intensities do not in general reflect the current effective population size. The method is applied to three class I (HLA-A, HLA-B and HLA-C) and two class II loci (HLA-DRB1 and HLA-DQA1) in the 1000 Genomes populations. Allowing for asymmetric balancing selection has only a slight effect on the results from the symmetric model. Mutations that restore symmetry of the selection model are preferentially retained because of the tendency of natural selection to maximize average fitness. However, slight differences in selective effects result in much longer persistence time of some alleles. Trans-species polymorphism (TSP), which is characteristic of MHC in vertebrates, is more likely when there are small differences in allelic fitness than when complete symmetry is assumed. Therefore, variation in allelic fitness expands the range of parameter values consistent with observations of TSP.

category: genetics


title: VTA-IC dopaminergic inputs enhances the salience to consolidate aversive but not appetitive taste recognition memory via D1 receptors

abstract: Taste memory involves storing information through plasticity changes in the neural network of taste, including the insular cortex (IC) and ventral tegmental area (VTA), a critical provider of dopamine. Although a VTA-IC dopaminergic pathway has been demonstrated, its role to consolidate taste recognition memory remains poorly understood. We found that photostimulation of dopaminergic neurons in the VTA or VTA-IC dopaminergic terminals of TH-Cre mice increases the salience of a novel taste stimulus regardless of its hedonic value, without altering their taste palatability. Importantly, the inhibition of the D1-like receptor into the IC impairs the salience to facilitate consolidation of an aversive taste recognition memory. Finally, our results showed that VTA photostimulation improves the salience to consolidate a conditioned taste aversion memory through the D1-like receptor into the IC. It is concluded that the dopamine activity from the VTA into IC is required to increase the salience enabling the consolidation of a taste recognition memory. Notably, the D1-like receptor activity into the IC is required to consolidate both innate and learned aversive taste memories but not appetitive taste memory.

category: animal behavior and cognition


title: Induction of virus-induced gene silencing and in planta validation in cucurbits using the CFMMV-Cm vector

abstract:
Virus-induced gene silencing (VIGS) has been employed for the high-throughput analysis of endogenous gene function. We developed a CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) for the efficient generation of viral transcripts in plants. Using the novel pCF93 vector, we identified genes related to male sterility in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. We previously reported reference-based and de novo transcriptomic profiling for the detection of differentially expressed genes between a male fertile line (DAH3615) and its near isogenic male sterile line (DAH3615-MS). Based on the RNA-seq results, we identified 38 de novo-exclusive differentially expressed genes (DEDEGs) that are potentially responsible for male sterility. Partial genes of 200[~]300bp were cloned into pCF93 which was then inoculated into DAH, a small type of watermelon that enables high-throughput screening with a small cultivation area. In this manner, we simultaneously characterized phenotypes associated with the 38 candidate genes in a common-sized greenhouse. Eight out of the 38 gene-silenced plants produced male sterile flowers with abnormal stamens and no pollens. Gene expression levels in flowers were validated via RT-qPCR. Stamen histological sections from male sterile floral buds and mature flowers showed developmental disruption and shrunken pollen sacs. Based on the current findings, we believe that the novel pCF93 vector and our VIGS system facilitate high-throughput analysis for the study of gene function in watermelons.

One sentence summaryThe CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) facilitates large-scale validation of male sterility-related gene functions in watermelon.

category: plant biology


title: Analytical kinetic model of native tandem promoters in E. coli

abstract:
Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites  dTSS, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS [≤] 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.

Author SummaryTandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.


title: Longitudinal changes of ADHD symptoms in association with white matter microstructure: a tract-specific fixel-based analysis

abstract:
BackgroundVariation in the longitudinal course of childhood attention deficit/hyperactivity disorder (ADHD) coincides with neurodevelopmental maturation of brain structure and function. Prior work has attempted to determine how alterations in white matter (WM) relate to changes in symptom severity, but much of that work has been done in smaller cross-sectional samples using voxel-based analyses. Using standard diffusion-weighted imaging (DWI) methods, we previously showed WM alterations were associated with ADHD symptom remission over time in a longitudinal sample of probands, siblings, and unaffected individuals. Here, we extend this work by further assessing the nature of these changes in WM microstructure by including an additional follow-up measurement (aged 18 - 34 years), and using the more physiologically informative fixel-based analysis (FBA).

MethodsData were obtained from 139 participants over 3 clinical and 2 follow-up DWI waves, and analyzed using FBA in regions-of-interest based on prior findings. We replicated previously reported significant models and extended them by adding another time-point, testing whether changes in combined ADHD and hyperactivity-impulsivity (HI) continuous symptom scores are associated with fixel metrics at follow-up.

ResultsClinical improvement in HI symptoms over time was associated with more fiber density at follow-up in the left corticospinal tract (lCST) (tmax=1.092, standardized effect[SE]=0.044, pFWE=0.016). Improvement in combined ADHD symptoms over time was associated with more fiber cross-section at follow-up in the lCST (tmax=3.775, SE=0.051, pFWE=0.019). Conclusions: Aberrant white matter development involves both lCST micro- and macrostructural alterations, and its path may be moderated by preceding symptom trajectory.

category: neuroscience


title: The spotlight of attention turns from rhythmic exploration-exploitation to a stable exploitation state

abstract: While traditional studies claim that visuospatial attention stays fixed at one location at a time, recent research has rather shown that attention rhythmically fluctuates between different locations at rates of prominent brain rhythms. However, little is known about the temporal dynamics of this fluctuation and, particularly, whether it changes over time. Thus, we addressed this question by investigating how visuospatial attention behaves over space and time. We recorded electroencephalographic activity of twenty-seven human participants while they performed a visuospatial cueing task, where attention was covertly oriented to the left or right visual field. In order to decode the spatial locus of attention from neural activity, we trained and tested a classifier on every timepoint of the orienting period, from the attentional cue to stimulus onset. This resulted in one temporal generalization matrix per participant, which was time-frequency decomposed to identify the sampling rhythm. Finally, a searchlight analysis was conducted to reveal the brain regions responsible for attention allocation. Our results show a dynamic evolution of the attentional spotlight, distinguishing between two states. In an early time window, attention explored both cued and uncued hemifield rhythmically at ~10 Hz. In a later time window attention focused on the cued hemifield. Classification was driven by occipital sources, while frontal regions exclusively became involved just before the spotlight settled onto the cued location. Together, our results define attentional sampling as a quasi-rhythmic dynamic process characterized by an initial rhythmic exploration-exploitation state, which is followed by a stable exploitation state.


title: Patient induced pluripotent stem cell-derived hepatostellate organoids establish a basis for liver pathologies in telomeropathies

abstract: Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells followed by generation of genotype-admixed hepatostellate organoids revealed a dominant phenotype in the parenchyma, with DC hepatocytes eliciting a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Thus, isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.


title: Filtration extraction method using microfluidic channel for measuring environmental DNA

abstract: The environmental DNA (eDNA) method, which is widely applied for biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, Biryu-Chip (BC), and a filtration/extraction method, BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of the extracted DNA was obtained with 1/20-1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we could perform on-site measurements of eDNA within 30 min using a mobile PCR device. Using the BC method, filtration and extraction could be performed easily and quickly. The PCR results obtained by the BC method were similar to those obtained by the Sterivex method. The BC method required fewer steps and therefore, the risk of DNA contamination could be reduced. When combined with a mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from a few 10 mL of the water sample, even on-site.


title: Factors affecting virus prevalence in honey bees in the Pacific-Northwest, USA

abstract:
Global efforts to assess honey bee health show viruses are major stressors that undermine colony performance. Identifying factors that affect virus incidence, such as management practices and landscape context, could aid in slowing virus transmission. Here we surveyed viruses in honey bees from 86 sites in the Pacific Northwest, USA, and tested effects of regional bee density, movement associated with commercial pollination, julian date, and hive management on virus prevalence. We also explored patterns of virus co-occurrence and spatial autocorrelation to identify whether local transmission was a primary driver of pathogen distribution. Our surveys found widespread prevalence of Deformed wing virus (DWV), Sacbrood virus (SBV), and Black queen cell virus (BQCV). BQCV and SBV were most prolific in commercial apiaries, while Chronic bee paralysis virus (CPBV) was more common in hobbyist apiaries than commercial apiaries. DWV was most common in urban landscapes and was best predicted by mite prevalence and julian date, while the incidence of both SBV and BQCV were best predicted by regional apiary density. We did not find evidence of additional spatial autocorrelation for any viruses, although high co-occurrence suggests parallel transmission patterns. Our results support the importance of mite management in slowing virus spread and suggest that greater bee density increases transmission. Our study provides support that viruses are widespread in honey bees and connects known mechanisms of virus transmission to the distribution of pathogens observed across the Pacific Northwest.

HighlightsThree viruses were widespread in honey bee populations across the Pacific Northwest, USA

Black queen cell and Sacbrood viruses were most common in high density hives

Deformed wing virus was most common in hives that had high mite loads

The presence of many viruses in bees suggests parallel or synergistic transmission


title: Recent Urban Development Reduces Bee Abundance and Diversity

abstract: Wild bee communities persist in cities despite major disruption of nesting and food resources by urban development. Bee diversity and abundance is key for urban agriculture and maintenance of plant diversity, and assessing what aspects of cities enhance bee populations will promote our capacity to retain and provision bee habitat. Here, we assessed how variation in land cover and neighborhood development history affected bee communities in the midwestern US urban landscape of Madison, Wisconsin. We sampled bee communities across 38 sites with relatively high (> 55%) or low (< 30%) levels of impervious surface, and assessed effects of land use and neighborhood development history on bee abundance and species richness. We show abundance and richness of bees was lower in recently developed neighborhoods, with particularly strong negative effects on soil nesting bees. Soil nesting bees and bee community richness decreased as cover of impervious surface increased, but above ground nesting bees were minimally impacted. Bee community similarity varied spatially and based on dissimilar local land cover, only for soil nesting bees, and the overall bee community. Impervious surface limited bee abundance and diversity, but new neighborhoods were associated with greater negative effects. We suggest that enhancing the structural diversity of new neighborhoods in urban ecosystems may imitate the structural benefits of older neighborhoods for bee populations.


title: Historical specimens and the limits of subspecies phylogenomics in the New World quails (Odontophoridae)

abstract: As phylogenomics focuses on comprehensive taxon sampling at the species and population/subspecies levels, incorporating genomic data from historical specimens has become increasingly common. While historical samples can fill critical gaps in our understanding of the evolutionary history of diverse groups, they also introduce additional sources of phylogenomic uncertainty, making it difficult to discern novel evolutionary relationships from artifacts caused by sample quality issues. These problems highlight the need for improved strategies to disentangle artifactual patterns from true biological signal as historical specimens become more prevalent in phylogenomic datasets. Here, we tested the limits of historical specimen-driven phylogenomics to resolve subspecies-level relationships within a highly polytypic family, the New World quails (Odontophoridae), using thousands of ultraconserved elements (UCEs). We found that relationships at and above the species-level were well-resolved and highly supported across all analyses, with the exception of discordant relationships within the two most polytypic genera which included many historical specimens. We examined the causes of discordance and found that inferring phylogenies from subsets of taxa resolved the disagreements, suggesting that analyzing subclades can help remove artifactual causes of discordance in datasets that include historical samples. At the subspecies-level, we found well-resolved geographic structure within the two most polytypic genera, including the most polytypic species in this family, Northern Bobwhites (Colinus virginianus), demonstrating that variable sites within UCEs are capable of resolving phylogenetic structure below the species level. Our results highlight the importance of complete taxonomic sampling for resolving relationships among polytypic species, often through the inclusion of historical specimens, and we propose an integrative strategy for understanding and addressing the uncertainty that historical samples sometimes introduce to phylogenetic analyses.


title: Functional divergence and spectral tuning of microbial rhodopsins from an ancestral proton pump

abstract: For billions of years, life has continuously adapted to dynamic physical conditions near the Earths surface. Fossils and other preserved biosignatures in the paleontological record are the most direct evidence for reconstructing the broad historical contours of this adaptive interplay. However, biosignatures dating to Earths earliest history are exceedingly rare. Here, we combine phylogenetic inference of primordial rhodopsin proteins with modeled spectral features of the Precambrian Earth environment to reconstruct the paleobiological history of this essential family of photoactive transmembrane proteins. Our results suggest that ancestral microbial rhodopsins likely acted as light-driven proton pumps and were spectrally tuned toward the absorption of green light, which would have enabled their hosts to occupy depths in a water column or biofilm where UV wavelengths were attenuated. Subsequent diversification of rhodopsin functions and peak absorption frequencies was enabled by the expansion of surface ecological niches induced by the accumulation of atmospheric oxygen. Inferred ancestors retain distinct associations between extant functions and peak absorption frequencies. Our findings suggest that novel information encoded by biomolecules can be used as "paleosensors" for conditions of ancient, inhabited niches of host organisms not represented elsewhere in the paleontological record. The coupling of functional diversification and spectral tuning of this taxonomically diverse protein family underscores the utility of rhodopsins as universal testbeds for inferring remotely detectable biosignatures on inhabited planetary bodies.


title: Earliest photic zone niches probed by ancestral microbial rhodopsins


title: Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis

abstract:
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.

Author summaryThe mitotic spindle is a microtubule (MT)-based molecular machine that mediates precise chromosome segregation during cell division. Both Drosophila and human cells assemble their spindles exploiting two main classes of MTs: MTs nucleated by the centrosomes (MT nucleating organelles) and MTs generated at or near the kinetochores (the chromosome-associated structures that bind the spindle MTs). Cells of both species can assemble a functional mitotic spindle in the complete absence of centrosomes, but the mechanisms underlying this process are still poorly understood. We used Drosophila S2 cells as model system to analyze spindle reassembly following colcemid-induced MT depolymerization. MT regrowth (MTR) after colcemid treatment was particularly informative as this drug disrupts the MT nucleating ability of the centrosomes but allows kinetochore-driven MTR (KDMTR). We analyzed KDMTR in normal cells and in cells subjected to RNA interference (RNAi)-mediated depletion of 8 different evolutionarily conserved proteins involved in spindle assembly, and identified proteins that either promote or delay KDMTR. These results coupled with the analysis of proteins localization during spindle reassembly allowed us to integrate the current model on the role of kinetochore-driven MT growth in spindle formation.


title: A 1-bp deletion in bovine QRICH2 causes low sperm count and immotile sperm with multiple morphological abnormalities

abstract:
BackgroundSemen quality and male fertility are monitored in artificial insemination bulls to ensure high insemination success rates. Only ejaculates that fulfill minimum quality requirements are processed and eventually used for artificial inseminations. We examined 70,990 ejaculates from 1343 Brown Swiss bulls to identify bulls from which all ejaculates were rejected due to low semen quality. This procedure identified a bull that produced twelve ejaculates with an aberrantly low number of sperm (0.2{+/-}0.2 x 109 sperm per ml) which were mostly immotile due to multiple morphological abnormalities.

ResultsThe genome of the bull was sequenced at 12-fold coverage to investigate a suspected genetic cause. Comparing the sequence variant genotypes of the bull with those from 397 fertile bulls revealed a 1-bp deletion in the coding sequence of QRICH2 encoding glutamine rich 2 as a compelling candidate causal variant. The 1-bp deletion causes a frameshift in translation and induces a premature termination codon (ENSBTAP00000018337.1:p.Cys1644AlafsTer52). The analysis of testis transcriptomes from 76 bulls showed that the transcript with the premature termination codon is subjected to nonsense-mediated mRNA decay. The 1-bp deletion resides on a 675 kb haplotype spanning 181 SNPs from the Illumina BovineHD Bead chip. The haplotype segregates at a frequency of 5% in the Brown Swiss cattle population. This analysis also identified another bull that carried the 1-bp deletion in the homozygous state. Semen analyses from the second bull confirmed low sperm concentration and immotile sperm with multiple morphological abnormalities primarily affecting the sperm flagellum and, to a lesser extent, the sperm head.

ConclusionsA recessive loss-of-function allele of bovine QRICH2 likely causes low sperm concentration and immotile sperm with multiple morphological abnormalities. Routine sperm analyses unambiguously identify homozygous bulls. A direct gene test can be implemented to monitor the frequency of the undesired allele in cattle populations.


title: Pre-metazoan origin of neuropeptide signalling

abstract: Neuropeptides are a diverse class of signalling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments and gene-structure comparisons we show that both precursors are present in bilaterians, cnidarians, ctenophores and sponges. We also found phoenixin and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. Phoenixin in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays we question the originally proposed GPR-173 (SREB3) as a phoenixin receptor. Our findings indicate that signalling by secreted neuropeptide homologs has pre-metazoan origins and thus evolved before neurons.

category: genomics


title: Invertebrate DNA methylation and gene regulation

abstract: As human activity alters the planet, there is a pressing need to understand how organisms adapt to environmental change. Of growing interest in this area is the role of epigenetic modifications, such as DNA methylation, in tailoring gene expression to fit novel conditions. Here, we reanalyzed nine invertebrate (Anthozoa and Hexapoda) datasets to validate a key prediction of this hypothesis: changes in DNA methylation in response to some condition correlate with changes in gene expression. While we detected both differential methylation and differential expression, there was no simple relationship between these differences. Hence, if changes in DNA methylation regulate invertebrate transcription, the mechanism does not follow a simple linear relationship.


title: Parental control: ecology drives plasticity in parental response to offspring signals

abstract: Different bird species have completely different parent-offspring interactions. When food is plentiful, the chicks that are begging the loudest are fed the most. When food is scarce, bird species instead feed the largest offspring. While this variation could be due to parents responding to signalling differently based on food availability, it could equally be due to offspring adjusting their behaviour, or to variation in information availability. We tested between these competing explanations experimentally, by manipulating food availability in a population of wild great tits, Parus major, while standardising offspring behaviour and size. We found that when food was more plentiful, parents were: (1) more likely to preferentially feed the chicks that were begging the most; and (2) less likely to preferentially feed larger chicks. In addition, we consistently found these same patterns, in a meta-analysis across 57 bird species. Overall, our results suggest that parents have more control over food distribution than offspring do, and that they flexibly adjust how they respond to both offspring signals and cues of offspring quality in response to food availability. Consequently, depending upon environmental conditions, predictably different signalling systems are favoured.


title: Swarm learning for decentralized artificial intelligence in cancer histopathology

abstract: Artificial Intelligence (AI) can extract clinically actionable information from medical image data. In cancer histopathology, AI can be used to predict the presence of molecular alterations directly from routine histopathology slides. However, training robust AI systems requires large datasets whose collection faces practical, ethical and legal obstacles. These obstacles could be overcome with swarm learning (SL) where partners jointly train AI models, while avoiding data transfer and monopolistic data governance. Here, for the first time, we demonstrate the successful use of SL in large, multicentric datasets of gigapixel histopathology images comprising over 5000 patients. We show that AI models trained using Swarm Learning can predict BRAF mutational status and microsatellite instability (MSI) directly from hematoxylin and eosin (H&E)-stained pathology slides of colorectal cancer (CRC). We trained AI models on three patient cohorts from Northern Ireland, Germany and the United States of America and validated the prediction performance in two independent datasets from the United Kingdom using SL-based AI models. Our data show that SL enables us to train AI models which outperform most locally trained models and perform on par with models which are centrally trained on the merged datasets. In addition, we show that SL-based AI models are data efficient and maintain a robust performance even if only subsets of local datasets are used for training. In the future, SL can be used to train distributed AI models for any histopathology image analysis tasks, overcoming the need for data transfer and without requiring institutions to give up control of the final AI model.

category: bioinformatics


title: Model-checking ecological state-transition graphs

abstract:
Model-checking is a methodology developed in computer science to automatically assess the dynamics of discrete systems, by checking if a system modelled as a state-transition graph satisfies a dynamical property written as a temporal logic formula. The dynamics of ecosystems have been drawn as state-transition graphs for more than a century, from state-and-transition models to assembly graphs. Thus, model-checking can provide insights into both empirical data and theoretical models, as long as they sum up into state-transition graphs. While model-checking proved to be a valuable tool in systems biology, it remains largely underused in ecology. Here we promote the adoption of the model-checking toolbox in ecology through its application to an illustrative example. We assessed the dynamics of a vegetation model inspired from state-and-transition models by model-checking Computation Tree Logic formulas built from a proposed catalogue of patterns. Model-checking encompasses a wide range of concepts and available software, mentioned in discussion, thus its implementation can be fitted to the specific features of the described system. In addition to the automated analysis of ecological state-transition graphs, we believe that defining ecological concepts with temporal logics could help clarifying and comparing them.

Author summaryEcologists have drawn state-transition graphs representing the dynamics of ecosystems for more than a century. Model-checking is an automated method for the analysis of such graphs developed in computer science and acknowledged by a Turing award in 2007. Ecologists appear to be mostly unaware of model-checking despite its successes in systems biology to assess the dynamics of biological networks.

We promote model-checking of ecological state-transition graphs through its application to an illustrative vegetation model. We exemplify the insights provided by model-checking by assessing management policies aiming to tackle savanna encroachment. We also provide a catalogue of patterns to help ecologists with the difficulty of formally expressing dynamical properties. We also discuss the wide range of model-checking concepts and available software, enabling to fit the specific features of the studied system, such as durations or probabilities.

Model-checking can be applied to both empirical data and theoretical models, as long as they sum up into state-transition graphs. It provides automated and accurate answers to complex questions that could barely be analysed through human examination, if not impossible to answer this way. In addition to the automated analysis of ecological state-transition graphs, we believe that formally defining ecological concepts within the model-checking framework could help in clarifying and comparing them.


title: STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci

abstract: Expansions of short tandem repeats (STRs) cause dozens of rare Mendelian diseases. However, STR expansions, especially those arising from repeats not present in the reference genome, are challenging to detect from short-read sequencing data. Such "novel" STRs include new repeat units occurring at known STR loci, or entirely new STR loci where the sequence is absent from the reference genome. A primary cause of difficulty detecting STR expansions is that reads arising from STR expansions are frequently mismapped or unmapped. To address this challenge, we have developed STRling, a new STR detection algorithm that counts k-mers (short DNA sequences of length k) in DNA sequencing reads, to efficiently recover reads that inform the presence and size of STR expansions. As a result, STRling can call expansions at both known and novel STR loci. STRling has a sensitivity of 83% for 14 known STR disease loci, including the novel STRs that cause CANVAS and DBQD2. It is the first method to resolve the position of novel STR expansions to base pair accuracy. Such accuracy is essential to interpreting the consequence of each expansion. STRling has an estimated 0.078 false discovery rate for known pathogenic loci in unaffected individuals and a 0.20 false discovery rate for genome-wide loci in unaffected individuals when using variants called from long-read data as truth. STRling is fast, scalable on cloud computing, open-source, and freely available at https://github.com/quinlan-lab/STRling.


title: Nonmetric ANOVA: a generic framework for analysis of variance on dissimilarity measures

abstract:
Classic Analysis of Variance (ANOVA; cA) tests the explanatory power of a partitioning on a set of objects. Nonparametric ANOVA (npA) extends to a case where instead of the object values themselves, their mutual distances are available. While considerably widening the applicability of the cA, the npA does not provide a statistical framework for the cases where the mutual dissimilarity measurements between objects are nonmetric. Based on the central limit theorem (CLT), we introduce nonmetric ANOVA (nmA) as an extension of the cA and npA models where metric properties (identity, symmetry, and subadditivity) are relaxed. Our model allows any dissimilarity measures to be defined between objects where a distinctiveness of a specific partitioning imposed on those are of interest. This derivation accommodates an ANOVA-like framework of judgment, indicative of significant dispersion of the partitioned outputs in nonmetric space. We present a statistic which under the null hypothesis of no differences between the mean of the imposed partitioning, follows an exact F-distribution allowing to obtain the consequential p-value. Three biological examples are provided and the performance of our method in relation to the cA and npA is discussed.

Significance StatementThe Nonmetric Analysis of Variance (nmANOVA) conveys a framework that allows a compatible type of ANOVA for the cases where the proper metric measurements between objects are either lost, unknown or however inaccessible. While classic ANOVA is based on the measurements of the data from a base datum, the nmANOVA is formulated on the dissimilarity outputs (not necessarily metric) defined between all objects. As the main goal of ANOVA in providing a statistical test for assessing the significance of a considered partitioning on the data, the nmANOVA is yielding a paralleled scheme of inference with 1) accommodating the outcomes dissimilarities into within and between groups statistics, 2) assessing their respective divergence with a parametric distribution, and 3) providing a resultant p-value indicative of evidences fore rejecting the null hypothesis.