title: A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis

abstract:
The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any non-essential gene, and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be co-transformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps.

ImportanceBacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific gRNA and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome-editing can be directed to any of nearly 4000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid, free of off-target effects, and facilitates a wide-range of genome manipulations.

"Healing is a matter of time, but it is sometimes also a matter of opportunity." - Hippocrates


title: Quantitative modeling of regular retinal microglia distribution

abstract:
1Microglia are resident immune cells in the central nervous system (CNS), showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglias morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated.

First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.

Author summaryMicroglia are resident immune cells in the central nervous system. It is known that the microglia cells show a regular distribution, but the mechanism underlying the regular distribution remains to be elucidated. In the present study, we quantitatively assayed the regularity of the microglia soma distribution using the image processing technique. Next, we formulated a mathematical model of cell distribution that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters using organ culture. As a result, we obtained parameters for cell migration, cell process dynamics, and extracellular ATP dynamics. Then we undertook numerical simulation of the mathematical model using the parameters obtained by the experiments. The resulting model simulations showed that direct cell-cell contact is the most important factor in generating regular spacing. Finally, we screened possible molecular pathways involved in the regular spacing of microglia, which confirmed the validity of the model.

category: cell biology


title: Force generation of KIF1C is impaired by pathogenic mutations

abstract:
Intracellular transport is essential for neuronal function and survival. The fastest neuronal transporter is the kinesin-3 KIF1C. Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients. However, neither the force generation of the KIF1C motor protein, nor the biophysical and functional properties of pathogenic mutant proteins have been studied thus far.

Here we show that full length KIF1C is a processive motor that can generate forces up to 5.7 pN. We find that KIF1C single molecule processivity relies on its ability to slip and re-engage under load and that its slightly reduced stall force compared to kinesin-1 relates to its enhanced probability to backslip. Two pathogenic mutations P176L and R169W that cause hereditary spastic paraplegia in humans maintain fast, processive single molecule motility in vitro, but with decreased run length and slightly increased unloaded velocity compared to the wildtype motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles.

Our results establish a baseline for the single molecule mechanics of Kif1C and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.


title: Stable coexistence in plant-pollinator-herbivore communities requires balanced mutualistic vs antagonistic interactions

abstract: Ecological communities consist of multiple species interacting in diverse ways. Understanding the mechanisms supporting coexistence requires accounting for such a diversity. Because most works focus either on mutualism or predation, how pollination and herbivory interactively determine the stable coexistence in plant-pollinator-herbivore communities is still poorly understood. Studying the typical three-species module of such communities, we determine the conditions allowing stable coexistence then investigate how its maintenance constrains the relative interaction strengths. Our results show that coexistence is possible if pollination is sufficiently strong relative to herbivory, while its stability is possible if herbivory is sufficiently strong relative to pollination. A balance between pollination and herbivory is therefore required. Interestingly, shared preferences for plant phenotypes, that would favor such balance, have been frequently reported in the empirical literature. The identified ecological trade-off between attracting pollinators and deterring herbivores therefore also appears as an emergent property of stable plant-pollinator-herbivore communities.

category: ecology


title: Parametric modulators of sex-biased conditioned fear responding

abstract: Pavlovian fear conditioning is a widely used behavioral paradigm for studying associative learning in rodents. Despite early recognition that subjects may engage in a variety of both conditioned and unconditioned responses, the last several decades have seen the field narrow its focus to measure freezing as the sole indicator of conditioned fear. We previously reported that female rats were more likely than males to engage in darting, an escape-like conditioned response that is associated with heightened shock reactivity. To determine how experimental parameters contribute to the frequency of darting in both males and females, we manipulated factors such as chamber size, shock intensity, and number of trials. To better capture fear-related behavioral repertoires in our animals, we developed ScaredyRat, an open-source custom Python tool that analyzes Noldus Ethovision-generated raw data files to identify Darters and quantify both conditioned and unconditioned responses. We find that like freezing, conditioned darting occurrences scale with experimental alterations. While most darting occurs in females, we find that with an extended training protocol, darting can emerge in males as well. Collectively, our data suggest that darting reflects a behavioral switch in conditioned responding that is a product of both an individual animals sex, shock reactivity, and experimental parameters, underscoring the need for careful consideration of sex as a biological variable in classic learning paradigms.

category: animal behavior and cognition


title: Darting across space and time: Parametric modulators of sex-biased conditioned fear responding


title: Extending the application of the SCA/Sectors method for the identification of domain boundaries and subtype specific residues in multi-domain biosynthetic proteins: Application to Polyketide Synthases

abstract:
Polypeptides with multiple enzyme domains, such as type I polyketide synthases, produce chemically complex compounds that are difficult to produce via conventional chemical synthesis and are often pharmaceutically or otherwise commercially valuable. Engineering polyketide synthases, via domain swapping and/or site directed mutagenesis, in order to generate novel polyketides, has tended to produce either low yields of product or no product at all. The success of such experiments may be limited by our inability to predict the key functional residues and boundaries of protein domains. Computational tools to identify the boundaries and the residues determining the substrate specificity of domains could reduce the trial and error involved in engineering multi-domain proteins. In this study we use statistical coupling analysis to identify networks of co-evolving residues in type I polyketide synthases, thereby predicting domain boundaries. We extend the method to predicting key residues for enzyme substrate specificity. We introduce bootstrapping calculations to test the relationship between sequence length and the number of sequences needed for a robust analysis. Our results show no simple predictor of the number of sequences needed for an analysis, which can be as few as a hundred and as many as a few thousand. We find that polyketide synthases contain multiple networks of co-substituting residues: some are intradomain but most multiple domains. Some networks of coupled residues correlate with specific functions such as the substrate specificity of the acyl transferase domain, the stereo chemistry of the ketoreductase domain, or domain boundaries that are consistent with experimental data. Our extension of the method provides a ranking of the likely importance of these residues to enzyme substrate specificity, allowing us to propose residues for further mutagenesis work. We conclude that analysis of co-evolving networks of residues is likely to be an important tool for re-engineering multi-domain proteins.

Author summaryMany important compounds such as antibiotics or food flavourings are produced naturally by molecular factories within plant, fungal and bacterial cells. These molecular factories typically comprise a complex of multiple interacting enzymes, each enzyme being a stage in a molecular production line. Often the enzymes are connected together as subsections of the same amino acid chain, i.e. protein, with the amino acid chain folding into the separate functional enzymatic domains that comprise the production line. Polyketide synthases are such multi-domain proteins, and their products often have antibacterial, antifungal and antitumoric effects. Engineering polyketide synthases thus has the potential to produce novel drug candidates. We applied and developed statistical approaches to detect where in an amino acid sequence the boundaries are between different domains, potentially allowing these regions to be swapped around for the synthesis of novel compounds. We used the same approaches to identify parts of the amino acid chain important for the function of different types of domain, pointing to how they might be modified to make novel compounds. These analyses agree with published experimental data and allow us to make novel predictions, which we expect to help experimentalists produce novel compounds of commercial and pharmaceutical interest.

category: bioengineering


title: Computational saturation mutagenesis of SARS-CoV-1 spike glycoprotein: stability, binding affinity, and comparison with SARS-CoV-2

abstract:
Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we performed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003-2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003-2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the S glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2.

Author SummarySevere acute respiratory syndrome coronavirus (SARS-CoV-1) is an RNA virus that undergoes frequent mutations, which may result in more virulent SARS-CoV-1 variants. To prevent another pandemic in the future, scientists must understand the mechanisms of viral mutations and predict if any variants could become a dominant. The infection of SARS-CoV-1 in cells is largely depending on the interactions of the viral Spike (S) and human angiotensin-converting enzyme 2 (hACE2). We applied a computational method to predict S missense mutations that will make SARS-CoV-1 more virulent. We are interested in the variants that can change SARS-CoV-1 spike protein stability and/or change the virus-receptor interactions. We mutated each residue of SARS-CoV-1 spike to all possible amino acids; we calculated the differences between the folding energy and binding energy of each variant and the wildtype and identified the target S mutations with significant effects on protein stability and protein-protein interaction. We found some viral mutations could destabilize S and weaken S-hACE2 binding of SARS-CoV-1 isolate. Our results show that the computational saturation mutagenesis is a reliable approach in the analysis and prediction of missense mutations.

category: bioinformatics


title: Rapid synaptic plasticity contributes to a learned conjunctive code of position and choice-related information in the hippocampus

abstract: To successfully perform goal-directed navigation, animals must know where they are and what they are doing--e.g., looking for water, bringing food back to the nest, or escaping from a predator. Hippocampal neurons code for these critical variables conjunctively, but little is known about how this where/what code is formed or flexibly routed to other brain regions. To address these questions, we performed intracellular whole-cell recordings in mouse CA1 during a cued, two-choice virtual navigation task. We demonstrate that plateau potentials in CA1 pyramidal neurons rapidly strengthen synaptic inputs carrying conjunctive information about position and choice. Plasticity-induced response fields were modulated by cues only in animals previously trained to collect rewards based on these cues. Thus, we reveal that gradual learning is required for the formation of a conjunctive population code, upstream of CA1, while plateau-potential-induced synaptic plasticity in CA1 enables flexible routing of the code to downstream brain regions.

category: neuroscience


title: Optogenetic stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsin variants yield opposite behavioral outcomes

abstract: Optogenetic manipulation of genetically-specified neuron populations has become a key tool in circuit neuroscience. The medial amygdala (MeA) receives pheromone information about conspecifics and has crucial functions in social behaviors; interestingly, this amygdalar structure contains a majority of GABAergic projection neurons. A previous study showed that optogenetic activation of MeA GABA neurons with channelrhodopsin-2H134R (ChR2) strongly enhanced inter-male aggression (Hong et al. 2014, Cell). When we attempted to reproduce these findings, accidentally using a faster channelrhodopsin variant (channelrhodopsin-2H134R,E123T or ChETA), we found the opposite results. We therefore systematically compared the behavioral outcome of optogenetic stimulation of MeApd GABA neurons with ChETA versus ChR2, employing two widely used AAV serotypes. This revealed that optogenetic stimulation with ChETA suppressed aggression, whereas optogenetic stimulation with ChR2 increased aggression. Recordings of membrane potential changes following optogenetic stimulation with ChETA versus ChR2 revealed larger plateau depolarizations, smaller action potential amplitudes, and larger local inhibition of neighboring inhibitory neurons with ChR2 as compared to ChETA. Our study shows that channelrhodopsin variants have to be chosen with care for in-vivo optogenetic experiments. Furthermore, the role of MeApd GABA neurons in aggression control should be re- evaluated.


title: Computational modeling predicts acidic microdomains in the glutamatergic synaptic cleft

abstract:
At chemical synapses, synaptic vesicles release their acidic contents into the cleft leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization, rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction (NMJ) as glutamate, adenosine triphosphate (ATP) and protons (H+) are released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase (PMCA) activity and predicts substantial cleft acidification but only for fractions of a millisecond following neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 milliseconds, as the PMCA removes H+ from the cleft in exchange for calcium ions (Ca2+) from adjacent pre- and post-synaptic compartments; thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis has little influence on the magnitude of acidification. Phosphate, but not bicarbonate buffering is effective at ameliorating the magnitude and time course of the acid spike, while both buffering systems are effective at ameliorating cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH disturbances accompanying neurotransmission.

SIGNIFICANCE STATEMENTAcid-base imbalances have surprisingly potent neurological effects highlighting the acute pH sensitivity of many neural mechanisms. Acid-Sensing Ion-Channels (ASICs), which open in response to acid shifts in extracellular pH, are an example of such a mechanism. However, while ASICs open during neurotransmission at conventional glutamatergic synapses, pH-sensitive electrodes and fluorophores show no signs of acidification at these synapses, only alkalinization. To resolve this paradox, we built a computational model which allows a glimpse beyond the experimental limitations of pH-sensitive electrodes and fluorophores. Our model reveals a highly dynamic pH landscape within the synaptic cleft, harboring deep but exceedingly rapid acid transients that give way to a prolonged period of alkalinization, thus reconciling ASIC activation with direct measurements of extracellular pH.


title: Co-localization of different Neurotransmitter Transporters on the same Synaptic Vesicle is Bona-fide yet Sparse

abstract: Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative single vesicle imaging, we show that a small population of neuronal SVs indeed contain different VTs to accomplish corelease. Surprisingly, this population is highly diverse (27 types), expressing distinct dual transporters suggesting corelease of various combinations of neurotransmitters. Using glutamatergic vesicles as an example, we demonstrate that transporter colocalization not only determine the transmitter type but also influences the SV content and synaptic quantal size. Thus, presence of diverse transporters on the same vesicle is bona-fide and, depending on the VT types, this may act as one mechanism to regulate neurotransmitter type, content and release in space and time.


title: Co-localization of different neurotransmitter transporters on synaptic vesicles is sparse except of VGLUT1 and ZnT3


title: Combination and competition between path integration and landmark navigation in the estimation of heading direction

abstract:
Successful navigation requires the ability to compute ones location and heading from incoming multisensory information. Previous work has shown that this multisensory input comes in two forms: body-based idiothetic cues, from ones own rotations and translations, and visual allothetic cues, from the environment (usually visual landmarks). However, exactly how these two streams of information are integrated is unclear, with some models suggesting the body-based idiothetic and visual allothetic cues are combined, while others suggest they compete. In this paper we investigated the integration of body-based idiothetic and visual allothetic cues in the computation of heading using virtual reality. In our experiment, participants performed a series of body turns of up to 360 degrees in the dark with only a brief flash (300ms) of visual feedback en route. Because the environment was virtual, we had full control over the visual feedback and were able to vary the offset between this feedback and the true heading angle. By measuring the effect of the feedback offset on the angle participants turned, we were able to determine the extent to which they incorporated visual feedback as a function of the offset error. By further modeling this behavior we were able to quantify the computations people used. While there were considerable individual differences in performance on our task, with some participants mostly ignoring the visual feedback and others relying on it almost entirely, our modeling results suggest that almost all participants used the same strategy in which idiothetic and allothetic cues are combined when the mismatch between them is small, but compete when the mismatch is large. These findings suggest that participants update their estimate of heading using a hybrid strategy that mixes the combination and competition of cues.

Author summarySuccessful navigation requires us to combine visual information about our environment with body-based cues about our own rotations and translations. In this work we investigated how these disparate sources of information work together to compute an estimate of heading. Using a novel virtual reality task we measured how humans integrate visual and body-based cues when there is mismatch between them -- that is, when the estimate of heading from visual information is different from body-based cues. By building computational models of different strategies, we reveal that humans use a hybrid strategy for integrating visual and body-based cues -- combining them when the mismatch between them is small and picking one or the other when the mismatch is large.


title: Simple, sensitive and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes, using loop mediated isothermal amplification combined with the electrochemical biosensing method

abstract:
BackgroundWolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia- based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity issues.

Methodology/Principal FindingsWe describe here the development of a LAMP combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers were more specific to wAlbB detection than those of the previous published ones if the assays were conducted with low-cost and non-specific detecting dyes. The detection capacity of our LAMP technique was 3.8 nM and the detection limit reduced to 2.16 fM when combined with the BIOSENSOR. Our study demonstrates that the BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification.

Conclusions/SignificanceOur results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.

Author SummaryMosquito-borne diseases such as dengue, chikungunya, Zika, and yellow fever are transmitted to humans mainly by the bites of Aedes aegypti mosquitoes. Controlling these diseases relies mostly on the use of insecticides, in which the efficiency has been reduced through development of insecticide resistance in mosquitoes. Wolbachia is the endosymbiotic bacteria that are naturally found in 40% of insects, including mosquitoes. The bacteria could protect their hosts from viral infections and could also cause sterility in host populations, therefore, providing an opportunity to use them for disease control. Application of a Wolbachia-based strategy needs simple, rapid and sensitive methods for detecting the bacteria in released mosquitoes. In this paper, we develop the combined methods of LAMP and BIOSENSORS for detecting wAlbB Wolbachia in mosquitoes. Our positive LAMP reaction can be visualized by color change from violet to blue at a sensitivity of [≥] 60 pg of genomic DNA. When used in combination with the BIOSENSOR method, the sensitivity increased a million fold without losing specificity. Our study indicates that both developed methods, either used in combination or stand-alone, are efficient and cost-effective, hence, it could be applied for routine surveys of Wolbachia in mosquito control programs that use Wolbachia-based approaches.

category: molecular biology


title: CellProfiler 4: Improvements in Speed, Utility and Usability

abstract:
BackgroundImaging data contains a substantial amount of information which can be difficult to evaluate by eye. With the expansion of high throughput microscopy methodologies producing increasingly large datasets, automated and objective analysis of the resulting images is essential to effectively extract biological information from this data. CellProfiler is a free, open source image analysis program which enables researchers to generate modular pipelines with which to process microscopy images into interpretable measurements.

ResultsHerein we describe CellProfiler 4, a new version of this software with expanded functionality. Based on user feedback, we have made several user interface refinements to improve the usability of the software. We introduced new modules to expand the capabilities of the software. We also evaluated performance and made targeted optimizations to reduce the time and cost associated with running common large-scale analysis pipelines.

ConclusionsCellProfiler 4 provides significantly improved performance in complex workflows compared to previous versions. This release will ensure that researchers will have continued access to CellProfilers powerful computational tools in the coming years.


title: The mutual repression between Pax2 and Snail factors regulates the epithelial/mesenchymal state during intermediate mesoderm differentiation

abstract:
The pronephros is the first renal structure in the embryo, arising after mesenchymal to epithelial transition (MET) of the intermediate mesoderm, where Pax2 induces epithelialisation of the mesenchyme. Here we show that, in the early embryo, Snail1 directly represses Pax2 transcription maintaining the intermediate mesoderm in an undifferentiated state. Reciprocally, Pax2 directly represses Snail1 expression to induce MET upon receiving differentiation signals. We also show that BMP7 acts as one such signal by downregulating Snail1 and upregulating Pax2 expression. This, together with the Snail1/Pax2 reciprocal repression, establishes a regulatory loop in a defined region along the anteroposterior axis, the bistability domain within the transition zone, where differentiation of the neural tube and the somites are known to occur. Thus, we show that the antagonism between Snail1 and Pax2 determines the epithelial/mesenchymal state during the differentiation of the intermediate mesoderm and propose that the bistability zone extends to the intermediate mesoderm, synchronizing the differentiation of tissues aligned along the mediolateral embryonic axis.

SummaryThe antagonism between Snail and Pax2 factors in the embryonic differentiation zone, tightly regulates the timing of mesodermal epithelialisation

category: developmental biology


title: A Comprehensive Overview of the Physical Health of the Adolescent Brain Cognitive Development Study (ABCD) Cohort at Baseline

abstract: Physical health in childhood is crucial for neurobiological as well as overall development, and can shape long-term outcomes into adulthood. The landmark, longitudinal Adolescent Brain Cognitive Development StudySM (ABCD study(R)), was designed to investigate brain development and health in almost 12,000 youth who were recruited when they were 9-10 years old and will be followed through adolescence and early adulthood. The overall goal of this paper is to provide descriptive analyses of physical health measures in the ABCD study at baseline, including but not limited to sleep, physical activity and sports involvement, and body mass index, and how these measures vary across demographic groups. This paper outlines how the physical health of the ABCD sample corresponds with that of the US population and highlights important avenues for health disparity research. This manuscript will provide important information for ABCD users and help guide analyses investigating physical health as it pertains to adolescent and young adult development.


title: Evolution of a minimal cell

abstract: Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life. Here, we report on how a synthetically constructed minimal cell contends with the forces of evolution compared to a non-minimized cell from which it was derived. Genome streamlining was costly, but 80% of fitness was regained in 2000 generations. Although selection acted upon divergent sets of mutations, the rates of adaptation in the minimal and non-minimal cell were equivalent. The only apparent constraint of minimization involved epistatic interactions that inhibited the evolution of cell size. Together, our findings demonstrate the power of natural selection to rapidly optimize fitness in the simplest autonomous organism, with implications for the evolution of cellular complexity.

category: evolutionary biology


title: Bayesian calibration, process modeling and uncertainty quantification in biotechnology

abstract: AO_SCPLOWBSTRACTC_SCPLOWHigh-throughput experimentation has revolutionized data-driven experimental sciences and opened the door to the application of machine learning techniques. Nevertheless, the quality of any data analysis strongly depends on the quality of the data and specifically the degree to which random effects in the experimental data-generating process are quantified and accounted for. Accordingly calibration, i.e. the quantitative association between observed quantities with measurement responses, is a core element of many workflows in experimental sciences. Particularly in life sciences, univariate calibration, often involving non-linear saturation effects, must be performed to extract quantitative information from measured data. At the same time, the estimation of uncertainty is inseparably connected to quantitative experimentation. Adequate calibration models that describe not only the input/output relationship in a measurement system, but also its inherent measurement noise are required. Due to its mathematical nature, statistically robust calibration modeling remains a challenge for many practitioners, at the same time being extremely beneficial for machine learning applications. In this work, we present a bottom-up conceptual and computational approach that solves many problems of understanding and implementing non-linear, empirical calibration modeling for quantification of analytes and process modeling. The methodology is first applied to the optical measurement of biomass concentrations in a high-throughput cultivation system, then to the quantification of glucose by an automated enzymatic assay. We implemented the conceptual framework in two Python packages, with which we demonstrate how it makes uncertainty quantification for various calibration tasks more accessible. Our software packages enable more reproducible and automatable data analysis routines compared to commonly observed workflows in life sciences. Subsequently, we combine the previously established calibration models with a hierarchical Monod-like differential equation model of microbial growth to describe multiple replicates of Corynebacterium glutamicum batch microbioreactor cultures. Key process model parameters are learned by both maximum likelihood estimation and Bayesian inference, highlighting the flexibility of the statistical and computational framework.


title: Rewiring the Three-Carbon Metabolism Abrogates Multiple MAPK-Induced Cellular Dysfunctions During Metabolic Disorder

abstract: Loss of membrane raft integrity, metabolic dysregulation and inflammation are hallmarks of chronic diseases and aging. It is not well understood how the stress response itself may contribute to the manifestation of these common traits. To explore this question, we screened the model organism S. cerevisiae, for the secretion of glycosylphosphatidylinositol-anchored proteins (GPI-APs) as a proxy for membrane raft instability. It is shown here that the multiple cellular dysfunctions previously described for a defect in the methylation pathway for phosphatidyl choline (PC) synthesis (opi3{Delta}) are linked with GPI-APs secretion. They collectively result from the sustained activation of the mitogen-activated protein kinase (MAPK) Hog1p. Through modifying the dihydroxyacetone phosphate / glycerol-3-phosphate ratio, activated MAPK promotes phospholipid gene de-repression and interferes with GPI anchor synthesis. Rewiring the three carbon metabolism, namely by deleting the mitochondrial glycerol phosphate dehydrogenase, abrogated the opi3{Delta} mutant pleiotropic phenotypes identifying key targets to counteract MAPK-induced cellular dysfunctions.


title: Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells

abstract: The migration of lymphatic endothelial cells (LECs) is key for the development of the complex and vast lymphatic vascular network that pervades most of the tissues in an organism. In zebrafish, arterial intersegmental vessels together with chemokines have been shown to promote lymphatic cell migration from the horizontal myoseptum (HM). Here we found that LECs departure from HM coincides with the emergence of mural cells around the intersegmental arteries, raising the possibility that arterial mural cells promote LEC migration. Our live imaging and cell ablation experiments revealed that LECs migrate slower and fail to establish the lymphatic vascular network in the absence of arterial mural cells. We determined that mural cells are a source for the C-X-C motif chemokine 12 (Cxcl12a and Cxcl12b) and vascular endothelial growth factor C (Vegfc). We showed that ERK, a downstream component of Vegfc-Vegfr3 singling cascade, is activated in migrating LECs and that both chemokine and growth factor signalling is required for the robust migration. Furthermore, Vegfc-Vegfr3 has a pro-survival role in LECs during the migration. Together, the identification of mural cells a source for signals that guide LEC migration and survival will be important in the future design for rebuilding lymphatic vessels in the disease contexts.


title: Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells

abstract: Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.


title: Spike protein cleavage-activation mediated by the SARS-CoV-2 P681R mutation: a case-study from its first appearance in variant of interest (VOI) A.23.1 identified in Uganda

abstract: Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences.

category: microbiology


title: Adaptive introgression and de novo mutations increase access to novel fitness peaks on the fitness landscape during a vertebrate adaptive radiation

abstract: Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 SNPs that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation.


title: The first genotypic fitness network in a vertebrate reveals that hybridization increases access to novel fitness peaks


title: Hybridization alters the shape of the genotypic fitness landscape, increasing access to novel fitness peaks during adaptive radiation


title: Logical modeling: Combining manual curation and automated parameterization to predict drug synergies

abstract: Treatment with drug combinations carries great promise for personalized therapy. We have previously shown that drug synergies targeting cancer can manually be identified based on a logical framework. We now demonstrate how automated adjustments of model topology and logic equations can greatly reduce the workload traditionally associated with logical model optimization. Our methodology allows the exploration of larger model ensembles that all obey a set of observations. We benchmark synergy predictions against a dataset of 153 targeted drug combinations. We show that well-performing manual models faithfully represent measured biomarker data and that their performance can be outmatched by automated parameterization using a genetic algorithm. The predictive performance of a curated model is strongly affected by simulated curation errors, while data-guided deletion of a small subset of edges can improve prediction quality. With correct topology we find some tolerance to simulated errors in the biomarker calibration data. With our framework we predict the synergy of joint inhibition of PI3K and TAK1, and further substantiate this prediction with observation in cancer cell cultures and in xenograft experiments.

category: systems biology


title: Blocking mitophagy does not improve fuel ethanol production in Saccharomyces cerevisiae

abstract:
Ethanol fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Shiroma and co-workers (2014) showed that blocking mitophagy by deletion of the mitophagy specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no differences in CO2 production, final ethanol titres or cell viability were observed after five rounds of ethanol fermentation, cell recycling and acid treatment, as commonly performed in sugarcane biorefineries. To test if S. cerevisiaes strain background influences this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial) and BY (laboratory), with and without a functional ATG32 gene, under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability and ethanol titres, among the three strains, we could not observe any improvement in fermentation performance related to the blocking of mitophagy. We conclude with caution that results obtained with Ginjo sake yeast is an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation.

ImportanceBioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is very well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease by-product formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. Shiroma and co-workers previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.12% increase in final ethanol titres during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to an improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.


title: B1-type cyclins control microtubule organization during cell division in Arabidopsis

abstract: Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support an important role in the regulation of GIP1 by CYCB1s.


title: Specialization of chromatin-bound nuclear pore complexes promotes yeast aging

abstract: The nuclear pore complex (NPC) mediates nearly all exchanges between nucleus and cytoplasm, and changes composition in many species as the organism ages. However, how these changes arise and whether they contribute themselves to aging is poorly understood. We show that in replicatively aging yeast cells attachment of DNA circles to NPCs drives the displacement of the NPCs nuclear basket and cytoplasmic complexes. Remodeling of the NPC resulted from the regulation of basket components by SAGA, rather than from damages. These changes affected NPC interaction with mRNA export factors, without affecting the residence of import factors or engaging the NPC quality control machinery. Mutations preventing NPC remodeling extended the replicative lifespan of the cells. Thus, our data indicate that DNA circles accumulating in the mother cell drive aging at least in part by triggering NPC specialization. We suggest that antagonistic pleiotropic effects of NPC specialization are key drivers of aging.


title: Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region

abstract: The N-terminus of S. aureus staphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SC{middle dot}ProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1[->]R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D with KD ~50 - 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1[->]R7) bound Frag D with KD ~7 - 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1[->]R7) for Frag D binding, with a 1:3 stoichiometry and KD ~7 - 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1[->]R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.

category: biochemistry


title: Optogenetic tools for public goods control in Saccharomyces cerevisiae

abstract:
Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light-control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in Saccharomyces cerevisiae to perturb both intercellular and interspecies interactions.

ImportanceRecent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition and resilience of microbial communities. In order to better understand the role of these interactions in governing community development it is critical to be able to alter them in a controlled manner. Optogenetically-controlled interactions offer advantages over static perturbations or chemically-controlled interactions as light can be manipulated in space and time and doesnt require the addition of nutrients or antibiotics. Here we report a system for rapidly achieving light-control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing Saccharomyces cerevisiae and serves as a proof-of-principle for implementing this approach in other consortia.


title: Prefrontal stimulation prior to motor sequence learning alters multivoxel patterns in the striatum and the hippocampus

abstract: Motor sequence learning (MSL) is supported by dynamical interactions between hippocampal and striatal networks that are thought to be orchestrated by the prefrontal cortex. In the present study, we tested whether individually-tailored theta-burst stimulation of the dorsolateral prefrontal cortex (DLPFC) prior to MSL, can modulate multivoxel response patterns in the stimulated cortical area, the hippocampus and the striatum. Response patterns were assessed with multivoxel correlation structure analyses of functional magnetic resonance imaging data acquired during task practice and during resting-state scans before and after learning/stimulation. Results revealed that, across stimulation conditions, MSL induced greater modulation of task-related DLPFC multivoxel patterns than random practice. A similar learning-related modulatory effect was observed on sensorimotor putamen patterns under inhibitory stimulation. Furthermore, MSL as well as inhibitory stimulation affected (posterior) hippocampal multivoxel patterns at post-intervention rest. Exploratory analyses showed that MSL-related brain patterns in the posterior hippocampus persisted into post-learning rest preferentially after inhibitory stimulation. These results collectively show that prefrontal stimulation can alter multivoxel brain patterns in deep brain regions that are critical for the MSL process. They also suggest that stimulation influenced early offline consolidation processes as evidenced by a stimulation-induced modulation of the reinstatement of task pattern into post-learning wakeful rest.


title: Transcriptional profiling of sequentially generated septal neuron fates

abstract: The septum is a ventral forebrain structure known to regulate innate behaviors. During embryonic development, septal neurons are produced in multiple proliferative areas from neural progenitors following transcriptional programs that are still largely unknown. Here, we use a combination of single cell RNA sequencing, histology and genetic models to address how septal neuron diversity is established during neurogenesis. We find that the transcriptional profiles of septal progenitors change along neurogenesis, coinciding with the generation of distinct neuron types. We characterize the septal eminence, a spatially distinct and transient proliferative zone composed of progenitors with distinctive molecular profiles, proliferative capacity and fate potential compared to the rostral septal progenitor zone. We show that Nkx2.1-expressing septal eminence progenitors give rise to neurons belonging to at least three morphological classes, born in temporal cohorts that are distributed across different septal nuclei in a sequential fountain-like pattern. Our study provides insight into the molecular programs that control the sequential production of different neuronal types in the septum, a structure with important roles in regulating mood and motivation.


title: Design, synthesis, and characterization of mG2P026 as a high contrast PET imaging ligand for metabotropic glutamate receptor 2

abstract: An array of triazolopyridines based on JNJ-46356479 (6) were synthesized as potential PET imaging ligands for metabotropic glutamate receptor 2 (mGluR2) in the brain. The selected candidates 8-11 featured an enhanced positive allosteric modulator (PAM) activity (37-fold max.) and an apparent mGluR2 agonist activity (25-fold max.) compared to compound 6. Radiolabeling of compounds 8 and 9 (also named mG2P026) was achieved via the Cu(I)-mediated radiofluorination in the automated TRACERLabTM FXF-N platform. Both [18F]8 and [18F]9 were obtained with satisfactory radiochemical yields (> 5%, non-decay corrected), high molar activity (> 180 GBq/mol), and excellent chemical and radiochemical purities (> 98%). Preliminary characterization of [18F]8 and [18F]9 in rats confirmed their excellent brain permeability with [18F]9 showing better brain heterogeneity and favorable binding kinetics. Pretreatment with different classes of PAMs enhanced the radioactivity uptake for both [18F]8 and [18F]9 at the regions of interest by 20.3-40.9% and 16.7-81.6%, respectively, due to their pharmacological effects. Further evaluation of [18F]9 in a nonhuman primate confirmed its superior brain heterogeneity in mapping mGlu2 receptors and its higher specific binding than [18F]6. Pretreatment with 0.5 mg/kg BINA led (2) to an enhanced brain uptake of [18F]9 by 3% in high tracer uptake regions that was consistent with the rat studies. Therefore, [18F]9 has the potential to be translated for human studies.


title: Global distribution and richness of Armillaria species inferred from public databases and amplicon sequencing datasets

abstract: Armillaria is a globally distributed fungal genus most notably recognized as economically important plant pathogens that are found predominantly in forest and agronomic systems. The genus sensu lato has more recently received attention for its role in woody plant decomposition and in mycorrhizal symbiosis with specific plants. Previous phylogenetic analyses suggest that around 50 species are recognized globally. Despite this previous work, no studies have analyzed the global species richness and distribution of the genus using data derived from fungal community sequencing datasets or barcoding initiatives. To assess the global diversity and species richness of Armillaria, we mined publicly available sequencing datasets derived from numerous primer regions for the ribosomal operon, as well ITS sequences deposited on Genbank, and clustered them akin to metabarcoding studies. Our estimates reveal that species richness ranges from 50 to 60 species, depending on whether the ITS1 or ITS2 marker is used. Eastern Asia represents the biogeographic region with the highest species richness. We also assess the overlap of species across geographic regions and propose some hypotheses regarding the drivers of variability in species diversity and richness between different biogeographic regions.


title: Global distribution and richness of Armillaria and related species inferred from public databases and amplicon sequencing datasets


title: A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

abstract: Argonaute proteins of the PIWI class complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

category: genetics


title: Frequent female song in blue tits: behavioural context suggests a role in intrasexual competition

abstract:
The blue tit (Cyanistes caeruleus) is an important avian model in evolutionary ecology (> 20,000 published scientific studies). Song, like in other songbird species, is generally described as a male trait and plays an important role in mate attraction and territory defence. Over the decades, there have been recurring reports of anecdotal female song but these have not led to any quantitative study of female song in blue tits. Our systematic sampling over three years revealed prolific female singing in a northern population of colour ringed blue tits. Daytime singing of females occurred throughout the breeding season during agonistic interactions, solo songs and alarm situations, and these contexts are similar to male song. Notably, female song was absent during the dawn chorus; the period around sunrise when males sing intensively just before mating. Female and male song overlapped substantially in acoustic structure (i.e. same song types, peak frequency or trill rates) but there were also significant differences in that females had smaller repertoires, shorter trills and lower vocal consistency. Differential selections pressures related with contextual (functional) differences in the role of male and female song could explain the observed differences in acoustic structure. The new finding of prolific female singing in such a well-studied species suggests we ought to revise our understanding of male and female vocal communication in this (and probably other) species. Identifying the selection pressures associated to the convergence versus divergence of male and female song may provide important insight in understanding birdsong evolution.

Lay summaryFemale song has been anecdotally reported in blue tits but there are no quantitative studies of singing behaviour, song structure or context. Here, we report frequent female singing in blue tits, associated mostly with agonistic interactions and alarm situations. Importantly, female song was not observed during dawn chorus, the period around sunrise when males sing intensively just before mating. Female and male song overlapped substantially in acoustic structure (i.e. same song types) but there were also significant differences (i.e. females sang with lower vocal consistency). We speculate that differences in context (function) of male and female song could explain the observed differences in acoustic structure. The new finding of prolific female singing in such a well-studied species suggests we ought to revise our understanding of male and female vocal communication.


title: Trends and Gaps on Philippine Scombrid Research: A bibliometric analysis

abstract: Philippine scombrids have been among the top priorities in fisheries research due primarily to their economic value worldwide. Assessment of the number of studies under general themes (diversity, ecology, taxonomy and systematics, diseases and parasites, and conservation) provides essential information to evaluate trends and gaps of research. This study presents a bibliometric evaluation of the temporal trends from 2000-2019 on Philippine scombrid research. Seven out of nine tuna species have SREA values equal to or higher than 1 with Thunnus albacares being the most researched tuna species. Twelve out of thirteen non-tuna species have SREA values less than one, thus indicating low research effort. It was apparent that there were significant differences in the number of studies in each thematic area except in  chemical analysis and  diseases and parasites between scombrid groups where there was low research effort observed. The research points at the uneven research distributions between scombrid groups in each thematic area. As locally published research are significantly behind foreign publications in terms of citation index, international collaborations by Filipino researchers have shown an increase research impact. Our study hopes to influence the local and international R&D agenda on Philippine scombrids and promote solidarity among nations towards its conservation and management.


title: Alevin-fry unlocks rapid, accurate, and memory-frugal quantification of single-cell RNA-seq data

abstract:
The rapid growth of high-throughput single-cell and single-nucleus RNA sequencing technologies has produced a wealth of data over the past few years. The available technologies continue to evolve and experiments continue to increase in both number and scale. The size, volume, and distinctive characteristics of these data necessitate the development of new software and associated computational methods to accurately and efficiently quantify single-cell and single-nucleus RNA-seq data into count matrices that constitute the input to downstream analyses.

We introduce the alevin-fry framework for quantifying single-cell and single-nucleus RNA-seq data. Despite being faster and more memory frugal than other accurate and scalable quantification approaches, alevin-fry does not suffer from the false positive expression or memory scalability issues that are exhibited by other lightweight tools. We demonstrate how alevin-fry can be effectively used to quantify single-cell and single-nucleus RNA-seq data, and also how the spliced and unspliced molecule quantification required as input for RNA velocity analyses can be seamlessly extracted from the same pre-processed data used to generate regular gene expression count matrices.


title: Metabolic and transcriptomic profiles of glioblastoma invasion revealed by comparisons between patients and corresponding orthotopic xenografts in mice

abstract: The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients tumors. The evolution of metabolic changes, followed in vivo longitudinally by 1H-Magnetic Resonance Spectroscopy (MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell-cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable for in vivo monitoring of progression in the non-enhancing infiltration zone of glioblastoma.

category: cancer biology


title: A novel post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons

abstract:
The causes of Parkinsons disease (PD) likely involve complex interactions between environmental factors and susceptibility genes with variants at the GBA locus encoding the glucocerebrosidase (GCase) enzyme being the strongest common genetic risk factor for PD. To understand GBA-related disease mechanisms, we used a novel multipart-enrichment proteomics and post-translational modification workflow to simultaneously identify peptides with phosphorylation, reversible cysteine-modifications or sialylated N-linked glycosylation, alongside unmodified proteins.

We identified large numbers of dysregulated proteins and post-translational modifications (PTMs) in heterozygous GBA-N370S PD patient induced pluripotent stem cells (iPSC)-derived dopamine neurons. Alterations in glycosylation status of lysosomal proteins identified disturbances in the autophagy-lysosomal pathway, concurrent with upstream perturbations in mTOR phosphorylation and activity in GBA-N370S iPSC-dopamine neurons. In addition, the strategy revealed several native and modified proteins encoded by PD-associated genes to be dysregulated in GBA-N370S neurons, enhancing our understanding of the wider role of GBA mutations on the neuronal proteome. Integrated pathway analysis of all datasets revealed impaired neuritogenesis in GBA-N370S PD iPSC-dopamine neurons and identified tau (MAPT) as a key mediator of this process. Using a functional assay, we confirmed neurite outgrowth deficits in GBA-N370S PD neurons and a central role for tau in this process. Furthermore, pharmacological restoration of GCase activity in GBA-N370S PD patient neurons rescued the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate novel neurodegeneration-associated pathways and identify phenotypes and potential drug targets in complex disease models.


title: Epileptiform GluN2B-driven excitation in hippocampus as a therapeutic target against temporal lobe epilepsy

abstract: NMDAR is an ionotropic glutamate receptor critically involved in excitatory synaptic transmission. The receptor properties are strongly determined by its subunit composition. One of the NMDAR subunits is GluN2B, which displays restricted and spatially different from other subunits expression in the mature brain. GluN2B-containing NMDARs are present in the hippocampus - a structure playing a major role in temporal lobe epilepsy (TLE). However, the contribution of GluN2B to pathophysiology of TLE has not been fully explored. Here, we report the functional alterations of GluN2B-containing NMDAR receptors in the hippocampus in distinct mouse models of temporal lobe epilepsy. In particular, we show the impact of GluN2B on excitatory feedback in granule cells. Based on these results, we propose a mechanism-oriented effective antiepileptic strategy that selectively antagonizes GluN2B-containing NMDARs with ifenprodil, a well-known GluN2B antagonist. Collectively, our research identifies GluN2B as one of the pivotal factors in pathogenesis of temporal lobe epilepsy and associated recurrent seizures. Furthermore, our study indicates the prospective antiepileptic properties of ifenprodil in TLE.


title: Electrical synapse asymmetry results from, and masks, neuronal heterogeneity

abstract: Electrical synapses couple inhibitory neurons across the brain, underlying a variety of functions that are modifiable by activity. Despite recent advances, many basic functions and contributions of electrical synapses within neural circuitry remain underappreciated. Among these is the source and impact of electrical synapse asymmetry. Using multi-compartmental models of neurons coupled through dendritic electrical synapses, we investigated intrinsic factors that contribute to synaptic asymmetry and that result in modulation of spike time between coupled cells. We show that electrical synapse location along a dendrite, input resistance, internal dendritic resistance, or directional conduction of the electrical synapse itself each alter asymmetry as measured by coupling between cell somas. Conversely, true synapse asymmetry can be masked by each of these properties. Furthermore, we show that asymmetry alters the spiking timing and latency of coupled cells by up to tens of milliseconds, depending on direction of conduction or dendritic location of the electrical synapse. These simulations illustrate that causes of asymmetry are multifactorial, may not be apparent in somatic measurements of electrical coupling, influence dendritic processing, and produce a variety of outcomes on spike timing of coupled cells. Our findings highlight aspects of electrical synapses that should be considered in experimental demonstrations of coupling, and when assembling networks containing electrical synapses.


title: Reliability of decision-making and reinforcement learning computational parameters

abstract: Computational models can offer mechanistic insight into cognition and therefore have the potential to transform our understanding of psychiatric disorders and their treatment. For translational efforts to be successful, it is imperative that computational measures capture individual characteristics reliably. To date, this issue has received little consideration. Here we examine the reliability of reinforcement learning and economic models derived from two commonly used tasks. Healthy individuals (N=50) completed a restless four-armed bandit and a calibrated gambling task twice, two weeks apart. Reward and punishment processing parameters from the reinforcement learning model showed fair-to-good reliability, while risk/loss aversion parameters from a prospect theory model exhibited good-to-excellent reliability. Both models were further able to predict future behaviour above chance within individuals. This prediction was better when based on participants own model parameters than other participants parameter estimates. These results suggest that reinforcement learning, and particularly prospect theory parameters, can be measured reliably to assess learning and decision-making mechanisms, and that these processes may represent relatively distinct computational profiles across individuals. Overall, these findings indicate the translational potential of clinically-relevant computational parameters for precision psychiatry.


title: Chemogenetic activation of midline thalamic nuclei fails to ameliorate memory deficits in two mouse models of Alzheimer's disease

abstract: One of the main features of Alzheimers disease is the progressive loss of memory, likely due to pathological changes within brain regions such as the hippocampus and entorhinal cortex. These structures are embedded within the extended memory circuit, an interconnected set of brain regions that are essential for episodic memory. The anterior thalamic nuclei (ATN) and thalamic nucleus reuniens (NRe) are both extensively and reciprocally connected with these important memory regions, so we sought to test the hypothesis that chemogenetically-enhancing neurotransmission through NRe and ATN would ameliorate memory deficits in two mechanistically-distinct mouse models of Alzheimers disease. Using the hAPP-J20 mouse model of amyloidopathy and the Tg4510 mouse model of tauopathy, we carried out stereotaxic injections of viral vectors to transduce hM3Dq (Gq)_mCherry into NRe or the anterio-dorsal/anterio-ventral nuclei of ATN, using mCherry as a control. At nine months (hAPP-J20) or six months (Tg4510) of age, mice underwent a behaviour battery of open field (OF), novel object recognition (NOR) and radial arm maze (RAM), with DREADD agonist 21 administered 30min prior to each behaviour test. Tissue was collected post-behaviour to confirm injection site and virus expression. Both Tg4510 and hAPP-J20 mice show marked hyperactivity in the OF, significant deficits in recognition memory, and a significant impairment in spatial reference and spatial working memory. Unexpectedly, chemogenetic activation of ATN or NRe did not significantly improve spatial memory impairments or reduce the observed hyperactivity, although NRe activation did modestly rescue recognition memory in J20 mice. This may be due to compensation elsewhere within the memory circuit, or that the pathological changes are too far advanced for behaviour reversal.


title: All-optical interrogation of neural circuits in behaving mice

abstract: Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such "all-optical" techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas - barrel cortex, visual cortex and hippocampus - using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.


title: PET imaging studies to investigate functional expression of mGluR2 using mG2P001

abstract: Metabotropic glutamate receptor 2 (mGluR2) has been extensively studied for the treatment of various neurological and psychiatric disorders. Understanding of the mGluR2 function is pivotal in supporting the drug discovery targeting mGluR2. Herein, the positive allosteric modulation of mGluR2 was investigated via the in vivo positron emission tomography (PET) imaging using 2-((4-(2-[11C]methoxy-4-(trifluoromethyl)phenyl)piperidin-1-yl)methyl)-1-methyl-1H-imidazo[4,5-b]pyridine ([11C]mG2P001).Distinct from the orthosteric compounds, pretreatment with the unlabeled mG2P001, a potent mGluR2 positive allosteric modulator (PAM), resulted in a significant increase instead of decrease of the [11C]mG2P001 accumulation in rat brain detected by PET imaging. Subsequent in vitro studies with [3H]mG2P001 revealed the cooperative binding mechanism of mG2P001 with glutamate and its pharmacological effect that contributed to the enhanced binding of [3H]mG2P001 in transfected CHO cells expressing mGluR2. The in vivo PET imaging and quantitative analysis of [11C]mG2P001 in non-human primates (NHPs) further validated the characteristics of [11C]mG2P001 as an imaging ligand for mGluR2. Self-blocking studies in primates enhanced accumulation of [11C]mG2P001 dose- and delivery-dependently. Altogether, these studies show that [11C]mG2P001 is a sensitive biomarker for mGluR2 expression and the binding is affected by the tissue glutamate concentration.


title: Distinct repertoire expansion patterns during affinity maturation of antigen responding camelid-derived single-domain VHH antibody

abstract:
Time-resolved antibody repertoire analysis is essential to elucidate the antibody maturation process during immunization. However, most analyses are merely snapshot comparisons of unimmunized and fully immunized animals. In this study, an alpaca was immunized with multiple antigen injections and its repertoire development was traced by high-throughput next-generation sequencing every week for 3 months. The use of alpaca-derived, single-domain antibody circumvents the problems associated with conventional antibodies including heavy-chain and light-chain matching and labor-intensive production. The sequences were processed into clusters and the antibodies were generated to determine whether the clusters included antigen-binding antibodies. For most of the antigen-responding clusters, repertoire development exhibited distinct time-dependent patterns compared with clusters unresponsive to antigens. Moreover, the sequences at the tips and root of the phylogenetic tree had strong and weak antigen-binding affinity, respectively. The foregoing characteristics could be exploited in the prediction of antigen-responding clusters and the physical properties of antibodies.

TeaserClusters of antigen-responding, single-domain antibodies exhibited distinct time-dependent repertoire development history

category: immunology


title: Distinct repertoire development history during affinity maturation of antigen-responding VHH antibody


title: Epigenetic regulation of frog innate immunity: Discovery of frog microRNAs associated with antiviral responses and ranavirus infection using a Xenopus laevis skin epithelial-like cell line

abstract: Epigenetic regulators such as microRNAs are emerging as conserved regulators of innate antiviral immunity in vertebrates, yet their roles in amphibian antiviral responses remain uncharacterized. We profiled changes in microRNA expressions in the Xenopus laevis skin epithelial-like cell line Xela DS2 in response to poly(I:C) - an analogue of double-stranded viral RNA and inducer of type I interferons - or frog virus 3 (FV3), an immunoevasive virus associated with amphibian mortality events. We sequenced small RNA libraries generated from untreated, poly(I:C)-treated, and FV3-infected cells. We detected 136 known X. laevis microRNAs and discovered 133 novel X. laevis microRNAs. Sixty-five microRNAs were differentially expressed in response to poly(I:C), many of which were predicted to target regulators of antiviral pathways such as cGAS-STING, RIG-I/MDA-5, TLR signaling, and type I interferon signaling, as well as products of these pathways (NF-{kappa}B-induced and interferon-stimulated genes). In contrast, only 49 microRNAs were altered by FV3 infection, fewer of which were predicted to interact with antiviral pathways. Interestingly, poly(I:C) treatment or FV3 infection downregulated transcripts encoding factors of the host microRNA biogenesis pathway. Our study is the first to suggest that host microRNAs regulate innate antiviral immunity in frogs, and sheds light on microRNA-mediated mechanisms of immunoevasion by FV3.


title: Rapid generation of homozygous fluorescent knock-in human cells using CRISPR/Cas9 genome editing and validation by automated imaging and digital PCR screening

abstract: We have previously described a protocol for genome engineering of mammalian cultured cancer cells with CRISPR/Cas9 to generate homozygous knock-ins of fluorescent tags into endogenous genes 1. Here, we are updating this protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high efficiency electroporation of optimized CRISPR/Cas9 reagents, screening of single cell derived clones by automated bright field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital PCR (dPCR) and automated data analysis. Compared to the original protocol 1, our current procedure (i) significantly increases the efficiency of tag integration, (ii) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (iii) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with dPCR. The increased efficiency of the new procedure reduces the number of clones that need to be analysed in- depth by more than ten-fold, and yields up to 20% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 days to 10 days during the overall [~]10 weeks procedure, allowing a single person to process up to 5 genes in parallel, assuming that validated reagents - e.g. PCR-primers, dPCR-assays, Western Blot antibodies - are available.


title: Promoting long-term forest landscape resilience in the Lake Tahoe basin

abstract: Rapid environmental changes expected in the 21st century challenge the resilience of wildlands around the world. The western portion of the Lake Tahoe basin (LTW) in California is an important ecological and cultural hotspot that is at risk of degradation from current and future environmental pressures. Historical uses, fire suppression, and a changing climate have created forest landscape conditions at risk of drought stress, destructive fire, and loss of habitat diversity. We prospectively modeled forest landscape conditions for a period of 100 years to evaluate the efficacy of five unique management scenarios in achieving desired landscape conditions across the 23,600 hectares of LTW. Management scenarios ranged from no management other than fire suppression to applying treatments consistent with historical fire frequencies and extent (i.e., regular and broadscale biomass reduction). We developed a decision support tool to evaluate environmental and social outcomes within a single framework to provide a transparent set of costs and benefits; results illuminated underlying mechanisms of forest resilience and provided actionable guidance to decision makers. Sixteen attributes were assessed in the model after assigning weights to each, derived through a survey of stakeholder priorities, so that the contribution of each attribute to evaluations of scenario performance was influenced by the combined priorities of stakeholders. We found that removing forest biomass across the landscape, particularly when accomplished using extensive fire-based removal techniques, led to highly favorable conditions for environmental quality and promoting overall landscape resilience. Environmental conditions resulting from extensive fire-based biomass removal also had nominal variation over time, in contrast with strategies that had less extensive and/or used physical removal techniques, namely thinning. Our analysis provided a transparent approach to data assessment, considering the priorities of stakeholders, to provide insights into the complexities of maintaining optimal conditions and managing landscapes to promote ecosystem resilience in a changing world.


title: Sensitive Detection of Site-wise Convergent Evolution in Large Protein Alignments with ConDor

abstract:
Evolutionary convergences are observed at all levels, from phenotype to DNA and protein sequences, and the changes observed at these different levels tend to be strongly correlated. Here we propose a simulation-based method to detect positions under convergent evolution in large protein alignments, without prior knowledge on the phenotype and environmental constraints. A phylogeny is inferred from the data and used in simulations to estimate the expected number of amino-acid changes in stable evolutionary constraints (null model) for each position. Similarly, we count the number of mutations towards the same amino acid in the data and test if they are occurring more often than expected.

We applied our method to two real datasets: HIV reverse transcriptase and fish rhodopsin, and to HIV-like simulated data. On the latter, with known convergent events and substitution model, we detected on average two third of these events, with a low fraction of false positives. With HIV data, one knows that drug resistance mutations (DRMs) are convergent. Even without any knowledge of patient treatment status, we retrieved more than 70% of positions corresponding to known DRMs. On the rhodopsin dataset, four substitutions are supposed to be convergent, as they change the maximum wavelength absorption of the photoreceptor and occurred several times independently during evolution. We detected three of them.

These results demonstrate the potential of the method to target specific mutations to be further studied experimentally or, for example, using a nonsynonymous/synonymous rate ratio approach. Our software named ConDor is available at http://condor.pasteur.cloud.


title: Toward an understanding of the chemical ecology of alternative reproductive tactics in the bulb mite (Rhizoglyphus robini)

abstract:
BackgroundUnder strong sexual selection, certain species evolve distinct intrasexual, alternative reproductive tactics (ARTs). In many cases, ARTs can be viewed as environmentally-cued threshold traits, such that ARTs coexist if their relative fitness alternates over the environmental cue gradient. Surprisingly, the chemical ecology of ARTs has been underexplored in this context. To our knowledge, no prior study has directly quantified pheromone production for ARTs in a male-polymorphic species. Here, we used the bulb mite--in which males are either armed fighters that kill conspecifics, or unarmed scramblers--as a model system to gain insight into the role of pheromones in the evolutionary maintenance of ARTs. Given that scramblers forgo investment into weaponry, we tested whether scramblers produce higher pheromone quantities than fighters, which would improve the fitness of the scrambler phenotype, e.g. through female mimicry to avoid aggression from competitors. To this end, we sampled mites from a rich and a poor nutritional environment and quantified their production of the female sex pheromone -acaridial through gas chromatography analysis.

ResultsWe found a positive relationship between pheromone production and body size, but males exhibited a steeper slope in pheromone production with increasing size than females. Females exhibited a higher average pheromone production than males. We found no significant difference in slope of pheromone production over body size between fighters and scramblers. However, scramblers reached larger body sizes and higher pheromone production than fighters, providing some evidence for a potential female mimic strategy adopted by large scramblers. Pheromone production was significantly higher in mites from the rich nutritional environment than the poor environment.

ConclusionFurther elucidation of pheromone functionality in bulb mites, and additional inter-and intrasexual comparisons of pheromone profiles are needed to determine if the observed intersexual and intrasexual differences in pheromone production are adaptive, if they are a by-product of allometric scaling, or diet-mediated pheromone production under weak selection. We argue chemical ecology offers a novel perspective for research on ARTs and other complex life-history traits.


title: Protracted speciation under the state-dependent speciation and extinction approach

abstract: How long does speciation take? What are the speciation processes that generated a species group? The answers to these important questions in evolutionary biology lie in the genetic difference not only among species, but also among lineages within each species. With the advance of genome sequencing in non-model organisms and the statistical tools to improve accuracy in inferring evolutionary histories among recently diverged lineages, we now have the lineage-level trees to answer these questions. However, we do not yet have an analytical tool for inferring speciation processes from these trees. What is needed is a model of speciation processes that generates both the trees and species identities of extant lineages. The model should allow calculation of the probability that certain lineages belong to certain species and have an evolutionary history consistent with the tree. Here we propose such a model and test the model performance on both simulated data and real data. We show that maximum likelihood estimates of the model are highly accurate and give estimates from real data that generate patterns consistent with observations. We discuss how to extend the model to account for different rates and types of speciation processes across lineages in a species group. By linking evolutionary processes on lineage level to species level, the model provides a new phylogenetic approach to study not just when speciation happened, but how speciation happened.


title: Penetrance estimation of SORL1 loss-of-function variants using a family-based strategy adjusted on APOE genotypes suggest a non-monogenic inheritance

abstract: For complex disorders, estimating the age-related penetrance associated with rare variants of strong effect is essential before a putative use for genetic counseling or disease prevention. However, rarity and co-occurrence with other risk factors make such estimations difficult. In the context of Alzheimer disease, we present a survival model to estimate the penetrance of SORL1 rare (allele frequency< 1%) Loss-of-Function variants (LoF) while accounting for APOE-{varepsilon}4, the main risk factor (allele frequency[~] 14% in Caucasians). We developed an efficient strategy to compute penetrance estimates accounting for both common and rare genetic variants based on available penetrance curves associated with common risk factors and using incomplete pedigree data to quantify the additional risk conferred by rare variants. Our model combines: (i) a baseline for non-carriers of SORL1 LoF variants, stratified by APOE genotypes derived from the Rotterdam study and (ii) an age-dependent proportional hazard effect for SORL1 LoF variants estimated from pedigrees with a proband carrying such a variant. We embed this model into an Expectation-Maximisation algorithm to accommodate for missing genotypes. Confidence intervals were computed by bootstraps. To correct for ascertainment bias, proband phenotypes were omitted. We obtained penetrance curves associated with SORL1 LoF variants at the digenic level. By age 70, we estimate a 100% penetrance of SORL1 LoF variants only among APOE-{varepsilon}4{varepsilon}4 carriers, while penetrance is 56%[40% - 72%] among {varepsilon}4 heterozygous carriers and 37%[26% - 51%] among {varepsilon}4 non-carriers. We conclude that rare SORL1 LoF variants should not be used for genetic counseling regardless of the APOE status.


title: RFU derived LRs for activity level assignments usingBayesian Networks

abstract: A comparative study has been carried out, comparing two different methods to estimate activity level likelihood ratios (LRa) using Bayesian Networks. The first method uses the sub-source likelihood ratio (log10LR{phi}) as a  quality indicator. However, this has been criticised as introducing potential bias from population differences in allelic proportions. An alternative method has been introduced that is based upon the total RFU of a DNA profile that is adjusted using the mixture proportion (Mx) which is calculated from quantitative probabilistic genotyping software (EuroForMix). Bayesian logistic regressions of direct transfer data showed that the two methods were comparable. Differences were attributed to sampling error, and small sample sizes of secondary transfer data. The Bayesian approach facilitates comparative studies by taking account of sampling error; it can easily be extended to compare different methods.