title: A revisit to universal single-copy genes in bacterial genomes

abstract: Universal single-copy genes (USCGs) are widely used for species classification and taxonomic profiling. Despite many studies, our understanding of USCGs in bacterial genomes might be out of date, especially how different the USCGs are in different studies, how well a set of USCGs can distinguish two bacterial species, how capable each USCG is in discriminating species, whether USCGs can separate different strains of a bacterial species, and so on. To fill the gaps, we studied USCGs in the most updated sequenced bacterial genomes. We showed that different USCG sets are quite different while coming from highly similar functional categories. We also found that although USCGs occur once in almost all bacterial genomes, each USCG does occur multiple times in certain genomes. We demonstrated that USCGs are reliable markers to distinguish different species and different strains of a bacterial species. The USCG ArgS is shown to be more powerful than other USCGs to tell different species in the same genus and different strains from the same species apart. Our study shed new light on the evolution of USCGs, which will facilitate their applications in phylogenomic and metagenomic studies.


title: Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade and the role of insertions and extensions in diversification

abstract: Ribonucleotide reductases (RNRs) are used by all organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6,779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade O. We employed small-angle X-ray scattering (SAXS), cryogenic-electron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from Synechococcus phage S-CBP4 and report the most minimal RNR architecture to-date. Using the catalytic barrel as a starting point for diversification, we traced the evolutionarily relatedness of insertions and extensions that confer the diversity observed in the RNR family. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study.

category: bioinformatics


title: GeneVector: Identification of transcriptional programs using dense vector representations defined by mutual information.

abstract: Deciphering individual cell phenotypes from cell-specific transcriptional processes requires high dimensional single cell RNA sequencing. However, current dimensionality reduction methods aggregate sparse gene information across similar cells, without directly measuring the relationships that exist between genes. By performing dimensionality reduction with respect to gene co-expression, low-dimensional features can model these gene-specific relationships and leverage shared signal to overcome sparsity. We describe GeneVector, a scalable framework for dimensionality reduction implemented as a vector space model using mutual information between gene expression. Unlike other methods, including principal component analysis and variational autoencoders, GeneVector uses latent space arithmetic in a lower dimensional gene embedding to identify transcriptional programs and classify cell types. Using four single cell RNA-seq datasets, we show that GeneVector was able to capture phenotype-specific pathways, perform batch effect correction, interactively annotate cell types, and identify pathway variation with treatment over time. GeneVector is available as an open source python package at https://github.com/nceglia/genevector.


title: Self-consistent solution to the Flory-Huggins model and applications in protein liquid-liquid phase separation

abstract: A self-consistent analytical solution for binodal concentrations of the 2-component Flory-Huggins model is derived. We show that this form extends the validity of the Ginzburg-Landau expansion away from the critical point to cover the whole phase space, from which an exponential scaling law of the dilute phase binodal concentration emerges. This approach allows us to fit experimental protein liquid-liquid phase separation data and extract parameters including the effective chain length and the solute-solvent interaction energy. We demonstrate explicitly the power of this approach by quantifying the interaction energy contributions of individual amino acids. This analytical framework can serve as a new way to decode the protein sequence grammar for liquid-liquid phase separation.

category: biophysics


title: TFIIF interacts with the Topoisomerase VI complex and selectively controls the expression of genes encoding PPR proteins involved in organellar RNA editing in Arabidopsis.

abstract: Communication between organelles and the nucleus is referred to as anterograde (nucleus to organelle) and retrograde (organelle to nucleus) signalling. In plants, the pentatricopeptide repeat (PPR) proteins represent a large family of nuclear-encoded proteins that are required for post-transcriptional control of chloroplast and mitochondria gene expression, and hence play a central role in the nuclear anterograde control of organelle genome expression. How PPR gene expression is controlled and regulated by retrograde signals is, however, still unknown. Here, we report a significant role for the general transcription factor TFIIF -subunit (TFIIF) in controlling PPR gene expression in Arabidopsis. First, we found that TFIIF interacts with the BIN4 subunit of the Topoisomerase VI (Topo VI). Transcriptome analysis of TFIIF and Topo VI mutant lines then revealed that many PLS-type PPR genes involved in RNA editing are reciprocally controlled by TFIIF and Topo VI. The misexpression of CLB19 and DYW1 genes in two allelic tfIIf mutants was associated with editing impairments in their plastid target RNAs rpoA and ndhD, respectively. Interestingly, we also detected a change in NDH activity in tfIIf plants. We also show that TFIIF and Topo VI coordinate the expression of NDH subunits encoded by the nuclear and plastid genomes. These results reveal the crucial role of the nuclear TFIIF and Topo VI complexes in controlling plastid genome expression at multiple levels of regulation, including the particular regulation of PPR gene expression.

category: plant biology


title: A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics

abstract: Accurate quantitation of antibody is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

category: immunology


title: Expression of the Bacillus thuringiensis vip3A insecticidal toxin gene is activated at the onset of stationary phase by VipR, an autoregulated transcription factor

abstract:
The Vegetative insecticidal protein Vip3A is produced by some Bacillus thuringiensis strains from the mid-log growth phase to sporulation. Although Vip3A is important for the entomopathogenicity of B. thuringiensis, the vip3A gene regulation is unknown. In the B. thuringiensis kurstaki HD1 strain, vip3A is carried by the pBMB299 plasmid, which is absent in the closely related strain B. thuringiensis kurstaki HD73. Using a transcriptional fusion between the vip3A promoter and lacZ, we observed that the HD73 strain is unable to express vip3A. This result suggests that a specific regulator is required for vip3A expression. Assuming that the regulator gene is located on the same plasmid as vip3A, we transferred the pBMB299 from the HD1 strain to the HD73 strain. We found that Vip3A was produced in the HD73 strain containing pBMB299, suggesting that the regulator gene is located on this plasmid. Using this heterologous host and promoter-lacZ transcription fusions, we confirmed that VipR is essential to activate vip3A expression at the onset of stationary phase. We demonstrated that vipR transcription is positively autoregulated and the determination of the vipR and vip3A promoters pinpointed a putative VipR target upstream from the Sigma A-specific -10 region of these two promoters. Surprisingly, this conserved sequence was also found upstream of cry1I and cry2 genes. Finally, we showed that vip3A and vipR expression is drastically increased in a {Delta}spo0A mutant unable to initiate sporulation. In conclusion, we have characterized a novel regulator involved in the entomopathogenic potency of B. thuringiensis through a sporulation-independent pathway.

ImportanceThe insecticidal properties of Bacillus thuringiensis are mainly due to Cry toxins which form a crystalline inclusion during sporulation. However, other proteins participate in the pathogenicity of the bacterium, notably the Vip3A toxins that are produced from vegetative growth to sporulation. The VipR regulator that activates vip3A gene expression at the onset of stationary phase is positively autoregulated and analysis of the promoter region of the vip3A and vipR genes reveals the presence of a highly conserved DNA sequence. This possible VipR target sequence is also found upstream of the cry2A and cry1I genes, suggesting that Cry toxins can be produced before the bacteria enter sporulation. Such a result could allow us to better understand the role of Cry and Vip3A toxins during the B. thuringiensis infectious cycle in insects, in addition to the primary role of the Cry toxins in the toxemia caused by ingestion of crystals.

category: microbiology


title: 5' transgenes drive leaky expression of 3' transgenes in inducible bicistronic vectors

abstract: Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences in a cell type of interest provide an invaluable tool for understanding the molecular regulation of cellular functions. DNA recombination employing the Cre-lox system is commonly used as a molecular switch for inducing the expression of recombinant proteins encoded within a bicistronic cassette. In such an approach, the two protein-coding sequences are separated by a 2A peptide or internal ribosome entry site (IRES), and expression is designed to be strictly Cre-dependent by using a lox-STOP-lox cassette or flip-excision (FLEX) switch. However, low-level or  leaky expression of recombinant proteins is often observed in the absence of Cre activity, potentially compromising the utility of this approach. To investigate the mechanism of leaky gene expression, we generated pCAG-lox-GFP-STOP-lox-Transgene A-2A-Transgene B vectors, which are designed to express nuclear-targeted GFP in the absence of Cre, and express both transgenes A and B after Cre-mediated recombination. We found that cells transfected with these bicistronic vectors exhibited low-level Cre-independent expression specifically of the transgene positioned 3' of the 2A peptide. We observed similar results in vivo by viral transduction of the adult mouse cerebral cortex with AAV-mutagenesis of putative transcription factor binding sites that the 5' transgene confers promoter-like activity that drives expression of the 3' transgene. Finally, we demonstrate that inclusion of an additional lox-STOP-lox cassette between the 2A sequence and 3' transgene dramatically reduces the extent of Cre-independent leaky gene expression. Our findings highlight that caution should be applied to the use of Cre-dependent bicistronic constructs when tight regulation of transgene expression is desired and provide a guide to preventing leaky gene expression when the expression of more than one protein is required.

category: molecular biology


title: Identification of Transient Intermediates During Spliceosome Activation by Single Molecule Fluorescence Microscopy

abstract:
Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA (snRNA) 3-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of yeast spliceosomes. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NineTeen Complex, NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both [Formula] and provide a kinetic framework for its formation and transformation during activation. Prior to [Formula] assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. Together our data suggest a Hfq-like function for Lsm2-8 in maintaining U2/U6 helix II integrity before it can be transferred to the NTC by transient formation of the [Formula] spliceosome.

Significance StatementThe spliceosome active site is created de novo during activation and involves numerous conformational and compositional changes. Here, we define a kinetic pathway for yeast spliceosome activation using single molecule fluorescence that includes transient intermediates not previously identified. Real-time measurements allow us to uncover rapid, reversible sampling interactions of the NineTeen Complex (NTC) that may prevent its accumulation on defective spliceosomes. By analogy with bacterial Hfq, we propose that the homologous Lsm2-8 proteins stabilize U2/U6 helix II during activation before the helix is transferred to the NTC in a short-lived spliceosome containing both Lsm2-8 and the NTC. Our data demonstrate how single molecule studies of activation can reveal kinetically-competent intermediates and complement cryo-EM studies of stalled or inhibited complexes.

category: biochemistry


title: Green emitting carbon quantum dots (GCQDs) to probe endocytic pathways in cells; for tissue and in vivo bioimaging

abstract: Small sized, carbon-based organic nanoparticles have recently gained attention due their advantage of biocompatibility, photostability and biological non-toxicity as compared to their inorganic counterparts. Herein, a new class of small (5-8 nm), green emitting fluorescent carbon quantum dots (GCQDs) were synthesized using organic substrates like citric acid and ascorbic acid in aqueous solvent containing water and ethanol. The very small size and bright green photoluminescence prompted their use for both in vitro and in vivo bioimaging. GCQDs were uptaken via clathrin mediated pathways in mouse kidney and liver primary cells. Similarly, they showed active uptake and distribution in the zebrafish embryo model system. The optical tunability and surface modification properties of these GCQDs provide a platform to be explored for them to emerge as a new class of targeted bioimaging entities, as well as tools for biomedical applications.

category: bioengineering


title: A retrospective molecular investigation of selected pigeon viruses between 2018-2021 in Turkey

abstract: A recent first detection of pigeon aviadenovirus-1 and pigeon circovirus co-infection associated with Young Pigeon Disease Syndrome (YPDS) in a pigeon flock in Turkey, prompted a study focused on documenting the distribution of Pigeon aviadenovirus (PiAdV-1 and PiAdV-2), Pigeon circovirus (PiCV), Columbid alphaherpesvirus 1 (pigeon herpesvirus (PiHV)) and Fowl aviadenovirus (FAdV) in the country. These viruses were selected as they are associated with severe disease in pigeons across the world. A total of 192 cloacal swabs were collected from young (<1 year old) pigeons from 16 different private pigeon flocks across Turkey, between 2018 and 2021 as part of routine diagnostic sampling. PiCV genetic material was the most frequently detected 4/16 (25%), PiAdV-1 and CoHV-1 DNA were both found in one flock each, while neither PiAdV-2 and FAdV were detected in any of the studied pigeon flocks. PiCV and PiHV genetic material were both detected in the same pigeon flocks cloacal samples as a co-infection with the identification of PiHV being a first in Turkey.


title: Genetic elements orchestrating Lactobacillus crispatus glycogen metabolism in the vagina

abstract: Glycogen in the female lower reproductive tract is a major carbon source for vaginal colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously we identified the pullulanase gene pulA in Lactobacillus crispatus, correlating with its ability to autonomously utilize glycogen for growth. Here we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacilllus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the pullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective pullulanase variant or in a pullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available human vaginal metagenome dataset indicates that 15 out of 272 samples, containing a Lactobacillus crispatus pulA gene, contain a defective variant of this gene. Another 23 out of 272 samples show large deletions or transposon insertions. Taken together, these results demonstrate that both environmental as well as genetic factors explain variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.


title: Anatomical and Palynological Investigation within flora of Family Asteraceae

abstract: A comprehensive microscopic investigation of the leaf epidermis, as well as anatomical and palynological research of selected Asteraceae species from the flora of Havali (Kahutta) Azad Jammu and Kashmir (AJK) Pakistan, was carried out. This study includes 11 plant species comprising of Senecio jacobea, Leucanthemum vulgare, Halianthus annuus, Erigeron bonariensis, Achillea millifolium, Halianthus Linnaeus, Taraxacum officinale, Anaphalius nepalensis, Erigeron Canadensis, and Tagetes erecta. All the species studied were amphistomatic, with four different forms of stomata i.e tetracytic, anomocytic, anisocytic, and tricytic. The main stomatal type was tetracytic, followed by anomocytic. The abaxial epidermis has a higher stomatal density than the adaxial epidermis. Highest stomatal density was present Tagetes erecta while lowest is present in Conyza canadensis. In lower epidermis stomatal index was higher in Taraxacum officinale followed by Halianthus annuus and Tagetes erecta while in upper epidermis highest index was shown by Halianthus annuus. For this study species of family Acteraceae were properly collected. Furthermore, variation in pollen can be seen. List of palynomorph which includes family name, botanical name, local name, English name, flower colour, season and pollen description for the logical arrangement of these species. The logical ordering of these species was guided by dust characteristics such as form and pollen morphology. Palynological data has been found to be too big for taxonomists to calculate and make appropriate observations on their findings.


title: A Chalcone Synthase-Like Bacterial Protein Catalyzes Heterocyclic C-Ring Cleavage of Naringenin to Alter Bioactivity Against Nuclear Receptors in Colonic Epithelial Cells

abstract: The human gut microbiota contributes enzymatic functions that are unavailable to host cells and play crucial roles in host metabolism, nutrient processing and regulating immune functions. As dietary compounds that are only partially absorbed, flavonoids are available for metabolism by gut microbiota, leading to diverse bioactive products. Combining prediction of enzyme promiscuity, metabolomics, and in vitro model systems, we identified a bacterial enzyme that can catalyze heterocyclic C-ring cleavage of naringenin. Culture experiments using a wild-type and mutant strain of Bacillus subtilis confirmed that the enzyme is a chalcone synthase-like polyketide synthase. The prediction-validation methodology developed in this work could be used to systematically characterize the products of gut bacterial flavonoid metabolism and identify the responsible enzymes and species. Further, we demonstrated that naringenin and its ring cleavage metabolites differentially engage the AhR and NR4A in intestinal epithelial cells. Our results suggest that the abundance of selected gut bacterial species impacts the profile of bioactive flavonoids and flavonoid-derived metabolites and thereby influences inflammatory responses in the intestine. These results are significant for understanding the mechanisms of gut microbiota-dependent effects of dietary flavonoids.


title: Placental mammal derived microRNAs alter pathways in the endometrial epithelia important for endometrial function.

abstract: We tested the hypothesis that a panel of placental mammal-specific miRNAs and their targets play important to establish receptivity to implantation and their dysregulated expression may be a feature in women with early pregnancy loss. Relative expression levels of miR-340-5p, -542-3p, and -671-5p all increased following treatment of Ishikawa cells with progesterone (10 g/ml) for 24 hrs (p < 0.05). RNA sequencing of these P4-treated cells identified co-ordinate changes to 6,367 transcripts of which 1713 were predicted targets of miR-340-5p, 670 of miR-542-3p, and 618 of miR-671-5p. Quantitative proteomic analysis of Ishikawa cells transfected with mimic or inhibitor (48 hrs: n=3 biological replicates) for each of the P4-regulated miRNAs was carried out to identify targets of these miRNAs. Excluding off target effects, mir-340-5p mimic altered 1,369 proteins while inhibition changed expression of 376 proteins (p < 0.05) of which, 72 were common to both treatments. A total of 280 proteins were identified between predicted (mirDB) and confirmed (in vitro) targets. In total, 171 proteins predicted to be targets by mirDB were altered in vitro by treatment with miR-340-5p mimic or inhibitor and were also altered by treatment of endometrial epithelial cells with P4. In vitro targets of miR-542-3p identified 1,378 proteins altered by mimic while inhibition altered 975 a core of 200 proteins were changed by both. 100 protein targets were predicted and only 46 proteins were P4 regulated. miR-671-mimic altered 1,252 proteins with inhibition changing 492 proteins of which 97 were common to both, 95 were miDB predicted targets and 46 were also P4-regulated. All miRNAs were detected in endometrial biopsies taken from patients during the luteal phase of their cycle, irrespective of prior or future pregnancy outcomes Expression of mir-340-5p showed an overall increase in patients who had previously suffered a miscarriage and had a subsequent miscarriage, as compared to those who had infertility or previous miscarriage and subsequently went on to have a life birth outcome. The regulation of these miRNAs and their protein targets regulate the function of transport and secretion, and adhesion of the endometrial epithelia required for successful implantation in humans. Dysfunction of these miRNAs (and therefore the targets they regulate) may contribute to endometrial-derived recurrent pregnancy loss in women.

category: cell biology


title: Proteostatic regulation of caveolins avoids premature oligomerisation and preserves ER homeostasis

abstract:
Caveolin-1 (CAV1) and CAV3 are membrane sculpting proteins driving formation of plasma membrane caveolae. Caveola formation is unique as it requires oligomerisation of newly synthesised caveolins through the biosynthetic-secretory pathway. Here, we combine structural, biochemical, and microscopy analyses to examine the early proteostasis of caveolin family members and mutants. We describe striking trafficking differences between newly synthesised caveolins, with CAV1 rapidly exported to the Golgi but CAV3 showing ER retention and targeting to lipid droplets. Only monomeric/low oligomeric caveolins are efficiently exported from the ER, with oligomers assembling beyond the cis-Golgi and disease-causing mutations leading to detrimental non-functional complexes. Caveolins in the ER are maintained at low levels by active proteasomal degradation, avoiding premature oligomerisation and ER stress. Increasing lipid availability, cholesterol for CAV1 and fatty acids for CAV3, enhances trafficking and reduces proteasomal degradation. In conclusion, we identify proteostatic mechanisms that modulate stability and trafficking of newly synthesised caveolins, protecting cells against ER stress but perturbed in caveolin-related disease.

SummaryUnderstanding the unique proteostasis of caveolins has important implications for cell biology and physiopathology. Combining structural, microscopy, and biochemical analyses, we uncover new insights into the mechanisms that differentiate the early biosynthetic steps of caveolin family members, isoforms, and pathogenic mutants.


title: Vinculin plays a role in neutrophil stiffening and transit through model capillary segments

abstract: Neutrophils are rapidly mobilized from the circulation to sites of inflammation. The mechanisms of neutrophil trafficking in the lung are distinct from those in the periphery, in part because the pulmonary capillaries are the primary site of neutrophil emigration rather than postcapillary venules. Since the diameter of a neutrophil is greater than the width of most pulmonary capillary segments, they must deform to transit through this capillary network, even at homeostasis. Resistance to deformation is primarily due to cortical actin that is rapidly assembled when a neutrophil is exposed to a priming or activation stimulus, resulting in neutrophil stiffening and subsequent sequestration within the pulmonary capillary network. In the current study, we use a microfluidic assay to characterize neutrophil transit through model capillary-like channels. Using techniques from single-particle tracking, we analyzed the cumulative distribution of neutrophil transit times and resolve population-based effects. We found that vinculin, an actin-binding adaptor protein, plays an essential role in neutrophil stiffening in response to formyl-Met-Leu-Phe (fMLP). Vinculin-deficient neutrophils lack the development of a population with slow transit through narrow channels that was observed in both wild-type murine bone marrow neutrophils and HoxB8-conditional progenitor-derived neutrophils. Atomic force microscopy studies provide further evidence that vinculin is required for neutrophil stiffening. Consistent with these findings, we observed that neutrophil sequestration in the lungs of mice is attenuated in the absence of vinculin. Together, our studies indicate that vinculin mediates actin-dependent neutrophil stiffening that leads to their sequestration in capillaries.


title: Projective LDDMM: Mapping Molecular Digital Pathology with Tissue MRI

abstract: Reconstructing dense 3D anatomical coordinates from 2D projective measurements has become a central problem in digital pathology for both animal models and human studies. We describe a new family of diffeomorphic mapping technologies called Projective LDDMM which generate diffeomorphic mappings of dense human MRI atlases at tissue scales onto sparse measurements at micron scales associated with histological and more general optical imaging modalities. We solve the problem of dense mapping surjectively onto histological sections by incorporating new technologies for crossing modalities that use non-linear scattering transforms to represent multiple radiomic-like textures at micron scales and incorporating a Gaussian mixture-model frame-work for modelling tears and distortions associated to each section. We highlight the significance of our method through incorporation of neuropathological measures and MRI, as relevant to the development of biomarkers for Alzheimers disease and one instance of the integration of imaging data across the scales of clinical imaging and digital pathology.


title: Cryo-electron tomography of the apicomplexan invasion machinery in its native state reveals rigid body motion of the conoid and docked secretory machinery

abstract: The apical complex is a conserved cytoskeletal structure that organizes the secretory and invasion machinery of all apicomplexan parasites, the causative agents of diseases such as malaria, toxoplasmosis, and cryptosporidiosis. The structure of the apical complex and its mechanism of motion, which is thought to be required for host cell invasion, is not well understood. Recent advances in cryo-electron tomography (cryo-ET) have allowed the high-resolution reconstruction of large complexes in their near-native states. Here, we use cryo-focused ion beam milling to generate a thin specimen of the coccidian apical complex, allowing us to visualize and compare its three-dimensional structure in the protruded and retracted states in situ by cryo-ET. Subtomogram averaging of the 8 nm repeats from the conoid fibers (specialized microtubules) revealed their polarity and unusual nine- protofilament arrangement with associated proteins that connect and likely stabilize the fibers. In spite of the open shape of the conoid fibers, neither the structure of the conoid fibers nor the architecture of the spiral-shaped conoid complex as a whole are altered during cycles of protrusion and retraction. Thus the conoid moves as a rigid body, and is not compressible like a spring, as has been previously suggested. Instead, we found that the apical polar rings (APR), previously considered a rigid structure to which the conoid is docked, dilates during conoid protrusion. Furthermore, we identified previously undescribed actin-like filaments connecting the conoid to the APR during protrusion, consistent with a role of actin in driving conoid movements. In addition, our data capture the parasites in the act of secretion during conoid extension, allowing us to identify a network of interactions between the apical complex cytoskeleton and the parasites secretory machinery.


title: Saturation mutagenesis of a predicted ancestral Syk-family kinase

abstract: Many tyrosine kinases cannot be expressed readily in E. coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and ZAP-70). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E.coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.


title: Combined measurements of proteome and cell turnover reveal the influences of tissue context and pathological aging on protein and organelle lifetimes

abstract: The lifespans of proteins can range from moments to years within mammalian tissues. Protein lifespan is relevant to organismal aging, as long-lived proteins can accrue damage over time. It is unclear how protein lifetime is shaped by tissue context, where both cell division and proteolytic degradation contribute to protein turnover. Here, we develop turnover and replication analysis by 15N isotope labeling (TRAIL) for parallel quantification of protein and cell lifetimes. We deploy TRAIL over 32 days in 4 mouse tissues to quantify cell proliferation with high precision and no toxicity and determine that protein lifespan varies independently of cell lifespan. Variation in protein lifetime is non-random: multiprotein complexes such as the ribosome have consistent lifetimes across tissues, while mitochondria, peroxisomes, and lipid droplets have variable lifetimes across tissues. To model the effects of aging on tissue homeostasis, we apply TRAIL to progeroid mice and uncover fat-specific alterations in cell lifetime and proteome composition, as well as a broad decrease in protein turnover flux. These data indicate that environmental factors influence protein turnover in vivo and provide a framework to understand proteome aging in tissue context.

category: systems biology


title: Immune Checkpoint Activity Regulates Polycystic Kidney Disease Progression

abstract: Innate and adaptive immune cells modulate Autosomal Dominant Polycystic Kidney Disease (ADPKD) severity, a common kidney disease with inadequate treatment options. ADPKD shares parallels with cancer where immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have shown that, in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of PD-1 on CD8+ T cells and PD-L1 on macrophages and epithelial cells in Pkd1RC/RC mice versus wildtypes, paralleling disease severity. PD-L1 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking two immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers. Together, our data suggests that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.


title: Mircubactin B rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis.

abstract: Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a complex protein machinery called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening >2,000 extracts from a diverse collection of actinobacteria, we identified a new compound, mirubactin B, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed by synthesising mirubactin B. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin B rescuing activity is not due simply to iron sequestration. The results demonstrate a novel antibacterial compound and add to growing evidence that bacterial siderophores have a range of activities beyond simple iron sequestration.


title: Functional Comparison Between Endogenous and Synthetic Notch Systems

abstract: The Notch pathway converts receptor-ligand interactions at the cell surface into a transcriptional response in the receiver cell. In recent years, synthetic Notch systems (SynNotch) that respond to different inputs and transduce different transcriptional responses have been engineered. One class of synNotch systems uses antibody-antigen interactions at the cell surface to induce the proteolytic cleavage cascade of the endogenous Notch autoregulatory core and the consequent release of a synNotch intracellular domain (ICD), converting surface antigen detection into a cellular response. However, whereas the activation of endogenous Notch requires ubiquitylation and subsequent endocytosis of the ligand ICD, these synNotch systems do not seem to have such a requirement because the synNotch ligands completely lack an ICD. This observation raises questions about existing models for the Notch activation mechanism. Here, we test how different structural and biochemical factors affect the dependence of endogenous and synthetic Notch activation on ligand ICD. We compare the behavior of antibody-antigen synNotch (aa-synNotch) to that of endogenous Notch, and to a synNotch system that uses induced FKBP-FRB dimerization with rapamycin (ff-synNotch), which still requires a ligand ICD. We found that differences in receptor-ligand affinity, in the identity of the transmembrane domain, or in the presence or absence of extracellular EGF repeats cannot explain the differences in ligand ICD requirement that distinguishes aa-synNotch from endogenous Notch or ff-synNotch. These findings suggest that the aa-SynNotch systems bypass the ligand ICD requirement because antigen-antibody pairs are able to promote other adhesive cell-cell interactions that provide the mechanical tension needed for ligand activation.


title: Growth phase estimation for abundant bacterial populations sampled longitudinally from human stool metagenomes.

abstract: Longitudinal sampling of the stool has yielded important insights into the ecological dynamics of the human gut microbiome. However, due to practical limitations, the most densely sampled time series from the human gut are collected at a frequency of about once per day, while the population doubling times for gut commensals are on the order of minutes-to-hours. Despite this, much of the prior work on human gut microbiome time series modeling has, implicitly or explicitly, assumed that day-to-day fluctuations in taxon abundances are related to population growth or death rates, which is likely not the case. Here, we propose an alternative model of the human gut as a continuous flow ecosystem at a dynamical steady state, where population dynamics occur internally and the bacterial population sizes measured in stool represent an endpoint of these internal dynamics. We formalize this idea as stochastic logistic growth of a population held at a constant dilution rate. We show how this model provides a path toward estimating the growth phases of gut bacterial populations in situ. We assess our model predictions against densely-sampled human stool metagenomic time series data. Consistent with our model, donors with slower defecation rates tended to harbor a larger proportion of taxa in later growth phases, while faster defecation rates were associated with more taxa in earlier growth phases. We discuss how these growth phase estimates may be used to better inform metabolic modeling in flow-through ecosystems, like animal guts or industrial bioreactors.

category: ecology


title: GNL3 is an evolutionarily-conserved stem cell gene influencing cell proliferation, animal growth, and regeneration in the hydrozoan Hydractinia

abstract: Nucleostemin (NS) is a vertebrate gene preferentially expressed in stem and cancer cells, which acts to regulate cell cycle progression, genome stability, and ribosome biogenesis. NS and its paralogous gene, GNL3-like (GNL3L), arose in the vertebrate clade after a duplication event from their orthologous gene, G protein Nucleolar 3 (GNL3). Research on invertebrate GNL3, however, has been limited. To gain a greater understanding of the evolution and functions of the GNL3 gene, we have performed studies in the hydrozoan cnidarian Hydractinia symbiolongicarpus, a colonial hydroid that continuously generates pluripotent stem cells throughout its life cycle and presents impressive regenerative abilities. We show that Hydractinia GNL3 is expressed in stem and germline cells. The knockdown of GNL3 reduces the number of mitotic and S-phase cells in Hydractinia larvae of different ages. Genome editing of Hydractinia GNL3 via CRISPR/Cas9 resulted in colonies with reduced growth rates, polyps with impaired regeneration capabilities, gonadal morphological defects, and low sperm motility. Collectively, our study shows that GNL3 is an evolutionarily-conserved stem cell and germline marker involved in cell proliferation, animal growth, regeneration, and sexual reproduction in Hydractinia, and sheds new light into the evolution of GNL3 and of stem cell systems.

category: evolutionary biology


title: Evolutionary analysis of p38 stress activated kinases in unicellular relatives of animals suggests an ancestral function in osmotic stress

abstract: p38 kinases are key elements of the cellular stress response in animals. They mediate the cell response to a multitude of stress stimuli, from osmotic shock to inflammation and oncogenes. However, it is unknown how such diversity of function in stress evolved in this kinase subfamily. Here, we show that the p38 kinase was already present in a common ancestor of animals and fungi. Later, in animals, it diversified into three JNK kinases and four p38 kinases. Moreover, we identified a fifth p38 paralog in fishes and amphibians. Our analysis shows that each p38 paralog has specific amino acid substitutions around the hinge point, a region between the N-terminal and C-terminal protein domains. We showed that this region can be used to distinguish between individual paralogs and predict their specific. Finally, we showed that the response to hyperosmotic stress in Capsaspora owczarzaki, a close unicellular relative of animals, follows a typical for the p38 kinases pattern of phosphorylation-dephosphorylation. At the same time, Capsasporas cells upregulate the expression of GPD1 protein resembling an osmotic stress response in yeasts. Overall, our results show that the ancestral p38 stress pathway originated in the root of opisthokonts, most likely as a cells reaction to salinity change in the environment. In animals, the pathway became more complex and incorporated more stimuli and downstream targets due to the p38 sequence evolution in the docking and substrate binding sites around the hinge region. Overall, this study improves our understanding of the p38 evolution and opens new perspectives for the p38 research.


title: Evolutionary analysis of p38 stress-activated kinases in unicellular relatives of animals suggests an ancestral function in osmotic stress


title: A novel de novo FEM1C variant as a potential cause of neurodevelopmental disorder with absent speech, pyramidal signs, and limb ataxia.

abstract: Maintaining protein homeostasis (proteostasis) requires the degradation of damaged or unwanted proteins and plays a crucial role in cellular function. The principal proteolytic component of the proteostasis network is the ubiquitin-proteasome system (UPS) which orchestrates protein degradation through ubiquitination of appropriate targets. Ubiquitination is mediated by an enzymatic cascade involving, i.e., E3 ubiquitin ligases, many of which belong to the cullin-RING ligases (CRLs) family. Genetic defects of the UPS are known causes of neurodevelopmental disorders, with >60 entities described so far. Using exome sequencing (ES) to diagnose a pediatric patient with global developmental delay, pyramidal signs, and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene, which encodes a CRL substrate receptor. The p.(Asp126His) variant, which alters a conserved amino acid (phyloP100way = 7.9), is located within a highly constrained coding region (CCR=98.6) and is predicted to be pathogenic by the majority (13/19) of in silico tools. To further assess its pathogenicity, we employed Caenorhabditis elegans nematode as a disease model. We found that the mutant vs. wild-type worms had impaired mobility as assessed by track length (p=0.0001), turn counts (p=0.0001), and omega bend counts (p=0.038). Furthermore, mutant worms had histologically normal muscle architecture but were sensitive to an acetylcholinesterase inhibitor - aldicarb which indicates that their locomotion defects result from synaptic defects rather than muscle dysfunction. We conclude that the disease in our patient may be the first reported case of a neurodevelopmental disorder caused by the FEM1C genetic defect.

category: genetics


title: Molecular and functional characterization of the Drosophila melanogaster conserved smORFome

abstract: Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 194 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, with [~]60 conserved in plants. Within these conserved smORFs, we observed remarkably heterogenous spatial and temporal expression patterns - indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains revealed a predicted enrichment of smORF polypeptides localizing to mitochondria. We conducted an embryonic ribosome profiling experiment and applied a machine learning approach, finding support for translation of 82 of these smORFs during embryogenesis. We further embarked on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.

category: genomics


title: Direct Visualization of Translesion DNA Synthesis Polymerase IV at the Replisome

abstract:
In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these TLS DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the {beta} sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the {beta} clamp but not its catalytic activity, and is reinforced by the presence of the {gamma} subunit of the {beta} clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.

SignificanceDamage to the nucleotide bases that make up the DNA in chromosomes creates a problem for their subsequent accurate duplication each time a cell divides. Typically, the cellular enzymatic machinery that replicates the DNA cannot copy a damaged base and specialized trans-lesion DNA polymerases, which are prone to making errors that result in mutations, are required to copy the damaged base, allowing replication to proceed. We demonstrate that the bacterial replisome, which is comprised of the enzymes required to replicate the chromosome, can associate with one of these specialized trans-lesion polymerases over long distances of replicated DNA. This association slows the speed of replication, thereby reducing the chance of mutations arising in the cell under conditions of stress.


title: Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology

abstract:
SignificanceAll-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications.

AimWe introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs.

ApproachThe system uses oblique trans-illumination, low-cost cameras, light-emitting diodes and off-the-shelf components (total < $15,000) to capture voltage, calcium and mechanical waves under electrical or optical stimulation.

ResultsOur results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and Vm - [Ca2+]i similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (Chanelrhodopsin2) only under very low green light (< 0.05mW/mm2). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrodes solution at room temperature.

ConclusionsAs multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.


title: Mutually exclusive spectral biclustering and its applications in cancer subtyping

abstract: Many soft biclustering algorithms have been developed and applied to various biological and biomedical data analyses. However, until now, few mutually exclusive (hard) biclustering algorithms have been proposed although they can be extremely useful for identify disease or molecular subtypes based on genomic or transcriptomic data. We considered the biclustering problem of expression matrices as a bipartite graph partitioning problem and developed a novel biclustering algorithm, MESBC, based on Dhillons spectral method to detect mutually exclusive biclusters. MESBC simultaneously detects relevant features (genes) and corresponding subgroups, and therefore automatically uses the signature features for each subtype to perform the clustering, improving the clustering performance. MESBC could accurately detect the pre-specified biclusters in simulations, and the identified biclusters were highly consistent with the true labels. Particularly, in setting with high noise, MESBC outperformed existing NMF and Dhillons method and provided markedly better accuracy. Analysis of two TCGA datasets (LUAD and BRAC cohorts) revealed that MESBC provided similar or more accurate prognostication (i.e., smaller p value) for overall survival in patients with breast and lung cancer, respectively, compared to the existing, gold-standard subtypes for breast (PAM50) and lung cancer (integrative clustering). In the TCGA lung cancer patients, MESBC detected two clinically relevant, rare subtypes that other biclustering or integrative clustering algorithms could not detect. These findings validated our hypothesis that MESBC could improve molecular subtyping in cancer patients and potentially facilitate better individual patient management, risk stratification, patient selection, therapeutic assignments, as well as better understanding gene signatures and molecular pathways for development of novel therapeutic agents.


title: An evolutionary perspective on the origin, conservation and binding partner acquisition of tankyrases

abstract: Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species exist. In this work we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identified the single-celled choanoflagellates as earliest origin of tankyrases. We further show that sequence and structural aspects of TNKSs are well conserved even between highly diverged species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners vary greatly between species, indicating that tankyrases have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, although their regulatory functions in cells have likely changed considerably throughout evolution.


title: MICOS Complex and Mitochondria Morphology Changes Across Aging in Cardiac Muscle

abstract: Cardiac disease remains a large cause of death among humans and therapies to treat it remain lacking. Mitochondria is heavily implicated in various critical functions of the cell not limited to the production of ATP. Critically, mitochondria break down across aging and heart also decrease in efficiency in aging. Key factors implicated in mitochondria morphology, such as the mitochondrial contact site and cristae organizing system (MICOS), role across aging remains to be seen in cardiac muscle. To better understand the relationship between mitochondria in heart, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the 3D networks in cardiac muscle samples of mice at aging intervals. Cardiac muscle showed some breaking down across aging, decreased areas, increased mitochondria, but less fragmentation and wide-spread changes across 2-year aging than previous studies in skeletal muscle. In studying cristae, the inner folds of mitochondria, we observed a loss of morphology across aging. This mimicked what was observed upon CRISPR/Cas9 knockdown of mitofilin, Chchd3, Chchd6 and Opa-1 which showed increased lamellar cristae and fragmented mitochondria while mitochondria length and volume decreased. We are the first to examine mitochondria changes in cardiac muscle across aging. Notably, we noticed differences in skeletal muscle, which we have previously studied, and cardiac muscle that suggests a differential response to mitochondrial aging in cardiac and skeletal muscle. In combination, these data suggest that, in the heart, loss of the MICOS complex may be implicated in the loss of function in mitochondria that is seen across aging.


title: Growth and adaptation mechanisms of tumour spheroids with time-dependent oxygen availability

abstract: Tumours are subject to external environmental variability. However, in vitro tumour spheroid experiments, used to understand cancer progression and develop cancer therapies, have been routinely performed for the past fifty years in constant external environments. Furthermore, spheroids are typically grown in ambient atmospheric oxygen (normoxia), whereas most in vivo tumours exist in hypoxic environments. Therefore, there are clear discrepancies between in vitro and in vivo conditions. We explore these discrepancies by combining tools from experimental biology, mathematical modelling, and statistical uncertainty quantification. Focusing on oxygen variability to develop our framework, we reveal key biological mechanisms governing tumour spheroid growth. Growing spheroids in time-dependent conditions, we identify and quantify novel biological adaptation mechanisms, including unexpected necrotic core removal, and transient reversal of the tumour spheroid growth phases.

category: cancer biology


title: Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS

abstract: Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.


title: Detection of local growth patterns in longitudinally imaged low-grade gliomas

abstract:
BackgroundDiffuse low-grade gliomas (LGGs) are primary brain tumors with infiltrative, anisotropic growth related to surrounding white and grey matter structures. In this study, we illustrate the use of deformation-based morphometry (DBM) as a simple and objective method to study the local change in growth patterns of LGGs.

MethodsAn imaging pipeline was developed involving the creation of patient-specific average templates and nonlinear registration of pre-treatment follow-up MRIs to the average template. Jacobian maps were derived and analyzed to identify areas of tissue expansion and contraction over time.

ResultsOur analysis demonstrates that tissue expansion occurs primarily around the edges of the tumor, while the lesion core and areas adjacent to obstacles, such as the skull, show no significant growth. Tumors also appeared to grow faster and predominantly in areas of white matter. Regions of the brain surrounding the lesion showed slight contraction over time, likely representing compression due to mass effect of the tumor.

ConclusionsWe demonstrate that DBM is a useful clinical tool to understand the long-term clinical course of an individuals tumor and identify areas of rapid growth, which can explain the clinical signs and symptoms, predict future symptoms, and guide targeted diagnostics and therapy.

HighlightsO_LILow-grade glioma expansion occurs primarily around the edges of the tumor.
C_LIO_LITumor cores and tissue next to obstacles show no significant growth over time.
C_LIO_LIDBM provides a clinically valuable assessment of local tumor growth and activity.
C_LI


title: Identifying Proteasome 26S Subunit, ATPase (PSMC) Family Genes as the Prognostic Indicators and Therapeutic Targets in Lung Adenocarcinoma

abstract: This study explored the prognostic and therapeutic potentials of multiple Proteasome 26S Subunit, ATPase (PSMC) family of genes (PSMC1-5) in lung adenocarcinoma (LUAD) diagnosis and treatment. All the PSMCs were found to be differentially expressed (upregulated) at the mRNA and protein levels in LUAD tissues. The promoter and multiple coding regions of PSMCs were reported to be differentially and distinctly methylated, which may serve in the methylation-sensitive diagnosis of LUAD patients. Multiple somatic mutations (alteration frequency: 0.6-2%) were observed along the PSMCs coding regions in LUAD tissues that could assist in the high-throughput screening of LUAD patients. A significant association between PSMCs overexpression and LUAD patients poor overall and relapse-free survival (p<0.05, HR:>1.3) and individual cancer stages (p<0.001) was discovered, which justifies PSMCs as the ideal targets for LUAD diagnosis. Multiple immune cells and modulators (i.e., CD274, IDO1) were found to be associated with PSMCs expression in LUAD tissues that could aid in formulating PSMC-based diagnostic measures and therapeutic interventions for LUAD. Functional enrichment analysis of neighbor genes of PSMCs in LUAD tissues revealed different genes (i.e., SLIRP, PSMA2, NUDSF3) previously known to be involved in oncogenic processes and metastasis co-expressed with PSMCs, which could also be investigated further. Overall, this study recommends that PSMCs and their transcriptional and translational products are potential candidates for LUAD diagnostic and therapeutic measure discovery. However, further laboratory research is needed to validate the findings of this experiment.


title: ALDH2 DEFICIENCY INCREASES SUSCEPTIBILITY TO BINGE ALCOHOL-INDUCED GUT LEAKINESS, ENDOTOXEMIA, AND ACUTE LIVER INJURY IN MICE THROUGH THE GUT-LIVER AXIS

abstract:
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme responsible for metabolizing toxic acetaldehyde to acetate and acts as a protective or defensive protein against various disease states associated with alcohol used disorder (AUD), including alcoholic liver disease (ALD), and elevated oxidative stress. We hypothesized that Aldh2-knockout (KO) mice are susceptible to binge alcohol-mediated liver injury than wild-type (WT) mice through increased gut leakiness and endotoxemia. Therefore, this study aimed to investigate the protective role of ALDH2 in binge alcohol-induced gut permeability, endotoxemia, and acute inflammatory liver injury by exposing Aldh2-KO or WT mice to a single oral dose of binge alcohol 3.5, 4.0, or 5.0 g/kg. Our findings showed for the first time that ALDH2 deficiency in Aldh2-KO mice increases their sensitivity to alcohol-induced oxidative and nitrative stress, enterocyte apoptosis, and nitration of gut tight junction (TJ) and adherent junction (AJ) proteins, leading to their degradation. These resulted in gut leakiness and endotoxemia in Aldh2-KO mice after exposure to a single dose of ethanol even at 3.5 g/kg, while no changes were observed in the corresponding WT mice. The elevated serum endotoxin (lipopolysaccharide, LPS) and/or bacterial translocation contributed to systemic inflammation, hepatocyte apoptosis, and subsequently acute liver injury, indicating the disruption in the gut-liver axis. Furthermore, treatment with Daidzin, an ALDH2 inhibitor, exacerbated ethanol-induced cell permeability and reduced TJ/AJ proteins in T84 human colonic cells. These changes were reversed by Alda-1, an ALDH2 activator, indicating a crucial role of ALDH2 in protecting against alcohol-induced epithelial barrier dysfunction. All these findings suggest that ALDH2 deficiency or gene mutation in humans is a risk factor to alcohol-mediated gut and liver injury, and ALDH2 could be an important therapeutic target against alcohol-associated tissue/organ damage.

HighlightsO_LIBinge alcohol increases oxidative and nitrative stress in the intestine and liver.
C_LIO_LIBinge alcohol causes gut leakiness, endotoxemia, and acute liver injury.
C_LIO_LILeaky gut is caused by elevated degradation of nitrated intestinal TJ/AJ proteins.
C_LIO_LIAldh2-KO mice are susceptible to binge-alcohol-induced leaky gut and liver injury.
C_LIO_LIALDH2 inhibition increases alcohol-induced T84 colonic epithelial cell permeability.
C_LI

category: pharmacology and toxicology


title: Regulation of metabolic health by dietary histidine in mice

abstract: The increasing prevalence of obesity is a serious threat to global health. Low protein (LP) diets are associated with a decreased risk of diabetes in humans, and a low protein diet promotes leanness and glycemic control in both rodents and humans. The effects of a LP diet on glycemic control are mediated by reduced dietary levels of the branched-chain amino acids (BCAAs). However, we have observed that reducing dietary levels of the other six essential amino acids leads to changes in body composition. Here, we find that dietary histidine plays a key role in the response to a LP diet. Specifically reducing dietary levels of histidine by 67% reduces weight gain of young, lean C57BL/6J mice, reducing both adipose and lean mass gain, without altering glucose metabolism. Specifically reducing dietary histidine rapidly reverses diet-induced obesity and hepatic steatosis in diet-induced obese mice, increasing insulin sensitivity; this normalization of metabolic health was associated not with caloric restriction or increased activity, but with increased energy expenditure that surprisingly did not require Fgf21. Histidine restriction started in mid-life promoted leanness and glucose tolerance in aged males, but did not affect frailty or lifespan in either sex. Finally, we demonstrate that variation in dietary histidine levels helps to explain body mass index differences in humans. Overall, our findings demonstrate that dietary histidine is a key regulator of weight and body composition in both mice and humans, and suggest that reducing dietary levels of histidine may be a highly translatable option for the treatment of obesity.

category: physiology


title: Unlocking the strength of inducible promoters in gram-negative bacteria

abstract:
Inducible promoters, such as the lac and tet promoters, are ubiquitous biotechnology tools. Inducible bacterial promoters have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulator proteins (e.g., LacI and TetR, and the -10 and -35 consensus sequences required to recruit the sigma ({sigma}) subunits of RNA polymerase to initiate transcription. Despite their widespread use in molecular biology, there remain problems with current inducible expression systems. Leaky transcription in the OFF state remains a particular challenge. Here we have updated the architecture of the lac and tet expression systems to improve their strength, control, and portability. We modified the genetic architecture of the lac and tet expression systems to contain consensus -10 and -35 sequence boxes to be strongly targeted by {sigma}70, to incorporate of a strong ribosome binding site recognized broadly by gram-negative bacteria, and to independently control of the transcriptional regulators by optimized constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which we demonstrate significantly improves the signal-to-background ratio of promoter measurement assays over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate-prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.

SignificanceMany of the latest advances in pharmaceuticals, materials, and foods involve the production of recombinant proteins from bacterial hosts. However, the regulated production of enzymes and functional protein products that are toxic to their microbial hosts remains a challenge. Our work provides new tools that enable tight control over expression of protein products in bacterial host strains. We show that our tools function not only in the broadly utilized Escherichia coli, but also in other gram-negative bacteria including the soil organism Pseudomonas putida and the marine bacterium Vibrio natriegens. Our technology will facilitate more efficient production of a broader range of protein products in diverse microbial hosts.

category: synthetic biology


title: Immune activation state modulates retrieval of infant engrams

abstract: Infantile amnesia is possibly the most ubiquitous form of memory loss in mammals. Despite its widespread relevance, little is known about the biological conditions for infantile amnesia to occur and its effect on the engram cells that encode a memory. We investigated how memories are stored in the brain throughout development by integrating engram labeling technology with mouse models of infantile amnesia. We discovered a sex-specific phenomenon in which male offspring in maternal immune activation models of autism spectrum disorder do not experience infantile amnesia. We rescued the same apparently forgotten infantile memories in mice by optogenetically reactivating dentate gyrus engram cells that were labeled during complex experiences in infant development. Further, we were able to permanently reinstate lost infantile memories by artificially updating the memory engram, demonstrating that infantile amnesia is a reversible process. Our findings suggest that immune activation during development modulates innate, and reversible, forgetting switches that determine whether infantile amnesia will occur.

category: neuroscience


title: Mechanisms and functions of respiration-driven gamma oscillations in the piriform cortex

abstract: Gamma oscillations are believed to underlie cognitive processes by shaping the formation of transient neuronal partnerships on a millisecond scale. These oscillations are coupled to the phase of breathing cycles in several brain areas, possibly reflecting local computations driven by sensory inputs sampled at each breath. Here, we investigated the mechanisms and functions of gamma oscillations in the piriform (olfactory) cortex of awake mice to understand their dependence on breathing and how they relate to local spiking activity. Mechanistically, we find that respiration drives gamma oscillations in the piriform cortex, which are inhibitory and result from recurrent connections between local excitatory and inhibitory neuronal populations. Moreover, respiration-driven inhibitory gamma oscillations are triggered by the activation of mitral/tufted cells in the olfactory bulb and are abolished during anesthesia. Functionally, we demonstrate that they locally segregate neuronal assemblies through a winner-take-all computation leading to sparse odor coding during each breathing cycle. Our results shed new light on the mechanisms of gamma oscillations, bridging computation, cognition and physiology.


title: Homogeneous inhibition is optimal for the phase precession of place cells in the CA1 field

abstract: Place cells are hippocampal neurons encoding the position of an animal in space. Studies of place cells are essential to understanding the processing and transmission of information by neural networks of the brain. An important characteristic of place cell spike trains is phase precession. When an animal is running through the place field, the discharges of the place cells shift from the ascending phase of the theta rhythm through the minimum to the descending phase. The role of excitatory inputs to CA1 pyramidal neurons along the Schaffer collaterals and the perforant pathway in phase precession is described, but the role of inputs from local interneurons to CA1 pyramidal cell is poorly understood. We have set the goal the contribution of field CA1 interneurons to the phase precession of place cells using mathematical methods. The CA1 field was chosen because it provides the largest set of experimental data required to build and verify the model. We have solved the optimization problem and found the parameters of the excitatory and inhibitory inputs to the pyramidal neuron of the CA1 field so that the neuron generates a spike train with the effect of phase precession. We have discovered that the uniform inhibition of field CA1 pyramidal neurons best explains the effect of phase precession. Among interneurons, axo-axonal neurons make the greatest contribution to the inhibition of pyramidal cells.


title: Phylogroup-specific variation shapes pangenome dynamics in Pseudomonas aeruginosa

abstract: Pseudomonas aeruginosa is a human opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C) of unevenly distributed size. Here, we assessed phylogroup-specific evolutionary dynamics in a collection of publicly available and newly sequenced genomes of P. aeruginosa. We explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups. We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of gene families to the pangenome, but show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these two types of cargo genes is often significantly correlated. Moreover, multiple inter- and intra-phylogroup interaction events occur at the accessory genome content level, suggesting that recombination events are frequent. Finally, we provide here a panel of phylogenetically diverse P. aeruginosa strains that can be used as a reference set for future functional analyses. Altogether, our results highlight distinct pangenome characteristics of the three phylogroups of P. aeruginosa, that are possibly influenced by variation in the abundance of CRISPR-Cas systems and that are shaped by the differential distribution of AMR and other defence systems.


title: Small-amplitude head oscillations result from a multimodal head stabilization reflex in hawkmoths

abstract: In flying insects, head stabilization is an important reflex which helps to reduce motion blur during fast aerial maneuvers. This reflex is multimodal and requires the integration of visual and antennal mechanosensory feedback, each operating as a negative-feedback control loop. As in any negative-feedback system, the head stabilization system possesses inherent oscillatory dynamics that depends on the rates and latencies of the sensorimotor components constituting the reflex. Consistent with this expectation, we observed small amplitude oscillations in the head motion (or head wobble) of the Oleander hawkmoth Daphnis nerii. We show here that these oscillations emerge from the inherent dynamics of the multimodal reflex that underlies gaze stabilization, and the amplitude of the head wobble is a function of both the visual feedback and antennal mechanosensory feedback from the Johnstons organs. The head wobble is thus an outcome of a multimodal, dynamically-stabilized head positioning reflex.


title: Modified MRI anonymization (de-facing) for improved MEG coregistration

abstract: Localising the sources of MEG/EEG signals often requires a structural MRI to create a head model, while ensuring reproducible scientific results requires sharing data and code. However, sharing of structural MRI data often requires removal of the face to help protect the identity of the individuals concerned. While automated de-facing methods exist, they tend to remove the whole face, which can impair methods for coregistering the MRI data with the EEG/MEG data. We show that a new, automated de-facing method that retains the nose maintains good MRI-MEG/EEG coregistration. Importantly, behavioural data show that this "face-trimming" method does not increase levels of identification relative to a standard de-facing approach, and has less effect on the automated segmentation and surface extraction sometimes used to create head models for MEG/EEG localisation. We suggest that this trimming approach could be employed for future sharing of structural MRI data, at least for those to be used in forward modelling (source reconstruction) of EEG/MEG data.


title: Altered basal ganglia output during self-restraint

abstract: Suppressing actions is essential for flexible behavior. Multiple neural circuits involved in behavioral inhibition converge upon a key basal ganglia output nucleus, the substantia nigra pars reticulata (SNr). To examine how changes in basal ganglia output contribute to self-restraint, we recorded SNr neurons during a proactive behavioral inhibition task. Rats responded to Go! cues with rapid leftward or rightward movements, but also prepared to cancel one of these movement directions as a Stop! cue might occur. This action restraint - visible as direction-selective slowing of reaction times - altered both rates and patterns of SNr spiking. Overall firing rate was elevated before the Go! cue, and this effect was driven by a subpopulation of direction-selective SNr neurons. In neural state space, this corresponded to a shift away from the restrained movement. Furthermore, SNr activity showed an increase in spike variability during proactive inhibition, similar to that reported with movement slowing in Parkinsons Disease. Increased spike variability corresponded to variable state-space trajectories, slowing reaction times via reduced preparation to move. These findings open new perspectives on basal ganglia mechanisms for movement preparation and cognitive control, in both healthy states and brain disorders.


title: Advancing antimicrobial resistance monitoring in surface waters with metagenomic and quasimetagenomic methods

abstract: Surface waters present a unique challenge for the monitoring of critically important antimicrobial resistance. Metagenomic approaches provide unbiased descriptions of taxonomy and antimicrobial resistance genes in many environments, but for surface water, culture independent data is insufficient to describe critically important resistance. To address this challenge and expand resistome reporting capacity of antimicrobial resistance in surface waters, we apply metagenomic and quasimetagenomic (enriched microbiome) data to examine and contrast water from two sites, a creek near a hospital, and a reservoir used for recreation and municipal water. Approximately 30% of the National Antimicrobial Resistance Monitoring Systems critically important resistance gene targets were identified in enriched data contrasted to only 1% in culture independent data. Four different analytical approaches consistently reported substantially more antimicrobial resistance genes in quasimetagenomic data compared to culture independent data across most classes of antimicrobial resistance. Statistically significant differential fold changes (p<0.05) of resistance determinants were used to infer microbiological differences in the waters. Important pathogens associated with critical antimicrobial resistance were described for each water source. While the single time-point for only two sites represents a small pilot project, the successful reporting of critically important resistance determinants is proof of concept that the quasimetagenomic approach is robust and can be expanded to multiple sites and timepoints for national and global monitoring and surveillance of antimicrobial resistance in surface waters.


title: EggVio: a user friendly and versatile pipeline for assembly and functional annotation of shallow depth sequenced samples

abstract:
1We introduce a homemade pipeline allowing to improve the quality of the metagenomic annotations carried out when using shallow depth metagenomic datasets. The main motivation being to be able to quantify more precisely, with greater certainty, the genes involved in bacterial interactions. The limitation in our experimental design is that we use a sequencing technique with a low throughput (miSeq) compared to the metagenomic standard (hiSeq) because we carry out a fairly large sampling (almost a hundred samples) in time series. This methodological constraint from our study means that the assembly of the sequences is not very exhaustive (less than 50% of the sequences manage to be assembled). In this chapter, we will therefore present a new pipeline designed to specifically deal with such kind of data. We used co-assembly and a sequence annotation strategy in order to recover the sequences that could not be mapped on the assembled contigs. In addition, in order to avoid adding too much noise, when rescuing reads, we have built an algorithm to define a threshold of e-value based on the noise of the sequence annotation learned from sequences mapped in the assembly.

We have selected several recent tools known to be effective for assembling, mapping and annotating these data. In addition, this pipeline was also built in order to be very user-friendly in terms of installation. In this idea of reproducibility, accessibility and transparency, we have designed an installation script to allow each user to install each tool required for the pipeline in a simple and reproducible way. Regarding the performances of this pipeline, we were able to show that the expected error rate (False discovery rate) for the annotation was close to 5%. Finally, we also used an actual dataset from a bioremediation site and showed that the representability of the samples seemed much better when we used our pipeline than when we used a classic metagenome assembly strategy.


title: Intrauterine inflammation leads to some sex and age-specific behavior and molecular differences in mice

abstract:
BackgroundPrenatal inflammation is associated with long-term adverse neurobehavioral outcomes in exposed children. Sex-specific differences in behavior have been observed in anxiety and learning; however, whether these differences manifest differently by age is unknown. This study assesses possible behavioral changes from in utero inflammation as a function of age in neonatal, juvenile, and adult animals. We also tested if the observed behavioral differences correlated to neonatal sex-specific neurogenesis gene expression changes in the hippocampus to suggest a mechanism for observed behavioral differences.

MethodsCD-1 timed pregnant dams were injected in utero with lipopolysaccharide (LPS, 50g/animal) or saline at embryonic day 15 (E15). Neonatal behavioral testing was performed on postnatal day (P) 5 on male and female pups born at term using the Ultrasonic Vocalization test (USV). Juvenile and mature animals of each sex were tested in Open Field (OF) and Barnes Maze (BM) on P28 and P67 (OF), and 32-36 and 70-75 (BM). A commercially available array designed to assess the expression of genes involved in mammalian neurogenesis was utilized for profiling gene expression in the hippocampal tissue isolated from LPS and saline-exposed P7 pups.

ResultsThere were no differences in stress responses measured by neonatal USV between LPS- and saline-exposed groups of either sex. In contrast, exposure to prenatal inflammation caused a male-specific increase in anxiety in mature but not juvenile animals. Juvenile LPS-exposed females had decreased movement in OF that was not present in adult animals. In addition, we observed improved memory retrieval in response to in utero LPS in the juvenile animals of both sexes. However, there was an impairment of long-term memory in adult LPS-exposed females. Finally, gene expression analyses revealed that LPS induced sex-specific changes in genes involved in hippocampal neurogenesis.

ConclusionsIntrauterine exposure to inflammation has age and sex-specific effects on anxiety and learning. These differences are not apparent in the neonatal period but begin to be evident in juvenile animals and evolve in adult animals. These sex-specific differences in learning may be correlated to sex-specific disruption of the expression of genes associated with neurogenesis in the hippocampus.

category: developmental biology


title: Mapping the risks of China's global coastal development to marine socio-ecological systems

abstract: Rapid coastal development continues to jeopardize the integrity of marine socio-ecological systems. China is now the largest bilateral creditor in the world, committing nearly half a trillion US dollars to overseas development finance since 2008. Meanwhile, there are growing concerns over the impacts of this boom in Chinese development finance on marine systems. Here, we quantify the risks of coastal development projects financed by China to marine biodiversity and coastal Indigenous communities. Ports present the greatest impact risks to marine systems, in terms of both magnitude and area at risk, with power plants, roads, and other facilities presenting relatively high localized risks. Risks are most prominent in Africa and the Caribbean, with coastal Indigenous communities in Western and Central Africa particularly vulnerable to the potential negative impacts of development. All projects present some risk to threatened marine species and potential critical habitats, but few present high risks to nearby marine protected areas. Most projects present additional risks to ecosystems that are already under increasing human pressures, but some are likely to introduce new risks to relatively intact ecosystems. "Bluing" future coastal development projects in Chinas overseas development finance portfolio will require more social and environmental safeguards, higher standards for host-country impact assessments, and greater integration of land-sea risk mitigation and management approaches.


title: The Devil Is In The Details: Landscape Features Are Insufficient To Explain Patterns Of Non-Native Fishes Distribution In North Patagonian Streams

abstract: Ecological communities are structured by combinations of factors known as habitat templates. These templates work as a filter allowing only species with particular traits or phenotypes to establish and persist excluding all others. Defining which habitat variables and spatial scales drive the assembly of freshwater communities is key to effective and efficient management of fluvial ecosystems. We took advantage of the relatively recent and well-studied history of salmonid introductions in Patagonia to evaluate if non-native species show different patterns of association with abiotic factors depending on the spatial scale of the environmental filter. We used a hierarchical approach to characterized environmental variables at the basin and reach scales to assess their influence on the presence, abundance and structure of the salmonid assemblages in breeding streams. We saw no evidence that presence/absence patterns of salmonid distribution were driven by landscape variables, except for those basins with physical environmental barriers to colonization. However, we did find evidence for relative abundances being influenced by climatic and geomorphological variables (e.g., precipitation and relief). Our results do not support a scenario in which any of the salmonid species modulates the distribution of the other species, suggesting that interference has played only a minor role in determining current fish distribution in fluvial systems of the region. Instead, current patterns of presence and abundance of salmonids are best explained as the product of environmental filters. Our findings contribute to our understanding of the ecology of individual species and provide insight into the mechanisms structuring fish assemblages in Southern Hemispheres lotic systems.


title: Cultural specialization as a double-edged sword: division into specialized guilds might promote cultural complexity at the cost of higher susceptibility to cultural loss

abstract: The transition to specialization of knowledge within populations could have facilitated the accumulation of cultural complexity in humans. Specialization allows populations to increase their cultural repertoire without requiring that members of that population increase their individual capacity to accumulate knowledge. However, specialization also means that domain-specific knowledge can be concentrated in small subsets of the population, making it more susceptible to loss. Here we use a model of cultural evolution to demonstrate that specialized populations can be more sensitive to stochastic loss of knowledge than populations without subdivision of knowledge, and that demographic and environmental changes have an amplified effect on populations with knowledge specialization. Finally, we suggest that specialization can be a double-edged sword; specialized populations may have an advantage in accumulating cultural traits but may also be less likely to expand and establish themselves successfully in new demes due to the increased cultural loss that they experience during the population bottlenecks that often characterize such expansions.


title: Rare CNVs and phenome-wide profiling: a tale of brain-structural divergence and phenotypical convergence

abstract: Copy number variations (CNVs) are rare genomic deletions and duplications that can exert profound effects on brain and behavior. Previous reports of pleiotropy in CNVs imply that they converge on shared mechanisms at some level of pathway cascades, from genes to large-scale neural circuits to the phenome. However, studies to date have primarily examined single CNV loci in small clinical cohorts. It remains unknown how distinct CNVs escalate the risk for the same developmental and psychiatric disorders. Here, we quantitatively dissect the impact on brain organization and behavioral differentiation across eight key CNVs. In 534 clinical CNV carriers from multiple sites, we explored CNV-specific brain morphology patterns. We extensively annotated these CNV-associated patterns with deep phenotyping assays through the UK Biobank resource. Although the eight CNVs cause disparate brain changes, they are tied to similar phenotypic profiles across [~]1000 lifestyle indicators. Our population-level investigation established brain structural divergences and phenotypical convergences of CNVs, with direct relevance to major brain disorders.


title: Honey bee functional genomics using symbiont-mediated RNAi

abstract: Bees are indispensable pollinators and model organisms for studying social behavior, development, and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a host RNA interference response that reduces expression of a targeted gene. To employ this FUGUES (FUnctional Genomics Using Engineered Symbionts) procedure, a double-stranded RNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by five days after colonization. This protocol can be accomplished in four weeks by bee researchers with microbiology and molecular cloning skills.


title: Quaternary Ammonium Salt-Modified Isabgol (Psyllium) Scaffold as an Antibacterial Dressing for Improved Wound Healing

abstract: Chronic wounds require suitable treatment and management strategies for proper healing. Among other causes, infection delays the healing of wounds and increases the risk of wound-related complications. Healing of chronic wounds requires an ingenious biomaterial that is biocompatible and anti-infective to achieve effective wound management. In this study, a wound dressing with inherent antibacterial and biocompatible properties was developed to assist the healing process. Natural polysaccharide Isabgol was chemically modified with Epoxy propyl trimethyl ammonium chloride to render antibacterial activity to the material. This is the first report of such chemical modification of this polymer for biomedical applications. The modified material was freeze-dried to obtain scaffolds. 13C NMR and FTIR analysis confirmed the modification of the Isabgol polymer chains with EPTMAC. The scaffold exhibits an organized porous structure that allows the exchange of gases and nutrients through the matrix, as confirmed by SEM analysis. The material possesses excellent swelling properties up to 17 times its initial weight that allows it to absorb wound exudates and maintain a moist environment at the wound site. The scaffold is biodegradable, and thermally and mechanically stable. The material is anti-infective and can prevent infections at the wound site, which is one of the major causes of delayed wound healing. The developed scaffolds have been proven to be biocompatible and suitable for use in blood contact applications. Finally, since Isabgol is a low-cost raw material, the quaternary ammonium-modified Isabgol scaffold can be an affordable wound dressing material.


title: Comparison of dry and wet electrodes for detecting gastrointestinal activity patterns from body surface electrical recordings

abstract:
ObjectiveGastrointestinal motility patterns can be mapped via electrical signals measured non-invasively on the body surface. However, long-term monitoring ([≥] 24 hr) may be hindered by skin-irritation inherent with traditional Ag/AgCl pre-gelled ("wet") electrodes. Therefore, the aim of this work was to investigate the practical utility of using dry electrodes for GI body-surface electrical measurements.

ApproachTo directly compare dry versus wet electrodes, we simultaneously recorded electrical signals from both types arranged in a 9 electrode (3 x 3) array during an {approx} 2.5 hr colonic meal-response study. Wavelet-based analyses were used to identify the signature post-meal colonic cyclic motor patterns. Signal quality was assessed for each electrode type through quantitative comparison of the dominant frequency, amplitude, signal-to-noise ratio (SNR), and signal energy vs time in the colonic frequency band. Blinded comparison of signal quality was carried out by four expert manual reviewers in order to assess the practical utility of each electrode type for identifying GI activity patterns.

Main resultsDry electrodes recorded high-quality GI signals comparable to that of wet electrodes, with dominant frequency in the range 2.85 - 3.25 cpm; peak-peak amplitudes of 120 {+/-} 40 {micro}V, and SNR in the range 7.5 - 11 dB. The CWT colonic frequency band energy versus time correlation coefficient value was [≥] 0.71 for the majority of studies (6 out of 7) indicating very good agreement between dry and wet electrode signals overall. Whereas wet electrodes were rated by expert reviewers as having slightly better signal quality for identifying GI activity patterns, dry electrodes caused no skin irritation and were thus better-tolerated by all subjects.

SignificanceDry electrodes are a viable option for long-term GI monitoring studies, offering a potentially more comfortable alternative to conventional wet electrode systems.


title: Engineered gene circuits with reinforcement learning allow bacteria to master gameplaying

abstract:
The engineering of living cells able to learn algorithms by themselves, such as playing board games --a classic challenge for artificial intelligence-- will allow complex ecosystems and tissues to be chemically reprogrammed to learn complex decisions. However, current engineered gene circuits encoding decision-making algorithms have failed to implement self-programmability and they require supervised tuning. We show a strategy for engineering gene circuits to rewire themselves by reinforcement learning. We created a scalable general-purpose library of Escherichia coli strains encoding elementary adaptive genetic systems capable of persistently adjusting their relative levels of expression according to their previous behavior. Our strains can learn the mastery of 3x3 board games such as tic-tac-toe, even when starting from a completely ignorant state. We provide a general genetic mechanism for the autonomous learning of decisions in changeable environments.

One-Sentence SummaryWe propose a scalable strategy to engineer gene circuits capable of autonomously learning decision-making in complex environments.