title: Structure and function of one unclassified-class glutathione S-transferase in Leptinotarsa decemlineata

abstract: Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation is through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agriculture pest of plants within Solanaceae have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 [A] by x-ray crystallography. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2,4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB could be inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.


title: Identification of novel chemical scaffolds that inhibit the growth of Mycobacterium tuberculosis in macrophages

abstract: Mycobacterium tuberculosis is an important global pathogen for which new drugs are urgently required. The ability of the organism to survive and multiply within macrophages may contribute to the lengthy treatment regimen with multiple drugs that are required to cure the infection. We screened the MyriaScreen II diversity library of 10,000 compounds to identify novel inhibitors of M. tuberculosis growth within macrophage-like cells using high content analysis. Hits were selected which inhibited the intramacrophage growth of M. tuberculosis without significant cytotoxicity to infected macrophages. We selected and prioritized compound series based on their biological and physicochemical properties and the novelty of the chemotypes. We identified five chemical classes of interest and conducted limited catalog structure-activity relationship studies to determine their tractability. We tested activity against intracellular and extracellular M. tuberculosis, as well as cytoxicity against murine RAW264.7 and human HepG2 cells. Benzene amide ethers, thiophene carboxamides and thienopyridines were only active against intracellular bacteria, whereas the phenylthiourea series was also active against extracellular bacteria. One member of a phenyl pyrazole series was moderately active against extracellular bacteria. We identified the benzene amide ethers as an interesting series for further work. These new compound classes serve as starting points for the development of novel drugs to target intracellular M. tuberculosis.

category: microbiology


title: Much more than a toxin: hydrogen cyanide is a volatile modulator of bacterial behaviour

abstract: Introductory paragraphBacteria communicate with each other and with other organisms in a chemical language comprising both diffusible and volatile molecules, and volatiles have recently gained increasing interest as mediators of bacterial interactions1. One of the first volatile compounds discovered to play a role in biotic interactions is hydrogen cyanide (HCN)2, a well-known toxin, which irreversibly binds to the key respiratory enzyme cytochrome C oxidase3. The main ecological function of this molecule was so far thought to lie in the inhibition of competing microorganisms4. Here we show that HCN is much more than a respiratory toxin and should be considered a major regulator of bacterial behaviour rather than a solely defensive secondary metabolite. Cyanogenesis occurs in both environmental and clinical Pseudomonas strains5,6. Using cyanide-deficient mutants in two Pseudomonas strains, we demonstrate that HCN functions as an intracellular and extracellular volatile signalling molecule, which leads to global transcriptome reprogramming affecting growth, motility, and biofilm formation, as well as the production of other secondary metabolites such as siderophores and phenazines. Our data suggest that bacteria are using endogenous HCN not only to control their own cellular functions, but are also able to remotely influence the behaviour of other bacteria sharing the same environment.


title: Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense

abstract:
The Nrf2/Keap1 axis plays a complex role in viral susceptibility, virus-associated inflammation and immune regulation. However, whether or how the Nrf2/Keap1 axis is involved in the interactions between equine lentiviruses and their hosts remains unclear. Here, we demonstrate that the Nrf2/Keap1 axis was activated during EIAV infection. Mechanistically, EIAV-Rev competitively binds to Keap1 and releases Nrf2 from Keap1-mediated repression, leading to the accumulation of Nrf2 in the nucleus and promoting Nrf2 responsive genes transcription. Subsequently, we demonstrated that the Nrf2/Keap1 axis represses EIAV replication via two independent molecular mechanisms: directly increasing antioxidant enzymes to promote effective cellular resistance against EIAV infection, and repression of Rev-mediated RNA transport through direct interaction between Keap1 and Rev. Together, these data suggest that activation of the Nrf2/Keap1 axis mediates a passive defensive response to combat EIAV infection. The Nrf2/Keap1 axis could be a potential target for developing the strategies for combating EIAV infection.

Author summaryHere we report that the Nrf2/Keap1 axis acts as an antiviral effector on EIAV replication through two-way regulation of Rev. On the one hand, EIAV-Rev activates the Nrf2/Keap1 axis by competitive interaction with Keap1, which promotes the transcription of cytoprotective genes implicated in the antiviral response. On the other hand, Keap1 restricts Rev/RRE-dependent RNA transport, leading to inhibition of viral protein synthesis and reduction in viral replication. This study highlights the importance of the Nrf2/Keap1 axis in controlling EIAV replication, and identifies this pathway as a potential new entry-point for treating lentivirus infection. Importantly, this report provides new insights into the regulation of viral replication by the Nrf2/Keap1 axis.


title: Intragenic proviral elements support transcription of defective HIV-1 proviruses

abstract:
HIV-establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription digital drop PCR identified Env and Nef transcripts which lacked 5 untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5UTR-deficient Env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5 rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH.

Author SummaryPeople living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes.


title: Streptococcus pneumoniae rapidly translocates from the nasopharynx through the cribriform plate to invade and inflame the dura

abstract: The entry routes and translocation mechanisms of bacterial pathogens into the central nervous system remain obscure. We report here that Streptococcus pneumoniae (Sp) or polystyrene microspheres, applied to the nose of a mouse, appeared in the meninges of the dorsal cortex within minutes. Recovery of viable bacteria from dissected tissue and fluorescence microscopy showed that up to at least 72h, Sp and microspheres were predominantly in the outer of the two meninges, the pachymeninx. No Sp were found in blood or cerebrospinal fluid. Evidence that this was not an artifact of the method of administration is that in mice infected by horizontal transmission, Sp were also predominantly in the meninges and absent from blood. Intravital imaging through the skull, and flow cytometry showed recruitment and activation of LysM+ cells in the dorsal pachymeninx at 5h and 10h following intranasal infection. Imaging of the cribriform plate suggested that both Sp and microspheres entered through its foramina via an inward flow of fluid connecting the nose to the pachymeninx. Our findings bring further insight into the invasion mechanisms of bacterial pathogens such as Sp into the central nervous system, but are also pertinent to the delivery of drugs to the brain, and the entry of air-borne particles into the cranium.


title: Iron Status Influences Mitochondrial Disease Progression in Complex I-Deficient Mice

abstract: Mitochondrial dysfunction caused by aberrant Complex I assembly and reduced activity of the electron transport chain is pathogenic in many genetic and age-related diseases. Mice missing the Complex I subunit NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4) are a leading mammalian model of severe mitochondrial disease that exhibit many characteristic symptoms of Leigh Syndrome including oxidative stress, neuroinflammation, brain lesions, and premature death. NDUFS4 knockout mice have decreased expression of nearly every Complex I subunit. As Complex I normally contains at least 8 iron-sulfur clusters and more than 25 iron atoms, we asked whether a deficiency of Complex I may lead to intracellular iron perturbations thereby accelerating disease progression. Consistent with this, iron supplementation accelerates symptoms of brain degeneration in these mice while iron restriction delays the onset of these symptoms and increases survival. NDUFS4 knockout mice display signs of iron overload in the liver including increased expression of hepcidin, and show changes in iron responsive element-regulated proteins consistent with increased intracellular iron that were prevented by iron restriction. These results suggest that perturbed iron homeostasis may contribute to pathology in Leigh Syndrome and possibly other mitochondrial disorders.

category: molecular biology


title: De novo biosynthesis of para-nitro-L-phenylalanine in Escherichia coli

abstract:
Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 {micro}M in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block.

HighlightsO_LIPara-nitro-L-phenylalanine (pN-Phe) is a valuable small molecule for its applications in genetic code expansion.
C_LIO_LIWe establish de novo biosynthesis of pN-Phe from glucose in E. coli, which is also the first example of a de novo pathway design for an unnatural but commonly used non-standard amino acid.
C_LIO_LIWe show the first use of an N-oxygenase enzyme in the de novo synthesis of a nitroaromatic product.
C_LIO_LIScreening of natural N-oxygenases and strain engineering resulted in final pN-Phe titers of 820 {+/-} 130 {micro}M in shake flask experiments with rich defined media.
C_LI

category: synthetic biology


title: Transcriptional interference in toehold switch-based RNA circuits

abstract: Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5 untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible for the ribosome. In the presence of a trigger RNA molecule protein production is turned on. Using antisense RNA against trigger molecules (anti-trigger RNA), gene expression can also be switched off again. We here study the utility and regulatory effect of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and anti-trigger transcription, whereas their cognate toehold switch, regulating expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is strongly influenced by transcriptional interference (TI) effects, leading to more than four-fold differences in expression levels. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. Based on this insight, we selected optimum promoter designs and arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers and two anti-triggers that function as a post-transcriptional RNA regulatory exclusive OR (XOR) gate.


title: Role for a lytic polysaccharide monooxygenase in cell wall remodelling

abstract: Peptidoglycan is a major constituent of the bacterial cell wall and an important determinant for providing protection to cells. Besides peptidoglycan (PG), many bacteria synthesize other glycans that become part of the cell wall. Streptomycetes grow apically, where they synthesize a glycan that is exposed at the outer surface, but how it gets there is unknown. Here we show that deposition of the apical glycan at the cell surface depends on two key enzymes, the endoglucanase CslZ and the lytic polysaccharide monooxygenase LpmP. Activity of these enzymes allows localized remodeling and degradation of the PG, and we propose that this facilitates passage of the glycan. The absence of both enzymes not only prevents morphological development, but also sensitizes strains to lysozyme. Given that lytic polysaccharide monooxygenases are commonly found in microbes, this newly identified biological role in cell-wall remodelling may be widespread.


title: Seasonal land use land cover change and the drivers in Kafta-Sheraro National Park, Tigray, Ethiopia

abstract: Kafta-sheraro national park (KSNP) is one of the homes of the African elephant has experienced extensive destruction of woodland following regular land use & land cover change in the past three decades, however, up to date, data and documentation detailing for these changes are not addressed. This study aims to evaluate the land use land cover change and drivers of change that occurred between 1988 and 2018. Landsat 5(TM), Landsat7 (ETM+), and Landsat 8 (OLI/TIRs) imagery sensors, field observation, and socio-economic survey data were used. The temporal and spatial Normalized difference vegetation index (NDVI) was calculated and tested the correlation between NDVI and precipitation/temperature. The study computed a kappa coefficient of the dry season (0.90) and wet season (0.845). Continuous decline of woodland (29.38%) and riparian vegetation (47.11%) whereas an increasing trend of shrub-bushland (35.28%), grassland (43.47%), bareland (27.52%), and cultivated land (118.36 km2) were showed over thirty years. More results showed bare land was expanded from wet to drier months, while, cultivated land and grazing land increased from dry to wet months. Based on the NDVI result high-moderate vegetation was decreased by 21.47% while sparse & non-vegetation was expanded by 19.8% & 1.7% (36.5 km2) respectively. Settlement & agricultural expansion, human-induced fire, firewood collection, gold mining, and charcoal production were the major proximate drivers that negatively affected the park resources. Around KSNP, the local community livelihood depends on farming, expansion of agricultural land is the main driver for woodland dynamics/depletion and this leads to increase resources competition and challenges for the survival of wildlife. Therefore, urgent sustainable conservation of park biodiversity via encouraging community participation in conservation practices and preparing awareness creation programs should be mandatory.

category: ecology


title: Functional and Highly Crosslinkable HIV-1 Envelope Glycoproteins Enriched in a Pretriggered Conformation

abstract:
Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (States 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the State-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize States 2/3. HIV-1 Env metastability has created challenges for defining the State-1 structure and developing immunogens mimicking this labile conformation. The availability of functional State-1 Envs that can be efficiently crosslinked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4 and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered State-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable Tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly crosslinkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state.

IMPORTANCEThe development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (State 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in State 1 and can be efficiently crosslinked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.


title: Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

abstract: The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of {beta}-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

category: biophysics


title: an anti-inflammatory activation sequence governs macrophage transcriptional dynamics during tissue injury

abstract:
Macrophages are essential for tissue repair and regeneration. Yet, the molecular programs, as well as the timing of their activation during and after tissue injury are poorly defined. Using a high spatio-temporal resolution single cell analysis of macrophages coupled with live imaging after sensory hair cell death in zebrafish, we find that the same population of macrophages transitions through a sequence of three major anti-inflammatory activation states. Macrophages first show a signature of glucocorticoid activation, then IL10 signaling and finally the induction of oxidative phosphorylation by IL4/Polyamine signaling. Importantly, loss-of-function of glucocorticoid and IL10 signaling shows that each step of the sequence is independently activated. Our results provide the first evidence that macrophages, in addition to a switch from M1 to M2, sequentially and independently transition though three anti-inflammatory pathways in vivo during tissue injury in a regenerating organ.

One-Sentence SummaryWe show that macrophages are sequentially activated by three different anti-inflammatory pathways during tissue injury.

category: immunology


title: Hippocampal-prefrontal theta coupling develops as mice become proficient in associative odorant discrimination learning

abstract: Learning and memory requires coordinated activity between different regions of the brain. Here we studied the interaction between infralimbic medial prefrontal cortex (mPFC) and hippocampal dorsal CA1 during associative odorant discrimination learning in the male mouse. We found that as the animal learns to discriminate odorants in a go-no go task the coupling of high frequency neural oscillations to the phase of theta oscillations (theta-referenced phase-amplitude coupling or tPAC) changes in a manner that results in divergence between rewarded and unrewarded odorant-elicited changes in the theta-phase referenced power (tPRP) for beta and gamma oscillations. In addition, in the proficient animal there was a decrease in the coordinated oscillatory activity between CA1 and mPFC in the presence of the unrewarded odorant. Furthermore, the changes in tPAC resulted in a marked increase in the accuracy for decoding contextual odorant identity from tPRP when the animal became proficient. Finally, we studied the role of Ca2+/calmodulin-dependent protein kinase II  (CaMKII), a protein involved in learning and memory, in oscillatory neural processing in this task. We find that the accuracy for decoding the contextual odorant identity from tPRP decreases in CaMKII knockout mice and that this accuracy correlates with behavioral performance. These results implicate a role for tPAC and CaMKII genotype in olfactory go-no go associative learning in the hippocampal-prefrontal circuit.

category: neuroscience


title: Neuronal glucose metabolism sets cholinergic tone and controls thermo-regulated signaling at the neuromuscular junction

abstract:
Cholinergic and sympathetic counter-regulatory networks control numerous physiologic functions including learning/memory/cognition, stress responsiveness, blood pressure, heart rate and energy balance. As neurons primarily utilize glucose as their primary metabolic energy source, we generated mice with increased glycolysis in cholinergic neurons by specific deletion of the fructose-2,6-phosphatase protein TIGAR. Steady-state and stable isotope flux analyses demonstrated increased rates of glycolysis, acetyl-CoA production, acetylcholine levels and density of neuromuscular synaptic junction clusters with enhanced acetylcholine release. The increase in cholinergic signaling reduced blood pressure and heart rate with a remarkable resistance to cold-induced hypothermia. These data directly demonstrate that increased cholinergic signaling through the modulation of glycolysis has several metabolic benefits particularly to increase energy expenditure and heat production upon cold exposure.

HighlightsO_LIDeficiency of a negative regulator of glycolysis (TIGAR) in cholinergic neurons increases the biosynthesis and content of the neurotransmitter acetylcholine.
C_LIO_LIIncreased cholinergic tone reduces blood pressure and heart rate while enhancing signaling at neuromuscular junction.
C_LIO_LIUpregulation of neuromuscular junction activation provides protection against the paralytic curare and cold-induced hypothermia.
C_LIO_LIModulation of cholinergic neuron glycolysis may provide a novel therapeutic approach for treatment of diseases stemming from reduced acetylcholine signaling such as myasthenia gravis and sarcopenic pre-synaptic dysfunction.
C_LI

category: cell biology


title: Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction

abstract:
BackgroundBiopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise.

MethodsReproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling.

ResultsReproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001s bioactivity : MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.

NOTEPlease see the end of the paper for links to ten files of supplementary information and for an independent peer review.

KEY POINTSO_LISynthetic hexapeptides have upregulated and downregulated nematode reproduction, following similar work in other nematode species and in fish and frogs.
C_LIO_LIPeptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro and influence circulating levels of ovine pituitary hormones in vivo via intracerebroventricular peptide infusion.
C_LIO_LIThe amino acid sequence of the 14mer master peptide arose from a (rat) bioassay- guided fractionation using ovine materials, whose aim was to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination.
C_LIO_LIThe bioactive peptides mimic the non-contiguous six-residue receptor-binding active face of a polypeptide hormone, which in mammals is a derivative of the secretory vesicle prohormone secretogranin II (SgII), taking the form of a novel, structually complex 70mer dubbed  SgII-70.
C_LIO_LIThe ability has been gained to reinforce or block a default reproductive and tissue- building OFF signal, using peptide mimetics.
C_LI

category: physiology


title: Synthesis of 4-methylvaleric acid, a precursor of pogostone, involves a 2-isobutylmalate synthase related to 2-isopropylmalate synthase of leucine biosynthesis

abstract:
O_LIWe show here that the side chain of pogostone, one of the major components of patchouli oil obtained from Pogostemon cablin and possessing a variety of pharmacological activities, is derived from 4-methylvaleric acid.
C_LIO_LIWe also show that 4-methylvaleric acid is produced through the one-carbon - ketoacid elongation pathway with the involvement of the key enzyme 2- isobutylmalate synthase (IBMS), a newly identified enzyme related to isopropylmalate synthase (IPMS) of Leu biosynthesis.
C_LIO_LISite-directed mutagenesis identified Met132 in the N-terminal catalytic region as affecting the substrate specificity of PcIBMS1. And even though PcIBMS1 possesses the C-terminal domain that in IPMS serves to mediate Leu inhibition, it is insensitive to Leu.
C_LIO_LIThe observation of the evolution of IBMS from IPMS, as well as previously reported examples of IPMS-related genes involved in making glucosinolates in Brassicaceae, acylsugars in Solanaceae, and flavor compounds in apple, indicate that IPMS genes represent an important pool for the independent evolution of genes for specialized metabolism.
C_LI

One Sentence SummaryWe describe a novel enzyme, 2-isobutylmalate synthase, that is related to 2-isopropylmalate synthase and is able to efficiently convert 4-methyl-2- oxovalerate to 2-isobutylmalate, a key intermediate in the synthesis of the pogostone precursor 4-methylvaleric acid.

category: plant biology


title: Adeno-associated virus (AAV) reduces cortical dendritic complexity in a TLR9-dependent manner

abstract: Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery in the nervous system. AAVs are widely used in research and are the basis of multiple FDA-approved gene therapies. Here, we find that the immune response to AAVs genome reduces dendritic complexity in mammalian cortex. Dendritic loss associated with AAV-mediated gene delivery occurs at experimentally-relevant titers, cannot be explained by responses to transgene expression or surgery, and is not restricted to a particular capsid serotype, encoded transgene, promoter, or production facility. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and upregulation of immune molecules that can limit dendritic complexity and synaptic transmission. Blocking detection of unmethylated CpG-rich DNA via Toll-like receptor 9 (TLR9) protects dendritic complexity, suggesting that immunodetection of a core feature of the AAV genome triggers dendritic loss. These results reveal previously unsuspected impacts of AAV on neuronal structure and function and identify TLR9 inhibitors as important tools to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.


title: Single-nucleus transcriptomic atlas of spinal cord neuron in human

abstract: Despite the recognized importance of spinal cord in sensory processing, motor behaviors and/or neural diseases, the underlying neuronal clusters remain elusive. Recently, several studies attempted to define the neuronal types and functional heterogeneity in spinal cord using single cell and/or single-nucleus RNA-sequencing in varied animal models. However, the molecular evidence of neuronal heterogeneity in human spinal cord has not been established yet. Here we sought to classify spinal cord neurons from human donors by high-throughput single-nucleus RNA-sequencing. The functional heterogeneity of identified cell types and signaling pathways that connecting neuronal subtypes were explored. Moreover, we also compared human results with previous single-cell transcriptomic profiles of mouse spinal cord. As a result, we generated the first comprehensive atlas of human spinal cord neurons and defined 18 neuronal clusters. In addition to identification of the new and functionally-distinct neuronal subtypes, our results also provide novel marker genes for previously known neuronal types. The comparation with mouse transcriptomic profiles revealed an overall similarity in the cellular composition of spinal cord between the two species. In summary, these results illustrate the complexity and diversity of neuronal types in human spinal cord and will provide an important resource for future researches to explore the molecular mechanism underlying several spinal cord physiology and diseases.


title: Regulation of Blos1 by IRE1 prevents the accumulation of Huntingtin protein aggregates

abstract:
Huntingtons Disease is characterized by accumulation of the aggregation-prone mutant Huntingtin (mHTT) protein. Here, we show that expression of mHTT in mouse cultured cells activates IRE1, the transmembrane sensor of stress in the endoplasmic reticulum, leading to degradation of the Blos1 mRNA and repositioning of lysosomes and late endosomes toward the microtubule organizing center. Overriding Blos1 degradation results in accumulation of larger mHTT aggregates and increased cell death. Although mHTT is degraded by macroautophagy when highly expressed, we show that prior to the formation of large aggregates, mHTT is degraded via an ESCRT-dependent, endosomal microautophagy pathway. This pathway is enhanced by Blos1 degradation and appears to protect cells from a toxic, less aggregated form of mHTT.

Condensed titleBlos1 regulation protects from Huntingtin protein aggregation


title: Single-cell transcriptomic analysis unveils spinal motor neuron subtype diversity underpinning the water-to-land transition in vertebrates

abstract: Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate motor movements in vertebrates. Distinct MN populations and their diversity has long been hypothesized to co-evolve with motor circuit to provide the neural basis from undulatory to ambulatory locomotion during aquatic-to-terrestrial transition of vertebrates. However, how these subtypes are evolved remains largely enigmatic. Using single-cell transcriptomics, we investigate heterogeneity in mouse MNs and discover novel segment-specific subtypes. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. We further uncovered that axial MNs are subdivided by three conserved and molecularly distinct subpopulations, defined by Satb2, Nr2f2 or Bcl11b expression. Although axial MNs are conserved from cephalochordates to humans, subtype diversity becomes prominent in land animals and appears to continue evolving in humans. Overall, our study provides a unified classification system for spinal MNs and paves the way towards deciphering how neuronal subtypes are evolved.

category: developmental biology


title: Osteoblasts pattern endothelium and somatosensory axons during zebrafish caudal fin organogenesis

abstract:
Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones, and endothelium. In adults, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development, we show the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation, and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.

Summary statementAnalysis of cellular interdependence during caudal fin development reveals roles for osteoblasts in patterning endothelium and somatosensory axon innervation.


title: The spread of the first introns in proto-eukaryotic paralogs

abstract: Spliceosomal introns are a unique feature of eukaryotic genes. Previous studies have established that many introns were present in the protein-coding genes of the last eukaryotic common ancestor (LECA). Intron positions shared between genes that duplicated before LECA could in principle provide insight into the emergence of the first introns. In this study we use ancestral intron position reconstructions in two large sets of duplicated families to systematically identify these ancient paralogous intron positions. We found that 20-35% of introns inferred to have been present in LECA were shared between paralogs. These shared introns, which likely preceded ancient duplications, were widespread across different functions, with the notable exception of nuclear transport. Since we observed a clear signal of pervasive intron loss prior to LECA, it is likely that substantially more introns were shared at the time of duplication than we can detect in LECA. The large extent of shared introns indicates an early origin of introns during eukaryogenesis and suggests an early origin of a nuclear structure, before most of the other complex eukaryotic features were established.

category: evolutionary biology


title: Comparative transcriptomic analysis of maize ear heterosis during the inflorescence meristem differentiation stage

abstract: Heterosis is widely used in many crops; however, its genetic mechanisms are only partly understood. Here, we sampled inflorescence meristem (IM) ears from the single-segment substitution maize (Zea mays) line lx9801hlEW2b, containing a heterotic locus hlEW2b associated with ear width, the receptor parent lx9801, the test parent Zheng58, and their corresponding hybrids. After transcriptomic analysis, 1638 genes were identified in at least one hybrid with nonadditively expressed patterns and different expression levels between the two hybrids. In particular, 2263 (12.89%) and 2352 (14.65%) genes showed allele-specific expression (ASE) in Zheng58 x lx9801 and Zheng58 x lx9801hlEW2b, respectively. A functional analysis showed that these genes were enriched in development-related processes and biosynthesis and catabolism processes, which are potentially associated with heterosis. Additionally, nonadditive expression and ASE may fine-tune the expression levels of crucial genes (such as WUS and KNOX that control IM development) controlling auxin metabolism and ear development to optimal states, and transcriptional variation may play important roles in maize ear heterosis. The results provide new information that increases our understanding of the relationship between transcriptional variation and heterosis formation during maize ear development, which may be helpful in clarifying the genetic and molecular mechanisms of heterosis.

category: genetics


title: Chromosome-scale genome assembly of the brown anole (Anolis sagrei), a model species for evolution and ecology

abstract: Rapid technological improvements are democratizing access to high quality, chromosome-scale genome assemblies. No longer the domain of only the most highly studied model organisms, now non-traditional and emerging model species can be genome-enabled using a combination of sequencing technologies and assembly software. Consequently, old ideas built on sparse sampling across the tree of life have recently been amended in the face of genomic data drawn from a growing number of high-quality reference genomes. Arguably the most valuable are those long-studied species for which much is already known about their biology; what many term emerging model species. Here, we report a new, highly complete chromosome-scale genome assembly for the brown anole, Anolis sagrei - a lizard species widely studied across a variety of disciplines and for which a high-quality reference genome was long overdue.

category: genomics


title: Muscle group specific transcriptomic and DNA methylation differences related to developmental patterning in FSHD

abstract: Muscle groups throughout the body are specialized in function and are specified during development by position specific gene regulatory networks. In developed tissue, myopathies affect muscle groups differently. Facioscapulohumeral muscular dystrophy, FSHD, affects upper body and tibialis anterior (TA) muscles earlier and more severely than others such as quadriceps. To investigate an epigenetic basis for susceptibility of certain muscle groups to disease, we perform DNA methylation and RNA sequencing on primary patient derived myoblasts from TA and quadricep for both control and FSHD2 as well as RNA-seq for myoblasts from FSHD1 deltoid, bicep and TA over a time course of differentiation. We find that TA and quadricep retain methylation and expression differences in transcription factors that are key to muscle group specification during embryogenesis. FSHD2 patients have differences in DNA methylation and expression related to SMCHD1 mutations and FGF signaling. Genes induced specifically in FSHD are more highly expressed in commonly affected muscle groups. We find a set of genes that distinguish more susceptible muscle groups including development-associated TFs and genes involved in WNT signaling. Adult muscle groups therefore retain transcriptional and DNA methylation differences associated with development, which may contribute to susceptibility in FSHD.


title: Reference genomes of 545 silkworms enable high-throughput exploring genotype-phenotype relationships

abstract: The silkworm Bombyx mori is a domestic insect for silk production and a lepidopteran model. The currently available genomes limit a full understanding of its genetic and phenotypic diversity. Here we assembled long-read genomes of 545 domestic and wild silkworms and constructed a high-resolution pan-genome dataset. We found that the silkworm population harbors extremely variable genomes containing 7,308 new gene families, 4,260 (22%) core gene families, and 3,432,266 non-redundant SVs. We deciphered a series of causal genes and variants associated with domestication, breeding, and ecological adaptation traits, and experimentally validated two of those genes using CRISPR-Cas9 or RNA interference. This unprecedented large-scale genomic resource allows for high-throughput screening of interesting traits for functional genomic research and breeding improvement of silkworms and may serve as a guideline for traits decoding in other species.


title: Insights into opium poppy (Papaver spp.) genetic diversity from genotyping-by-sequencing analysis

abstract: Opium poppy (Papaver somniferum) is one of the worlds oldest medicinal plants and a versatile model system to study secondary metabolism. However, our knowledge of its genetic diversity is limited, restricting utilization of the available germplasm for research and crop improvement. We used genotyping-by-sequencing to investigate the extent of genetic diversity and population structure in a collection of poppy germplasm consisting of 91 accessions originating in 30 countries of Europe, North Africa, America, and Asia. We identified five genetically distinct subpopulations using discriminate analysis of principal components and STRUCTURE analysis. Most accessions obtained from the same country were grouped together within subpopulations, likely a consequence of the restriction on movement of poppy germplasm. Alkaloid profiles of accessions were highly diverse, with morphine being dominant. Phylogenetic analysis identified genetic groups that were largely consistent with the subpopulations detected and that could be differentiated broadly based on traits such as number of branches and seed weight. These accessions and the associated genotypic data are valuable resources for further genetic diversity analysis, which could include definition of poppy core sets to facilitate genebank management and use of the diversity for genetic improvement of this valuable crop.


title: Real-world size of objects serves as an axis of object space

abstract:
Our mind can represent various objects from the physical world metaphorically into an abstract and complex high-dimensional object space, with a finite number of orthogonal axes encoding critical object features. Previous fMRI studies have shown that the middle fusiform sulcus in the ventral temporal cortex separates the real-world small-size map from the large-size map. Here we asked whether the feature of objects real-world size constructed an axis of object space with deep convolutional neural networks (DCNNs) based on three criteria of sensitivity, independence and necessity that are impractical to be examined altogether with traditional approaches. A principal component analysis on features extracted by the DCNNs showed that objects real-world size was encoded by an independent component, and the removal of this component significantly impaired DCNNs performance in recognizing objects. By manipulating stimuli, we found that the shape and texture of objects, rather than retina size, co-occurrence and task demands, accounted for the representation of the real-world size in the DCNNs. A follow-up fMRI experiment on humans further demonstrated that the shape, but not the texture, was used to infer the real-world size of objects in humans. In short, with both computational modeling and empirical human experiments, our study provided the first evidence supporting the feature of objects real-world size as an axis of object space, and devised a novel paradigm for future exploring the structure of object space.

TeaserThis work provides the first evidence illuminating the feature of objects real-world size as an axis of the object space for object recognition with a mutually-inspired paradigm of computational modelling and biological observation.


title: Redundant functions of the SLC5A transporters Rumpel Kumpel and Bumpel in ensheathing glial cells

abstract: Neuronal processing is energy demanding, and relies on sugar metabolism as an energy source. To provide a constant metabolite supply neurons and glial cells express many glucose and lactate transporters of the solute carrier (SLC) 5A family. Here we dissect the partially redundant functions of three highly related glia specific Drosophila genes encoding SLC5A proteins, Rumpel, Bumpel and Kumpel. While knockdown of rumpel causes several behavioral phenotypes, they are less prominent in rumpel mutants. bumpel and kumpel mutants are viable and fertile, lacking discernible phenotypes. However, in bumpel kumpel double mutants and to an even greater extent in rumpel bumpel kumpel triple mutants oogenesis is disrupted at the onset of the vitollegenic phase. This indicates at least partially redundant functions between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporter proteins, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, our results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior.


title: De novo variants in Chinese ASD trios reveal genetic basis underlying autism without developmental delay and intellectual disabilities

abstract: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that causes a range of social communication and behavioral impairments. ASD typically manifests in young children, often with developmental delay or intellectual disabilities (DD/ID) as comorbidities. Accruing evidence indicates that ASD is highly heritable and genomewide studies on ASD cohorts have defined numerous genetic contributors. Notably, most of these studies have been performed with individuals of European and Hispanic ancestry and thus there is a paucity of genetic analyses of ASD in the East Asian population. Here, we performed whole-exome sequencing on 772 ASD trios from China, combining with a previous study of 369 Chinese ASD trios, to identify de novo variants in a total of 1141 ASD trios. We found that ASD probands without DD/ID carried less disruptive de novo variants, including protein-truncating and missense variants, than ASD with DD/ID. Surprisingly, we showed that expression of genes with de novo variants found in ASD probands without DD/ID were enriched in a specific group of neural progenitor cells, suggesting a potential mechanism underlying high-functioning autism. Importantly, some ASD risk genes from this study are not present in the current ASD gene database, suggesting that there are novel genetic contributors to ASD in East Asian populations. We validated one such novel ASD risk gene - SLC35G1 by showing that mice harboring heterozygous deletion of Slc35g1 exhibited defects in social interaction behaviors. Together, this work nominates novel ASD risk genes and indicates that ASD genetic studies in different geographic populations are essential to reveal the comprehensive genetic architecture of ASD.


title: Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation

abstract: The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the 2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22s functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest the motor domain 2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of 2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.


title: Different methods for niche and fitness differences computation offer contrasting explanations of species coexistence

abstract: In modern coexistence theory, species coexistence can either arise via stabilizing mechanisms that increase niche differences or equalizing mechanisms that reduce fitness differences. Having a common currency for interpreting these mechanisms is essential for synthesizing knowledge across different studies and systems. Several methods for quantifying niche and fitness differences exist, but it remains unknown to what extent these methods agree on the reasons why species coexist. Here, we apply four common methods to quantify niche and fitness differences to one simulated and two empirical data sets. We ask if different methods result in different insights into what drives species coexistence. We find that different methods disagree on the effects of resource supply rates (simulated data), and of plant traits or phylogenetic distance (empirical data), on niche and fitness differences. More specifically, these methods often do not agree better than expected by chance. We argue for (1) a better understanding of what connects and sets apart different methods, and (2) the simultaneous application of multiple methods to enhance a more complete insight into why species coexist.


title: Genotype-specific benefits and risks of a mutualist association during natural epidemics

abstract:
O_LIWhile pathogenic and mutualistic microbes are ubiquitous across ecosystems and often co-occur within hosts, how they interact to determine patterns of disease in genetically diverse host populations is unknown.
C_LIO_LITo test whether microbial mutualists provide protection against pathogens, and whether this is influenced by host genotype, we conducted a field experiment in three naturally-occurring epidemics of a fungal pathogen, Podosphaera plantaginis, infecting a host plant, Plantago lanceolata, in the [A]land Islands, Finland. In each population, we collected epidemiological data on experimental plants, originating from six allopatric populations, which had been inoculated with a mixture of mutualistic mycorrhizal fungi or a non-mycorrhizal control.
C_LIO_LIInoculation with mycorrhizal fungi introduced benefits to host from every population, such as increased host growth and reduced disease severity. Host genotypes that were more susceptible to the pathogen received stronger protective effects from mycorrhizal fungi. However, inoculation with mycorrhizal fungi also increased host infection risk.
C_LIO_LIOur results show that mycorrhizal fungi introduce growth and defensive benefits, and shift infection risks in host populations under pathogen attack. Understanding how mutualists alter host susceptibility to disease will be important for predicting infection outcomes in ecological communities and in agriculture.
C_LI

Plain Language SummaryBeneficial,  mycorrhizal fungi in roots help plants grow and can protect them from disease caused by other, pathogenic microbes. This study shows that mycorrhizal fungi influence patterns of plant disease during pathogen outbreaks in a natural landscape.


title: Mycorrhizal fungi influence disease during epidemics in plant populations


title: The extended Price equation: migration and sex

abstract: The Price equation provides a general partition of evolutionary change into two components. The first is usually thought to represent natural selection and the second, transmission bias. Here, we provide a new derivation of the generalised equation, which contains a largely ignored third term. Unlike the original Price equation, this extension can account for migration and mixed asexual and sexual reproduction. The notation used here expresses the generalised equation explicitly in terms of fitness, rendering this otherwise difficult third term more open to biological interpretation and use. This re-derivation also permits fundamental results, derived from the Price equation, to be more easily generalised. We take Hamiltons rule as a case study, and provide an exact, total expression that allows for population structures like haplodiploidy. Our analysis, more generally, makes clear the previously hidden assumptions in similar fundamental results, highlighting the caution that must be taken when interpreting them.


title: Inevitability of Red Queen evolution driven by organismic complexity and simple feedback via environmental modification

abstract: Evolution in complex high-dimensional phenotype spaces can be very different than the caricature of uphill evolutionary trajectories in a low-dimensional fitness landscape. And slight modifications of the environment can have large consequences for the future evolution. Here, the simplest approximation of evolution, an almost-always clonal population evolving by small effect mutations under deterministic "adaptive" dynamics, is studied. The complexities of organisms and their interactions with their environments are caricatured by population growth rates being smoothly varying random functions in very high dimensional phenotype spaces. In a fixed environment, there are huge numbers of fitness maxima, yet evolutionary trajectories wander around amongst saddles, gradually slowing down but still wandering widely and without committing to any maximum. But with even very small changes of the environment caused by the phenotypic changes, after an initial transient the evolution continues forever without further slowing down. In this Red Queen "phase" the apparent rate of increase of the fitness saturates (at a feedback strength-dependent rate) and the trajectories perpetually wander over large phenotypic distances. Organismic complexities, caricatured by a large number of constraints on the molecular-level phenotype, together with the simplest possible interactions of the organisms with their environment, are shown to be sufficient to cause such Red Queen dynamics. Arguments are made for the ubiquity of such behavior.


title: A genetic bottleneck of mitochondrial DNA during human lymphocyte development

abstract: Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Mutations that accumulate in mitochondrial DNA (mtDNA) in somatic cells have been implicated in cancer, degenerative diseases, and the aging process. However, the mechanisms used by somatic cells to maintain proper functions despite their mtDNA mutation load are poorly understood. Here, we analyzed somatic mtDNA mutations in more than 30,000 human single peripheral and bone marrow mononuclear cells and observed a significant overrepresentation of homoplastic mtDNA mutations in B, T and NK lymphocytes despite their lower mutational burden than other hematopoietic cells. The characteristic mutational landscape of mtDNA in lymphocytes were validated with data from multiple platforms and individuals. Single-cell RNA-seq and computational modeling demonstrated a stringent mitochondrial bottleneck during lymphocyte development likely caused by lagging mtDNA replication relative to cell proliferation. These results illuminate a potential mechanism used by highly metabolically active immune cells for quality control of their mitochondrial genomes.


title: Improved Characterization of Balancing Selection Genome Wide and a Detailed Look at HLA Genes

abstract: Balancing selection occurs when multiple alleles are kept at elevated frequencies in equilibrium due to opposing evolutionary pressures. A new statistical method was developed to test for selection using efficient Bayesian techniques. Selection signals in three different data sets, generated with variable sequencing technologies, were compared: clinical trios, HLA NGS typed samples, and whole-genome long-read samples. Genome-wide, selection was observed across multiple gene families whose biological functions favor diversification, revealing established targets as well as 45 novel genes under selection. Using high-resolution HLA typing and long-read sequencing data, for the characterization of the MHC, revealed strong selection in expected peptide-binding domains as well as previously understudied intronic and intergenic regions of the MHC. Surprisingly, SIRPA, demonstrated dramatic selection signal, second only to the MHC in most settings. In conclusion, employing novel statistical approaches and improved sequencing technologies is critical to properly analyze complex genomic regions.


title: Improved detection of evolutionary selection highlights potential bias from different sequencing strategies in complex genomic-regions


title: Rod-genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor compositions and progressive retinal degeneration

abstract:
The neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of rod photoreceptors in mammals. Mutations in NRL have been associated with autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in zebrafish, a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model by CRISPR-Cas9 technology and observed a surprising phenotype characterized by the reduction but not total elimination of rods and the over-grown of green-cones. By tracing the developmental process of rods, we discovered two waves of rod genesis in zebrafish, emerging at the embryonic stage with an nrl-dependent pattern and the post-embryonic stage with an nrl-independent pattern, respectively. Through bulk and single-cell RNA sequencing, we constructed the gene expression profiles for the whole retinal tissues and each of the retinal cell types in WT and nrl knockout zebrafish. We detected the rod/green-cone intermediate photoreceptors in nrl knockout zebrafish, suggesting that there may be a kind of rod/green-cone bipotent precursors and its fate choice between rod and green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator for nrl-independent rods, based on the cell-type-specific expression pattern and the retinal phenotype of nrl/mafba double knockout zebrafish. Furthermore, the altered photoreceptor compositions and abnormal gene expression caused progressive retinal degeneration and subsequent regeneration in nrl knockout zebrafish. Our work revealed a novel function of mafba gene in rod development and established a more suitable model for the developmental processes and regulatory mechanisms of rod and green-cone photoreceptors in zebrafish.

Author SummaryVision is mediated by two types of light-sensing cells named rod and cone photoreceptors in animal eyes. Abnormal generation, dysfunction or death of photoreceptor cells can all cause irreversible vision problems. NRL is the most important gene for the occurrence and function of rod cells in mice and humans. Surprisingly, we found that in zebrafish, a popular animal model used for mimicking human diseases and testing treatments, there are two types of rod cells and breaking the function of nrl gene only affects the generation of rod cells at the embryonic stage but not the juvenile and adult stages. The rod cells produced later are proved to be driven by the mafba gene, which has not been reported to play a role in rod cells. In addition to the reduction of rod cells, deletion of nrl also results in the occurrence of rod/green-cone hybrid cells and the increasing number of green-cones. The combination of changes at cellular and molecular levels finally lead to a disease condition named retinal degeneration. These findings add new knowledge to the research field and highlight the conserved and species-specific regulatory mechanisms of photoreceptor development and maintenance.


title: Clonal hematopoiesis is associated with increased toxicity in large B-cell lymphoma patients treated with chimeric antigen receptor T cell therapy

abstract:
To explore the role of clonal hematopoiesis (CH) on chimeric antigen receptor (CAR) T-cell therapy outcomes, we performed targeted deep-sequencing on 114 large B-cell lymphoma patients treated with anti-CD19 CAR T-cells. We detected CH in 42 (36.8%) pre-treatment patient samples, most frequently in PPM1D (19/114) and TP53 (13/114) genes. The incidence of grade [≥]3 immune-effector cell-associated neurotoxicity syndrome (ICANS) was higher in CH-positive patients compared to CH-negative patients (45.2% vs. 25.0%, p=0.038). Higher toxicities with CH were primarily driven by three CH genes, DNMT3A, TET2 and ASXL1 (DTA mutations). The incidence of grade [≥]3 ICANS [58.9% vs. 25%, p=0.02] and grade [≥]3 cytokine release syndrome [17.7% vs. 4.2%, p=0.08] were higher in patients with DTA mutations than those without CH. The estimated 24-month cumulative incidence of therapy-related myeloid neoplasms after CAR-T therapy was higher in patients with CH than those without CH (19% [95%CI: 5.5-38.7] vs. 4.2% [95%CI: 0.3-18.4], p=0.028).

Statement of SignificanceOur study reveals that clonal hematopoiesis mutations, especially those associated with inflammation (DNMT3A, TET2, ASXL1), are associated with severe grade toxicities in lymphoma patients receiving anti-CD19 chimeric antigen receptor therapy. Further studies to investigate the mechanisms and interventions to improve toxicities in the context of CH are warranted.


title: Using TrackMate to Analyze Drosophila Larval and Adult Locomotion

abstract:
Drosophila adult and larvae exhibit sophisticated behaviors that are widely used in development, synaptic transmission, sensory physiology, and learning and memory research. Many of these behaviors depend on locomotion, the ability of an animal to move. However, the statistical analysis of locomotion is not trivial. Here we use an open-source Fiji plugin TrackMate to track the locomotion of Drosophila adults and larvae. We build optimal experimental setups to rapidly process recordings by Fiji and analyze by TrackMate. We also provide tips for analyzing non-optimal recordings. TrackMate extracts the X and Y positions of an animal on each frame of an image sequence or a video. This information allows for generating moving trajectories, calculating moving distances, and determining preference indices in two-choice assays. Notably, this free-cost analysis method does not require programming skills.

Summary statementThis study uses an open-source Fiji plugin TrackMate to computationally analyze Drosophila adult and larval behavioral assays, which does not require programming skills.

category: animal behavior and cognition


title: Enhanced keratin extraction from wool waste using a deep eutectic solvent

abstract:
In this study, the solubilisation of waste coarse wool as a precursory step for the large scale valorisation of keratin was investigated using a green deep eutectic solvent (DES) based on L-cysteine and lactic acid. The investigation was undertaken via the response surface methodology and based on the Box-Behnken design for four process variables of temperature (70-110 {degrees}C), dissolution time (2-10 h), the mass of L-cysteine (0.5-2.5 g) in 20 mL of lactic acid, and wool load in the DES (0.2-0.6 g). The effect of variations in temperature was established to be the most significant process variable influencing keratin yield from waste coarse wool in the current work. An optimum keratin yield (93.77 wt.%) was obtained at the temperature of 105 {degrees}C, 8 h dissolution time, with 1.6 g L-cysteine in 20 mL of lactic acid using 0.5 g of wool. This study suggests L-cysteine and lactic acid as a green solvent with the potential to scale up keratin recovery from waste wool without significant destruction in the structure of the recovered keratin.

HighlightsO_LIKeratin recovery from wool using deep eutectic solvent was assessed
C_LIO_LIThe basis for the use of the new deep eutectic solvent was discussed
C_LIO_LIThe effects of the process variables on keratin yield were explored
C_LIO_LIKeratin recovered was optimised and characterised.
C_LI

category: bioengineering


title: Predicting Knee Adduction Moment Response to Gait Retraining with Minimal Clinical Data

abstract:
Knee osteoarthritis is a progressive disease mediated by high joint loads. Foot progression angle modifications that reduce the knee adduction moment (KAM), a surrogate of knee loading, have demonstrated efficacy in alleviating pain and improving function. Although changes to the foot progression angle are overall beneficial, KAM reductions are not consistent across patients. Moreover, customized interventions are time-consuming and require instrumentation not commonly available in the clinic. We present a model that uses minimal clinical data to predict the extent of first peak KAM reduction after toe-in gait retraining. For such a model to generalize, the training data must be large and variable. Given the lack of large public datasets that contain different gaits for the same patient, we generated this dataset synthetically. Insights learned from ground-truth datasets with both baseline and toe-in gait trials (N=12) enabled the creation of a large (N=138) synthetic dataset for training the predictive model. On a test set of data collected by a separate research group (N=15), the first peak KAM reduction was predicted with a mean absolute error of 0.134% body weight * height (%BW*HT). This error is smaller than the test sets subject average standard deviation of the first peak during baseline walking (0.306 %BW*HT). This work demonstrates the feasibility of training predictive models with synthetic data and may provide clinicians with a streamlined pathway to identify a patient-specific gait retraining outcome without requiring gait lab instrumentation.

Author SummaryGait retraining as a conservative intervention for knee osteoarthritis shows great promise in extending pain-free mobility and preserving joint health. Although customizing a treatment plan for each patient may help to ensure a therapeutic response, this procedure cannot yet be performed outside of the gait laboratory, preventing research advances from becoming a part of clinical practice. Our work aims to predict the extent to which a patient with knee osteoarthritis will benefit from a non-invasive gait retraining therapy using measures that can be easily collected in the clinic. To overcome a lack of normative databases for gait retraining, we generated data synthetically based on limited ground-truth examples, and provided experimental evidence for the models ability to generalize to new subjects by evaluating on data collected by a separate research group. Our results can contribute to a future in which predicting the therapeutic benefit of a potential treatment can determine a custom treatment path for any patient.


title: Metrics of High Cofluctuation and Entropy to Describe Control of Cardiac Function in the Stellate Ganglion

abstract: Cardiac neural control requires dynamic adaptation of mechanical and electrical indices to meet blood flow demands. The control system receives inputs to coordinate cardiac function, producing "functional" outputs such as blood pressure and heart rate. Bilateral stellate ganglia (SG) integrate inputs and produce efferent cardiopulmonary sympathetic outputs. We investigate network processing of cardiopulmonary transduction by SG neuronal populations in porcine with chronic pacing-induced heart failure and control subjects during extended in-vivo extracellular microelectrode recordings. We derive network-level spatiotemporal dynamic signatures based on linking neuronal population cofluctuation and examine differences in "neural specificity" of SG network activity to cardiac cycle phases. Information processing and cardiac control in chronic heart failure by the SG, relative to controls, exhibits: i) more frequent, short-lived, high magnitude cofluctuations, ii) greater variation in neural specificity to cardiac cycles, and iii) neural network activity and cardiac control linkage that depends on disease state and cofluctuation magnitude.


title: Nanocrown electrodes for robust and scalable intracellular recordings of cardiomyocytes for cardiotoxicity screening

abstract: Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. However, the conventional patch clamp techniques for measuring iAPs are either laborious and low throughput or not suitable for measuring electrically connected cardiomyocytes. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. Nanoelectrodes show great promise, but the large variability in success rate, signal strength, and the low throughput of device fabrication have hindered them from being broadly adopted for proarrhythmia drug assessment. In this work, we developed vertically-aligned and semi-hollow nanocrown electrodes that are mechanically robust and made through a scalable fabrication process. Nanocrown electrodes achieve >99% success rates in obtaining intracellular access through electroporation, allowing reliable and simultaneous iAP recordings from up to 57 human pluripotent stem-cell-derived cardiomyocytes (hPSC-CMs). The accuracy of nanocrown electrode recordings is validated by simultaneous patch clamp recording from the same cell. Nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four to five incremental drug doses. In this way, the dose-response data is self-referencing, which avoids the cell-to-cell variations inherent to hPSC-CMs. We are hopeful that this technology development is a step towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.


title: Nanocrown electrodes for reliable and robust intracellular recording of cardiomyocytes and cardiotoxicity screening


title: An in vitro - agent based modelling approach to optimisation of culture medium for generating muscle cells

abstract: Many of the protocols in contemporary tissue engineering remain insufficiently optimised. Methodologies for culturing the complex structures of muscle tissue are particularly lacking, both in terms of quality and quantity of mature cells. Here, we analyse images from in vitro experimentation to quantify the effects of the composition of culture media on mouse-derived myoblast behaviour and myotube cell quality. Computational modelling was then used to predict the optimum range of media compositions for culturing. We define a metric of uniformity of myonuclei distribution as an early indicator of cell quality and difference in myonuclei density over time as an indicator of cell quantity. Analysis of live and static images of muscle cell differentiation revealed that changes in culture media result in significant changes in indicators of cell quantity and quality as well as changes in myoblast migratory behaviour. By describing cell behaviours as a set of functions of media composition we designed a model for predicting cell quality. Cell behaviours were taken directly from experimental images or inferred using Approximate Bayesian Computation and applied as inputs to an agent-based model of cell differentiation which predicted cell quality indicators. Our results suggest that culturing muscle cells in a neural cell differentiation medium reduces the quantity of cell fusion, but does not diminish cell quality. We also show that, while high concentrations of serum are detrimental to cell development, increasing serum concentration raises the total amount of myoblast fusion. This implies a trade-off between the quantity and quality of cells produced when choosing a culture medium. Our model provided a good prediction of experimental results for media with 5% serum provided the background cell proliferation rate was known.


title: SVAT: Secure Outsourcing of Variant Annotation and Genotype Aggregation

abstract:
BackgroundSequencing of thousands of samples provides genetic variants with allele frequencies spanning a very large spectrum and gives invaluable insight for genetic determinants of diseases. Protecting the genetic privacy of participants is challenging as only a few rare variants can easily re-identify an individual among millions. In certain cases, there are policy barriers against sharing genetic data from indigenous populations and stigmatizing conditions.

ResultsWe present SVAT, a method for secure outsourcing of variant annotation and aggregation, which are two basic steps in variant interpretation and detection of causal variants. SVAT uses homomorphic encryption to encrypt the data at the client-side. The data always stays encrypted while it is stored, in-transit, and most importantly while it is analyzed. SVAT makes use of a vectorized data representation to convert annotation and aggregation into efficient vectorized operations in a single framework. Also, SVAT utilizes a secure re-encryption approach so that multiple disparate genotype datasets can be combined for federated aggregation and secure computation of allele frequencies on the aggregated dataset.

ConclusionsOverall, SVAT provides a secure, flexible, and practical framework for privacy-aware outsourcing of annotation, filtering, and aggregation of genetic variants. SVAT is publicly available for download from https://github.com/harmancilab/SVAT

category: bioinformatics


title: Beyond the reach of homology: successive computational filters find yeast pheromone genes

abstract: The mating of fungi depends on pheromones that mediate communication between two mating types. Most species use short peptides as pheromones, which are either unmodified (e.g., -factor in Saccharomyces cerevisiae) or C-terminally farnesylated (e.g., a-factor in S. cerevisiae). Peptide pheromones have been found by genetics or biochemistry in small number of fungi, but their short sequences and modest conservation make it impossible to detect homologous sequences in most species. To overcome this problem, we used a four-step computational pipeline to identify candidate a-factor genes in sequenced genomes of the Saccharomycotina, the fungal clade that contains most of the yeasts: we require that candidate genes have a C-terminal prenylation motif, are fewer than 100 amino acids long, contain a proteolytic processing motif upstream of the potential mature pheromone sequence, and that closely related species contain highly conserved homologs of the potential mature pheromone sequence. Additional manual curation exploits the observation that many species carry more than one a-factor gene, encoding identical or nearly identical pheromones. From 332 fungal genomes, we identified strong candidate pheromone genes in 238 genomes, covering 13 clades that are separated from each other by at least 100 million years, the time required for evolution to remove detectable sequence homology. For one small clade, the Yarrowia, we demonstrated that our algorithm found the a-factor genes: deleting all four related genes in the a-mating type of Yarrowia lipolytica prevents mating.


title: Evaluation of Vicinity-based Hidden Markov Models for Genotype Imputation

abstract: The decreasing cost of DNA sequencing has led to a great increase in our knowledge about genetic variation. While population-scale projects bring important insight into genotype-phenotype relationships, the cost of performing whole-genome sequencing on large samples is still prohibitive. In-silico genotype imputation coupled with genotyping-by-arrays is a cost-effective and accurate alternative for genotyping of common and uncommon variants. Imputation methods compare the genotypes of the typed variants with the large population-specific reference panels and estimate the genotypes of untyped variants by making use of the linkage disequilibrium patterns. Most accurate imputation methods are based on the Li-Stephens hidden Markov model, HMM, that treats the sequence of each chromosome as a mosaic of the haplotypes from the reference panel. Here we assess the accuracy of local-HMMs, where each untyped variant is imputed using the typed variants in a small window around itself (as small as 1 centimorgan). Locality-based imputation is used recently by machine learning-based genotype imputation approaches. We assess how the parameters of the local-HMMs impact the imputation accuracy in a comprehensive set of benchmarks and show that local-HMMs can accurately impute common and uncommon variants and can be relaxed to impute rare variants as well. The source code for the local HMM implementations is publicly available at https://github.com/harmancilab/LoHaMMer.


title: Evolutionary strategies applied to artificial gene regulatory networks

abstract: Evolution optimizes cellular behavior throughout sequential generations by selecting the successful individual cells in a given context. As gene regulatory networks (GRNs) determine the behavior of single cells by ruling the activation of different processes - such as cell differentiation and death - how GRNs change from one generation to the other might have a relevant impact on the course of evolution. It is not clear, however, which mechanisms that affect GRNs effectively favor evolution and how. Here, we use a population of computational robotic models controlled by artificial gene regulatory networks (AGRNs) to evaluate the impact of different genetic modification strategies in the course of evolution. The virtual agent senses the ambient and acts on it as a bacteria in different phototaxis-like tasks - orientation to light, phototaxis, and phototaxis with obstacles. We studied how the strategies of gradual and abrupt changes on the AGRNs impact evolution considering multiple levels of task complexity. The results indicated that a gradual increase in the complexity of the performed tasks is beneficial for the evolution of the model. Furthermore, we have seen that larger gene regulatory networks are needed for more complex tasks, with single-gene duplication being an excellent evolutionary strategy for growing these networks, as opposed to full-genome duplication. Studying how GRNs evolved in a biological environment allows us to improve the computational models produced and provide insights into aspects and events that influenced the development of life on earth.


title: De novo prediction of RNA-protein interactions with Graph Neural Networks

abstract: RNA-binding proteins (RBPs) are key co-and post-transcriptional regulators of gene expression, playing a crucial role in many biological processes. Experimental methods like CLIP-seq have enabled the identification of transcriptome-wide RNA-protein interactions for select proteins, however the time and resource intensive nature of these technologies call for the development of computational methods to complement their predictions. Here we leverage recent, large-scale CLIP-seq experiments to construct a de novo predictor of RNA-protein interactions based on graph neural networks (GNN). We show that the GNN method allows not only to predict missing links in an RNA-protein network, but to predict the entire complement of targets of previously unassayed proteins, and even to reconstruct the entire network of RNA-protein interactions in different conditions based on minimal information. Our results demonstrate the potential of modern machine learning methods to extract useful information on post-transcriptional regulation from large data sets.


title: Experimental Studies of Carpenter Bee (Xylocopa: Apidae) Thorax Mechanics During Defensive Buzzing

abstract: Bees and other Hymenoptera utilize thorax vibration to realize an extensive range of behaviors ranging from flight to pollination. Strong indirect flight muscles contract to deform the thoracic walls and the resulting oscillation is sustained through a mechanism called stretch activation. While the mechanics of the insect thorax and muscles have been studied extensively during flight, relatively little is known about the thorax mechanics during non-flight behaviors. In this work, we investigate the thorax mechanics of the carpenter bee Xylocopa californica during defensive buzzing. During defensive buzzing, the insect folds its wings over its abdomen and rapidly fires it flight muscles, resulting in a loud audible buzz and large forces intended to deter predators. We devised a novel experiment to measure thorax oscillation and directional force production from a defensively buzzing carpenter bee. The largest peak forces were on average 175 mN and were oriented with the insects dorsal-ventral muscle group. Peak forces oriented with the insects dorsal-longitudinal muscle group averaged 117 mN. Thorax velocities were about 90 mm s^-1 p-p and velocity amplitude was positively correlated to peak force. Thorax oscillation frequency averaged 132 Hz but was highly variable both within individuals and across the tested population. From our measurements, we estimated the peak mechanical power required by defensive buzzing at 8.7 mW, which we hypothesize is greater than the power required during flight. Overall, this study provides insight into the function and capabilities of the Hymenopteran indirect flight muscle during non-flight behaviors.


title: A cell-based screen identifies HDAC inhibitors and PLK inhibitor as activators of RIG-I signaling

abstract: Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

category: cancer biology


title: A cell-based screen identifies HDAC inhibitors as activators of RIG-I signaling


title: Tracking chromatin state changes using μMap photo-proximity labeling

abstract: Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects.1,2 Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health.1 However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study.3 Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-Map method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics.4 We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-Map improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.


title: Survival Genie: A web platform for single cell data, gene ratio, and cell composition based survival analyses across adult and pediatric cancers

abstract:
The genomics data-driven identification of gene signatures and pathways has been routinely explored for predicting cancer survival and making decisions related to targeted treatments. A large number of packages and tools have been developed to correlate gene expression/mutations to the clinical outcome but lack the ability to perform such analysis based on pathways, gene sets, and gene ratios. Furthermore, in this single-cell omics era, the cluster markers from cancer single-cell transcriptomics studies remain an underutilized prognostic option. Additionally, no bioinformatics online tool evaluates the associations between the enrichment of canonical cell types and survival across cancers. Here we have developed Survival Genie, a web tool to perform survival analysis on single-cell RNA-seq (scRNA-Seq) data and a variety of other molecular inputs such as gene sets, genes ratio, tumor infiltrating immune cells proportion, gene expression profile scores, and tumor mutation burden. For a comprehensive analysis, Survival Genie contains 53 datasets of 27 distinct malignancies from 11 different cancer programs related to adult and pediatric cancers. Users can upload scRNA-Seq data or gene sets and select a gene expression partitioning method (i.e., mean, median, quartile, cutp) to determine the effect of expression levels on survival outcomes. The tool provides comprehensive results including box plots of low and high-risk groups, Kaplan-Meier plots with univariate Cox proportional hazards model, and correlation of immune cell enrichment and molecular profile.

The analytical options and comprehensive collection of cancer datasets make Survival Genie a unique resource to correlate gene sets, pathways, cellular enrichment, and single-cell signatures to clinical outcomes to assist in developing next-generation prognostic and therapeutic biomarkers. Survival Genie is open-source and available online at https://bbisr.shinyapps.winship.emory.edu/SurvivalGenie/.


title: Survival Genie, a web platform for survival analysis across pediatric and adult cancers.


title: Robust W1282X-CFTR rescue by a small molecule GSPT1 degrader

abstract: With the approval of elexacaftor/tezacaftor/ivacaftor (trade name Trikafta), the vast majority of people with cystic fibrosis (CF) are eligible for CF transmembrane conductance regulator (CFTR) modulator therapy. Remaining individuals have premature termination codons or rare CFTR variants with limited treatment options. Although clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, these cells can be expanded by overexpression of mouse Bmi-1 and human TERT (hTERT). We therefore used this approach to develop two non-CF and three CF (F508del/F508del, F508del/S492F, W1282X/W1282X) nasal cell lines and two W1282X/W1282X bronchial cell lines. Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The F508del/F508del and F508del/S492F cell lines robustly responded to Trikafta, which was mirrored in the parent primary cells and the cell donors clinical response. CC-90009, a novel cereblon E3 ligase modulator targeting the GSPT1 protein, rescued ~20% of wildtype CFTR function in our panel of W1282X/W1282X cell lines and primary cells. Intriguingly, CC-90009 also diminished epithelial sodium channel function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirror primary cell responses to CFTR modulators and illustrate novel therapeutic approaches for the W1282X CFTR variant.

category: pathology


title: Biotechnology data analysis training with Jupyter Notebooks

abstract: Biotechnology has experienced innovations in analytics and data processing. As the volume of data and its complexity grows, new computational procedures for extracting information are developed. However, the rate of change outpaces the adaptation of biotechnology curricula, necessitating new teaching methodologies to equip biotechnologists with data analysis abilities. To simulate experimental data, we created a virtual organism simulator (silvio) by combining diverse cellular and sub-cellular microbial models. silvio was utilized to construct a computer-based instructional workflow with important steps during strain characterization and recombinant protein expression. The instructional workflow is provided as a Jupyter Notebook with comprehensive explanatory text of biotechnological facts and experiment simulations using silvio tools. The students conduct data analysis in Python or Excel. This instructional workflow was separately implemented in two distance courses for Masters students in biology and biotechnology. The concept of using virtual organism simulations that generate coherent results across different experiments can be used to construct consistent and motivating case studies for biotechnological data literacy.

category: scientific communication and education