title: Optimized Data-Independent Acquisition Approach for Proteomic Analysis at Single-Cell Level

abstract: Single-cell proteomic analysis provides valuable insights into cellular heterogeneity allowing the characterization of the cellular microenvironment which is difficult to accomplish in bulk proteomic analysis. Currently, single-cell proteomic studies utilize data-dependent acquisition (DDA) mass spectrometry (MS) coupled with a TMT labelled carrier channel. Due to the extremely imbalanced MS signals among the carrier channel and other TMT reporter ions, the quantification is compromised. Thus, data-independent acquisition (DIA)-MS should be considered as an alternative approach towards single-cell proteomic study since it generates reproducible quantitative data. However, there are limited reports on the optimal workflow for DIA-MS-based single-cell analysis. Herein, we report an optimized DIA workflow for single-cell proteomics using Orbitrap Lumos Tribrid instrument. We utilized a breast cancer cell line (MDA-MB-231) and induced drug resistant polyaneuploid cancer cells (PACCs) to evaluate our established workflow. We found that a short LC gradient was preferable for peptides extracted from single cell level with less than 2 ng sample amount. The total number of co-searching peptide precursors was also critical for protein and peptide identifications at nano- and sub-nano-gram levels. Post-translationally modified peptides could be identified from a nano-gram level of peptides. Using the optimized workflow, up to 1,500 protein groups were identified from a single PACC corresponding to 0.2 ng of peptides. Furthermore, about 200 peptides with phosphorylation, acetylation, and ubiquitination were identified from global DIA analysis of 100 cisplatin resistant PACCs (20 ng). Finally, we used this optimized DIA approach to compare the whole proteome of MDA-MB-231 parental cells and induced PACCs at a single-cell level. We found the single-cell level comparison could reflect real protein expression changes and identify the protein copy number. Our results demonstrate that the optimized DIA pipeline can serve as a reliable quantitative tool for single-cell as well as sub-nano-gram proteomic analysis.

category: systems biology


title: Quantification of macromolecular biomass composition for constraint-based metabolic modeling

abstract: Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM iML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.


title: Dysfunction of grey matter NG2 glial cells affects neuronal plasticity and behavior

abstract: NG2 glia represent a distinct type of macroglial cells in the CNS and are unique among glia because they receive synaptic input from neurons. They are abundantly present in white and grey matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of grey matter NG2 glia and their synaptic input are ill defined yet. Here we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Focussing on the hippocampus, we found that loss of the Kir4.1 potentiated synaptic depolarizations of NG2 glia and enhanced the expression of myelin basic protein. Notably, while mice with targeted deletion of the K+ channel in NG2 glia showed impaired long term potentiation at CA3-CA1 synapses, they demonstrated improved spatial memory as revealed by testing new object location recognition. Our data demonstrate that proper NG2 glia function is critical for normal brain function and behavior.

category: neuroscience


title: A nitrite-oxidizing bacterium constitutively consumes atmospheric hydrogen

abstract: Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) of the genus Nitrospira contribute to nitrification in diverse natural environments and engineered systems. Nitrospira are thought to be well-adapted to substrate limitation owing to their high affinity for nitrite and capacity to use alternative energy sources. Here, we demonstrate that the canonical nitrite oxidizer Nitrospira moscoviensis oxidizes hydrogen (H2) below atmospheric levels using a high-affinity group 2a nickel-iron hydrogenase [Km(app) = 32 nM]. Atmospheric H2 oxidation occurred under both nitrite-replete and nitrite-deplete conditions, suggesting low-potential electrons derived from H2 oxidation promote nitrite-dependent growth and enable survival during nitrite limitation. Proteomic analyses confirmed the hydrogenase was abundant under both conditions and indicated extensive metabolic changes occur to reduce energy expenditure and growth under nitrite-deplete conditions. Respirometry analysis indicates the hydrogenase and nitrite oxidoreductase are bona fide components of the aerobic respiratory chain of N. moscoviensis, though they transfer electrons to distinct electron carriers in accord with the contrasting redox potentials of their substrates. Collectively, this study suggests atmospheric H2 oxidation enhances the growth and survival of NOB in amid variability of nitrite supply. These findings also extend the phenomenon of atmospheric H2 oxidation to a seventh phylum (Nitrospirota) and reveal unexpected new links between the global hydrogen and nitrogen cycles.

category: microbiology


title: Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting

abstract: Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the trans-Golgi network (TGN) by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the endoplasmic reticulum (ER). Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule biogenesis. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation although to different extents in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for secretory granule biogenesis and sorting by self-aggregation.

category: cell biology


title: Extracellular Vesicles Facilitate Large-Scale, Homogenizing Dynamic Exchange of Proteins and RNA Among Cultured Chinese Hamster Ovary (CHO) and Human Cells

abstract: Cells in culture are viewed as unique individuals in a large population communicating through extracellular molecules and, more recently extracellular vesicles (EVs). Our data here paints a different picture: the homogenizing effect of large-scale exchange of cellular material through EVs. We show that Chinese Hamster Ovary (CHO) cells dynamically produce and uptake EVs, to exchange proteins and RNAs at large-scale. To visualize the dynamic production and cellular uptake of EVs, we used correlative confocal microscopy and scanning electron microscopy, as well as flow cytometry to interrogate labeled cells. We employed cells expressing fluorescent proteins (GFP, miRFP703) and tagged cells with protein and RNA dyes. Flow cytometry was used to quantify the exchange of cellular RNA between cells through EVs. This EV-mediated dynamic exchange observed in CHO cultures was also observed in cultures of the human CHRF-288-11 cell line and of primary human hematopoietic stem and progenitor cells. This study demonstrates an underappreciated native cell communication and protein/RNA exchange mechanism mediated by EVs spanning cell type and lines, suggesting the proximity of cells in normal and tumor tissues may also result in prolific cellular exchange. This exchange would be expected to homogenize the cell-population cytoplasm and dynamically regulate cell proliferation and cellular state.

category: bioengineering


title: Unveiling the links between peptide identification and differential analysis FDR controls by means of a practical introduction to knockoff filters

abstract: In proteomic differential analysis, FDR control is often performed through a multiple test correction (i.e., the adjustment of the original p-values). In this protocol, we apply a recent and alternative method, based on so-called knockoff filters. It shares interesting conceptual similarities with the target-decoy competition procedure, classically used in proteomics for FDR control at peptide identification. To provide practitioners with a unified understanding of FDR control in proteomics, we apply the knockoff procedure on real and simulated quantitative datasets. Leveraging these comparisons, we propose to adapt the knockoff procedure to better fit the specificities of quantitive proteomic data (mainly very few samples). Performances of knockoff procedure are compared with those of the classical Benjamini-Hochberg procedure, hereby shedding a new light on the strengths and weaknesses of target-decoy competition.

category: bioinformatics


title: An ancestral function of strigolactones as symbiotic rhizosphere signals

abstract: In flowering plants, carotenoid-derived strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), a previously unidentified SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating that it is ancestral in land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

category: plant biology


title: Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

abstract: Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

category: synthetic biology


title: Neurons undergo pathogenic metabolic reprograming in models of familial ALS

abstract: Normal cellular function requires a rate of ATP production sufficient to meet demand. In most neurodegenerative diseases (including Amyotrophic Lateral Sclerosis, ALS), mitochondrial dysfunction is postulated raising the possibility of impaired ATP production and a need for compensatory maneuvers to sustain the ATP production/demand balance. We find in our rodent models of familial ALS (fALS), impairment in neuronal glycolytic flux with maintained or enhanced activity of the citric acid cycle. This rewiring of metabolism is associated with normal ATP levels and redox status, supporting the notion that mitochondrial function is not compromised in neurons expressing fALS genes. Genetic loss-of-function manipulation of individual steps in the glycolysis and the pentose phosphate pathway blunt the negative phenotypes seen in various fALS models. We propose that neurons adjust fuel utilization in the setting of neurodegenerative disease-associated mitochondrial dysfunction in a baleful manner and targeting this process can be healthful.


title: Fine multi-coil electronic control of transcranial magnetic stimulation: effects of stimulus orientation and intensity

abstract:
BackgroundTranscranial magnetic stimulation (TMS) coils allow only a slow, mechanical adjustment of the stimulating electric field (E-field) orientation in the cerebral tissue. Fast E-field control is needed to synchronize the stimulation with the ongoing brain activity. Also, empirical models that fully describe the relationship between evoked responses and the stimulus orientation and intensity are still missing.

ObjectiveWe aimed to (1) develop a TMS transducer for manipulating the E-field orientation electronically with high accuracy at the neuronally meaningful millisecond-level time scale and (2) devise and validate a physiologically based model describing the orientation selectivity of neuronal excitability.

MethodsWe designed and manufactured a two-coil TMS transducer. The coil windings were computed with a minimum-energy optimization procedure, and the transducer was controlled with our custom-made electronics. The electronic E-field control was verified with a TMS characterizer. The motor evoked potential amplitude and latency of a hand muscle were mapped in 3{degrees} steps of the stimulus orientation in 16 healthy subjects for three stimulation intensities. We fitted a logistic model to the motor response amplitude.

ResultsThe two-coil TMS transducer allows one to manipulate the pulse orientation accurately without manual coil movement. The motor response amplitude followed a logistic function of the stimulus orientation; this dependency was strongly affected by the stimulus intensity.

ConclusionThe developed electronic control of the E-field orientation allows exploring new stimulation paradigms and probing neuronal mechanisms. The presented model helps to disentangle the neuronal mechanisms of brain function and guide future non-invasive stimulation protocols.


title: TMS with fast and accurate electronic control: measuring the orientation sensitivity of corticomotor pathways


title: The Hybrid Drive: a chronic implant device combining tetrode arrays with silicon probes for layer-resolved ensemble electrophysiology in freely moving mice

abstract: Understanding the function of brain cortices requires simultaneous investigation at multiple spatial and temporal scales and to link neural activity to an animals behavior. A major challenge is to measure within- and across-layer information in actively behaving animals, in particular in mice that have become a major species in neuroscience due to an extensive genetic toolkit. Here we describe the Hybrid Drive, a new chronic implant for mice that combines tetrode arrays to record within-layer information with silicon probes to simultaneously measure across-layer information. The design of our device combines up to 14 tetrodes and 2 silicon probes, that can be arranged in custom arrays to generate unique areas-specific (and multi-area) layouts. We show that large numbers of neurons and layer-resolved local field potentials can be recorded from the same brain region across weeks without loss in electrophysiological signal quality. The drives lightweight structure ({approx} 3.5 g) leaves animal behavior largely unchanged during a variety of experimental paradigms. We demonstrate how the data collected with the Hybrid Drive allow state-of-the-art analysis in a series of experiments linking the spiking activity of CA1 pyramidal layer neurons to the oscillatory activity across hippocampal layers. Our new device fits a gap in the existing technology and increases the range and precision of questions that can be addressed about neural computations in freely behaving mice.


title: Alpha blocking and 1/fβ spectral scaling in resting EEG can be accounted for by a sum of damped alpha band oscillatory processes

abstract:
The dynamical and physiological basis of alpha band activity and 1/f{beta} noise in the EEG are the subject of continued speculation. Here we conjecture, on the basis of empirical data analysis, that both of these features may be economically accounted for through a single process if the resting EEG is conceived of being the sum of multiple stochastically perturbed alpha band damped linear oscillators with a distribution of dampings (relaxation rates). The modulation of alpha-band and 1/f{beta} noise activity by changes in damping is explored in eyes closed (EC) and eyes open (EO) resting state EEG. We aim to estimate the distribution of dampings by solving an inverse problem applied to EEG power spectra. The characteristics of the damping distribution are examined across subjects, sensors and recording condition (EC/EO). We find that there are robust changes in the damping distribution between EC and EO recording conditions across participants. The estimated damping distributions are found to be predominantly bimodal, with the number and position of the modes related to the sharpness of the alpha resonance and the scaling ({beta}) of the power spectrum (1/f{beta}). The results suggest that there exists an intimate relationship between resting state alpha activity and 1/f{beta} noise with changes in both governed by changes to the damping of the underlying alpha oscillatory processes. In particular, alpha-blocking is observed to be the result of the most weakly damped distribution mode becoming more heavily damped. The results suggest a novel way of characterizing resting EEG power spectra and provides new insight into the central role that damped alpha-band activity may play in characterising the spatio-temporal features of resting state EEG.

Author summaryThe resting human electroencephalogram (EEG) exhibits two dominant spectral features: the alpha rhythm (8-13 Hz) and its associated attenuation between eyes-closed and eyes-open resting state (alpha blocking), and the 1/f{beta} scaling of the power spectrum. While these phenomena are well studied a thorough understanding of their respective generative processes remains elusive. By employing a theoretical approach that follows from neural population models of EEG we demonstrate that it is possible to economically account for both of these phenomena using a singular mechanistic framework: resting EEG is assumed to arise from the summed activity of multiple uncorrelated, stochastically driven, damped alpha band linear oscillatory processes having a distribution of relaxation rates or dampings. By numerically estimating these damping distributions from eyes-closed and eyes-open EEG data, in a total of 136 participants, it is found that such damping distributions are predominantly bimodal in shape. The most weakly damped mode is found to account for alpha band power, with alpha blocking being driven by an increase in the damping of this weakly damped mode, whereas the second, and more heavily damped mode, is able to explain 1/f{beta} scaling present in the resting state EEG spectra.


title: Humans and mice fluctuate between external and internal modes of sensory processing

abstract: 2Perception cycles through periods of enhanced and reduced sensitivity to external information. Here, we asked whether such infra-slow oscillations arise as a noise-related epiphenomenon of limited processing capacity or, alternatively, represent a structured mechanism of perceptual inference. Using two large-scale datasets, we found that humans and mice waver between alternating intervals of externally- and internally-oriented modes of sensory analysis. During external mode, perception was more sensitive to external sensory information, whereas internal mode was characterized by enhanced biases toward perceptual history. Computational modeling indicated that dynamic changes in mode are governed by two interlinked factors: (i), the integration of subsequent stimuli over time and, (ii), infra-slow anti-phase oscillations in the perceptual impact of external sensory information versus internal predictions that are provided by perceptual history. Between-mode fluctuations may benefit perception by enabling the generation of stable representations of the environment despite an ongoing stream of noisy sensory inputs.


title: Multiomics reveals persistence of obesity-associated immune cell phenotypes in adipose tissue during weight loss and subsequent weight regain.

abstract: Most individuals do not maintain weight loss, and weight regain increases cardio-metabolic risk beyond that of obesity. Adipose inflammation directly contributes to insulin resistance; however, immune-related changes that occur with weight loss and weight regain are not well understood. Single cell RNA-sequencing was completed with CITE-sequencing and biological replicates to profile changes in murine immune subpopulations following obesity, weight loss, and weight cycling. Weight loss normalized glucose tolerance, however, type 2 immune cells did not repopulate adipose following weight loss. Many inflammatory populations persisted with weight loss and increased further following weight regain. Obesity drove T cell exhaustion and broad increases in antigen presentation, lipid handing, and inflammation that persisted with weight loss and weight cycling. This work provides critical groundwork for understanding the immunological causes of weight cycling-accelerated metabolic disease. Thus, we have created an open-access interactive portal for our processed data to improve accessibility for the research community.

category: immunology


title: Exon junction complex-associated multi-adapter RNPS1 nucleates splicing regulatory complexes to maintain transcriptome surveillance

abstract: The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing, mRNA export, mRNA translation, and nonsense-mediated mRNA decay (NMD) via various interacting peripheral proteins. The EJC-binding protein RNPS1 might serve two functions: it suppresses mis-splicing of cryptic splice sites and activates NMD in the cytoplasm. When analyzing the transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines, we find no evidence for RNPS1 being a globally essential NMD factor. However, various aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that about half of these RNPS1-dependent splicing events was fully or partially rescued by the expression of the isolated RRM domain of RNPS1, whereas other splicing events are regulated by its C-terminal domain. We identified many splicing-regulatory factors, including SR proteins and U1 snRNP components, that specifically interact with the C-terminus or with the RRM of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.

category: molecular biology


title: Constitutive signal bias mediated by the human GHRHR splice variant 1

abstract:
Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to {beta}-arrestins. Based on the cryo-electron microscopy structures of SV1 in the apo state or in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Significance StatementThe mechanism of functional changes induced by alternative splicing of GHRHR is largely unknown. Here, we demonstrate that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The cryo-electron microscopy structures of SV1 and molecular dynamics simulations reveal the different functionalities between GHRHR and SV1 at the near-atomic level, i.e., the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Our findings provide valuable insights into functional diversity of class B1 GPCRs which may aid in the design of better therapeutic agents against certain cancers.


title: Spatial and temporal expression of PORCN is highly dynamic in the developing mouse cochlea.

abstract:
The mammalian organ of Corti is a highly specialized sensory organ of the cochlea with a fine-grained pattern that is essential for auditory function. The sensory epithelium, the organ of Corti consists of a single row of inner hair cells and three rows of outer hair cells that are intercalated by support cells in a mosaic pattern. Previous studies show that the Wnt pathway regulates proliferation, promotes medial compartment formation in the cochlea, differentiation of the mechanosensory hair cells and axon guidance of Type II afferent neurons. WNT ligand expressions are highly dynamic throughout development but are insufficient to explain the roles of the Wnt pathway. We address a potential way for how WNTs specify the medial compartment by characterizing the expression of Porcupine (PORCN), an O-acyltransferase that is required for WNT secretion. We show PORCN expression across embryonic ages (E)12.5 - E14.5, E16.5, and postnatal day (P)1. Our results showed enriched PORCN in the medial domains during early stages of development, indicating that WNTs have a stronger influence on patterning of the medial compartment. PORCN was rapidly downregulated after E14.5, following the onset of sensory cell differentiation; residual expression remained in some hair cells and supporting cells. On E14.5 and E16.5, we also examined the spatial expression of Gsk3{beta}, an inhibitor of canonical Wnt signaling to determine its potential role in radial patterning of the cochlea. Gsk3{beta} was broadly expressed across the radial axis of the epithelium; therefore, unlikely to control WNT-mediated medial specification. In conclusion, the spatial expression of PORCN enriches WNT secretion from the medial domains of the cochlea to influence the specification of cell fates in the medial sensory domain.

HighlightsO_LIWnt ligands are broadly expressed during cochlear development.
C_LIO_LIPORCN expression is highly dynamic during early cochlear development
C_LIO_LIPORCN becomes restricted to the medial domains along the longitudinal axis.
C_LIO_LIWnt medial specification is regulated at the level of WNT ligand secretion.
C_LI

category: developmental biology


title: GinJinn2: Object detection and segmentation for ecology and evolution

abstract:
O_LIProper collection and preparation of empirical data still represent one of the most important, but also expensive steps in ecological and evolutionary/systematic research. Modern machine learning approaches, however, have the potential to automate a variety of tasks, which until recently could only be performed manually. Unfortunately, the application of such methods by researchers outside the field is hampered by technical difficulties, some of which, we believe, can be avoided.
C_LIO_LIHere, we present GinJinn2, a user-friendly toolbox for deep learning-based object detection and instance segmentation on image data. Besides providing a convenient command-line interface to existing software libraries, it comprises several additional tools for data handling, pre- and postprocessing, and building advanced analysis pipelines.
C_LIO_LIWe demonstrate the application of GinJinn2 for biological purposes using four exemplary analyses, namely the evaluation of seed mixtures, detection of insects on glue traps, segmentation of stomata, and extraction of leaf silhouettes from herbarium specimens.
C_LIO_LIGinJinn2 will enable users with a primary background in biology to apply deep learning-based methods for object detection and segmentation in order to automate feature extraction from image data.
C_LI

category: ecology


title: Direct Observation of Anisotropy-Driven Formation of Amyloid Protein Core-Shell Structures in Real-time by Super-resolution Microscopy

abstract: The misfolding of proteins and their aggregation in the form of fibrils or amyloid-like spherulites are involved in a spectrum of pathological abnormalities. Our current understanding of protein amyloid aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalisation Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of a microscopically identical aggregates, e.g. amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

category: biophysics


title: Direct Observation of Heterogeneous Formation of Amyloid Spherulites in Real-time by Super-resolution Microscopy


title: Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

abstract: SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.


title: High-Spatiotemporal-Resolution Nanopore Sequencing of SARS-CoV-2 and Host Cell RNAs

abstract: Recent studies have disclosed the genome, transcriptome and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for non-canonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (MOI=0.1) applied for the infection. In this study, we used direct cDNA and direct RNA nanopore sequencings for the generation of high-coverage, high-temporal-resolution transcriptomic datasets on SARS-CoV-2 and on primate host cells infected with a high virus titer (MOI=5). Sixteen sampling time points ranging from 1 to 96h with a varying time resolution and three biological replicates were used in the experiment for both the infected and the non-infected cells.

category: genomics


title: Platelet proteome analysis reveals an early hyperactive phenotype in SARS-CoV-2-infected humanized ACE2 mice

abstract:
Coronavirus disease-2019 (COVID-19) provokes a hypercoagulable state with increased incidence of thromboembolism and mortality. Platelets are major effectors of thrombosis and hemostasis. Suitable animal models are needed to better understand COVID-19-associated coagulopathy (CAC) and underlying platelet phenotypes. Here, we assessed K18-hACE2 mice undergoing a standardized SARS-CoV-2 infection protocol to study dynamic platelet responses via mass spectrometry-based proteomics. In total, we found significant changes in >1,200 proteins. Strikingly, protein alterations occurred rapidly by 2 days post-infection (dpi) and preceded outward clinical signs of severe disease. Pathway enrichment analysis of 2dpi platelet proteomes revealed that SARS-CoV-2 infection upregulated complement-coagulation networks (F2, F12, CFH, CD55/CD59), platelet activation-adhesion-degranulation proteins (PF4, SELP, PECAM1, HRG, PLG, vWF), and chemokines (CCL8, CXCL5, CXCL12). When mice started to lose weight at 4dpi, pattern recognition receptor signaling (RIG-I/MDA5, CASP8, MAPK3), and interferon pathways (IFIT1/IFIT3, STAT1) were predominant. Interestingly, SARS-CoV-2 spike protein in the lungs was observed by immunohistochemistry, but in platelets was undetected by proteomics. Similar to patients, K18-hACE2 mice during SARS-CoV-2 infection developed progressive lymphohistiocytic interstitial pneumonia with platelet aggregates in the lungs and kidneys. In conclusion, this model recapitulates activation of coagulation, complement, and interferon responses in circulating platelets, providing valuable insight into platelet pathology during COVID-19.

Key PointsO_LISARS-CoV-2-infected humanized ACE2 mice recapitulate platelet reprogramming towards activation-degranulation-aggregation.
C_LIO_LIComplement/coagulation pathways are dominant in platelets at 2 days post-infection (dpi), while interferon signaling is dominant at 4dpi.
C_LI


title: Free for all, or free-for-all? A content analysis of Australian university open access policies

abstract: Recent research demonstrates that Australia lags in providing open access to research outputs. In Australia, while the two major research funding bodies require open access of outputs from projects they fund, these bodies only fund a small proportion of research conducted. The major source of research and experimental development funding in Australian higher education is general university, or institutional, funding, and such funds are not subject to national funder open access policies. Thus, institutional policies and other institutional supports for open access are important in understanding Australias OA position. The purpose of this paper is, therefore, to understand the characteristics of Australian institutional open access policies and to explore the extent they represent a coherent and unified approach to delivering and promoting open access in Australia. Open access policies were located using a systematic web search approach and then their contents were analysed. Only half of Australian universities were found to have an open access policy. There was a wide variation in language used, expressed intent of the policy and expectations of researchers. Few policies mention monitoring or compliance and only three mention consequences for non-compliance. While it is understandable that institutions develop their own policies, when language is used which does not reflect national and international understandings, when requirements are not clear and with consequences, policies are unlikely to contribute to understanding of open access, to uptake of the policy, or to ease of transferring understanding and practices between institutions. A more unified approach to open access is recommended.

category: scientific communication and education


title: A kiwellin protein-like fold containing rust effector protein localizes to chloroplast and suppress cell death in plants

abstract:
The effector proteins expressed by plant pathogens are one of the essential components of the host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the lack of conserved features and huge diversity in their primary sequence. In the present study, extensive secretome analysis was performed in sixteen major plant fungal pathogens to find the conserved features in the candidate secretory effector proteins (CSEPs) using homology and ab initio modeling approaches. Interestingly, a variable number of plant kiwellin proteins fold like secretory proteins were found in all the major rust fungal pathogens. Many of them are predicted as potential effector proteins. For instance, 26 out of 35 Kiwellin like proteins identified in Puccinia striiformis race 104E 137A were predicted as potential effector proteins. In addition, a kiwellin predicted effector gene, Pst_13960, from the Indian Puccinia striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pst_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the kiwellin fold in rust fungi. Further analysis of these proteins predicted these candidates to contain N-terminal Intrinsically disordered regions (IDRs) putatively associated with chloroplast translocation as deletion of region abolished the chloroplast localization of Pstr_13960. Overall, the current study reports the presence of kiwellin like proteins in rust fungi that act as a novel effector in plants.

Author SummaryRust fungi are one of the most devastating plants infecting pathogens. These pathogens secrete several distinct proteins like effector proteins that help the pathogens in the establishment of infection by suppressing cell death induced by the plants. Despite being important, these effector proteins remain unexplored due to the lack of conserved features. Currently, different methods are being used to characterize them however, could not describe their specific function fully due to a lack of knowledge of the functional domain. Recent advancement in effector protein tertiary structure characterization using NMR (Nuclear magnetic resonance) and X-ray crystallography has been very helpful in identifying the conserved structural features defining functionality. However, these techniques are quite complicated and may take a lot of time and labor. On the other hand, the computational approaches for structural prediction of the effectors may help to identify known folds or domains with few efforts but at a significant level. Therefore, such computational approaches can be efficiently implemented in the preliminary screening of the candidates. In the present study using the computational structure prediction method, we were able to find several conserved novel kiwellin folds containing effectors, in different rust fungi. We characterized one of the candidates and it showed interference with artificially induced cell death in plants. This study highlights the novel function of the kiwellin like effector proteins of the rust fungi that are already identified to play a role in host defense against plant pathogens.


title: PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes

abstract:
Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine- associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a {beta}-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a {Delta}prkA mutant to nearly wild-type levels. While a {Delta}prkA mutant is defective for PG synthesis during cell wall stress, a double {Delta}reoM {Delta}prkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a {Delta}prkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence.

AUTHOR SUMMARYMany antibiotics target bacterial cell wall biosynthesis, justifying continued study of this process and the ways bacteria respond to cell wall insults, especially in the context of infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are master regulators of cell wall stress responses in bacteria and are conserved in several major pathogens, including Listeria monocytogenes, Staphylococcus aureus, and Mycobacterium tuberculosis. We previously showed that the PASTA kinase in L. monocytogenes, PrkA, is essential for the response to cell wall stress and virulence. PrkA is also necessary for adaptation to the cytosol, which L. monocytogenes requires during its infectious lifecycle. In this work, we combined orthogonal phosphoproteomic and genetic screens to identify PrkA substrates in L. monocytogenes. We show that phosphoregulation of one candidate from both screens, ReoM, increases peptidoglycan synthesis and that this regulation is required for cytosolic survival and virulence. We also demonstrate that the PASTA kinase-ReoM pathway regulates cell wall stress responses in another significant pathogen, methicillin-resistant S. aureus. Additionally, we uncover a PrkA-independent role for ReoM in vivo in L. monocytogenes, suggesting a need for nuanced modulation of peptidoglycan synthesis during infection. Cumulatively, this study provides new insight into how bacterial pathogens control cell wall synthesis during infection.


title: SARS-CoV-2 hijacks p38β/MAPK11 to promote viral protein translation

abstract: SARS-CoV-2, the causative agent of the COVID-19 pandemic, drastically modifies the cells that it infects. One such effect is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammation pathways that are dysregulated in severe COVID-19 cases. Inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduces both cytokine production and viral replication. Here, we applied a systems biology approach to better understand interactions between the p38/MAPK pathway and SARS-CoV-2 in human lung epithelial cells. We found several components of the p38/MAPK pathway positively and negatively impact SARS-CoV-2 infection and that p38{beta} is a required host factor for SARS-CoV-2 that acts by promoting viral protein translation in a manner that prevents innate immune sensing. Furthermore, we combined chemical and genetic perturbations of p38{beta} with quantitative phosphoproteomics to identify novel, putative p38{beta} substrates in an unbiased manner, with broad relevance beyond SARS-CoV-2 biology.


title: Regulation of let-7 microRNAs by a two-step turnover pathway is of critical pathophysiological significance in humans

abstract: MicroRNAs are critical regulators of diverse developmental and physiological processes, and they themselves get regulated both at the level of biogenesis and turnover. We demonstrate that the ribonuclease XRN2 can degrade the mature forms of certain let-7 family members in multiple human cancer cell lines, without affecting their precursors. XRN2 depletion results in a reduction in the expression of a number of oncogenes, and diminishes the proliferative and metastatic potential of cancer cells. The clinical relevance of these observations is also verified in tumour transcriptomics data from public RNA-sequencing datasets, where XRN2 mRNA expression is inversely correlated with the levels of a large number of miRNAs, including let-7 members, and high XRN2 mRNA levels are associated with poor survival in hepatocellular carcinoma, lung adenocarcinoma, and glioblastoma. We demonstrate that the miRNA is released by an as-yet unidentified proteinaceous  miRNA release factor from the grasp of Argonaute before its degradation. Our analyses of the patient-derived transcriptomics data also show that XRN2, via its regulation of let-7, affects multiple pathways in a consistent manner across epithelial and glial cell lineages, and thus, is of critical pathophysiological significance.


title: XRN2-mediated regulation of let-7 microRNAs is of critical pathophysiological significance in Humans


title: Development of a universal nanobody-binding Fab module for fiducial-assisted cryo- EM studies of membrane proteins

abstract: With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon CDR grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryo-EM structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ~50 kDa. Using an additional anti-Fab nanobody further facillitated reliable initial 3D structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, humanized, and can be conveniently expressed in E. coli in large amounts, it may not only be useful for structural biology, but also for biomedical applications.


title: Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors

abstract: Essential functions of mitogen-activated protein kinases (MAPKs) depend on their capacity to selectively phosphorylate a limited repertoire of substrates. MAPKs harbor a conserved groove located outside of the catalytic cleft that binds to short linear sequence motifs found in substrates and regulators. However, the weak and transient nature of these "docking" interactions poses a challenge to defining MAPK interactomes and associated sequence motifs. Here, we describe a yeast-based genetic screening pipeline to evaluate large collections of MAPK docking sequences in parallel. Using this platform we analyzed a combinatorial library based on the docking sequences from the MAPK kinases MKK6 and MKK7, defining features critical for binding to the stress-activated MAPKs JNK1 and p38. We subsequently screened a library consisting of ~12,000 sequences from the human proteome, revealing a large number of MAPK-selective interactors, including many not conforming to previously defined docking motifs. Analysis of p38/JNK1 exchange mutants identified specific docking groove residues mediating selective binding. Finally, we verified that docking sequences identified in the screen could function in substrate recruitment in vitro and in cultured cells. Collectively, these studies establish an approach for characterization of MAPK docking sequences and provide a resource for future investigation of signaling downstream of p38 and JNK MAP kinases.


title: Interactomic analysis reveals a new homeostatic role for the HIV restriction factor TRIM5α in mitophagy

abstract: The protein TRIM5 has multiple roles in anti-retroviral defense, but the mechanisms underlying TRIM5 action are unclear. Here, we used an APEX2-based proteomics approach to identify TRIM5-interacting proteins. Analysis of the TRIM5 interactome found proteins participating in a wide variety of cellular functions including regulating antiviral signaling pathways. We used this data set to uncover a novel role for TRIM5 in mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in multiple human diseases. Mitochondrial damage triggered the relocalization of TRIM5 to ER-mitochondria contact sites where TRIM5 colocalized with markers of autophagy initiation and autophagosome biogenesis. Furthermore, we found that TRIM5 knockout attenuated both Parkin-dependent and Parkin-independent mitophagy by preventing the recruitment of autophagy regulators FIP200 and ATG13 to unhealthy mitochondria. Finally, TRIM5 knockout cells showed reduced mitochondrial function under basal conditions and were more susceptible to uncontrolled immune activation and cell death in response to mitochondrial damage than were wild type cells. Taken together, our studies have identified a homeostatic role for a protein previously recognized exclusively for its antiviral actions.


title: Vps54 regulates lifespan and locomotor behavior in adult Drosophila melanogaster

abstract: Vps54 is an integral subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). A destabilizing missense mutation in Vps54 causes the age-progressive motor neuron (MN) degeneration, muscle weakness, and muscle atrophy observed in the wobbler mouse, an established animal model for human MN disease. It is currently unclear how the disruption of Vps54, and thereby the GARP complex, leads to MN and muscle phenotypes. To develop a new tool to address this question, we have created an analogous model in Drosophila by generating novel loss-of-function alleles of the fly Vps54 ortholog (scattered/scat). We find that null scat mutant adults are viable but have a significantly shortened lifespan. Like phenotypes observed in the wobbler mouse, we show that scat mutant adults are male sterile and have significantly reduced body size and muscle area. Moreover, we demonstrate that scat mutant adults have significant age-progressive defects in locomotor function. Interestingly, we see sexually dimorphic effects, with scat mutant adult females exhibiting significantly stronger phenotypes. Finally, we show that scat interacts genetically with rab11 in MNs to control age-progressive muscle atrophy in adults. Together, these data suggest that scat mutant flies share mutant phenotypes with the wobbler mouse and may serve as a new genetic model system to study the cellular and molecular mechanisms underlying MN disease.

category: genetics


title: Genetic Characterization and Curation of Diploid A-Genome Wheat Species

abstract:
The A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum. monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.) and T. urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC), using genotyping-by-sequencing (GBS) and identified 13,089 curated SNPs. Genomic analysis detected misclassified and duplicated accessions with most duplicates originated from the same or a nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were duplicates supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that the T. urartu as the closest A-genome diploid to wheat through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races , {beta}, and {gamma} described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring domestication selection signature (Btr1) on the short arm of chromosome 3Am at ~ 70 Mb. We established A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 80% of allelic variants in the A-genome collection and constitute a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

One-sentence summaryGenotyping of gene bank collections of diploid A-genome relatives of wheat uncovered relatively higher genetic diversity and unique evolutionary relationships which gives insight to the effective use of these germplasm for wheat improvement.


title: Homologous recombination induced by a replication fork barrier requires cooperation between strand invasion and strand annealing activities

abstract: Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. Elucidating the mechanism of recombination between repeated sequences in the context of replication stress is essential to understanding how genome rearrangements occur. To gain insight into this process, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Remarkably, we show that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, as well as Mph1/Rad5 fork remodelers, Mre11/Exo1 short and long-range resection machineries, Rad1-Rad10 nuclease and DNA polymerase {delta}. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 structure-selective nucleases. Physical analysis of replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats at stalled replication forks that can actively contribute to genomic rearrangements.


title: Genome of a novel Sediminibacterium discovered in association with two species of freshwater stream Cyanobacteria in Southern California

abstract: Here we report the discovery of a novel Sediminibacterium sequenced from laboratory cultures of freshwater stream cyanobacteria from sites in Southern California, grown in BG11 media.


title: Machine learning enables discovery of DNA-carbon nanotube sensors for serotonin

abstract:
DNA-wrapped single walled carbon nanotube (SWNT) conjugates have remarkable optical properties leading to their use in biosensing and imaging applications. A critical limitation in the development of DNA-SWNT sensors is the current inability to predict unique DNA sequences that confer a strong analyte-specific optical response to these sensors. Here, near-infrared (nIR) fluorescence response datasets for ~100 DNA-SWNT conjugates, narrowed down by a selective evolution protocol starting from a pool of ~1010 unique DNA-SWNT candidates, are used to train machine learning (ML) models to predict new unique DNA sequences with strong optical response to neurotransmitter serotonin. First, classifier models based on convolutional neural networks (CNN) are trained on sequence features to classify DNA ligands as either high response or low response to serotonin. Second, support vector machine (SVM) regression models are trained to predict relative optical response values for DNA sequences. Finally, we demonstrate with validation experiments that integrating the predictions of ensembles of the highest quality CNN classifiers and SVM regression models leads to the best predictions of both high and low response sequences. With our ML approaches, we discovered five new DNA-SWNT sensors with higher fluorescence intensity response to serotonin than obtained previously. Overall, the explored ML approaches introduce an important new tool to predict useful DNA sequences, which can be used for discovery of new DNA-based sensors and nanobiotechnologies.

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title: Time evolution of PEG-shedding and serum protein coronation determines the cell uptake kinetics and delivery of lipid nanoparticle formulated mRNA

abstract: Development of efficient lipid nanoparticle (LNP) vectors remains a major challenge towards broad clinical translation of RNA therapeutics. New lipids will be required, but also better understanding LNP interactions with the biological environment. Herein, we model protein corona formation on PEG-ylated DLin-MC3-DMA LNPs and identify time-dependent maturation steps that critically unlock their cellular uptake and mRNA delivery. Uptake requires active serum proteins and precedes after a significant ([~]2 hours) lag-time, which we show can be eliminated by pre-incubating LNPs for 3-4 hours in serum-containing media. This indicates an important role of protein corona maturation for the pharmacokinetic effects of these LNPs. We show, using single-nanoparticle imaging, NMR diffusometry, SANS, and proteomics, that the LNPs, upon serum exposure, undergo rapid PEG-shedding ([~]30 minutes), followed by a slower rearrangement of the adsorbed protein layer. The PEG-shedding coincides in time with high surface abundance of Apolipoprotein A-II, whereas the LNPs preferentially bind Apolipoprotein E when their maximum uptake-competent state is reached. Finally, we show that pre-incubation of the LNPs enables rapid uptake and allows pulse-chase video-microscopy colocalization experiments with sufficiently short pulse durations to gain improved mechanistic understanding of how intracellular trafficking events determine delivery efficacy, emphasizing early endosomes as important delivery-mediating compartments.


title: Progerin-Expressing Endothelial Cells are Unable to Adapt to Shear Stress

abstract: Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. HGPS patients experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study we examined how progerin-expressing ECs adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control ECs to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in ECs, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that ECs either expressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). ECs overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing ECs had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, pre-alignment of cells to shear stress prior to progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of EC to properly adapt to shear stress.


title: A Network Approach to Identify Biomarkers of Differential Chemotherapy Response Using Patient-Derived Xenografts of Triple-Negative Breast Cancer

abstract: Triple negative breast cancer (TNBC) is a highly heterogeneous set of diseases that has, until recently, lacked any FDA-approved, molecularly targeted therapeutics. Thus, systemic chemotherapy regimens remain the standard of care for many. Unfortunately, even combination chemotherapy is ineffective for many TNBC patients, and side-effects can be severe or lethal. Identification of predictive biomarkers for chemotherapy response would allow for the prospective selection of responsive patients, thereby maximizing efficacy and minimizing unwanted toxicities. Here, we leverage a cohort of TNBC PDX models with responses to single-agent docetaxel or carboplatin to identify biomarkers predictive for differential response to these two drugs. To demonstrate their ability to function as a preclinical cohort, PDX were molecularly characterized using whole-exome DNA sequencing, RNAseq transcriptomics, and mass spectrometry-based total proteomics to show proteogenomic consistency with TCGA and CPTAC clinical samples. Focusing first on the transcriptome, we describe a network-based computational approach to identify candidate epithelial and stromal biomarkers of response to carboplatin (MSI1, TMSB15A, ARHGDIB, GGT1, SV2A, SEC14L2, SERPINI1, ADAMTS20, DGKQ) and docetaxel (ITGA7, MAGED4, CERS1, ST8SIA2, KIF24, PARPBP). Biomarker panels are predictive in PDX expression datasets (RNAseq and Affymetrix) for both taxane (docetaxel or paclitaxel) and platinum-based (carboplatin or cisplatin) response, thereby demonstrating both cross expression platform and cross drug class robustness. Biomarker panels were also predictive in clinical datasets with response to cisplatin or paclitaxel, thus demonstrating translational potential of PDX-based preclinical trials. This network-based approach is highly adaptable and can be used to evaluate biomarkers of response to other agents.

category: cancer biology


title: Apoplastic class III peroxidases PRX62 and PRX69 regulate ROS-homeostasis and cell wall associated extensins linked to root hair growth at low-temperature in Arabidopsis thaliana

abstract: Root Hairs (RHs) growth is highly influenced by endogenous as well as by external environmental signals that coordinately regulate its final cell size. RHs actively expand the root surface responsible for nutrient uptake and water absorption. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to low temperature was linked to a reduced nutrient availability in the media. Here, we explored the molecular basis of this strong RH growth response by using the Genome Wide Association Studies (GWAS) approach on Arabidopsis thaliana natural accessions. We identified the poorly characterized PEROXIDASE 62 (PRX62) as a key protein triggering this conditional growth under a moderate low-temperature stress. In addition, we identified the related protein PRX69 as an important factor in this developmental process. The prx62 prx69 double mutant and the PRX62 and PRX69 over-expressing lines showed contrasting RH phenotypes, peroxidase activities and cyt/apoReactive Oxygen Species (ROS) levels. Strikingly, a cell wall protein extensin (EXT) reporter revealed the effect of peroxidase activity on the EXT cell wall association at 10{degrees}C in the RH apical zone. EXT cell wall insolubilization was enhanced at 10{degrees}C, which was completely abolished under the PRX inhibitor salicylhydroxamic acid (SHAM) treatment. Finally, we demonstrated that the Root Hair defective 6-like 4 (RSL4) bHLH family transcription factor directly controls the expression of PRX69. Collectively, our results indicate that both PRX62 and PRX69 are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low-temperature.


title: Computational metabolomics illuminates the lineage-specific diversification of resin glycoside acylsugars in the morning glory (Convolvulaceae) family

abstract: Acylsugars are a class of plant defense compounds produced across many distantly related families. Members of the horticulturally important morning glory (Convolvulaceae) family produce a diverse sub-class of acylsugars called resin glycosides (RGs), which comprise oligosaccharide cores, hydroxyacyl chain(s), and decorating aliphatic and aromatic acyl chains. While many RG structures are characterized, the extent of structural diversity of this class in different genera and species is not known. In this study, we asked whether there has been lineage-specific diversification of RG structures in different Convolvulaceae species that may suggest diversification of the underlying biosynthetic pathways. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed from root and leaf extracts of 26 species sampled in a phylogeny-guided manner. LC-MS/MS revealed thousands of peaks with signature RG fragmentation patterns with one species producing over 300 signals, mirroring the diversity in Solanaceae-type acylsugars. A novel RG from Dichondra argentea was characterized using Nuclear Magnetic Resonance spectroscopy, supporting previous observations of RGs with open hydroxyacyl chains instead of closed macrolactone ring structures. Substantial lineage-specific differentiation in utilization of sugars, hydroxyacyl chains, and decorating acyl chains was discovered, especially among Ipomoea and Convolvulus - the two largest genera in Convolvulaceae. Adopting a computational, knowledge-based strategy, we further developed a high-recall workflow that successfully explained ~72% of the MS/MS fragments, predicted the structural components of 11/13 previously characterized RGs, and partially annotated ~45% of the RGs. Overall, this study improves our understanding of phytochemical diversity and lays a foundation for characterizing the evolutionary mechanisms underlying RG diversification.


title: Course-based Undergraduate Research Module for Enzyme Discovery Using Protein Structure Prediction

abstract: A major challenge for science educators is teaching foundational concepts while introducing their students to current research. Here we describe an active learning module developed to teach protein structure fundamentals while supporting ongoing research in enzyme discovery. It can be readily implemented in both entry-level and upper-division college biochemistry or biophysics courses. Pre-activity lectures introduced fundamentals of protein secondary structure and provided context for the research projects, while a homework assignment familiarized students with 3D visualization of biomolecules using UCSF Chimera, a free protein structure viewer. The activity is an online survey in which students compare structure elements in papain, a well-characterized cysteine protease from Carica papaya, to novel homologous proteases identified from the genomes of an extremophilic microbe (Halanaerobium praevalens) and two carnivorous plants (Drosera capensis and Cephalotus follicularis). Students were then able to identify, with varying levels of accuracy, a number of structural features in cysteine proteases that could expedite the identification of novel or biochemically interesting cysteine proteases for experimental validation in a university laboratory. Student responses to a post-activity survey were largely positive and constructive, describing points in the activity that could be improved and indicating that the activity helped them learn about protein structure.


title: Voltage imaging identifies spinal circuits that modulate locomotor adaptation in zebrafish

abstract: Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We mapped the spiking, subthreshold dynamics, relative timing, and sub-cellular electrical propagation across large populations of simultaneously recorded cells. We validated the approach by confirming properties of known sub-types, and we characterized a yet undescribed sub-population of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail-beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.


title: Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

abstract:
The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

HIGHLIGHTSO_LIE3 ligase UFD-2 stimulates ubiquitylation activity of CHIP/CHN-1
C_LIO_LIUFD-2 binding promotes dimerization of CHIP/CHN-1 U-box domains and utilization of E2 enzymes
C_LIO_LIHSP70/HSP-1 by latching the U-box and TPR domains stabilizes the autoinhibitory state of CHIP/CHN-1, limiting interactions with E2s and UFD-2
C_LIO_LIAssembly with UFD-2 enables CHIP/CHN-1 to regulate lipid metabolism by ubiquitylation of S-Adenosylhomocysteinase
C_LI


title: The ataxia protein sacsin is required for integrin trafficking and synaptic organization

abstract: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS, which manifest as a childhood-onset cerebellar ataxia. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament (IF) disorganization, and loss of Purkinje neurons. It is unclear how the loss of SACS causes these deficits, or why they manifest as cerebellar ataxia. We employed a multi-omics approach to characterize molecular and cellular deficiencies in SACS knockout (KO) cells. We identified alterations in microtubule structure and dynamics, protein trafficking, and mislocalization of synaptic and focal adhesion proteins. Targeting PTEN, a negative regulator of focal adhesions, rescued several cellular phenotypes in SACS KO cells. We found sacsin interacts with proteins implicated in vesicle transport, including HSP proteins, and interactions between structural and cell adhesion proteins were diminished in SACS KO cells. In all, this study suggests that trafficking and localization of synaptic adhesion proteins is a causal molecular deficiency in ARSACS.


title: Trade-driven trapping dampens the biodiversity benefits of forest restoration in Southeast Asia

abstract: Tropical forest restoration stands to deliver important conservation gains in lowland Southeast Asia, which has suffered some of the worlds highest rates of forest degradation and loss. This promise, however, may be undermined by defaunation driven by ubiquitous wildlife trapping in the region, particularly for forest birds that are part of the multi-million-dollar pet trade. To date, quantification of the impacts of trade-driven trapping on rates of biodiversity recovery from forest restoration has been limited. Here, we use a unique long-term survey dataset to ask how trade-driven trapping may interfere with the expected recovery of avian community under forest restoration, at a flagship ecosystem restoration site in the lowland rainforests of Sumatra, Indonesia. We show that tropical forest restoration is associated with the increases in the abundance of 88% of bird species over time. However, impacts of trapping within more accessible areas of the forest meant that this recovery was dampened for 74% of bird species, relative to levels expected as a result of the magnitude of forest recovery observed. Most species (80%) showed increasingly positive relationships between abundance and site remoteness over the period, a pattern that was found for both species targeted for the pet trade (85% of species) and those trapped opportunistically or as  bycatch (78% of species). We emphasize the urgency of tackling the emerging threat of pet trade to Southeast Asias avian diversity, not least to ensure the effectiveness of efforts towards forest restoration.


title: Ancestral protein topologies draw the rooted bacterial tree of life

abstract: Aminoacyl tRNA synthetases (aaRSs) are among the proposed proteins present in the Last Universal Common Ancestor (LUCA). There are two types of glycyl tRNA synthetases (GlyRSs), from which the archaeal-eukaryal type is the one suggested to be present in LUCA. Here we solved the crystal structure of a complete bacterial glycyl tRNA synthetase (bacGlyRS) and show that indeed, bacGlyRS carries several structural signals that point it at the origin of all aaRSs. Furthermore, if bacGlyRS is ancestral, it should help to build a reliable Tree of Life (ToL). Given the modular nature of protein evolution, we used only two sub-domain segments with duplicated ancestral topologies, no detected orthologs and an assumed limited horizontal gene transfer (HGT). These motifs correspond to the non-specific RNA binding regions of contemporary bacGlyRS, archaeal CCA-adding enzyme (arch-CCAadd), and eukaryotic rRNA processing enzyme (euk-rRNA). The calculated, rooted bacterial ToL agrees with several phyla relationships unaccounted by the available trees.

category: evolutionary biology


title: Bacterial glycyl tRNA synthetase offers glimpses of ancestral protein topologies


title: Half a century of stable antibiotic resistance in livestock-associated Staphylococcus aureus and its dynamic readaptation to humans

abstract: Mobile genetic elements (MGEs) are agents of horizontal gene transfer in bacteria, but can also be vertically inherited by daughter cells. Establishing the dynamics that led to contemporary patterns of MGEs in bacterial genomes is central to predicting the emergence and evolution of novel and resistant pathogens. Here we develop existing methods to fully characterise the evolutionary history of MGEs in methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex (CC) 398. CC398 is the dominant MRSA in European livestock, and a growing cause of human infections. We reconstruct MGE dynamics using a collection of 1,180 CC398 genomes, sampled from livestock and humans, over 27 years. We find that the emergence of livestock-associated CC398 coincided with the acquisition of a Tn916 transposon carrying a tetracycline resistance gene, which has been stably vertically inherited for 57 years. This was followed by the acquisition of a type V SCCmec that carries methicillin, tetracycline and heavy metal resistance genes, which has been maintained by vertical and horizontal transmission for 35 years, with occasional truncations and replacements with type IV SCCmec. In contrast, a class of prophages that carry a human immune evasion gene cluster, that are largely absent from livestock-associated CC398, have been repeatedly gained and lost in both human- and livestock-associated CC398. These contrasting dynamics mean that when livestock-associated MRSA infects humans, adaptation to the human host outpaces loss of antibiotic resistance. In addition, stable inheritance means that antibiotic resistance in livestock-associated MRSA may persist despite ongoing reductions in antibiotic and zinc oxide use in farming.


title: Human readaptation outpaces loss of antibiotic resistance in livestock-associated MRSA


title: Cross-scale Analysis of Temperature Compensation in the Cyanobacterial Circadian Clock System

abstract: Clock proteins maintain constant enzymatic activity regardless of temperature, even though thermal fluctuation is accelerated as temperature increases. We investigated temperature influences on the dynamics of KaiC, a temperature-compensated ATPase in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated very slightly in a temperature-dependent manner. Our mutation studies revealed that internal motions of KaiC include several contributions of opposing temperature sensitivities. To take advantage of this balancing effect, the motional frequency of local dynamics in KaiC needs to exceed [~]0.3 ps-1. Some of the mutation sites may be in a pathway through which the motional frequency in the C-terminal domain of KaiC is fed back to the active site of ATPase in its N-terminal domain. The temperature-compensating ability at the dynamics level is likely crucial for circadian clock systems, into which the clock proteins are incorporated, to achieve reaction- or even system-level temperature compensation of the oscillation frequency.


title: Interferon-γ Increases Sensitivity to Chemotherapy and Provides Immunotherapy Targets in Models of Metastatic Castration-Resistant Prostate Cancer

abstract: Interferon-{gamma} (IFN{gamma}) is a cytokine with limited evidence of benefit in cancer clinical trials to date. However, it could potentially play a role in potentiating anti-tumor immunity in the immunologically "cold" metastatic castration-resistant prostate cancer (mCRPC) by inducing antigen presentation pathways and concurrently providing targets for immune checkpoint blockade therapy. Moreover, it could additionally increase sensitivity to chemotherapy based on its pleiotropic effects on cell phenotype. Here, we show that IFN{gamma} treatment induced expression of major histocompatibility class-I (MHC-I) genes and PD-L1 in prostate cancer cells in vitro. Furthermore, IFN{gamma} treatment led to a decrease in E-cadherin expression with a consequent increase in sensitivity to chemotherapy in vitro. In an in vivo murine tumor model of spontaneous metastatic prostate cancer, IFN{gamma} systemic pretreatment upregulated the expression of HLA-A and decreased E-cadherin expression in the primary tumor, and more importantly in the metastatic site led to increased apoptosis and limited micrometastases in combination with paclitaxel treatment compared to diffuse metastatic disease in control and monotherapy treatment groups. These findings suggest that IFN{gamma} may be useful in combinatorial regimens to induce sensitivity to immunotherapy and chemotherapy in hepatic metastases of mCRPC.


title: Melanomagenesis driven by a kinase-dead mutant BRAF lacking codons 600-618

abstract: While considerable success has been achieved with treating BRAF-V600E mutant tumors in several organ systems, tumors expressing non-V600E BRAF mutations remain a significant clinical challenge. Such atypical BRAF mutations are classified based on their kinase activity, RAS-dependence and their requirement for dimerization to function. We identified an unusually large in-frame deletion in a melanoma patient, encompassing codons 600-618 in the kinase domain. This patient experienced rapid disease progression when treated with dual BRAF/MEK inhibition. To determine whether and how such a large in-frame BRAF deletion might be activating, we ectopically expressed it in HEK293 cells and monitored BRAF pathway activation and response to BRAF and MEK inhibitors. We demonstrate that this mutation results in a kinase-deficient BRAF mutant which, nevertheless, activates MEK/ERK signaling in a dimerization-dependent manner, placing this mutation among the class III BRAF mutants.


title: FKBPL and FKBP8 regulate DLK degradation and neuronal responses to axon injury

abstract: DLK is a key regulator of axon regeneration and degeneration in response to neuronal injury. To understand the molecular mechanisms controlling the DLK function, we performed yeast two-hybrid screening analysis and identified FKBPL as a DLK-binding protein that bound to the kinase domain and inhibited the kinase enzymatic activity of DLK. FKBPL regulated DLK stability through ubiquitin-dependent DLK degradation. We tested other members in the FKBP protein family and found that FKBP8 also induced DLK degradation as FKBPL did. We found that Lysine 271 residue in the kinase domain of DLK was a major site of ubiquitination and SUMO3-conjugation and responsible for FKBP8-mediated degradation. In vivo overexpression of FKBP8 delayed progression of axon degeneration and neuronal death following axotomy in sciatic and optic nerves, respectively, although axon regeneration efficiency was not enhanced. This research identified FKBPL and FKBP8 as new DLK-interacting proteins that regulated DLK stability by MG-132 or bafilomycin A1-sensitive protein degradation.

category: neuroscience


title: Role of miR-181c in Diet-Induced Obesity Through Regulation of Lipid Synthesis in Liver

abstract: We recently identified a nuclear-encoded miRNA (miR-181c) in cardiomyocytes that can translocate into mitochondria to regulate mitochondrial gene (mt-COX1), and influence obesity-induced cardiac dysfunction through mitochondrial pathway. Liver plays a pivotal role during obesity. Therefore, we hypothesized that miR-181c plays an important role in pathophysiological complications associated with obesity. We used miR-181c/d-/- mice to study the miR-181c role in lipogenesis in hepatocytes during diet-induced obesity (DIO). Indirect calorimetric measurements were made during the 26 weeks high fat diet (HFD) exposure. qPCR was performed to examine the gene expression involved in lipid synthesis. Here, we show that miR-181c/d-/- mice are not protected against all metabolic consequences of HFD exposure. After 26 weeks of the HFD, miR-181c/d-/- mice had a significantly higher body fat (%) compared to WT. Glucose tolerance tests showed hyperinsulinemia and hyperglycemia, indicative of insulin insensitivity in the miR-181c/d-/- mice. HFD-fed miR-181c/d-/- mice had higher serum and liver triglyceride levels compared to HFD-fed WT mice. qPCR data demonstrated that several genes which are regulated by isocitrate dehydrogenase 1 (IDH1) were upregulated in miR-181c/d-/- liver compared to WT liver. Furthermore, an AAV-8 was used to deliver miR-181c, in vivo, to validate the potential role of miR-181c in the liver. miR-181c delivery attenuate the lipogenesis by downregulating the same lipid synthesis genes in the liver. In hepatocytes, miR-181c regulates lipid biosynthesis by targeting IDH1. Taken together, the data indicate that overexpression of miR-181c can be beneficial for various lipid metabolism disorders.


title: Novel roles of small extracellular vesicles in regulating the quiescence and proliferation of neural stem cells

abstract: Neural stem cells (NSCs) quiescence plays pivotal roles in securing sustainable neurogenesis and avoiding stemness exhaustion in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals, and environmental stimuli. Though the mechanisms of the transitions between NSC states have been extensively investigated, they remain to be fully elucidated. Exosomes are small extracellular vesicles (sEVs) containing functional units such as proteins, microRNAs, and mRNAs. It has already been demonstrated that sEVs actively participate in cancer cell proliferation and metastasis. However, the role of sEVs in NSC quiescence has not been investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed expression level fluctuations of NSCs sEV protein cargo at different proliferative conditions. We also identified functional clusters of gene ontology annotations from differentially expressed proteins in three sources of exosomes. Moreover, the use of exosome inhibitors revealed the contribution of exosomes to NSC quiescence at the entrance into quiescence, as well as in quiescence maintenance. Exosome inhibition delayed the entrance into quiescence by proliferating NSCs and allowed quiescent NSCs to exit from the G0 phase of the cell cycle. Protein translation was significantly upregulated in both quiescent NSCs and quiescent-induced NSCs via the exosome inhibition. Our results demonstrated that NSC exosomes are involved in regulating the quiescence of NSCs and provide a functional prediction of NSCs exosome protein cargos in terms of cell-cycle regulation and protein synthesis.


title: Riceberry rice bran protein hydrolyzed fractions induced apoptosis, senescence and G1/S cell cycle arrest in human colon cancer cell lines

abstract: Riceberry rice bran is the part of rice that has been scrubbed out during coloring process. There are various health benefits with high protein content and antioxidant ability. The hydrolyzed rice bran consists of diverse peptides that provide various bioactive properties. This work aimed to study the effect of hydrolyzed riceberry rice bran extracted on colon cancer cell lines (HT-29 and SW-620) compared to normal cell (PCS-291-010). The MTT assay result showed that our extract has less cytotoxicity on normal cell (PCS-291-010, IC50 = 6,680.00 g/ml) compared to the colon cancer cell lines and has more effect on metastatic cancer cell line (SW-620, IC50 = 5,492.31 g /ml) than non-metastatic cancer cell line (HT-29, IC50 =6,040.76 g/ml). According to the DNA fragmentation pattern analysis, the ladder pattern indicated that the rice bran extract can induce the apoptosis process in SW-620 cell line. Confirmed the pattern of apoptotic cell by AO/PI double stain test and quantified apoptotic cells by Annexin V. For the cell senescence analysis, SA-{beta}-gal staining technique was performed at 24 h after treatments, HT-29 reached maximum senescence rate at 85.74% while SW-620 had only 17.23% of senescence. And a result of cell cycle analysis, HT-29 were decreased the number of cells in S, M/G2 phase, and increased the number of cells in G0/G1 phase. Furthermore > 50 kDa peptide fraction separated from HRBE has a potent anti-cancer cells (SW-620, IC50 = 4,908 g/ml). In conclusion, the hydrolyzed riceberry rice bran extract can inhibit colon cancer cell lines with less effect on normal cell. The extracts could induce apoptosis process in metastatic cancer cell and induce senescence process in non-metastatic cancer cell. This observed information will be useful and applicable for medical research and colon cancer treatment in the future.


title: Neuron-specific coding sequences are the most highly conserved in the mammalian brain

abstract:
Neurons have become highly diverse in cell size, morphology, phenotype and function in mammalian evolution, whereas glial cells are much less varied in size and types across species. This difference in diversity suggests that neuron-specific coding and regulatory sequences have admitted more variation in evolution, that is, are less evolutionarily conserved than those expressed in glial cells. Here we calculate values of dN/dS from Ensembl98 for coding sequences expressed specifically in neurons, astrocytes, oligodendrocytes, microglia and endothelial cells in the brain across 92 mammalian species with reference to either mouse or human. Surprisingly, we find that protein-coding sequences that are specifically expressed in neurons are far less variable than those specific to other cell types in the brain. We next analyze phastCons values of neuronal and non-neuronal regulatory sequences and find that neuronal cis-regulatory DNA elements are more conserved than their non-neuronal counterpart, and neuron-specific promoter sequences are at least as conserved as other cell type-specific promoter sequences. Moreover, neuron-specific coding sequences are as highly conserved across mammalian species as ATPase coding sequences, the benchmark of evolutionary conservation, followed by heart and skeletal muscle-specific sequences. Neuronal diversity in mammalian evolution thus arises despite high levels of negative selection on neuron-specific protein-coding sequences. We propose that such strong evolutionary conservation is imposed by excitability, which continually exposes cells to the risk of excitotoxic death, and speculate that variability of neuronal cell sizes arises as a consequence of variability in levels of activity, possibly constrained by energy supply to the developing brain. In support of this hypothesis, we find, using the Allen Cell Type Database, that larger cortical neurons do have lower firing rates.

Significance StatementThe majority of cells in the mammalian body, including glial cells in the brain, maintain a constant size across species from mice to elephants. The exception is neurons, whose size (the sum of the soma, axon, and dendrite) varies over 100-fold both within each brain and across species, often becoming larger in larger brains. What evolutionary mechanism allows neurons to be so exceptionally diverse in size? We show that neuron-specific genes are some of the most highly conserved in mammalian evolution, which implicates alternate causes of diversity in neuronal cell size beyond heritable genetics. In addition, we show that firing rates and neuronal cell sizes are negatively correlated. We propose a novel direction of research into how neuronal cell size diversity might be a self-organized response to variations in levels of activity, which are an exclusive property of excitable cells like neurons.


title: Cannabidiol activates neuronal Kv7 channels

abstract: Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously-expressed human Kv7.2/7.3 channels by CBD. Using patch clamp recordings, we found that CBD at low concentrations activates Kv7.2/7.3 channels at subthreshold voltages, with 100 nM CBD producing a doubling of current at -50 mV. CBD shifted the voltage-dependence of channels in the hyperpolarizing direction, producing a shift in the midpoint of activation by [~]-14 mV at 300 nM. CBD also effectively enhanced native M-current in both mouse superior cervical ganglion neurons and rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD seems likely to contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.


title: Constitutive signaling by the C-terminal fragment of polycystin-1 is mediated by a tethered peptide agonist

abstract:
Mutation of the PKD1 gene, encoding polycystin-1 (PC1), is the primary cause of autosomal dominant polycystic kidney disease. PC1 is an 11-transmembrane domain protein that binds and modulates the activity of multiple heterotrimeric G protein families and is thought to function as a non-canonical G protein-coupled receptor (GPCR). PC1 shares a conserved GPCR autoproteolysis inducing [GAIN] domain with the adhesion family of GPCRs, that promotes an auto-catalytic, cis-cleavage at the GPCR proteolysis site (GPS) located proximal to the first transmembrane domain. GPS cleavage divides these receptors into two associated  subunits, the extracellular N-terminal (NTF) and transmembrane C-terminal (CTF) fragments. For the adhesion GPCRs, removal of the NTF leads to activation of G protein signaling as a result of the exposure and subsequent intramolecular binding of the extracellular N-terminal stalk of the CTF, i.e., the tethered cryptic ligand or tethered agonist model. Here, we test the hypothesis that PC1-mediated signaling is regulated by an adhesion GPCR-like, tethered agonist mechanism. Using cell-based reporter assays and mutagenesis of PC1 expression constructs, we show that the CTF form of PC1 requires the stalk for signaling activation and synthetic peptides derived from the PC1 stalk sequence can re-activate signaling by a  stalk-less CTF. In addition, we demonstrate that ADPKD-associated missense mutations within the PC1 stalk affect signaling and can inhibit GPS cleavage. These results provide a foundation for beginning to understand the molecular mechanism of G protein regulation by PC1 and suggest that a tethered agonist-mediated mechanism can contribute to PKD pathogenesis.

SIGNIFICANCE STATEMENTMutations of the PKD1 gene, encoding polycystin-1, are the predominant cause of autosomal dominant polycystic kidney disease (ADPKD), a systemic disease that is the 4th leading cause of kidney failure. Polycystin-1 functions as an atypical GPCR capable of binding or activating heterotrimeric G proteins, which is essential for preventing renal cystogenesis. However, little is known regarding its regulation. Polycystin-1 shares structural features with the Adhesion family of GPCRs. In this work, we combined mutagenesis and cellular signaling assays which demonstrated that constitutive activation of signaling by polycystin-1 involves an Adhesion GPCR-like molecular mechanism. This study provides new knowledge regarding the structure-function relationships of polycystin-1 which will stimulate additional areas of investigation and reveal novel avenues of therapeutic intervention for ADPKD.

category: biochemistry