title: Evaluation of the α-synuclein PET radiotracer (d3)-MODAG-001 in pigs

abstract:
BackgroundA positron emission tomography (PET) radiotracer to neuroimage -synuclein aggregates would be a crucial addition for early diagnosis and treatment development in disorders such as Parkinsons disease, where elevated aggregate levels is a histopathological hallmark. The radiotracer (d3)-[11C]MODAG-001 has recently shown promise for visualization of -synuclein pre-formed fibrils (-PFF) in rodents. We here test the radiotracer in a pig model where proteins are intracerebrally injected immediately before scanning. Four pigs were injected in one hemisphere with 150 {micro}g -PFF, and in the other hemisphere, either 75 {micro}g -PFF or human brain homogenate from either dementia with Lewy bodies (DLB) or Alzheimers disease (AD) was injected. All pigs underwent one or two (d3)-[11C]MODAG-001 PET scans, quantified with the non-invasive Logan graphical analysis using the occipital cortex as a reference region.

ResultsThe -PFF and AD homogenate injected brain regions had high uptake of (d3)-[11C]MODAG-001 compared to the occipital cortex or cerebellum. BPND values in 150 {micro}g -PFF injected regions was 0.78, and in the AD homogenate injected regions was 0.73. By contrast, the DLB homogenate injected region did not differ in uptake and clearance compared to the reference regions. The time-activity curves and BPND values in the 150 {micro}g and 75 {micro}g injected region of -PFFs show a dose-dependent effect, and the PET signal could be blocked by pretreatment with unlabeled MODAG-001.

ConclusionWe find that both -PFF and AD brain homogenates give rise to increased binding of (d3)-[11C]MODAG-001 when injected into the pig brain. Despite its limited specificity for cerebral -synuclein pathology, (d3)-[11C]MODAG-001 shows promise as a lead tracer for future radiotracer development.


title: An orderly sequence of autonomic and neural events at transient arousal changes

abstract: Resting-state functional magnetic resonance imaging (rsfMRI) allows the study of functional brain connectivity based on spatially structured variations in neuronal activity. Proper evaluation of connectivity requires removal of non-neural contributions to the fMRI signal, in particular hemodynamic changes associated with autonomic variability. Regression analysis based on autonomic indicator signals has been used for this purpose, but may be inadequate if neuronal and autonomic activity covary. To investigate this potential co-variation, we performed rsfMRI experiments while concurrently acquiring electroencephalography and autonomic indicator signals, including heart rate, respiratory depth, and peripheral vascular tone. We identified a recurrent and systematic pattern of fMRI, EEG, and autonomic changes coincidental with intermittent arousal, suggesting arousal modulation. The temporal relationship between the various signals indicated combined neural and autonomic contribution to the fMRI signal, both of which involve widespread brain areas. The fMRI changes included brief signal reductions in salience and default-mode networks, and the thalamus, followed by a biphasic global change. These results suggest that proper measurement of functional connectivity with fMRI requires accounting for the full spectrum of autonomic and neural changes, as well as their co-variation during arousal state transitions.

category: neuroscience


title: The genetics of specific cognitive abilities

abstract: Most research on individual differences in performance on tests of cognitive ability focuses on general cognitive ability (g), the highest level in the three-level Cattell-Horn-Carroll (CHC) hierarchical model of intelligence. About 50% of the variance of g is due to inherited DNA differences (heritability) which increases across development. Much less is known about the genetics of the middle level of the CHC model, which includes 16 broad factors such as fluid reasoning, processing speed, and quantitative knowledge. We provide a meta-analytic review of 863,041 monozygotic-dizygotic twin comparisons from 80 publications for these middle-level factors, which we refer to as specific cognitive abilities (SCA). Twin comparisons were available for 11 of the 16 CHC domains. The average heritability across all SCA is 55%, similar to the heritability of g. However, there is substantial differential heritability and the SCA do not show the dramatic developmental increase in heritability seen for g. We also investigated SCA independent of g (g-corrected SCA, which we refer to as SCA.g). A surprising finding is that SCA.g remain substantially heritable (53% on average), even though 25% of the variance of SCA that covaries with g has been removed. Our review frames expectations for genomic research that will use polygenic scores to predict SCA and SCA.g. Genome-wide association studies of SCA.g are needed to create polygenic scores that can predict SCA profiles of cognitive abilities and disabilities independent of g. These could be used to foster childrens cognitive strengths and minimise their weaknesses.

category: genetics


title: Neural decoding from surface high-density EMG signals: influence of anatomy and synchronization on the number of identified motor units

abstract:
ObjectiveHigh-density surface electromyography (HD-sEMG) allows the reliable identification of individual motor unit (MU) action potentials. Despite the accuracy in decomposition, there is a large variability in the number of identified MUs across individuals and exerted forces. Here we present a systematic investigation of the anatomical and neural factors that determine this variability.

ApproachWe investigated factors of influence on HD-sEMG decomposition, such as synchronization of MU discharges, distribution of MU territories, muscle-electrode distance (MED - subcutaneous fat thickness), maximum anatomical cross-sectional area (ACSAmax), and fiber CSA. For this purpose, we recorded HD-sEMG signals, ultrasound, magnetic resonance imaging, and muscle biopsy of the biceps brachii muscle from two groups of participants - untrained-controls (UT=14) and strength-trained (>3 years of training, ST=16) - while they performed isometric ramp contractions with elbow flexors (at 15, 35, 50 and 70% maximum voluntary torque - MVT). We assessed the correlation between the number of accurately detected MUs by HD-sEMG decomposition and each measured parameter, for each target force level. Multiple regression analysis was then applied.

Main resultsST subjects showed lower MED (UT: 4.8 {+/-} 1.4 vs. ST: 3.7 {+/-} 0.8 mm) associated to a greater number of identified motor units (UT: 21.3 {+/-} 10.2 vs. ST: 29.2 {+/-} 11.8 MUs/subject). Both groups showed a negative correlation between MED and the number of identified MUs at low forces (r= -0.6, p=0.002 at 15% MVT). Moreover, the number of identified MUs was positively correlated to the distribution of MU territories (r=0.56, p=0.01) and ACSAmax (r=0.48, p=0.03) at 15% MVT. By accounting for all anatomical parameters, we were able to partly predict the number of decomposed MUs at low but not at high forces.

SignificanceOur results confirmed the influence of subcutaneous tissue on the quality of HD-sEMG signals and demonstrated that MU spatial distribution and ACSAmax are also relevant parameters of influence for current decomposition algorithms.

category: bioengineering


title: LRLoop: Feedback loops as a design principle of cell-cell communication

abstract: Genome-wide gene expression profiling, and single-cell RNA-seq in particular, allows the predictions of molecular mechanisms regulating cell-cell communication based on the expression of known ligand-receptor pairs. Currently available techniques for predicting ligand-receptor interactions are one-directional from sender to receiver cells. Here we describe LRLoop, a new method for analyzing cell-cell communication that is based on bi-directional ligand-receptor interactions, where two pairs of ligand-receptor interactions are identified that are responsive to each other, and thereby form a closed feedback loop. We assessed LRLoop using both bulk and single-cell datasets and found our method significantly reduces the false positive rate seen with existing methods. Finally, we applied LRLoop to the single-cell datasets obtained from retinal development and discovered many new bi-directional ligand-receptor interactions among individual cell types that potentially control proliferation, neurogenesis and/or cell fate specification.

category: bioinformatics


title: Blocking Gi/o-coupled signaling eradicates cancer stem cells and sensitizes breast tumors to HER2-targeted therapies to inhibit tumor relapse

abstract: Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.

category: cancer biology


title: A chromosome-level phased Citrus sinensis genome facilitates understanding Huanglongbing tolerance mechanisms at the allelic level in an irradiation induced mutant

abstract: Sweet orange (SWO) originated from introgressive hybridization of pummelo and mandarin resulting in a highly heterozygous genome. Here, we assembled a chromosome-level phased Valencia SWO (DVS) genome with [~]98.5% completeness, high accuracy (QV=50.6), and the highest annotation BUSCO completeness (99.2%) thus far in citrus. DVS harbors a high level of allelic variances and enables study of allelic somatic structural mutations and corresponding allelic expression alteration in two SWO mutants, one with high Huanglongbing tolerance (T19) and one more sensitive (T78). In T78, a large deletion on the pummelo-origin chr8 causes regional allelic expression absence. In T19, seven upregulated genes are located at one terminal of a translocated segment, including three genes related to heat shock protein (HSP) regulation. Furthermore, 68 of 133 HSPs are significantly upregulated in T19, which may be related to its enhanced HLB tolerance by preventing phloem necrosis. The DVS will advance allelic level studies in citrus.

category: plant biology


title: Neuroaffective profiles are associated with e-cigarette use

abstract:
IntroductionIdentifying the psychophysiological underpinnings of cue-induced compulsive nicotine use will provide new targets for relapse prevention treatments. We tested whether neuroaffective responses to motivationally relevant stimuli are associated with cue-induced nicotine self-administration. We hypothesized that smokers with stronger neuroaffective responses to nicotine-related cues than to pleasant stimuli (C>P) are more vulnerable to cue-induced nicotine self-administration than smokers with stronger neuroaffective responses to pleasant stimuli than to nicotine-related cues (P>C).

MethodsSmokers (N=36) looked at pleasant, unpleasant, neutral, and nicotine-related images signaling that an electronic nicotine delivery system (ENDS) was immediately available for use. We measured event-related potentials (a direct measure of cortical activity) and computed the amplitude of the late positive potential, a robust index of motivational salience. We used k-means cluster analysis to identify individuals characterized by the C>P or the P>C neuroaffective profile. We compared the ENDS use frequency in the two groups using quantile regression for counts.

ResultsCluster analysis assigned 18 smokers to the C>P profile and 18 smokers to the P>C profile. Smokers with the C>P neuroaffective profile used the ENDS significantly more often than smokers with the P>C profile. Significant differences in the number of puffs persisted across different quantiles.

ConclusionsThese results support the hypothesis that individual differences in the tendency to attribute motivational salience to drug-related cues underlie vulnerability to cue-induced drug self-administration.

ImplicationsBy linking brain reactivity profiles to nicotine self-administration, we identified a neuroaffective biomarker that could guide the development of personalized treatments to prevent smoking relapse.


title: A longitudinal CA2/3 to CA1 circuit for initiating context-dependent associative learning

abstract: Associating environmental context with emotional experience is a vital brain function that requires the activity in both dorsal and ventral hippocampus. While only ventral hippocampus connects with the amygdala, a hub for fear learning, it remains unclear how the two hippocampal areas interact during contextual fear conditioning (CFC). We found that projections from dorsal CA2/CA3 (dCA2/3) to the dorsal part of ventral CA1 (vCA1d) contributed significantly to CFC. Deep-brain Ca2+ imaging revealed a CFC-enhanced difference in neuronal activities evoked by conditioned vs. neutral environmental context in both dCA2/3 and vCA1d areas. Notably, contextual fear retrieval correlated with changes in conditioned context-evoked activity in vCA1d, but not in dCA2/3. Furthermore, slice recordings showed that CFC potentiated dCA2/3-to-vCA1d projection strength, in line with coordinated elevation of monosynaptic excitation and reduction of disynaptic inhibition mediated by somatostatin-expressing interneurons. Thus, vCA1d represents a critical site for initiating emotional association with contextual information from dCA2/3.


title: Neural responses to broadband visual flicker in marmoset primary visual cortex

abstract: Temporal information is ubiquitous in natural vision and must be represented accurately in the brain to allow us to interact with a constantly changing world. Recent studies have employed a random stimulation paradigm to map the temporal response function (TRF) to luminance changes in the human EEG. This approach has revealed that the visual system, when presented with broadband visual input, actively selects distinct temporal frequencies, and retains their phase-information for prolonged periods of time. This non-linear response likely originates in primary visual cortex (V1), yet, so far it has not been investigated on a neural level. Here, we characterize the steady-state response to random broadband visual flicker in marmoset V1. In two experiments, we recorded from i) marmosets passively stimulated under general anesthesia, and ii) awake marmosets, under free viewing conditions. Our results show that LFP coupling to the stimulus was broadband and unselective under anesthesia, whereas in awake animals, it was restricted to two distinct frequency components, in the alpha and beta range. Within these frequency bands, coupling adhered to the receptive field (RF) boundaries of the local populations. The responses outside the RF did not provide evidence for a propagation of stimulus information across the cortex, contrary to results in human EEG studies. This result may be explained by short fixation durations, warranting further investigation. In summary, our findings show that during awake behavior V1 neural responses to broadband information are selective for distinct frequency bands, and that this selectivity is likely controlled actively by top-down mechanisms.


title: NMNAT2 in cortical glutamatergic neurons exerts both cell and non-cell autonomous influences to shape cortical development and to maintain neuronal health

abstract: Here we show that deleting NMNAT2 from cortical glutamatergic neurons (NMNAT2 cKO) results in progressive axonal loss, neuroinflammation, small hippocampi and enlarged ventricles. Interestingly, dramatic neuroinflammation responses were observed around the long-range axonal tracts of NMNAT2 cKO cortical neurons. In addition to the neurodegenerative-like phenotype, we also found the absence of whisker-representation patterns "barrels" in the primary somatosensory cortex of NMNAT2 cKO mice. These observations suggest that NMNAT2 is required in developing cortical circuits and in maintaining the health of cortical neurons. Unbiased transcriptomic analysis suggests that NMNAT2 loss in cortical neurons after axonal outgrowth phase upregulates mitochondria function while greatly reducing synaptogenesis pathways. Complete loss of Sarm1 function in NMNAT2 cKO mice restores barrel map formation and axonal integrity and abolishes the inflammatory response. Interestingly, reducing Sarm1 function in NMNAT2 cKO mice by deleting only one copy of Sarm1 restores barrel map formation but did not diminish the neurodegenerative-like phenotype. Only complete loss of Sarm1 prevents neurodegeneration and inflammatory responses.


title: Light/dark and temperature cycling modulate metabolic electron flow in Pseudomonas aeruginosa biofilms

abstract:
Sunlight drives phototrophic metabolism, which affects redox conditions and produces substrates for non-phototrophs. These environmental parameters fluctuate daily due to Earths rotation, and non-phototrophic organisms can therefore benefit from the ability to respond to, or even anticipate, such changes. Circadian rhythms, such as diurnal changes in body temperature, in host organisms can also affect local conditions for colonizing bacteria. Here, we investigated the effects of light/dark and temperature cycling on biofilms of the opportunistic pathogen Pseudomonas aeruginosa PA14. We grew biofilms in the presence of a respiratory indicator dye and found that greater dye reduction occurred in biofilm zones that formed during dark intervals and at lower temperatures. This pattern formation occurred with cycling of blue, red, or far-red light, and a screen of mutants representing potential sensory proteins identified two with defects in pattern formation specifically under red light cycling. We also found that the physiological states of biofilm subzones formed under specific light and temperature conditions were retained during subsequent condition cycling. Light/dark and temperature cycling affected expression of genes involved in primary metabolic pathways and redox homeostasis, including those encoding electron transport chain components. Consistent with this, we found that cbb3-type oxidases contribute to dye reduction under light/dark cycling conditions. Together, our results indicate that cyclic changes in light exposure and temperature have lasting effects on redox metabolism in biofilms formed by a non-phototrophic, pathogenic bacterium.

IMPORTANCEOrganisms that do not obtain energy from light can nevertheless be affected by diurnal changes in light exposure. Many aspects of animal and fungal physiology fluctuate in response to these changes, including body temperature and the activities of antioxidant and other redox enzymes that play roles in metabolism. Whether redox metabolism is affected by light/dark and temperature cycling in bacteria that colonize such circadian organisms has not been studied in detail. Here we show that growth under light/dark and temperature cycling leads to rhythmic changes in redox metabolism in Pseudomonas aeruginosa and identify proteins involved in this response. P. aeruginosa is a major cause of healthcare-associated infections and designated as a serious threat by the CDC due to its recalcitrance during treatments. Our findings have the potential to inform therapeutic strategies that incorporate controlled light exposure or consider P. aeruginosas responses to conditions in the host.

category: microbiology


title: Antiviral DExD/H-box helicase 60 selectively inhibits translation from type II internal ribosome entry sites

abstract: During viral infection, competition ensues between viruses and their host cells to control the protein synthesis machinery. In response, certain host defense proteins globally limit mRNA translation. However, this is also detrimental for host protein synthesis. Here we describe an interferon-stimulated helicase, DDX60, that specifically inhibits translation from type II viral internal ribosome entry sites (IRESs). IRESs are RNA structures that enable mRNAs to recruit ribosomes directly, bypassing translation initiation using a 5 cap. DDX60 was previously observed to inhibit replication of a reporter hepatitis C virus (HCV). We show that DDX60 likely does not inhibit HCV replication, but surprisingly, inhibits the type II IRES used in the reporter HCV genomic RNA. Using firefly luciferase mRNAs translationally driven by different viral IRESs or a 5 cap analog, we show that DDX60 selectively reduces translation driven by type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not other IRES types or a 5 cap analog. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not poliovirus or bovine enterovirus 1 (type I IRES) replication. Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit poliovirus replication. Finally, we demonstrate that DDX60 specifically reduces polysome binding on type II IRES mRNA, but not 5 cap-dependent mRNA. Our data demonstrate that the cellular defense factor DDX60 counteracts viral takeover of host translation by blocking ribosome access to type II IRES elements specifically.


title: Spike Protein-independent Attenuation of SARS-CoV-2 Omicron Variant in Laboratory Mice

abstract: Despite being more transmissible, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant was found to cause milder diseases in laboratory animals, often accompanied by a lower viral load compared to previous variants of concern. This study revealed the structural basis for a robust interaction between the receptor binding domain of the Omicron spike protein and mouse ACE2. Pseudovirus bearing the Omicron spike protein efficiently utilized mouse ACE2 for entry. By comparing viral load and disease severity among laboratory mice infected by a natural Omicron variant or recombinant ancestral viruses bearing either the entire Omicron Spike or only the N501Y/Q493R mutations in its spike, we found that mutations outside the spike protein in the Omicron variant may be responsible for the observed lower viral load. Together, our results indicated that a post-entry block to the Omicron variant exists in laboratory mice.


title: Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program

abstract:
Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurposes multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitive short liner motif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early activation of Hh transcription factor GLI-1 and upregulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120 Hh SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an -TRP120 Hh SLiM antibody.

TRP120 Hh SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection.

Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

Author summary

Ehrlichia chaffeensis is an obligately intracellular bacterium that preferentially infects and replicates within mononuclear phagocytes and survives intracellularly by modulating cellular signaling pathways to subvert innate immune defenses. This investigation reveals the complex and expanding role that the E. chaffeensis TRP120 moonlighting effector and SLiM ligand mimetics have on immune subversion and infection through activation and regulation of evolutionarily conserved signaling pathways. Herein, we define a TRP120 Hh SLiM mimetic that induces Hh signaling and regulates the anti-apoptotic protein BCL-2 to prevent sequential activation of caspase 9 and 3, promoting E. chaffeensis infection. This study defines a novel prokaryotic SLiM mimetic that repurposes evolutionarily conserved eukaryotic signaling pathways to promote survival of an intracellular bacterium.


title: A comprehensive phenotypic screening strategy to identify modulators of cargo translocation by the bacterial Type IVB secretion system

abstract:
Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage co-culture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii, notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines.

ImportanceMulti-drug-resistant pathogens are an emerging threat to human health. Since conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of "smarter" therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogens Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii, our study represents the first step in our pursuit towards precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.


title: The Brazilian population of Fusarium oxysporum f. sp. cubense is not structured by VCG or by geographic origin

abstract: Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is considered one of the most destructive diseases of bananas. Paradoxically, knowledge of the genetics of the pathogen population in the Americas is very limited. In this study, a collection of 178 monosporic isolates from several banana producing regions, located in different climatic zones along a South to North transect in Brazil, was formed to assess the genetic structure of the population of Foc. The isolates underwent pathogenicity tests, PCR diagnosis for the detection of Tropical race 4 and screening to SIX homologs. The VCG of 119 isolates was determined by pairing against 17 testers. A group of 158 isolates was selected for microsatellite genotyping. There was moderate diversity of Foc in Brazil. Eight VCGs were identified: 0120, 0122, 0124, 0125, 0128, 01215, 01220, and 01222, of which 78% of isolates belong to a single VCG, while 22% of isolates belong to complexes of VCGs. The distribution of VCGs is uneven and independent of the banana genotype. VCGs were correlated with homologs of the SIX genes and varied according to geographic regions. Four SSR loci were polymorphic and on average 7.5 alleles were detected per locus. Thirty-five multilocus genotypes (MLGs) were identified. There was no association between VCG and MLGs and no genetic structure of the population of Foc in Brazil was detected.


title: Transcriptome Mining Reveals a Spectrum of RNA Viruses in Primitive Plants

abstract:
Current knowledge of plant viruses stems largely from those affecting economically important plants. Yet, plant species in cultivation represent a small and bias subset of the plant kingdom. Here, we describe virus diversity and abundance from a survey of 1079 transcriptomes from species across the breadth of the plant kingdom (Archaeplastida) by analysing open-source data from the One Thousand Plant Transcriptomes Initiative (1KP). We identified 104 potentially novel viruses, of which 40% comprised single-stranded positive-sense RNA viruses across eight orders, including members of the Hepelivirales, Tymovirales, Cryppavirales, Martellivirales and Picornavirales. One-third of the newly described viruses comprised double-stranded RNA viruses from the orders Durnavirales and Ghabrivirales. The remaining were negative-sense RNA viruses from the Rhabdoviridae, Aspiviridae, Yueviridae, Phenuiviridae and the newly proposed Viridisbunyaviridae. Our analysis considerably expands the known host range of 13 virus families to include lower plants (e.g., Benyviridae and Secoviridae) and four virus families to include algae hosts (e.g., Tymoviridae and Chrysoviridae). The discovery of the first 30 kDa movement protein in a non-vascular plant, suggests that the acquisition of plant virus movement proteins occurred prior to the emergence of the plant vascular system. More broadly, however, a co-phylogeny analysis revealed that the evolutionary history of these families is largely driven by cross-species transmission events. Together, these data highlight that numerous RNA virus families are associated with older evolutionary plant lineages than previously thought and that the scarcity of RNA viruses found in lower plants to date likely reflects a lack of investigation rather than their absence.

ImportanceOur knowledge of plant viruses is mainly limited to those infecting economically important host species. In particular, we know little about those viruses infecting primitive plant lineages such as the ferns, lycophytes, bryophytes and charophytes. To expand this understanding, we conducted a broad-scale viral survey of species across the breadth of the plant kingdom. We find that primitive plants harbour a wide diversity of RNA viruses including some that are sufficiently divergent to comprise a new virus family. The primitive plant virome we reveal offers key insights into the evolutionary history of core plant virus gene modules and genome segments. More broadly, this work emphasises that the scarcity of viruses found in these species to date likely reflects the absence of research in this area.


title: Changes in interactions over ecological time scales influence single cell growth dynamics in a metabolically coupled marine microbial community

abstract: Microbial communities thrive in almost all habitats on earth. Within these communities, cells interact through the release and uptake of metabolites. These interactions can have synergistic or antagonistic effects on individual community members. The collective metabolic activity of microbial communities leads to changes in their local environment. As the environment changes over time, the nature of the interactions between cells can change. We currently lack understanding of how such dynamic feedbacks affect the growth dynamics of individual microbes and of the community as a whole. Here we study how interactions mediated by the exchange of metabolites through the environment change over time within a simple marine microbial community. We used a microfluidic-based approach that allows us to disentangle the effect cells have on their environment from how they respond to their environment. We found that the interactions between two species - a degrader of chitin and a cross-feeder that consumes metabolic by-products - changes dynamically over time as cells modify their environment. Cells initially interact positively and then start to compete at later stages of growth. Our results demonstrate that interactions between microbes are not static and depend on the state of the environment, emphasizing the importance of disentangling how modifications of the environment affects species interactions. This experimental approach can shed new light on how interspecies interactions scale up to community level processes in natural environments.


title: IMPACT OF PHYLLOSPHERE METHYLOBACTERIUM ON HOST RICE LANDRACES

abstract: The genus Methylobacterium includes widespread plant-associated bacteria that dominate the plant phyllosphere (occupying leaf surfaces), consume plant-secreted methanol, and can produce plant growth promoting metabolites. However, despite the potential to increase agricultural productivity, their impact on host fitness in the natural environment is relatively poorly understood. Here, we conducted field experiments with three traditionally cultivated rice landraces from north eastern India. We inoculated seedlings with native vs. non-native phyllosphere Methylobacterium strains, finding significant impacts on plant growth and grain yield. However, these effects were variable: whereas some Methylobacterium isolates were beneficial for their host, others had no impact or were no more beneficial than the bacterial growth medium. Host plant benefits were not consistently associated with Methylobacterium colonization, altered phyllosphere microbiome composition, changes in early expression of plant stress response pathways, or bacterial auxin production. We provide the first clear demonstration of the benefits of phyllosphere Methylobacterium for rice yield under field conditions, and highlight the need for further analysis to understand the mechanisms underlying these benefits. Given that the host landrace-Methylobacterium relationship is not generalizable, future agricultural applications will require careful testing to identify coevolved host- bacterium pairs that may enhance the productivity of high-value rice varieties.


title: Multi-excitation Raman Spectroscopy Complements Whole Genome Sequencing for Rapid Detection of Bacterial Infection and Resistance in WHO Priority Pathogens

abstract: Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.


title: Influenza A virus reassortment in mammals gives rise to genetically distinct within-host sub-populations

abstract: Influenza A virus (IAV) genetic exchange through reassortment has the potential to accelerate viral evolution and has played a critical role in the generation of multiple pandemic strains. For reassortment to occur, distinct viruses must co-infect the same cell. The spatio-temporal dynamics of viral dissemination within an infected host therefore define opportunity for reassortment. Here, we used wild type and synonymously barcoded variant viruses of a pandemic H1N1 strain to examine the within-host viral dynamics that govern reassortment in guinea pigs, ferrets and swine. The first two species are well-established models of human influenza, while swine are a natural host and a frequent conduit for cross-species transmission and reassortment. Our results show reassortment to be pervasive in all three hosts but less frequent in swine than in ferrets and guinea pigs. In ferrets, tissue-specific differences in the opportunity for reassortment are also evident, with more reassortants detected in the nasal tract than the lower respiratory tract. While temporal trends in viral diversity are limited, spatial patterns are clear, with heterogeneity in the viral genotypes detected at distinct anatomical sites revealing extensive compartmentalization of reassortment and replication. Our data indicate that the dynamics of viral replication in mammals allow diversification through reassortment but that the spatial compartmentalization of variants likely shapes their evolution and onward transmission.


title: A transcription factor and a phosphatase regulate temperature-dependent morphogenesis in a fungal plant pathogen

abstract:
Naturally fluctuating temperatures provide a constant environmental stress that requires adaptation. Some fungal pathogens respond to heat stress by producing new morphotypes that maximize their overall fitness. The fungal wheat pathogen Z. tritici responds to heat stress by switching from its yeast-like blastospore form to hyphae or chlamydospores. The regulatory mechanisms underlying this switch are unknown. Here, we demonstrate that a differential heat stress response is ubiquitous in Z. tritici populations around the world. We used QTL mapping to identify a single locus associated with the temperature-dependent morphogenesis and we found two genes, the transcription factor ZtMsr1 and the protein phosphatase ZtYvh1, regulating this mechanism. We find that ZtMsr1 regulates repression of hyphal growth and induces chlamydospore formation whereas ZtYvh1 is required for hyphal growth. We next pinpointed that chlamydospore formation is a response to the intracellular osmotic stress generated by the heat stress. This intracellular stress stimulates the CWI and HOG MAPK pathways resulting in hyphal growth. If cell wall integrity is however compromised, ZtMsr1 represses the hyphal development program and might induce the chlamydospore-inducing genes as a stress-response survival strategy. Taken together, these results suggest a novel mechanism through which morphological transitions are orchestrated in Z. tritici - a mechanism possibly also present in other pleomorphic fungi.

IMPORTANCETemperature is an environmental signal constantly monitored by pleomorphic fungi. Our experiments showed that yeast-to-hyphal or yeast-to-chlamydospore transitions are ubiquitous heat stress responses in Z. tritici. QTL mapping allowed us to identify a transcription factor and a protein phosphatase contributing to temperature-dependent morphogenesis. We showed that intracellular osmolarity is the pivotal signal inducing these transitions. We propose a regulatory network controlling Z. tritici morphogenesis, which may have broad implications for temperature sensing of fungal pathogens.

category: molecular biology


title: Expression and secretion of circular RNAs in the parasitic nematode, Ascaris suum

abstract: Circular RNAs (circRNAs) are a recently identified RNA species with emerging functional roles as microRNA (miRNA) and protein sponges, regulators of gene transcription and translation, and as modulators of fundamental biological processes including immunoregulation. circRNAs have been found in a variety of species including plants, animals, and model genetic organisms such as the free-living nematode Caenorhabditis elegans. Relevant to this study, circRNAs have recently been described in the parasitic nematode, Haemonchus contortus, suggesting they may have functionally important roles in parasites. Given this involvement in regulating biological processes, a better understanding of their role in parasites could be leveraged for future control efforts. Here, we report the use of next-generation sequencing to identify 1,997 distinct circRNAs expressed in adult female stages of the gastrointestinal parasitic nematode, Ascaris suum. We describe spatial expression in the ovaries and body wall muscle, and also report circRNA presence in extracellular vesicles (EVs) secreted by the parasite into the external environment. Further, we used an in-silico approach to predict that a subset of Ascaris circRNAs bind both endogenous parasite miRNAs as well as human host miRNAs, suggesting they could be functional as both exogenous and endogenous miRNA sponges to alter gene expression. There was not a strong correlation between Ascaris circRNA length endogenous miRNA interactions, indicating Ascaris circRNAs are enriched for Ascaris miRNA binding sites, but that human miRNAs were predicted form a more thermodynamically stable bond with Ascaris circRNAs. These results suggest that secreted circRNAs could be interacting with host miRNAs at the host-parasite interface and influencing host gene transcription. Lastly, although we previously found that therapeutically relevant concentrations of the anthelmintic drug ivermectin inhibited EV release from parasitic nematodes, we did not observe a direct effect on Ascaris circRNAs expression or secretion.

category: cell biology


title: Analysis of Sexual vs Asexual Reproduction Using Four Measurements

abstract: The prevalence of sexual reproduction has long been an outstanding problem of evolutionary biology. In accordance with the mathematical approach employed by past researchers, we propose a mathematical framework to address this problem. We define and derive four measurements, diversity measure DVM, diffusion measure DFM, optimality measure OPM, and survivability measure SVM to compare sexual reproduction with asexual reproduction. We show that DVM increases exponentially in sexual reproduction, while only linearly in asexual reproduction. Hence, sexual reproduction allows species more opportunity to adapt. We also show that DFM is bounded in sexual reproduction and OPM is inversely related to DFM. Thus, sexual reproduction leads to smaller DFM and hence a larger OPM. We further show that SVM is a monotonic increasing function of OPM. Hence, sexual reproduction is better by virtue of producing a more homogeneous population.

category: evolutionary biology


title: A tissue specific atlas of gene promoter DNA methylation variability and the clinical value of its assessment

abstract:
BackgroundComplex diseases have multifactorial etiologies making clinically actionable diagnostic markers difficult to identify. Novel tools with higher diagnostic yield and utility in driving personalized care are needed.

MethodsWe utilized Illumina methylation array data from over 2400 samples to assess DNA methylation patterns in 20 distinct cell types ranging from sperm to brain as well as various disease states. We generated a simple analysis pipeline for DNA methylation data that focuses on intra-individual methylation variability within gene promoters. The analysis is designed, not to identify single causative gene alterations but instead focuses on any movement away from "healthy" methylation. This approach identifies altered regulation across multiple genes in related pathways thus enabling us to detect shifts in gene regulatory activity associated with distinct tissues and phenotypes. We explored three distinct questions in our assessment. 1) Are patterns of epigenetic variability tissue specific? 2) Do diseased tissues exhibit altered variability patterns compared to normal tissue? 3) Can epigenetic variability be detected in complex disease?

ResultsUnsupervised clustering analyses established that patterns of epigenetic variability are tissue specific and that these patterns are at least as predictive of tissue type as differential methylation analysis. We demonstrated the ability to use these patterns to differentiate between healthy and diseased tissue with unsupervised clustering even in cases of complex multifactorial diseases. We applied this method to the clinical use case of male infertility and found that men undergoing intrauterine insemination (IUI) with the lowest number of epigenetically dysregulated promoters in their sperm were almost twice as likely to father a child than men participating in IUI with the highest number of dysregulated promoters (p=0.011). We saw no significant difference in birth rates between groups of men with high and low numbers of dysregulated promoters undergoing in vitro fertilization (IVF), indicating IVF as a better treatment than IUI to achieve live birth in the presence of multi-pathway dysregulation in sperm.

ConclusionsThis study demonstrates that patterns of epigenetic variability can differentiate between tissue types. While intuitive, this finding has never been demonstrated previously and suggests that specific epigenetic variability patterns may be used to predict phenotypic changes in disease states as these are, by definition, functional changes to cellular phenotypes. We demonstrate that the variability of gene regulatory marks are distinct between healthy and diseased tissue. This is particularly apparent at genes known to be important to cell function of the tissue of interest. While in some cases these regional alterations can be seen across the entire genome, more often the regulatory alterations that define a pathological phenotype are restricted to genes of known importance to a particular tissue. Importantly, in the case of sperm, we found that these patterns of variability did have utility in predicting infertile patients who would conceive through intrauterine insemination (IUI). We would propose that this discriminatory ability is due to the fact that the signature can be assessed in an n-of-1 context and that the patterns of variability identify any shift away from regulatory normalcy in pathways known to be impactful in the tissue of interest, and not only assessing the presence or absence of rare genetic variants. While the data presented here are encouraging, more work needs to be performed in other tissues to determine when, and in what context, these findings could be clinically actionable.

category: genomics


title: High-performance GPCR optogenetics based on molecular properties of animal opsins, MosOpn3 and LamPP

abstract: Optogenetics for GPCR signaling is highly valuable but still requires effective and versatile tools with performance evaluation from molecular properties. Here we investigated performance of two animal opsins, mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP) in optical manipulation in vivo by using C. elegans. MosOpn3 introduced in a nociceptor neurons induced avoidance responses light-dependently with a retinal isomer ubiquitously present in every tissue, like ChR2 and unlike canonical vertebrate opsins. Remarkably, the sensitivity is ~7000 times higher than the case of ChR2 in the light-induced behavior. LamPP introduced in motor neurons induced violet light-dependent stop and green light-dependent go, demonstrating color-dependent manipulation of behaviors using LamPP. Furthermore, our molecular engineering extended the usability of MosOpn3 and LamPP to different signaling cascades and kinetics. Current findings demonstrated that the availability of two animal opsins is equivalent to that of ChR2 in terms of retinal requirement, providing solid strategies for GPCR optogenetics.

category: biochemistry


title: Identification of Unusual Oxysterols Biosynthesised in Human Pregnancy by Charge-Tagging and Liquid Chromatography - Mass Spectrometry

abstract: The aim of this study was to identify sterols, oxysterols and any down-stream metabolites in placenta, umbilical cord blood plasma, maternal plasma and amniotic fluid to enhance our knowledge of the involvement of these molecules in pregnancy. We confirm the identification of 20S- hydroxycholesterol in human placenta, previously reported in a single publication, and propose a pathway from 22R-hydroxycholesterol to a C27 bile acid of probable structure 3{beta},20R,22R- trihydroxycholest-5-en-(25R)26-oic acid. The pathway is evident not only in placenta, but pathway intermediates are also found in umbilical cord plasma, maternal plasma and amniotic fluid but not non-pregnant women.


title: Belayer: Modeling discrete and continuous spatial variation in gene expression from spatially resolved transcriptomics

abstract: Spatially resolved transcriptomics (SRT) technologies simultaneously measure gene expression and spatial location of cells in a 2D tissue slice, enabling the study of spatial patterns of gene expression. Current approaches to model spatial variation in gene expression assume either that gene expression is determined by discrete cell types or that gene expression varies continuously across a tissue slice. However, neither of these modeling assumptions adequately represent spatial variation in gene expression: the first assumption ignores continuous variation within a spatial region containing cells of the same type, while the second assumption does not allow for discontinuous changes in expression, e.g., due to a sharp change in cell type composition. We propose a model of spatial patterns in gene expression that incorporates both discontinuous and continuous spatial variation in gene expression. Specifically, we model the expression of a gene in a layered tissue slice as a piecewise linear function of a single spatial coordinate with potential discontinuities at tissue layer boundaries. We formulate the problem of inferring tissue layer boundaries and gene expression functions for all genes simultaneously. We derive a dynamic programming algorithm to find the optimal boundaries of tissue layers when these boundaries are lines parallel to a coordinate axis. We generalize this algorithm to arbitrarily curved tissue layers by transforming the tissue geometry to be axis-aligned using the theory of conformal maps from complex analysis. We implement these algorithms in a method called Belayer. Applying Belayer to simulated data and to spatial transcriptomics data from the human dorsolateral prefrontal cortex, we demonstrate that Belayer achieves both higher accuracy in clustering tissue layers compared to state-of-the-art SRT clustering methods, and higher accuracy in identifying cortical layer marker genes compared to commonly-used methods for identification of spatially varying genes.


title: CRSP: Comparative RNA-seq pipeline for species lacking both of sequenced genomes and reference transcripts

abstract:
RNA sequencing (RNA-seq) has been a widely used high-throughput method to characterize transcriptomic dynamics spatiotemporally. However, typical RNA-seq data analysis pipelines depend on either a sequenced genome or annotated reference transcripts. This restriction makes the use of RNA-seq for species lacking both of sequenced genomes and reference transcripts challenging. To solve this problem, we developed CRSP, an RNA-seq pipeline integrating a comparative species strategy but not depending on a specific sequenced genome or reference transcripts. Benchmarking suggests the CRSP tool can achieve high accuracy to quantify gene expression levels.

AvailabilityCRSP and documents are available at: https://github.com/pjiang1105/CRSP.


title: CRSP: Comparative RNA-seq pipeline for species lacking both a reference genome and annotated transcriptome


title: Genesis of bursting activity: Role of inhibitory constraints on persistent Na+ currents

abstract: Some neurons express a plateau of spike activity greatly outlasting a stimulus, whereas others do not. This difference could be due to differential expression of ion channels (e.g., persistent Na+, and Ca2+) or similar expression of channels that differ in their properties. We compared three Aplysia neurons with varying capacity to generate plateau potentials: B51 with self-terminating plateaus, B64 with plateau potentials that do not self-terminate, and the regular spiking neuron B8. Our results indicate the three neurons expressed outward currents IA and ID, voltage-gated Ca2+ currents ICaL and ICaR, and persistent inward INaP. The most notable difference observed was a larger IA, ID, and IKCa currents in B8, and the plateau generating B64 did not express IKCa. Computational models suggest outward currents suppress and temporally constrain the plateau potential and that inward Ca2+ currents suppress plateau potentials when coupled with IKCa.


title: A highly attenuated SARS-CoV-2 related pangolin coronavirus variant has a 104nt deletion at the 3'-terminus untranslated region

abstract: SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3-terminus untranslated region (3-UTR). We further report the characterization of the GX_P2V variant in in vitro and in vivo infection models. In cultured Vero and BGM cells, the GX_P2V variant produced minimal cell damage and small plaques. The GX_P2V variant infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V variant is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely due to the 104-nt deletion in the HVR in the 3-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.


title: Deep tissue infection by an invasive human fungal pathogen requires novel lipid-based suppression of the IL-17 response

abstract: Candida albicans is the most common cause of human fungal infection, but the mechanisms of invasive pathogenesis remain poorly defined. Here we identify an unexpected mechanism: lipid-mediated immunosuppression. Through forward genetics, we found that C. albicans secretes a lipase, Lip2, that is critical for invasive disease. Murine infection with C. albicans strains that lack Lip2 display an exaggerated host IL-17 response that leads to fungal clearance from solid organs and host survival. IL-17 signaling is required for Lip2 action. The lipase activity of Lip2 inhibits IL-17 production indirectly through suppression of IL-23 production by tissue resident dendritic cells. We conclude that C. albicans suppresses antifungal IL-17 defense in solid organs by altering the tissue lipid milieu.


title: N-phosphonacetyl-L-aspartate enhances type I interferon anti-viral responses through activation of non-canonical NOD2 signaling

abstract:
Type I interferon production and the expression of interferon-stimulated genes (ISGs) are key components of an innate immune response to many microbial pathogens. Dysregulation of this response can result in uncontrolled infection, inflammation, and autoimmune disease. Understanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights into infection control strategies and autoimmune disease therapies. Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that acts as both a bacterial sensor protein and a mediator of antiviral responses. Antibacterial functions of NOD2 are enhanced by treatment with the small molecule inhibitor of pyrimidine biosynthesis N-phosphonacetyl-L-aspartate (PALA), though how this might function in the host antiviral response remains unknown. Therefore, we tested the ability of PALA to enhance NOD2-dependent antiviral responses. Alone, PALA treatment of macrophages was not sufficient to induce interferon {beta} (IFN{beta}) production or ISG expression. Instead, PALA synergized with IFN{beta} stimulation to enhance expression and activation of interferon-stimulated gene factor 3 (ISGF3) and induce the upregulation of a subset of ISGs in co-treated cells. Furthermore, PALA treatment of epithelial cells resulted in impaired viral replication of the herpesvirus, human cytomegalovirus. Induction of the PALA-enhanced antiviral response required activation of non-canonical NOD2 signaling mediated by mitochondrial antiviral-signaling protein (MAVS) and interferon response factor 1 (IRF1), rather than the classical receptor-interacting serine/threonine protein kinase 2 (RIP2) pathway or other IRFs previously reported to mediate NOD2 antiviral responses. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses and antiviral activity.

Significance StatementUnderstanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights to improve infection control strategies and autoimmune disease therapies. This work demonstrates that the pyrimidine synthesis inhibitor N-phosphonacetyl-L-aspartate synergizes with type I interferon to enhance antiviral responses through activation of a non-canonical NOD2 signaling pathway. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses.


title: Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells

abstract: Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We have previously developed a scalable monolayer system to differentiate human embryonic stem cell (hESC) into mature skeletal muscle cells (SkMC) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both hESC-SkMC fusion index and expression of various skeletal muscle markers including the motor protein Myosin Heavy Chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled to improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery to further enhance muscle development or treat muscle diseases.


title: Salp blooms increase carbon export 5-fold in the Southern Ocean

abstract: The Southern Ocean (SO) contributes substantially to the global biological carbon pump (BCP). Salps in the SO, in particular Salpa thompsoni, are keystone grazers that produce large, fast-sinking fecal pellets with high export potential. In a first study of this kind, we conducted Lagrangian experiments to quantify the salp bloom impacts on export pathways by contrasting locations differing in salp bloom presence/absence. We show that blooms increased particle export by ~5-fold, and exported up to 46% of net primary production out of the euphotic zone. BCP efficiency increased from 5% in non-salp areas to up to 28% in salp areas, which is among the highest recorded in the global ocean. Using SO salp abundances from KRILLBASE, we estimate they can consume ~ 13% of regional production, mediating 13-40% of the SO BCP. Consideration in models forecasting the SO BCP is recommended considering long-term increases in SO salp abundances.

category: ecology


title: Finding mutations in all the wrong places: Prevalence of knock-down resistance F1534S 1 mutations among Aedes albopictus (family: Culicidae, order: Diptera) in North Carolina

abstract:
BackgroundKnock-down resistance (kdr) mutations in the voltage gated sodium channel gene of Aedes species mosquitoes are biomarkers for resistance to pyrethroid insecticides. In the United States, few studies have reported kdr mutations among Aedes albopictus (family: Culicidae, order: Diptera, Skuse, 1895) populations. In this study we sought to explore the potential for permethrin-impregnated uniforms worn by military servicemembers to drive kdr emergence.

Methods and ResultsWe collected 538 Aedes albopictus mosquitoes, including 156 from 4 sites at Fort Bragg (exposed), North Carolina and 382 from 15 sites in Wake County (control), North Carolina to compare the prevalence of kdr mutations. Of those successfully sequenced, we identified 12 (3.0%) mosquitoes with kdr mutations, all of which were attributed to variants at position 1534 within domain 3. All mutations were found in mosquitoes collected at Wake County sites; no mutations were identified at Fort Bragg. There was a focus of mutations observed at the Wake County sites with approximately 92% (11 of 12) of the mosquitoes with the mutation coming from one site, where kdr mutations represented 24.4% (11 of 45) of all mosquitoes collected.

ConclusionsOur study did not show any evidence that universal implementation of permethrin-impregnated uniforms drives the development of resistance. In contrast, we observed highly focal resistance in a suburban area of Raleigh, which may be attributable to peri-domestic mosquito control activities that involve area dispersal of pyrethroid insecticides. More robust surveillance is needed to monitor the emergence and spread of resistance.

AUTHOR SUMMARYResistance to commonly employed insecticides among Aedes albopictus (family: Culicidae, order: Diptera, Skuse, 1895) mosquitoes poses as a substantial public health threat. In this study we sought to explore the potential for permethrin-impregnated uniforms worn by military servicemembers to drive emergence of resistance to pyrethroid insecticides by collecting and testing mosquitoes from both military and civilian sites. Overall, we did not identify mosquitoes harboring resistance at Ft. Bragg, but did find a focus of resistance in sub-urban Raleigh, which may be driven by commercial, peri-domestic mosquito control activities. These results suggest that resistance to pyrethroid insecticides may be more prevalent in the United States than previously known, but highly heterogenous. More robust surveillance is needed to monitor the emergence and spread of resistance.


title: The NOD/RIPK2 signaling pathway contributes to osteoarthritis susceptibility

abstract: Introductory paragraphOsteoarthritis (OA) is a debilitating disease characterized by loss of homeostasis of the joint with consequent remodeling of tissue architecture1. The molecular pathways that limit disease onset or progression are unknown2-5, and consequently no disease-modifying drugs are available1,6-9. We sought genes that contribute to dominant forms of hereditary OA with the aim of identifying pathways whose activity level contributes to OA susceptibility. We found seven independent alleles affecting the NOD/RIPK2 pathway. To determine if altered signaling is sufficient to confer heightened OA susceptibility, mice carrying the OA-associated hyperactive Ripk2104Asp allele were generated. Knees of heterozygous Ripk2104Asp mice exhibit no overt signs of joint remodeling. Nevertheless, the mice respond to injury with markedly advanced post-traumatic OA. Uninjured heterozygous Ripk2104Asp mice appear primed to develop OA: their knees exhibit elevated NOD/RIPK2 pathway activity, localized inflammation, and altered expression of extracellular matrix genes linked to OA. In contrast to the joint, the mice display no evidence of systemic elevated inflammation. Elevated NOD/RIPK2 signaling confers vulnerability to OA.


title: The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

abstract:
The Hsp70 family of molecular chaperones is well-conserved and expressed in all organisms. In budding yeast, cells express four highly similar cytosolic Hsp70s Ssa1, 2, 3 and 4 which arose from gene duplication. Ssa1 and 2 are constitutively expressed while Ssa3 and 4 are induced upon heat shock. Recent evidence suggests that despite their amino acid similarity, these Ssas have unique roles in the cell. Here we examine the relative importance of Ssa1-4 in the regulation of the enzyme ribonucleotide reductase (RNR). We demonstrate that cells expressing either Ssa3 or Ssa4 as their sole Ssa are compromised for their resistance to DNA damaging agents and activation of DDR transcription. In addition, we show that the steady state levels and stability of RNR small subunits Rnr2 and Rnr4 are reduced in Ssa3 or Ssa4-expressing cells, a result of decreased Ssa-RNR interaction. Interaction between the Hsp70 co-chaperone Ydj1 and RNR is correspondingly decreased in cells only expressing Ssa3 and 4. Through studies of Ssa2/4 domain swap chimeras, we determined that the C-terminal domain of Ssas are the source of this functional specificity. Taking together, our work suggests a distinct role for Ssa paralogs in regulating DNA replication mediated by C-terminus sequence variation.

Author SummaryCells require molecular chaperones to fold proteins into their active conformation. A major mystery however is why cells express so many highly-related and apparently redundant Hsp70 paralogs. We examined the role of four Hsp70 paralogs in budding yeast (Ssa1, 2, 3 and 4) on the activity of the ribonucleotide reductase (RNR complex). Importantly, we demonstrate there is selectivity of RNR subunits for Ssa1 and Ssa2 subunits, which is dictated by the co-chaperone Ydj1. Taken together, our work provides new insight into the functional specificity of Hsp70 paralogs using a native client protein.


title: Novel method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

abstract:
Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Long-read whole genome sequencing is prohibitively expensive for mtDNA heteroplasmy detection and often does not recapitulate the full mtDNA length.

Here we describe a method to target, multiplex and sequence full-length, native single-molecule the human mitochondrial genome utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks as barcodes with long-read sequencing, we implemented a protocol in an optimal setting for both high or low integrity genomic DNA to target the circular mitochondrial genome with extremely high coverage. Our analytical pipeline efficiently detects single nucleotide heteroplasmy, physically determines phase and can accurately disentangle complex deletion patterns. This workflow is a unique tool for studying mtDNA variation in health and disease, and will accelerate mitochondrial research.


title: Hyperoxia improves repeated-sprint ability and the associated training load in athletes

abstract: 1This study investigated the impact of hyperoxic gas breathing (HYP) on repeated-sprint ability (RSA) and on the associated training load (TL). Thirteen team- and racquet-sport athletes performed 6-s all-out sprints with 24-s recovery until exhaustion (power decrement [≥] 15% for two consecutive sprints) under normoxic (NOR: FIO2 0.21) and hyperoxic (HYP: FiO2 0.40) conditions in a randomized, single-blind and crossover design. The following variables were recorded throughout the tests: mechanical indices, arterial O2 saturation (SpO2), oxygenation of the vastus lateralis muscle with near-infrared spectroscopy, and electromyographic activity of the vastus lateralis, rectus femoris and gastrocnemius lateralis muscles. Session TL (work x rate of perceived exertion) and neuromuscular efficiency (work/EMG) were calculated. Compared with NOR, HYP increased SpO2 (2.7 {+/-} 0.8%, Cohens effect size ES 0.55), the number of sprints (14.5 {+/-} 8.6%, ES 0.28), the total mechanical work (13.6 {+/-} 6.8%, ES 0.30) and the session TL (19.4 {+/-} 7.0%, ES 0.33). Concomitantly, HYP increased the amplitude of muscle oxygenation changes during sprints (25.2 {+/-} 11.7%, ES 0.36) and recovery periods (26.1 {+/-} 11.4%, ES 0.37), as well as muscle recruitment (9.9 {+/-} 12.1%, ES 0.74) and neuromuscular efficiency (6.9 {+/-} 9.0%, ES 0.24). We conclude that breathing a hyperoxic mixture enriched to 40% O2 improves the total work performed and the associated training load during an open-loop RSA session in trained athletes. This ergogenic impact may be mediated by metabolic and neuromuscular alterations.

category: physiology


title: Proteomics profiling reveals regulation of immune response to Salmonella Typhimurium infection in mice

abstract:
Regulation of the immune response to Salmonella typhimurium (S. Typhimurium) infection is a complex process, influenced by genetic and environmental factors. Different inbred mouse strains show distinct levels of resistance to S. Typhimurium infection, ranging from susceptible (e.g., C57BL/6J) to resistant (e.g., DBA/2J) strains. However, the underlying molecular mechanisms contributing to the host response remain elusive. In this study, we present a comprehensive proteomics profiling of the spleen tissue from C57BL/6J and DBA/2J strains with different doses of S. Typhimurium infection by tandem tag mass coupled with two-dimensional liquid chromatography-tandem mass spectrometry. We identified and quantified 3,986 proteins, resulting in 475 differentially expressed proteins between C57BL/6J and DBA/2J strains. Functional enrichment analysis revealed that the mechanisms of innate immune responses to S. Typhimurium infection are associated with several signaling pathways, including the interferon signaling pathway. Our proteomic data also discovered a plausible gene in a genomic region that control different levels of resistance to S. Typhimurium infection. We further revealed the roles of macrophage cells and pro-inflammatory cytokines in the mechanisms under the natural resistance to S. Typhimurium. In summary, our results provide new insights into the genetic regulation of the immune response to S. Typhimurium infection in mice.

Author SummarySalmonella infection (salmonellosis) is a common zoonotic disease that mainly propagates through contaminated food and drink. Various mouse strains display prominent disparities in responses to Salmonella invasion. Elucidating the heterogeneous immune reactions between different mouse strains can shed light on the fundamental molecular mechanisms of the innate immune system. Here, we employed a combination of proteomics and systems biology approaches to provide an unprecedented panorama of the inextricably interlaced immune signaling pathways in response to Salmonella infection in mice. Our results revealed the dynamics of cell signaling molecules elicited by inflammation and established new connections among them. We also identified a new candidate gene involved in the combat with pathogens. Our proteomic data and results contribute to understanding the intricate interactions of immune responses to Salmonella infection from molecular to systemic levels.

category: immunology


title: MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells

abstract: The MCM2-7 hetero-hexamer is the replicative DNA helicase that plays a central role in eukaryotic DNA replication. In proliferating cells, the expression level of the MCM2-7 hexamer is kept high, which safeguards the integrity of the genome. However, how the MCM2-7 hexamer is assembled in living cells remains unknown. Here, we revealed that the MCM-binding protein (MCMBP) plays a critical role in the assembly of this hexamer in human cells. MCMBP associates with MCM3 which is essential for maintaining the level of the MCM2-7 hexamer. Acute depletion of MCMBP demonstrated that it contributes to MCM2-7 assembly using nascent MCM3. Cells depleted of MCMBP gradually ceased to proliferate because of reduced replication licensing. Under this condition, p53-positive cells exhibited arrest in the G1 phase, whereas p53-null cells entered the S phase and lost their viability because of the accumulation of DNA damage, suggesting that MCMBP is a potential target for killing p53-deficient cancers.


title: Dynamic reorganization and selective segregation of mitochondria under DarT-mediated mtDNA damage

abstract: Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Two key processes of fission and fusion are critical for the distribution, segregation and maintenance of mitochondria and their genomes (mtDNA). While recent work has begun to elucidate the importance of mitochondrial organization for mitochondrial genome maintenance, the impact of mtDNA perturbations on mitochondrial dynamics and/ or organellar maintenance remains less understood. To address this, we develop a tool to induce mitochondria-specific DNA damage, using a mitochondrial-targeted base modifying bacterial toxin, DarT. Following damage induction, we observe dynamic reorganization of the mitochondrial network. We find that mtDNA damage predominantly affects rates of fusion, likely driven by mitochondrial dysfunction arising due to DNA damage. Changes in network organization are associated with loss of mtDNA, independent of mitophagy. Unexpectedly, we find that perturbation to exonuclease function of the mtDNA replicative polymerase, Mip1, results in rapid loss of mtDNA. Our data suggest that, under damage, the partitioning of defective mtDNA and organelle can be de-coupled, with an emphasis on mitochondrial segregation even in the absence of its DNA. Together, our works reveals the importance of genome maintenance on mitochondrial function, that can act as a modulator of organelle organization and segregation.


title: Impacts of climate change on California's rangeland production: sensitivity and future projection

abstract: Rangelands support many important ecosystem services and are highly sensitive to climate change. Understanding temporal dynamics in rangeland gross primary production (GPP) and how it may change under projected climate change, including more frequent and severe droughts, is critical for ranching communities to cope with future changes. Covering ~10% of Californias climatologically and topographically diverse landscapes, annual rangelands express varying sensitivity to precipitation fluctuation and warming. Herein, we examined how climate regulates temporal dynamics of annual GPP in Californias annual rangeland across scales, based on 20 years of satellite record derived GPP at 500-meter resolution since 2001. We built gradient boosted regression tree models for 23 ecoregion subsections in our study area, relating annual GPP with 30 climatic variables and disentangling the partial dependence of GPP on each climate variable. Our analysis showed that GPP was most sensitive to growing season precipitation amount; GPP decrease as much as 200 g C/m2/yr when growing season precipitation decreased from 400 to 100 mm/yr in one of the driest subsections. We also found that years with more evenly distributed growing season precipitation had higher GPP. Warmer winter minimum air temperature enhanced GPP in approximately two-thirds of the subsections. In contrast, average growing season mean and maximum air temperatures showed a negative relationship with annual GPP. When forced by downscaled future climate projections, changes in future rangeland productivity at the ecoregion subsection scale were more remarkable than at the state level; this suggests rangeland productivity responses to climate change will be highly variable at the local level. Further, we found large uncertainty in precipitation projections among the four climate models used in this study. Specifically, drier models predicted a larger degree of reduction in GPP, especially in drier subsections. Our machine learning-based analysis highlights key regional differences in GPP vulnerability to climate and provides insights into the intertwining and potentially counteracting effects of seasonal temperature and precipitation regimes. This work demonstrates the potential of using remote sensing to enhance field-based rangeland monitoring and, combined with machine learning, to inform adaptive management and conservation within the context of weather extremes and climate change.


title: The influence of biological and statistical properties of CpGs on epigenetic predictions of eighteen traits

abstract:
BackgroundCpG methylation levels can help to explain inter-individual differences in phenotypic traits. Few studies have explored whether identifying CpG subsets based on biological and statistical properties can maximise predictions while minimising array content.

MethodsVariance component analyses and penalised regression (epigenetic predictors) were used to test the influence of (i) the number of CpGs considered, (ii) mean CpG methylation variability and (iii) methylation QTL status on the variance captured in eighteen traits by blood DNA methylation. Training and test sets comprised [≤]4,450 and [≤]2,578 unrelated individuals from Generation Scotland, respectively.

ResultsAs the number of CpG sites under consideration decreased, so too did the estimates from the variance components and prediction analyses. Methylation QTL status and mean CpG variability did not influence variance components. However, relative effect sizes were 15% larger for epigenetic predictors based on CpGs with methylation QTLs compared to sites without methylation QTLs. Relative effect sizes were 45% larger for predictors based on CpGs with mean beta-values between 10%-90% compared to those using hypo- or hypermethylated CpGs (beta-value [≤]10% or [≥]90%).

ConclusionArrays with fewer CpGs could reduce costs, leading to increased sample sizes for analyses. Our results show that reducing array content can restrict prediction metrics and careful attention must be given to the biological and distribution properties of CpGs in array content selection.


title: Leukotrienes promote stem cell self-renewal and chemoresistance in acute myeloid leukemia

abstract:
Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.

Key PointsO_LIAML blasts overexpress and induce ALOX5 expression in response to daunorubicin
C_LIO_LI5-Lipoxygenase (ALOX5) promotes blast survival and LSC self-renewal.
C_LIO_LIThe effects of ALOX5 on AML blast survival and self-renewal are mediated by the LTB4-BLTR signaling axis.
C_LI


title: Construction of in silico protein-protein interaction networks across different topologies using machine learning

abstract: AO_SCPLOWBSTRACTC_SCPLOWProtein-protein interactions (PPIs) are essential to understanding biological pathways as well as their roles in development and disease. Computational tools have been successful at predicting PPIs in silico, but the lack of consistent and reliable frameworks for this task has led to network models that are difficult to compare and, overall, a low level of trust in the PPI predictions. To better understand the underlying mechanisms that underpin these models, we designed B4PPI, an open-source framework for benchmarking that accounts for a range of biological and statistical pitfalls while facilitating reproducibility. We use B4PPI to shed light on the impact of network topology and how different algorithms deal with highly connected proteins. By studying functional genomics-based and sequence-based models (the two most popular approaches) on human PPIs, we show their complementarity as the former performs best on lone proteins while the latter specialises in interactions involving hubs. We also show that algorithm design has little impact on performance with functional genomic data. We replicate our results between both human and S. Cerevisiae data and demonstrate that models using functional genomics are better suited to PPI prediction across species. With rapidly increasing amounts of sequence and functional genomics data, our study provides a systematic foundation for future construction, comparison and application of PPI networks.


title: A mitochondrial blood-based patient stratification candidate biomarker for Parkinson's disease

abstract:
Parkinsons disease (PD) is the most common neurodegenerative movement disorder and neuroprotective interventions remain elusive. High throughput biomarkers aimed to stratify patients based on shared etiology is one critical path to the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific mitochondrial DNA (mtDNA) damage accumulation in neuronal and in vivo PD models, as well as human PD postmortem brain tissue. In this study, to investigate mtDNA damage as a potential blood biomarker for PD, we describe a novel Mito DNADX assay that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, compatible with assessing large cohorts of patient samples. We found that levels of mtDNA damage were increased in blood derived from early-stage idiopathic PD patients or those harboring the pathogenic LRRK2 G2019S mutation compared to age-matched healthy controls. Given that increased mtDNA damage was also found in non-manifesting LRRK2 mutation carriers, mtDNA damage may begin to accumulate prior to a clinical PD diagnosis. LRRK2 kinase inhibition mitigated mtDNA damage in idiopathic PD models and patient-derived cells. The latter observations further substantiate a mechanistic role for wild-type LRRK2 kinase activity in idiopathic PD and support mtDNA damage reversal as a suitable approach to slow PD-related pathology. In light of recent advances in the field of precision medicine, the analysis of mtDNA damage as a blood-based patient stratification biomarker should be included in future clinical trials.

One Sentence SummaryBlood test identifies Parkinsons patients most likely to respond to mitochondria-targeted therapeutics facilitating a precision medicine approach.


title: Combination of an inject-and-transfer system for serial femtosecond crystallography

abstract: Serial femtosecond crystallography (SFX) enables the determination of the room-temperature crystal structure of macromolecules without causing radiation damage and provides time-resolved molecular dynamics by pump-probe experiments. In the SFX experiment, the injector-based sample delivery method continuously provides fresh crystals to X-rays, and the fixed-target scanning method can be programmed to move the crystals to the desired location. This study introduces a combination of the inject-and-transfer system (BITS) method for sample delivery for SFX experiments, a hybrid injection, and a fixed-target scanning method. BITS allows solution samples to be reliably deposited on an ultraviolet ozone (UVO)-treaed polyimide films at flow rates as low as 1 nl/min. In application of BITS in SFX experiment, the lysozyme crystal samples were embedded in a viscous lard medium and injected at a 50-100 nl/min flow rate through a syringe needle onto an UVO-treated polyimide film mounted on a fixed-target scan stage. The deposited crystal sample on film were raster scanned to XFEL by motion stage in the horizontal and vertical directions. Using this method, we successfully determined the room-temperature structure of lysozyme at 2.1 [A] resolution. This method can be applied to the SFX experiments.

category: biophysics


title: Glypican-3 (GPC3) is associated with MCPyV-negative status and impaired outcome in Merkel Cell Carcinoma

abstract:
BackgroundMerkel cell carcinoma (MCC) is an aggressive skin cancer, related to the Merkel Cell Polyomavirus (MCPyV) in 80% of cases. Immune checkpoint inhibitors provide sustained benefit in about half of MCC patients with advanced disease. Glypican-3 (GPC3) is an oncofetal tumor antigen that is an attractive target for chimeric antigen receptor T cell therapy due to its highly restricted expression on normal tissue and high prevalence in several solid tumors. GPC3 was previously found to be expressed in MCC but its association with tumor characteristics or prognosis has not been reported.

ObjectivesTo investigate the expression of GPC3 in MCC by immunohistochemistry (IHC) and its association with tumor characteristics, MCPyV status, and patient outcome.

MethodsThe GC33 antibody clone was validated for GPC3 IHC staining of tumor specimens in comparison to an established GPC3 IHC antibody. A tissue microarray of tumors collected from an ongoing cohort of MCC patients was stained for GPC3 by IHC using GC33 antibody. Association of GPC3 positivity with baseline characteristics, MCPyV status (quantitative PCR) and outcome (death from MCC, recurrence) were assessed by Fishers exact tests and Cox regression analysis.

ResultsAmong 62 tumors from 59 patients, 42 samples (67.7%) were GPC3-positive. GPC3 expression was more frequently observed in females (p=0.048) and MCPyV-negative tumors (p=0.021). In the multivariate analysis, GPC3 expression was associated with increased death from disease (CSS) (hazard ratio [HR] 4.05, 95% CI 1.06-15.43), together with advanced age (HR 4.85, 95% CI 1.39-16.9) and male gender (HR 4.64, 95% CI 1.31-16.41).

ConclusionsGPC3 expression is frequently expressed in MCC tumors, especially MCPyV-negative cases, and is associated with increased risk of death. The high prevalence of surface GPC3 makes it a putative drug target.


title: A Fast And Intuitive Method For Calculating Dynamic Network Reconfiguration And Node Flexibility

abstract: AO_SCPLOWBSTRACTC_SCPLOWDynamic interactions between brain regions, either during rest or performance of cognitive tasks, have been studied extensively using a wide variance of methods. Although some of these methods allow elegant mathematical interpretations of the data, they can easily become computationally expensive or difficult to interpret and compare between subjects or groups. Here, we propose an intuitive and computationally efficient method to measure dynamic reconfiguration of brain regions, also termed flexibility. Our flexibility measure is defined in relation to an a-priori set of biologically plausible brain modules (or networks) and does not rely on a stochastic data-driven module estimation, which, in turn, minimizes computational burden. The change of affiliation of brain regions over time with respect to these a-priori template modules is used as an indicator of brain network flexibility. We demonstrate that our proposed method yields highly similar patterns of whole-brain network reconfiguration (i.e., flexibility) during a working memory task as compared to a previous study that uses a data-driven, but computationally more expensive method. This result illustrates that the use of a fixed modular framework allows for valid, yet more efficient estimation of whole-brain flexibility, while the method additionally supports more fine-grained flexibility analyses restricted to biologically plausible brain networks.


title: Single-cell transcriptome landscape of zebrafish liver reveals hepatocytes and immune cell interactions in understanding nonalcoholic fatty liver disease

abstract: Zebrafish have emerged as an attractive animal model for studying nonalcoholic fatty liver disease (NAFLD). However, little is known about the cell types and intercellular interactions in zebrafish liver. Here, we established a liver atlas that consists of 10 cell types using single-cell RNA sequencing. By examining the heterogeneity of hepatocytes and analyzing the expression of NAFLD-associated genes in the specific cluster, we provide a potential target cell model to study NAFLD. Additionally, our analysis identified two distinct resident macrophages with inflammatory and noninflammatory functions and characterized the successive stepwise development of T cell subtypes in the liver. Importantly, we uncovered possible molecular mechanisms and revealed the central regulation of macrophages on target cells of fatty liver by analyzing the cellular interaction between hepatocytes and immune cells. Our data provide valuable information for future research on NAFLD in zebrafish.


title: Unsupervised encoding selection through ensemble pruning for biomedical classification

abstract:
BackgroundOwing to the rising levels of multi-resistant pathogens, antimicrobial peptides, an alternative strategy to classic antibiotics, got more attention. A crucial part is thereby the costly identification and validation. With the ever-growing amount of annotated peptides, researchers employed artificial intelligence to circumvent the cumbersome, wet-lab-based identification and automate the detection of promising candidates. However, the prediction of a peptides function is not limited to antimicrobial efficiency. To date, multiple studies successfully classified additional properties, e.g., antiviral or cell-penetrating effects. In this light, ensemble classifiers are employed to utilize the advantages of peptide encodings; hence, further improving the prediction. Although we recently presented a workflow to significantly diminish the initial encoding choice, an entire unsupervised encoding selection, considering various machine learning models, is still lacking.

ResultsWe developed a workflow, automatically selecting encodings and generating classifier ensembles by employing sophisticated pruning methods. We observed that the Pareto frontier pruning is a good method to create encoding ensembles for the datasets at hand. In addition, encodings combined with the Decision Tree classifier as the base model are often superior. However, our results also demonstrate that none of the ensemble building techniques is outstanding for all datasets.

ConclusionThe workflow conducts multiple pruning methods to evaluate ensemble classifiers composed from a wide range of peptide encodings and base models. Consequently, researchers can use the workflow for unsupervised encoding selection and ensemble creation. Ultimately, the extensible workflow can be used as a plugin for the PEPTIDE REACToR, further establishing it as a versatile tool in the domain.


title: An AI-assisted Tool For Efficient Prostate Cancer Diagnosis

abstract: Pathologists diagnose prostate cancer by core needle biopsy. For low-grade and low-volume cases, the pathologists look for the few malignant glands out of hundreds within a core. They may miss the few malignant glands, resulting in repeat biopsies or missed therapeutic opportunities. This study developed a multi-resolution deep learning pipeline detecting malignant glands in core needle biopsies to help pathologists effectively and accurately diagnose prostate cancer in low-grade and low-volume cases. The pipeline consisted of two stages: the gland segmentation model detected the glands within the sections and the multi-resolution model classified each detected gland into benign vs. malignant. Analyzing a gland at multiple resolutions provided the classification model to exploit both morphology information (of nuclei and glands) and neighborhood information (for architectural patterns), important in prostate gland classification. We developed and tested our pipeline on the slides of a local cohort of 99 patients in Singapore. The images were made publicly available, becoming the first digital histopathology dataset of prostatic carcinoma patients of Asian ancestry. Our pipeline successfully classified the core needle biopsy parts (81 parts: 50 benign and 31 malignant) into benign vs. malignant. It achieved an AUROC value of 0.997 (95% CI: 0.987 - 1.000). Moreover, it produced heatmaps highlighting the malignancy of each gland in core needle biopsies. Hence, our pipeline can effectively assist pathologists in core needle biopsy analysis.


title: Capacity and tradeoffs in neural encoding of concurrent speech during Selective and Distributed Attention

abstract:
Speech comprehension is severely compromised when several people talk at once, due to limited perceptual and cognitive resources. Under some circumstances listeners can employ top-down attention to prioritize the processing of task-relevant speech. However, whether the system can effectively represent more than one speech input remains highly debated.

Here we studied how task-relevance affects the neural representation of concurrent speakers under two extreme conditions: when only one speaker was task-relevant (Selective Attention), vs. when two speakers were equally relevant (Distributed Attention). Neural activity was measured using magnetoencephalography (MEG) and we analysed the speech-tracking responses to both speakers. Crucially, we explored different hypotheses as to how the brain may have represented the two speech streams, without making a-priori assumptions regarding participants internal allocation of attention.

Results indicate that neural tracking of concurrent speech did not fully mirror their instructed task-relevance. When Distributed Attention was required, we observed a tradeoff between the two speakers despite their equal task-relevance, akin to the top-down modulation observed during Selective Attention. This points to the systems inherent limitation to fully process two speech streams, and highlights the complex nature of attention, particularly for continuous speech.


title: Creativity in Verbal Associations is Linked to Semantic Control

abstract: While memory is known to play a key role in creativity, previous studies have not isolated the critical component processes and networks. We asked participants to generate links between words that ranged from strongly related to completely unrelated in long-term memory, delineating the neurocognitive processes that underpin more unique versus stereotypical patterns of retrieval. Less creative responses to strongly associated word pairs were associated with greater engagement of episodic memory: in highly familiar situations, semantic and episodic stores converge on the same information. This pattern of retrieval was associated with greater engagement of core default mode network. In contrast, more creative responses to weakly associated word pairs were associated with the controlled retrieval of less dominant semantic information and greater recruitment of the semantic control network, which overlaps with the dorsomedial subsystem of default mode network. Consequently, although both creative/controlled and stereotypical/more automatic patterns of retrieval are associated with activation within default mode network, these processes show little overlap in activation. These findings show that creativity emerges from controlled aspects of semantic cognition.