title: Serum profiles of metabolomic and GDF-15 differentiate idiopathic pulmonary fibrosis from other interstital lung diseases: a preliminary study

abstract:
Objective1.Explore the differential metabolites and metabolic pathways between different interstitial lung disease (ILD) and healthy controls, so as to find out new biomarkers and metabolic pathways of ILD and provide new biological targets for IPF diagnosis and treatment.2.Detect the levels of serum related fibrosis and metabolic indexes of ILD, explore the relationship between these indexes and the types and general parameters of ILD diseases, and focus on GDF-15 on the basis of metabonomics, explore new metabolic pathways related to ILD, and further expound the pathogenesis of idiopathic pulmonary fibrosis.

Method1.According to the inclusion criteria and exclusion criteria, the ILD patients admitted to our hospital from January 1, 2021 to January 1, 2022 were signed with informed consent, and all of them had clear multidisciplinary diagnosis. At the same time, the physical examination in the health management center of our hospital was selected as the healthy control group. General clinical data of ILD patients and healthy controls were collected, including gender, age, onset, clinical manifestations, clinical biochemistry, lung function and other related data. Collect patients peripheral venous blood, keep serum samples, and analyze the serum of ILD patients and healthy controls by liquid chromatography-mass spectrometry (LC-MS) to obtain differential metabolites, metabolic maps and related data. According to the diagnosis, the patients were divided into Idiopathic pulmonary fibrosis (IPF), connective tissue disease-associated interstitial lung disease (CTD-ILD), other interstitial lung disease (ILD) patients and healthy control group. R statistical software was used to observe whether there are differential metabolites in IPF group and healthy control group, CTD-ILD group and healthy control group, other ILD groups and healthy control group, IPF group and CTD-ILD group, IPF group and other ILD groups, CTD-ILD group and other ILD groups by using positive and negative ion flow charts and score charts of orthogonal partial least squares-discriminant analysis. And the potential biomarkers were found through the S-plot diagram of OPLS-DA and the variable weight value VIP>1. ROC curve analysis of differential metabolites was carried out by SPSS21.0 software to analyze the value of differential diagnosis and find out new biomarkers with differential diagnosis significance. Based on KEGG database, the metabolic pathway enrichment analysis of differential metabolites was carried out to find out the main metabolic pathways. 2.Serum levels of type I collagen, KL-6, IL-1{beta}, TNF-, IGF-1 and GDF-15 were detected by Enzyme linked immunosorbent assay (ELISA) in all patients and healthy controls. Using SPSS21.0 statistical software and R statistical software, using T-test, variance analysis or nonparametric test method, according to different diagnoses (IPF/CTD-ILD/ other ILD/ healthy control group, ILD/ healthy control group, IPF/ other), the data were analyzed and compared in subgroups. Pearson correlation analysis was used to analyze the correlation between serum biomarkers and other factors such as lung function, metabolomics and so on. ROC curve was used to analyze the diagnostic efficiency of each index, and the best critical value, sensitivity and specificity were found out.

Results1.Analysis of general clinical data and clinical indicators: 26 cases of IPF, 21cases of CTD-ILD, 23 cases of others ILD and 20 cases of healthy control group were included, respectively. There were significant differences in age, sex and smoking history among the four groups (P < 0.05), and the differences were statistically significant. There were statistically significant differences among IPF group, CTD-ILD group and other ILD groups in cough and expectoration symptoms, eosinophil count percentage, creatine kinase, creatine kinase isoenzyme, C-reactive protein and carbon monoxide diffusion capacity (DLco) (P < 0.05). 2. Metabonomics analysis: LC-MC total ion flow chart of serum samples and orthogonal partial least squares discriminant analysis (OPLS-DA) of serum samples showed the difference of ion peak intensity, and there were different metabolites among each group. The OPLS-DA model has good quality evaluation and reliable data analysis. There are 193, 115, 101, 188, 148 and 169 differential metabolites in IPF group and healthy control group, IPF group and CTD-ILD group, CTD-ILD group and other ILD groups, others ILD groups and healthy control group respectively. There is a correlation between the differential metabolites of each ILD subgroup and the healthy control group. The most relevant metabolic pathways in IPF mainly include choline metabolic pathway, metabolic pathway and linoleic acid metabolic pathway in cancer. The metabolic pathways most related to CTD-ILD and other ILD include choline metabolic pathway and retrograde neural signal metabolic pathway in cancer. Metabolic pathway and linoleic acid metabolic pathway may distinguish IPF from other ILD. Metabolites involved in metabolic pathway are L-Glutamate, PE(18:3(6Z,9Z,12Z)/P-18:0) and PC (18: 2 (9z, 12z)/20: 2 (11z, 14z)), respectively. There are three differential metabolites of linoleic acid pathway, namely 12,13-EpOME, 13S-HODE and PC(18:2(9Z,12Z)/20:2(11Z,14Z)). There are 35 kinds of differential metabolites in IPF group and healthy control group, and the top three places of AUC are 3- sulfodeoxycholic acid, 20,22-dihydrodigitalis glycoside, ({+/-}14) (15)-EET-SI, with AUC values of 0.985, sensitivity of 100% and specificity of 92.3%. L- acetylcarnitine, 5-hydroxydodecanoate and L-Glutamate are involved in significant enrichment pathways, among which the ROC curve area of L-Glutamate is the largest, with AUC value of 0.921, cutoff value of 40145.490, sensitivity of 80%, specificity of 96%, and participation in metabolic pathways. 3. Serum metabolism-related detection indexes: The six serum detection indexes of IPF group and CTD-ILD group are higher than those of healthy control group, and the difference is statistically significant (P < 0.05), but the difference between IPF group and CTD-ILD group is not statistically significant (P>0.05). The expression of GDF-15 in IPF group and non-IPF ILD group was higher than that in healthy control group, with significant difference, but there was no significant difference between IPF group and non-IPF ILD group. Type I collagen and IL-1{beta} were negatively correlated with DLco. KL-6 is negatively correlated with the measured values of VC and DLco; IGF-1 was negatively correlated with FVC, VC and DLco (all P<0.05). GDF-15 was positively correlated with type I collagen, KL-6, IL-1{beta}, TNF- and IGF-1, and the difference was statistically significant (P < 0.05). ROC curve shows that the area under ROC curve (AUC) of six serum indexes in IPF group is greater than 0.9; In CTD-ILD group, the AUC of IGF-1 was greater than 0.9, the cutoff value was 35.081ng/ml, the sensitivity was 90%, the specificity was 76%, and the area under ROC curve was greater than 0.8, and the difference was statistically significant. In other ILD groups, the ROC curve area of these six indicators ranged from 0.6 to 0.8, among which IGF-1, GDF-15 and TNF- had diagnostic value. In IPF, L-Glutamate was positively correlated with type I collagen, KL-6, IGF-1, IL-1{beta}, TNF- and GDF-15, and the difference was statistically significant (P < 0.05), with the highest correlation with IGF-1.

ConclusionIn this study, the serum samples of IPF patients, CTD-ILD patients, other interstitial lung diseases and healthy controls were analyzed by LM-MS and ELISA, and the following conclusions were drawn. 1.There are differences in serum metabolites in different groups. IPF has three main metabolic pathways, namely choline metabolic pathway, metabolic pathway and linoleic acid metabolic pathway in cancer, among which metabolic pathway and linoleic acid metabolic pathway are different from other groups. There are 35 differential metabolites in IPF and healthy control group with the area under ROC curve larger than 0.80, among which the metabolites with the highest diagnostic value are 3- sulfodeoxycholic acid, 20,22-dihydrodigitalis glycoside and ({+/-}) 14 (15)-EET-SI. The AUC value of L- glutamic acid is the highest among the metabolites that participate in the enrichment pathway significantly. 2. The expressions of type I collagen, KL-6, IL-1{beta}, TNF-, IGF-1 and GDF-15 are different in different ILDs, which have potential diagnostic and differential diagnostic value, and are correlated with lung function indexes. In IPF, GDF-15 is positively correlated with L-Glutamate, which may be involved in metabolic pathway to further promote pulmonary fibrosis.


title: An E3 ligase network engages GCN1 to promote elongation factor-1α degradation on stalled ribosomes

abstract: How cells monitor the status of translating ribosomes is a major question in gene regulation. Elongating ribosomes frequently stall during mRNA translation, resulting in context- dependent activation of quality control pathways. However, surveillance mechanisms that specifically respond to stalled ribosomes with an elongation factor occupying the GTPase center have not been identified. By employing ternatin-4, an allosteric elongation factor-1 (eEF1A) inhibitor, we unveil an E3 ligase network that triggers ubiquitination and degradation of eEF1A on stalled ribosomes. A CRISPRi screen revealed two E3 ligases of unknown function, RNF14 and RNF25, which are both essential for ternatin-induced eEF1A degradation. Based on quantitative proteomics analysis, we find that RNF14 and RNF25 promote ubiquitination of eEF1A and a discrete set of ribosomal proteins. By forming a complex with RNF14, the ribosome collision sensor GCN1 plays an essential role in eEF1A degradation. Our findings illuminate a translation elongation checkpoint that monitors the ribosomal GTPase center.

category: cell biology


title: The effect of migration and variation on populations of Escherichia coli adapting to complex fluctuating environments

abstract: Migration, a critical evolutionary force, can have contrasting effects on adaptation. It can aid as well as impede adaptation. The effects of migration on microbial adaptation have been studied primarily in simple constant environments. Very little is known about the effects of migration on adaptation to complex, fluctuating environments. In our study, we subjected replicate populations of Escherichia coli, adapting to complex and unpredictably fluctuating environments to different proportions of clonal ancestral immigrants. Contrary to the results from simple/constant environments, the presence of clonal immigrants reduced all measured proxies of fitness. However, migration from a source population with a greater variance in fitness resulted in no change in fitness w.r.t the no-migration control, except at the highest level of migration. Thus, the presence of variation in the immigrants could counter the adverse effects of migration in complex and unpredictably fluctuating environments. Our study demonstrates that the effects of migration are strongly dependent on the nature of the destination environment and the genetic makeup of immigrants. These results enhance our understanding of the influences of migrating populations, which could help better predict the consequences of migration.

category: evolutionary biology


title: Introgression between highly divergent sea squirt genomes: an adaptive breakthrough?

abstract:
Human-mediated introductions are reshuffling species distribution on a global scale. Consequently, an increasing number of allopatric taxa are now brought into contact, promoting introgressive hybridization between incompletely isolated species and new adaptive gene transfer. The broadcast spawning marine species, Ciona robusta, has been recently introduced in the native range of its sister taxa, Ciona intestinalis, in the English Channel and North-East Atlantic. These sea squirts are highly divergent, yet hybridization has been reported by crossing experiments and genetic studies in the wild. Here, we examined the consequences of secondary contact between C. intestinalis and C. robusta in the English Channel. We produced genomes phased by transmission to infer the history of divergence and gene flow, and analyzed introgressed genomic tracts. Demographic inference revealed a history of secondary contact with a low overall rate of introgression. Introgressed tracts were short, segregating at low frequency, and scattered throughout the genome, suggesting traces of past contacts during the last 30 ky. However, we also uncovered a hotspot of introgression on chromosome 5, characterized by several hundred Kb-long C. robusta haplotypes segregating in C. intestinalis, that introgressed during contemporary times the last 75 years. Although locally more frequent than the baseline level of introgression, C. robusta alleles are not fixed, even in the core region of the introgression hotspot. Still, LD patterns and haplotype-based tests suggest this genomic region is under recent positive selection. We further detected in the hotspot an over-representation of candidate SNPs lying on a cytochrome P450 gene with a high copy number of tandem repeats in the introgressed alleles. Cytochromes P450 are a superfamily of enzymes involved in detoxifying exogenous compounds, constituting a promising avenue for functional studies. These findings support that introgression of an adaptive allele is possible between very divergent genomes and that anthropogenic hybridization can provide the raw material for adaptation of native lineages in the Anthropocene.

Author summaryIntrogression, the transfer of genetic material by hybridization between taxa, is increasingly recognized to sometimes persist for long during species divergence. However, the evolutionary consequences of human-induced introgression remain largely unknown, especially in the marine realm. While some argue it poses a threat to the genome integrity of native species, others consider it has a great potential to fuel adaptation. In this work, we quantify the magnitude and genomic distribution of introgression after secondary contact between a native sea squirt and its divergently related sister species recently introduced in the English Channel. The genome-wide pattern suggests introgression is mostly impeded between these two incompatible genomes that only exchanged a few very short chromosome tracts sparsely distributed in the native genome. We nonetheless found a hotspot of long tracts that recently introgressed in a single region of the genome, with a clear footprint of recent positive selection. In the center of the hotspot, we further detected a promising candidate gene for adaptive introgression: a cytochrome P450 detoxifying enzyme with a high copy number in the introgressed allele. Therefore, our results support that adaptive introgression can remain possible between very divergent genomes and that anthropogenic hybridization can provide the raw material for the adaptation of native lineages in the Anthropocene.


title: Repeat sequences limit the effectiveness of LGT and favoured the evolution of meiotic sex in early eukaryotes

abstract:
The transition from prokaryotic lateral gene transfer to eukaryotic meiotic sex is poorly understood. Phylogenetic evidence suggests that it was tightly linked to eukaryogenesis, which involved an unprecedented rise in both genome size and the density of genetic repeats. Expansion of genome size raised the severity of Mullers ratchet, whilst limiting the effectiveness of lateral gene transfer (LGT) at purging deleterious mutations. In principle, an increase in recombination length combined with higher rates of LGT could solve this problem. Here we show using a computational model that this solution fails in the presence of genetic repeats prevalent in early eukaryotes. The model demonstrates that dispersed repeat sequences allow ectopic recombination, which leads to the loss of genetic information and curtails the capacity of LGT to prevent mutation accumulation. Increasing recombination length in the presence of repeat sequences exacerbates the problem. Mutational decay can only be resisted with homology along extended sequences of DNA. We conclude that the transition to homologous pairing along linear chromosomes was a key innovation in meiotic sex, which was instrumental in the expansion of eukaryotic genomes and morphological complexity.

Relevance StatementThe origin of meiotic sex is a long-standing evolutionary enigma. This novel mechanism of reproduction replaced lateral gene transfer (LGT), the uptake and recombination of pieces of environmental DNA seen in bacteria and archaea. We link its origin to the expanded genome size and proliferation of genetic repeats found in early eukaryotes. Both factors led to high levels of mutation accumulation and gene loss under LGT which could not be retarded through increases in the rate of LGT or the length of DNA recombined. Meiotic sex with homologous pairing of long chromosome sized pieces of DNA promoted purifying selection and supressed ectopic recombination. It permitted the evolution of the expanded genome needed for the evolution of complex eukaryotic life.


title: Acetaldehyde makes a distinct mutation signature in single-stranded DNA

abstract: Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG[->]TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the genotoxic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA is highly mutagenic and makes strand-biased mutations on guanines (G[->]T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Pol{zeta}. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption.

category: genetics


title: Periodic spatial patterning with a single morphogen

abstract: Multicellular development employs periodic spatial patterning to generate repetitive structures such as digits, vertebrae, and teeth. Turing patterning has long provided a key paradigm for understanding such systems. The simplest Turing systems are believed to require at least two signals, or morphogens, that diffuse and react to spontaneously generate periodic patterns. Here, using mathematical modeling, we show that a minimal circuit comprising an intracellular positive feedback loop and a single diffusible morphogen is sufficient to generate stable, long-range spatially periodic cellular patterns. The model considers cells as discrete entities as a key feature, and incorporates transient boundary conditions. Linear stability analysis reveals that this single-morphogen Turing circuit can support a broad range of spatial wavelengths, including fine-grain patterns similar to those generated by classic lateral inhibition systems. Further, signals emanating from a boundary can initiate and stabilize propagating modes with a well-defined spatial wavelength. Once formed, patterns are self-sustaining and robust to noise. Finally, while noise can disrupt patterning in pre-patterned regions, its disruptive effect can be overcome by a bistable intracellular circuit loop, or by considering patterning in the context of growing tissue. Together, these results show that a single morphogen can be sufficient for robust spatial pattern formation, and should provide a foundation for engineering pattern formation in the emerging field of synthetic developmental biology.

category: synthetic biology


title: A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome

abstract: CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. DPP8 and DPP9 (DPP8/9) inhibitors, including Val-boroPro (VbP), accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin, controls CARD8 activation. In unstressed cells, the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. In VbP-stressed cells, the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Overall, this work shows that CARD8 NTs susceptibility to 20S proteasome-mediated degradation controls inflammasome activation.

category: immunology


title: Decoding functional high-density lipoprotein particle surfaceome interactions

abstract:
High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport in the human body and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDLs journey throughout the body, its function is mediated through interactions with cell surface receptors on different cell types. Using four different cellular model systems HDL is interacting with, we comparatively analyzed their surfaceomes to define the HDL receptome. Surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAEC) revealed the main currently known HDL-receptor scavenger receptor B1 (SCRB1), as well as 154 shared cell surface receptors representing potential HDL interaction receptor candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF modulated surfaceome of HAEC using auto-CSC technology. VEGF-A treatment led to a remodeling of the surfaceome of HAEC cells including the previously reported higher surfaceome abundance of SCRB1 upon treatment. 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using HATRIC-based ligand receptor capturing (HATRIC-LRC) technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK), which we show is directly contributing to endothelial HDL binding and uptake. Subsequent proximity ligation assays (PLA) demonstrate furthermore the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence functional HDL binding and uptake.

List of human gene and protein names discussed in the paperABCA1: Phospholipid-transporting ATPase ABCA1 (ABCA1)

ABCA7: Phospholipid-transporting ATPase ABCA7 (ABCA7)

ABCG1: ATB-binding cassette G1 (ABCG1)

ACVR2A: Activin receptor type-2A (AVR2A)

APOA1: Apolipoprotein A1 (APOA1)

APOB: Apolipoprotein B (APOB)

APOC3: Apolipoprotein C-III (APOC3)

APOH: Beta-2-glycoprotein 1 (APOH)

APOM: Apolipoprotein M (APOM)

ATP5MF: Ecto-F1-ATPase (ATPK)

CD4: T-cell surface glycoprotein CD4 (CD4)

CD14: Monocyte differentiation antigen CD14 (CD14)

CD36: platelet glycoprotein 4 (CD36)

EGFR: Epidermal growth factor receptor (EGFR)

ITGAM: Integrin alpha-M (ITAM)

ITGAV: Integrin alpha-V (ITAV)

KDR: Vascular endothelial growth factor receptor 2 (VGFR2)

KIT: Mast/stem cell growth factor receptor Kit (KIT)

MEGF8: Multiple epidermal growth factor-like domains protein 8 (MEGF8)

MEGF10: Multiple epidermal growth factor-like domains protein 10 (MEGF10)

MERTK: tyrosine-protein kinase Mer (MERTK)

MRC2: C-type mannose receptor 2 (MRC2)

MSR1: Macrophage scavenger receptor types I and II (MSRE)

PCDHAC1: Protocadherin alpha-C1 (PCDC1)

PLTP: Phospholipid transfer protein (PLTP)

S1PR1: sphingosine 1-phosphate receptor 1 (S1PR1)

S1PR2: sphingosine 1-phosphate receptor 2 (S1PR2)

S1PR3: sphingosine 1-phosphate receptor 3 (S1PR3)

SCARA3: Scavenger receptor class A member 3 (SCAR3)

SCARB1: Scavenger receptor B1 (SCRB1)

SLC8A3: Sodium/calcium exchanger 2 (NAC2)

TF: Transferrin (TRFE)

TFRC: Transferrin receptor protein 1 (TFR1)

VEGFA: vascular endothelial growth factor A (VEGF-A)

Significance statementA molecular understanding of the high-density lipoprotein (HDL) receptome would provide the rational basis for modulating reverse cholesterol transport. Here we used cellular model systems mimicking tissues involved in HDL-trafficking and reverse cholesterol transport to define the HDL receptome. The receptor tyrosine-protein kinase Mer (MERTK) was identified as a novel mediator of HDL binding and uptake in human aortic endothelial cells. Endothelial MERTK resides within the surfaceome proximal to SCRB1 representing a potential co-receptor. The molecular nanoscale organization of the cell-specific surfaceome may influence cellular HDL interactions and functionality.

category: molecular biology


title: Cytosolic peptide accumulation activates the NLRP1 and CARD8 inflammasomes

abstract: NLRP1 and CARD8 are related sensors that form inflammasomes, but the danger signals that they detect are not fully established. These proteins undergo autoproteolysis, generating repressive N-terminal (NT) and inflammatory C-terminal (CT) fragments. The proteasome- mediated degradation of the NT releases the CT from autoinhibition, but the CT is then sequestered in a complex with the full-length sensor and DPP9. Here, we show that cytosolic peptide accumulation activates these inflammasomes. We found that a diverse array of peptides accelerates NT degradation, and those with N-terminal XP sequences also destabilize the ternary complexes. Peptides interfere with many biological processes, including protein folding. We show that unrelated agents that disrupt protein folding also induce NT degradation, but do not cause inflammasome activation because DPP9 sequesters the CT fragments in the absence of XP peptides. Overall, these results indicate that NLRP1 and CARD8 detect protein misfolding that is associated with peptide accumulation.


title: Systematic analysis of cilia characteristics and Hedgehog signaling in five immortal cell lines

abstract: Dysfunction of the primary cilium, a microtubule-based signaling organelle, leads to genetic conditions called ciliopathies. Hedgehog (Hh) signaling is mediated by the primary cilium in vertebrates and is therefore implicated in ciliopathies; however, it is not clear which immortal cell lines are the most appropriate for modeling pathway response in human disease; therefore we systematically evaluated Hh in five commercially available, immortal mammalian cell lines: ARPE-19, HEK293T, hTERT RPE-1, NIH/3T3, and SH-SY5Y. All of the cell lines ciliated adequately for our subsequent experiments, except for SH-SY5Y which were excluded from further analysis. hTERT RPE-1 and NIH/3T3 cells relocalized Hh pathway components Smoothened (SMO) and GPR161 and upregulated Hh target genes in response to pathway stimulation. In contrast, pathway stimulation did not induce target gene expression in ARPE-19 and HEK293T cells, despite SMO and GPR161 relocalization. These data indicate that human hTERT RPE-1 cells and murine NIH/3T3 cells, but not ARPE-19 and HEK293T cells, are suitable for modeling the role of Hh signaling in ciliopathies.


title: Hand cold pressor test induces thermogenesis in upper thoracic regions as measured by skin surface infrared thermography

abstract:
BackgroundCold exposure may cause health problems and impaired productivity in outdoor or cold-temperature workers. The cold pressor test (CPT) is a laboratory procedure that measures cardiovascular and thermoregulatory responses to acute cold exposure such as metabolic activity in brown adipose tissue. How the body responds to acute cold exposure of a hand is not completely understood. We tested the hypothesis that the upper thorax produces heat during a single hand-CPT, which restores warmth to the cold-expose appendage.

ObjectivesThe objective was to measure skin temperature changes in the upper thoracic regions and the cold-exposed appendage during a CPT. The secondary objective was to determine if cardiovascular or psychological responses during CPT accounted for skin temperature changes.

Methods50 healthy participants immersed their right hand up to wrist level in 4 {degrees}C water for three minutes. Surface skin temperatures were imaged by infrared thermography at baseline, during CPT, and in the recovery phase. Sublingual oral temperature and water bath temperature were recorded throughout the test. Cardiovascular responses were monitored by continuous finger pulse-wave plethysmography. Peak pain and peak stress were reported by the participants on a Likert scale.

ResultsCPT increased the systolic blood pressure (+22 mmHg, p < 0.001), diastolic blood pressure (+15 mmHg, p < 0.001) and heart rate (+7 beats per minute, p = 0.024). During CPT, skin temperature increased on thoracic regions including mediastinal (+0.5{degrees}C, p < 0.021), sternal (+0.5{degrees}C, p < 0.002), right supraclavicular (+0.3{degrees}C, p < 0.042) and left supraclavicular (+0.3{degrees}C, p < 0.016) regions. During CPT, the hand was cooler on ventral (-14.6{degrees}C p < 0.001) and dorsal (-15.2{degrees}C p < 0.001) sides, and warmed up during recovery. The ventral forearm, dorsal forearm, antecubital fossa, and adjacent medial epicondyle region were significantly cooler throughout the recovery time. The oral temperature did not change during CPT. There were no correlations between the change in mediastinal skin temperature and the sex of the participant, or changes in cardiovascular parameters, peak pain, or peak stress values.

ConclusionsLocalized hand cooling caused a rapid warming of the thorax, dissipation of cold in the forearm, and rewarming of the hand during recovery. Thermoregulation was not dependant on pain, stress, sex, or cardiovascular changes between participants. By understanding thermoregulation, better approaches can be developed to mitigate the negative impacts of cold exposure.

category: physiology


title: Parallel processing, hierarchical transformations, and sensorimotor associations along the 'where pathway

abstract: Visually guided behaviors require the brain to transform ambiguous retinal images into object-level spatial representations and implement sensorimotor transformations. These processes are supported by the dorsal  where pathway. However, the specific functional contributions of areas along this pathway remain elusive due in part to methodological differences across studies. We previously showed that macaque caudal intraparietal (CIP) area neurons possess robust three-dimensional (3D) visual representations, carry choice-related and presaccadic activity, and exhibit experience-dependent sensorimotor associations (Chang et al., 2020b). Here, we used a common experimental design to reveal parallel processing, hierarchical transformations, and the formation of sensorimotor associations along the  where pathway by extending the investigation to V3A, a major feedforward input to CIP. Higher-level 3D representations and choice-related activity were more prevalent in CIP than V3A. Both areas contained presaccadic activity that predicted the direction/timing of eye movements. Intriguingly, the time-course of presaccadic activity in CIP aligned with the temporally integrated V3A output. Sensorimotor associations between 3D orientation and saccade direction preferences were strongest in CIP and moderated by choice signals in both areas. Together, the results explicate parallel representations, hierarchical transformations, and functional associations of visual and presaccadic signals at a key juncture in the  where pathway.

category: neuroscience


title: Simultaneously spatiotemporal gene expression and chromatin accessibility for mouse brain development

abstract: Brain are complex biological tissues which function relies on coordinated anatomical and molecular structure comprised by a large number of specialized cells. The spatial architecture of brain which is key to the understanding of its physiological and pathological significance is formed during embryo development. However, the molecular basis for discrete neuroanatomical domains particularly in the context of spatial organization of the brain is inadequate. Here, we introduced Spatial-ATAC-RNA-seq by crisscross indexing (SPARC-seq), a method for parallel profiling of gene expression and chromatin accessibility with spatial information retained in developing mouse brain. Our study has established a direct means to spatially determine the coordination between chromatin accessibility and transcriptome, identified the chromatin potential to define cell fate determination of brain structure, and uncovered spatiotemporal regulatory principles during mammalian brain development.

category: developmental biology


title: Modeling the effect of cooperativity in ternary complex formation and targeted protein degradation mediated by heterobifunctional degraders

abstract:
Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degradation (TPD) by HBFs offers a promising approach to modulating disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase--including the protein-protein interaction (PPI) between the POI and the ligase--contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmacodynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model predicts that there is an optimal range of cooperativity values for efficient degradation, which depends on the kinetic rates of the other steps in the process. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data, and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.

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category: biochemistry


title: An agent based model of intracellular ice formation and propagation in small tissues

abstract:
Successful cryopreservation of tissues and organs would be a critical tool to accelerate drug discovery and facilitate myriad life saving and quality of life improving medical interventions. Unfortunately success in tissue cryopreservation is quite limited, and there have been no reports of successful long term organ cryopreservation. One principal challenge of tissue and organ cryopreservation is the propagation of damaging intracellular ice. Understanding the probability that cells in tissues form ice under a given cryopreservation protocol would greatly accelerate protocol design, enabling rational model-based decisions of all aspects of the cryopreservation procedure. Established models of intracellular ice formation (IIF) in individual cells have previously been extended to small linear (one-cell-wide) arrays to establish the theory of intercellular ice propagation in tissues. However these small-scale lattice-based tissue ice propagation models have not been extended to more realistic tissue structures, and do not account for intercellular forces that arise from the expansion water into ice that may cause mechanical disruption of tissue structures during freezing. To address these shortcomings, here we present the development and validation of a lattice-free agent-based stochastic model of ice formation and propagation in small tissues. We validate our Monte Carlo model against Markov chain models in the linear two-cell and four-cell arrays presented in the literature, as well as against new Markov chain results for 2 x 2 arrays. Moreover we expand the existing model to account for the solidification of water into ice in cells. We then use literature data to inform a model of ice propagation in hepatocyte disks, spheroids, and tissue slabs. Our model aligns well with previously reported experiments, and demonstrates that the mechanical effects of individual cells freezing can be captured.

Author summaryThe widespread ability to successfully store, or cryopreserve, tissues and organs in liquid nitrogen temperatures would be game changing for human and animal medicine and drug discovery. However, success is limited to a select number of small tissues, and no organs can currently be stored in a frozen or solid state and survive thawing. One major contributor to damage during this process is the formation of intracellular ice, and its associated cell level damage. This ice formation is complicated in tissues by the number of intercellular connections facilitating intercellular ice propagation. Previous researchers have developed and experimentally validated simple one dimensional models of ice propagation in tissues, but these fail to capture complex tissue geometries, and have many fewer intercellular connections compared to three dimensional tissues. In this paper, we adopt previous models of ice formation and propagation to a model capable of capturing arbitrary cell orientations in three dimensions, allowing for realistic tissue structures to be modelled. We validated this tool on simple models and with experimental data, and then test it on three structures made of digital liver cells: disks, spheroids, and slabs. We show that we can capture new information about the interaction of cooling the tissue, the formation of intracellular ice, the movement of ice from one cell to another, and the mechanical disruption that occurs during this process. This allows for novel insights into a mechanism of damage during cryopreservation that is cooling rate and tissue structure dependent.

category: biophysics


title: Detection of Fortunate Molecules Induce PArticle Resolution Shift (PAR-shift) towards Single-molecule Limit in SMLM: A Technique for Resolving Molecular Clusters in Cellular System

abstract: Molecules capable of emitting a large number of photons (also known as fortunate molecules) are crucial for achieving resolution close to a single molecule limit (the actual size of a single molecule). We propose a long-exposure single molecule localization microscopy (leSMLM) technique that enables detection of fortunate molecules, which is based on the fact that detecting a relatively small subset of molecules with large photon emission increases its localization [Formula]. Fortunate molecules have the ability to emit a large burst of photons over a prolonged time (> average triplet-state lifetime). So, a long exposure time allows the time window necessary to detect these elite molecules. The technique involves the detection of fortunate molecules to generate enough statistics for a quality reconstruction of the target protein distribution in a cellular system. Studies show a significant PArticle Resolution Shift (PAR-shift) of about 6 nm and 11 nm towards Single-molecule-limit (away from diffraction-limit) for an exposure time window of 60 ms and 90 ms, respectively. In addition, a significant decrease in the fraction of fortunate molecules (single molecules with small localization precision) is observed. Specifically, 8.33% and 3.43% molecules are found to emit in 30 - 60 ms and 60 - 90 ms, respectively, when compared to SMLM. The long exposure has enabled better visualization of Dendra2HA molecular cluster, with sub-clusters within a large cluster. Thus, the proposed technique (leSMLM) facilitates a better study of cluster formation in fixed samples. Overall, the method enables better spatial resolution at the cost of relatively poor temporal resolution.


title: 3D Reconstructions of Mouse Skeletal Muscle and Heart Muscle Reveal a Decrease in the MICOS Complex and Altered Mitochondrial Networks

abstract: Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria cristae are essential for the production of ATP. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. Methods: We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels were also measured through 3D reconstruction. CRISPR/Cas9 KO was performed to examine changes in mitochondria upon loss of the mitochondrial contact site and cristae organizing system (MICOS) complex and optic atrophy 1 (OPA-1). Metabolomics were used to find key metabolite pathways changed upon off of the MICOS complex. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: To our knowledge, studies of mitochondrial changes in skeletal muscle throughout aging remains limited. 3D reconstructions of nanotunnels elucidated novel patterns of mitochondrial aging in skeletal muscle. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction.


title: 3D Reconstructions of Mouse Skeletal Muscle Reveal a Decrease in the MICOS Complex and Altered Mitochondrial Networks


title: Optimized transcriptional signature for evaluation of MEK/ERK pathway baseline activity and long-term modulations in ovarian cancer

abstract: Ovarian cancer is the most aggressive and lethal of all gynecologic malignancies. High activity of the MEK/ERK signaling pathway is tightly associated with tumor growth, high recurrence rate, and treatment resistance. Several transcriptional signatures were proposed recently for evaluation of MEK/ERK activity in tumor tissue. In the present study, we validated the performance of a robust multi-cancer MPAS 10-gene signature in various experimental models and publicly available sets of ovarian cancer samples. Expression of four MPAS genes (PHLDA1, DUSP4, EPHA2, and SPRY4) displayed reproducible responses to MEK/ERK activity modulations across several experimental models in vitro and in vivo. Levels of PHLDA1, DUSP4, and EPHA2 expression were also significantly associated with baseline levels of MEK/ERK pathway activity in multiple human ovarian cancer cell lines and ovarian cancer patient samples available from the TCGA database. High EPHA2 expression, platinum therapy resistance, and advanced age at diagnosis were associated with poor overall patient survival. Taken together, our results demonstrate that performance of transcriptional signatures is significantly affected by tissue specificity and aspects of particular experimental models. We therefore propose that gene expression signatures derived from comprehensive multi-cancer studies should be always validated for each cancer type.

category: cancer biology


title: Bipartite recruitment of PCH2 and COMET to the synaptonemal complex drives chromosome axis reconstruction leading to crossover restriction

abstract: Chromosome axis-associated HORMA domain proteins (HORMADs), e.g., ASY1 in Arabidopsis, are crucial for meiotic recombination. ASY1, as other HORMADs, is assembled on the axis at early meiosis and depleted when homologous chromosomes synapse. Puzzlingly, both processes are catalyzed by AAA+ ATPase PCH2 together with its cofactor COMET. Here, we show that the ASY1 remodeling complex is temporally and spatially differently assembled. While PCH2 and COMET appear to directly interact in the cytoplasm in early meiosis, PCH2 is recruited by the transverse filament protein ZYP1 and brought to the ASY1-bound COMET assuring the timely removal of ASY1 during chromosome synapsis. Since we found that the PCH2 homolog TRIP13 also binds to the ZYP1 homolog SYCP1 in mouse, we postulate that this mechanism is conserved among eukaryotes. Deleting the PCH2 binding site of ZYP1 led to a failure of ASY1 removal. Interestingly, the placement of one obligatory crossover per homologous chromosome pair, compromised by ZYP1 depletion, is largely restored in this engineered zyp1 allele suggesting that crossover assurance is promoted by synapsis. In contrast, the engineered zyp1 allele, similar to the zyp1 null mutant, showed elevated type I crossover numbers indicating that PCH2-mediated eviction of ASY1 from the axis restricts crossover formation.


title: Global Spatial Transcriptome of Macaque Brain at Single-Cell Resolution

abstract: Global profile of gene expression at single-cell resolution remains to be determined for primates. Using a recently developed technology ("Stereo-seq"), we have obtained a comprehensive single-cell spatial transcriptome map at the whole-brain level for cynomolgus monkeys, with [~]600 genes per cell for 10 m-thick coronal sections (up to 15 cm2 in size). Large-scale single-nucleus RNA-seq analysis for [~]1 million cells helped to identify cell types corresponding to Stereo-seq gene expression profiles, providing a 3-D cell type atlas of the monkey brain. Quantitative analysis of Stereo-seq data revealed molecular fingerprints that mark distinct neocortical layers and subregions, as well as domains within subcortical structures including hippocampus, thalamus, striatum, cerebellum, hypothalamus and claustrum. Striking whole-brain topography and coordinated patterns were found in the expression of genes encoding receptors and transporters for neurotransmitters and neuromodulators. These results pave the way for cellular and molecular understanding of organizing principles of the primate brain.


title: A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

abstract: Gene editing using engineered nucleases frequently produces on- and off-target indels in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain genetically heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of pre-cultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo-manipulation platform establishes a novel paradigm to control genetic heterogeneity in HSC gene editing and therapy.


title: Ubiquitin ligase TRAIP plays an essential role during the S-phase of unperturbed cell cycle in the resolution of DNA replication - transcription conflicts

abstract: Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, for the first time, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We find that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.


title: Fate and state transitions during human blood vessel organoid development

abstract: Blood vessel organoids (BVOs) derived from human pluripotent stem cells have emerged as a novel system to understand human vascular development, model disorders, and develop regenerative therapies. However, it is unclear which molecular states constitute BVOs and how cells differentiate and self-organize within BVOs in vitro and after transplantation. Here we reconstruct BVO development over a time course using single-cell transcriptomics. We observe progenitor states that bifurcate into endothelial and mural fates, and find that BVOs do not acquire definitive arterio-venous endothelial identities in vitro. Chromatin accessibility profiling identifies gene regulatory network (GRN) features associated with endothelial and mural fate decisions, and transcriptome-coupled lineage recording reveals multipotent progenitor states within BVOs. We perform single-cell genetic perturbations within mosaic BVOs to dissect the impact of transcription factor (TF) and receptor depletion on cell differentiation, and highlight multiple TFs including MECOM and ETV2 as strong-effect regulators of human BVO development. We show that manipulation of VEGF and Notch signaling pathways alters BVO morphogenesis and endothelial GRNs, and induces arteriovenous-like state differentiation. We analyze matured BVOs after transplantation using scRNA-seq, and observe matured endothelium with clear arteriovenous specification. We also observe off-target cell fates with bone and adipocyte features, suggesting multipotent states reside within the BVOs in vitro that expand and diversify in less restrictive conditions. Finally, we map vascular disease associated genes to BVO cell states to highlight the potential of BVOs for disease modeling. Altogether, our data and analyses provide the first comprehensive cell state atlas of BVO development and illuminate both the power and limitation of BVOs for translational research.


title: A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification

abstract: Developmental control of gene expression critically depends on distal cis-regulatory elements including enhancers which interact with promoters to activate gene expression. To date no global experiments have been conducted that identify their cell type and cell stage-specific transcription stimulatory activity within one developmental pathway and in a chromatin context. Here, we describe a high-throughput method that identifies thousands of differentially active cis-elements able to stimulate a minimal promoter at five stages of hematopoietic progenitor development from embryonic stem cells, which can be adapted to any ES cell derived cell type. Exploring this new resource, we show that blood cell-specific gene expression is controlled by the concerted action of thousands of differentiation stage-specific sets of cis-elements which respond to cytokine signals that terminate at signalling responsive transcription factors. Our work presents a major advance in our understanding of developmental gene expression control in the hematopoietic system and beyond.

category: genomics


title: Small Molecule Modulation of the Archetypal UbiB protein COQ8

abstract: Small molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize inhibitor and activator molecules for the archetypal UbiB member, COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays, and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Second, using 1H-13C HMQC NMR and hydrogen-deuterium exchange mass spectrometry, we reveal that the CoQ precursor mimetic, 2-propylphenol (2-PP), modulates the quintessential UbiB KxGQ domain to increase COQ8A nucleotide affinity and ATPase activity. Our newfound chemical tools promise to lend new mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.


title: Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity

abstract: FMRFamide and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFamide activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFamide potency in one of its native targets, the excitatory FMRFamide-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of function, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFamide, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching DEG/ENaC superfamily, incorporating mutagenesis and experimental dissection of function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFamide activity at FaNaCs and illuminates evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.


title: Inhalable polymer nanoparticles for versatile mRNA delivery and mucosal vaccination

abstract:
An inhalable platform for mRNA therapeutics would enable minimally invasive and lung targeted delivery for a host of pulmonary diseases. Development of lung targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung. We optimized biodegradable poly(amine-co-ester) polyplexes for mRNA delivery using end group modifications and polyethylene glycol. Our polyplexes achieved high transfection of mRNA throughout the lung, particularly in epithelial and antigen-presenting cells. We applied this technology to develop a mucosal vaccine for SARS-CoV-2. Intranasal vaccination with spike protein mRNA polyplexes induced potent cellular and humoral adaptive immunity and protected K18-hACE2 mice from lethal viral challenge.

One-sentence summaryInhaled polymer nanoparticles (NPs) achieve high mRNA expression in the lung and induce protective immunity against SARS-CoV-2.

category: bioengineering


title: Analysis of calcium dynamics for dim-light responses in rod and cone photoreceptors

abstract:
Rod and cone photoreceptors in vertebrate eyes are fundamental sensory neurons underlying vision. They use a sophisticated signal transduction pathway consisting of a series of biochemical processes to convert the absorption of light into an electrical response. Several of these processes are modulated by feedback mechanisms that depend on the intracellular Ca2+ concentration. In this work we use a representative phototransduction model with fast buffering to study how the Ca2+ dynamics affects the light response of mouse rod and cone photoreceptors. We obtain analytic solutions for dim-light stimulation that reveal how the dynamics of the Ca2+ concentration and the electrical current are correlated. We show how modifying the Ca2+ dynamics via buffering affects amplitude and waveform of flash and step responses. We infer that under normal physiological conditions the change in the Ca2+ concentration is proportional to the current. We show that damped oscillations appear if the Ca2+ dynamics becomes slowed down and delayed with respect to the current, e.g. through the application of buffers. We find that oscillations do not require the presence of slow buffers. We show that the phase space of the dim-light response is controlled by the ratio between the effective rate ca that controls the Ca2+ dynamics, and the dark turnover rate of cyclic GMP {beta}d. Finally, we investigate how the light response is affected by modifying the extracellular Ca2+ concentration. In summary, we provide a comprehensive analysis that reveals how the Ca2+ dynamics affects the dim-light response in rods and cones.

SignificanceThe signal transduction pathway of vertebrate rod and cone photoreceptors transforms light into an electrical response. It is regulated by several feedback processes that depend on the intracellular Ca2+ concentration. Despite of the importance of this feedback for the photoreceptor functioning, it is not well studied how modifying the Ca2+ dynamics affects the light response, for example by adding buffers. Here we derive analytic results for dim-light stimulations that provide insight into how the Ca2+ dynamics and the electrical current response are correlated. Our analysis can be used to infer properties of the Ca2+ dynamics from the observed current response, and provides insight if direct measurements of the Ca2+ dynamics in the small outer segment of a photoreceptors are not available.


title: From neural border to migratory stage: A comprehensive single cell roadmap of the timing and regulatory logic driving cranial and vagal neural crest emergence

abstract: Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification towards definitive neural crest. Here, we combine ultra-dense single cell transcriptomes with machine-learning and large-scale experimental validation to provide a comprehensive GRN underlying neural crest fate diversification from induction to early migration stages. During gastrulation, a transient neural border zone state precedes choice between neural crest and placodes following a "dual convergence model". Transcription factor connectome and bifurcation analyses demonstrate the early emergence of neural crest fates at neural plate stage, alongside an unbiased multipotent lineage persisting until after epithelial-mesenchymal transition. We decipher the circuits driving cranial and vagal neural crest formation and provide a broadly applicable strategy for investigating SC transcriptomes in vertebrate GRNs in development, evolution and disease.


title: Premature translation of the zygotic genome activator Zelda is not sufficient to precociously activate gene expression

abstract: Following fertilization, the unified germ cells rapidly transition to a totipotent embryo. Maternally deposited mRNAs encode the proteins necessary for this reprogramming as the zygotic genome remains transcriptionally quiescent during the initial stages of development. The transcription factors required to activate the zygotic genome are among these maternally deposited mRNAs and are robustly translated following fertilization. In Drosophila, the mRNA encoding Zelda, the major activator of the zygotic genome, is not translated until one hour after fertilization. Here we demonstrate that zelda translation is repressed following fertilization by the TRIM-NHL protein Brain tumor (BRAT). BRAT also regulates Zelda levels in the larval neuroblast lineage. In the embryo, BRAT-mediated translational repression is regulated by the Pan Gu (PNG) kinase, which is triggered by egg activation. The PNG kinase phosphorylates translational regulators, suggesting that PNG kinase activity alleviates translational repression of zelda by BRAT and coupling translation of zelda with that of other regulators of early embryonic development. Using the premature translation of zelda in embryos lacking BRAT activity, we showed that early translation of a zygotic genome activator is not sufficient to drive precocious gene expression. Instead, Zelda-target genes showed increased expression at the time they are normally activated. We propose that transition through early development requires the integration of multiple processes, including the slowing of the nuclear division cycle and activation of the zygotic genome. These processes are coordinately controlled by PNG kinase-mediated regulation of translation.


title: Endothelial-derived sphingolipids are required for vascular development and systemic lipid homeostasis

abstract: Serine palmitoyl transferase (SPT), the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL), is needed for embryonic development, physiological homeostasis, and response to stress. The functions of de novo SL synthesis in vascular endothelial cells (EC), which line the entire circulatory system, are not well understood. Here we show that the EC de novo synthesis not only impacts vascular development but also maintains normal SL metabolic homeostasis in the circulatory system and peripheral organs. Mice with an endothelial-specific gene knockout of SPTLC1 (Sptlc1 ECKO), an essential subunit of the SPT complex, exhibited EC-intrinsic effects including reduced EC proliferation and tip/stalk cell differentiation, resulting in delayed retinal vascular development. In addition, Sptlc1 ECKO mice had reduced pathological retinal neovascularization in the oxygen-induced retinopathy model, suggesting that EC SL produced from the de novo pathway are needed for efficient VEGF signaling within the vascular system. Post-natal deletion of the EC Sptlc1also showed cell-extrinsic effects, including rapid reduction of several SL metabolites in plasma, red blood cells and peripheral organs (lung and liver) but not in the retina, part of the central nervous system (CNS). In the liver, EC de novo SL synthesis was required for acetaminophen-induced ceramide elevation and hepatotoxicity. These results suggest that EC-derived SL metabolites are in constant flux between the vasculature, circulatory elements, and parenchymal cells of non-CNS organs. Taken together, our data point to the central role of the endothelial SL biosynthesis in maintaining vascular development and neovascular proliferation, non-CNS tissue metabolic homeostasis and hepatocyte response to stress.


title: PRMT5 as a Novel Druggable Vulnerability for EWSR1-ATF1-driven Clear Cell Sarcoma

abstract: Clear cell sarcoma of soft tissue (CCSST) is an ultra-rare sarcoma with poor prognosis presently with no cure. It is characterized by a balanced t(12;22) (q13;q12) chromosomal translocation, resulting in a fusion of the Ewings sarcoma gene EWSR1 with activating transcription factor 1 (ATF1) to give an oncogene EWSR1-ATF1. Unlike normal ATF1, whose transcription activity is dependent on phosphorylation, EWSR1-ATF1 is constitutively active to drive ATF1-dependent gene transcription to cause tumorigenesis. No EWSR1-ATF1-targeted therapies have been identified due to the challenges in targeting intracellular transcription factors. To identify potential druggable targets for CCSST, we show that protein arginine methyltransferase 5 (PRMT5) is a novel enzyme in enhancing EWSR1-ATF1-mediated gene transcription to sustain CCSST cell proliferation. Genetic silencing of PRMT5 in CCSST cells resulted in severely impaired cell proliferation and EWSR1-ATF1-driven transcription. Furthermore, the clinical-stage PRMT5 inhibitor JNJ-64619178 potently and efficaciously inhibited CCSST cell growth in vitro and in vivo. These results provide new insights into PRMT5 as a transcription regulator and warrant JNJ-64619178 for further clinical development to treat CCSST patients.


title: Single cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis

abstract: Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with ageing and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using two murine models of injury and repair we show that obstructive injury generates senescent epithelia which persist after resolution of the original injury, promote ongoing fibrosis and impede adaptive repair. Depletion of senescent cells with ABT263 reduces fibrosis in reversed ureteric obstruction and after renal ischaemia-reperfusion injury. We validate these findings in humans, showing that senescence and fibrosis persist after relieved renal obstruction. We next characterise senescent epithelia in murine renal injury using single cell RNA-Seq. We extend our classification to human kidney and liver disease and identify conserved pro-fibrotic proteins which we validate in vitro and in human disease. We demonstrate that one such molecule, Protein Disulfide Isomerase Family A Member 3 (PDIA3), is essential for TGF-beta mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduces kidney fibrosis during ongoing renal injury and as such represents a new potential therapeutic pathway. Analysis of the signalling pathways of senescent epithelia connects senescence to organ fibrosis, permitting rational design of anti-fibrotic therapies.


title: Jupytope: Computational extraction of structural properties of viral epitopes

abstract:
Epitope residues located on viral surface proteins are of immense interest in immunology and related applications such as vaccine development, disease diagnosis and drug design. Most tools rely on sequence based statistical comparisons, such as information entropy of residue positions in aligned columns to infer location and properties of epitope sites. To facilitate cross-structural comparisons of epitopes on viral surface proteins, a python-based extraction tool implemented with Jupyter notebook is presented (Jupytope). Given a viral antigen structure of interest, a list of known epitope sites and a reference structure, the corresponding epitope structural properties can quickly be obtained. The tool integrates biopython modules for commonly used software such as NACCESS, DSSP as well as residue depth and outputs a list of structure derived properties such as dihedral angles, solvent accessibility, residue depth and secondary structure that can be saved in several convenient data formats. To ensure correct spatial alignment, Jupytope takes a list of given epitope sites and their corresponding reference structure and aligns them before extracting the desired properties. Examples are demonstrated for epitopes of Influenza and SARS-CoV2 viral strains. The extracted properties assist detection of two Influenza subtypes and show potential in distinguishing between four major clades of SARS-CoV2, as compared with randomized labels. The tool will facilitate analytical and predictive works on viral epitopes through the extracted structural information.

Key MessagesO_LIJupytope combines existing 3D-structural software to extract the properties of viral epitopes into a convenient text or csv file format
C_LIO_LIThe structural properties serve as parameters or features that quantitatively capture viral epitopes
C_LIO_LIAssociation of structural properties to viral subtypes (for Influenza) or clades (SARS-CoV2) is demonstrated with a simple XGBoost model
C_LIO_LIStructure datasets mapped to SARS-CoV2 WHO clades and Pango lineages, as well as chain annotations are available for download
C_LI

category: bioinformatics


title: Heterotypic vaccination responses against SARS-CoV-2 Omicron BA.2

abstract: The Omicron sub-lineage BA.2 of SARS-CoV-2 has recently become dominant across many areas in the world in the on-going waves of COVID-19. Compared to the ancestral/wild-type (WT) virus, Omicron lineage variants, both BA.1 and BA.2, contain high number of mutations, especially in the spike protein, causing significant immune escape that leads to substantial reduction of vaccine and antibody efficacy. Because of this antigenic drift, BA.2 exhibited differential resistance profile to monoclonal antibodies than BA.1. Thus, it is important to understand whether the immunity elicited by currently available vaccines are effective against the BA.2 subvariant. We directly tested the heterotypic vaccination responses against Omicron BA.2, using vaccinated serum from animals receiving WT- and variant-specific mRNA vaccine in lipid nanoparticle (LNP) formulations. Omicron BA.1 and BA.2 antigen showed similar reactivity to serum antibodies elicited by two doses of WT, B.1.351 and B.1.617 LNP-mRNAs. Neutralizing antibody titers of B.1.351 and B.1.617 LNP-mRNA were ~2-fold higher than that of WT LNP-mRNA. Both homologous boosting with WT LNP-mRNA and heterologous boosting with BA.1 LNP-mRNA substantially increased waning immunity of WT vaccinated mice against both BA.1 and BA.2 subvariants. The BA.1 LNP-mRNA booster was ~3-fold more efficient than WT LNP-mRNA at elevating neutralizing antibody titers of BA.2. Together, these data provided a direct preclinical evaluation of WT and variant-specific LNP-mRNAs in standard two-dose and as boosters against BA.1 and BA.2 subvariants.


title: In silico analysis predicts a limited impact of SARS-CoV-2 variants on CD8 T cell recognition

abstract: Since the start of the COVID-19 pandemic, mutations have led to the emergence of new SARS-CoV-2 variants, and some of these have become prominent or dominant variants of concern. This natural course of development can have an impact on how protective the previously naturally or vaccine induced immunity is. Therefore, it is crucial to understand whether and how variant specific mutations influence host immunity. To address this, we have investigated how mutations in the recent SARS-CoV-2 variants of interest and concern influence epitope sequence similarity, predicted binding affinity to HLA, and immunogenicity of previously reported SARS-CoV-2 CD8 T cell epitopes. Our data suggests that the vast majority of SARS-CoV-2 CD8 T cell recognized epitopes are not altered by variant specific mutations. Interestingly, for the CD8 T cell epitopes that are altered due to variant specific mutations, our analyses show there is a high degree of sequence similarity between mutated and reference SARS-CoV-2 CD8 T cell epitopes. However, mutated epitopes, primarily derived from the spike protein, in SARS-CoV-2 variants Delta, AY.4.2 and Mu display reduced predicted binding affinity to their restriction element. These findings indicate that the recent SARS-CoV-2 variants of interest and concern have limited ability to escape memory CD8 T cell responses raised by vaccination or prior infection with SARS-CoV-2 early in the pandemic. The overall low impact of the mutations on CD8 T cell cross-recognition is in accordance with the notion that mutations in SARS-CoV-2 are primarily the result of receptor binding affinity and antibody selection pressures exerted on the spike protein, unrelated to T cell immunity.


title: Intranasal Nanoemulsion Adjuvanted S-2P Vaccine Demonstrates Protection in Hamsters and Induces Systemic, Cell-Mediated and Mucosal Immunity in Mice

abstract: With the rapid progress made in the development of vaccines to fight the SARS-CoV-2 pandemic, almost >90% of vaccine candidates under development and a 100% of the licensed vaccines are delivered intramuscularly (IM). While these vaccines are highly efficacious against COVID-19 disease, their efficacy against SARS-CoV-2 infection of upper respiratory tract and transmission is at best temporary. Development of safe and efficacious vaccines that are able to induce robust mucosal and systemic immune responses are needed to control new variants. In this study, we have used our nanoemulsion adjuvant (NE01) to intranasally (IN) deliver stabilized spike protein (S-2P) to induce immunogenicity in mouse and hamster models. Data presented demonstrate the induction of robust immunity in mice resulting in 100% seroconversion and protection against SARS-CoV-2 in a hamster challenge model. There was a significant induction of mucosal immune responses as demonstrated by IgA- and IgG-producing memory B cells in the lungs of animals that received intranasal immunizations compared to an alum adjuvanted intramuscular vaccine. The efficacy of the S-2P/NE01 vaccine was also demonstrated in an intranasal hamster challenge model with SARS-CoV-2 and conferred significant protection against weight loss, lung pathology, and viral clearance from both upper and lower respiratory tract. Our findings demonstrate that intranasal NE01-adjuvanted vaccine promotes protective immunity against SARS-CoV-2 infection and disease through activation of three arms of immune system: humoral, cellular, and mucosal, suggesting that an intranasal SARS-CoV-2 vaccine may play a role in addressing a unique public health problem and unmet medical need.


title: A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability

abstract: mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing enhanced stability, improved manufacturability, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of long-lived bone marrow-resident antibody secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability at room temperature for at least 6 months when lyophilized. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.


title: Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant protect Vero E6 cells against infection by viral pseudotyped particles expressing SARS-CoV-2, SARS-CoV or MERS-CoV Spike protein.

abstract: Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses (HuCoV) that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surface and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with [~]97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S- pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:16 dilution, their infectivity was dramatically reduced by [~]98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.

category: microbiology


title: Topologically engineered antibodies and Fc-fusion proteins: a new class of multifunctional therapeutic candidates for SARS-CoV-2, cancer, and other disease

abstract: The ability of antibodies and Fc-fusion proteins to bind multiple targets cooperatively is limited by their topology. Here we describe our discovery that ACE2 Fc-fusion proteins spontaneously cross-dimerize, forming topologically distinct "superdimers" that demonstrate extraordinary SARS-CoV-2 intra-spike cooperative binding and potently neutralize Omicron B.1.1.529 at least 100-fold better than eight clinically authorized antibodies. We also exploited cross- dimerization to topologically engineer novel superdimeric antibodies and Fc-fusion proteins with antibody-like plasma half-lives to address cancer and infectious disease therapy. These include bispecific ACE2-antibody superdimers that potently neutralize all major SARS-CoV-2 variants, and bispecific anti-cancer and anti-viral antibody superdimers that are more potent than two-antibody cocktails. Superdimers are efficiently produced from single cells, providing a new therapeutic approach to many disease indications.


title: A CNN model for predicting binding affinity changes between SARS-CoV-2 spike RBD variants and ACE2 homologues

abstract: The cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the association of its receptor binding domain (RBD) with human angiotensin converting enzyme 2 (hACE2) as the first crucial step. Efficient and reliable prediction of RBD-hACE2 binding affinity changes upon amino acid substitutions can be valuable for public health surveillance and monitoring potential spillover and adaptation into non-human species. Here, we introduce a convolutional neural network (CNN) model trained on protein sequence and structural features to predict experimental RBD-hACE2 binding affinities of 8,440 variants upon single and multiple amino acid substitutions in the RBD or ACE2. The model achieves a classification accuracy of 83.28% and a Pearson correlation coefficient of 0.85 between predicted and experimentally calculated binding affinities in five-fold cross-validation tests and predicts improved binding affinity for most circulating variants. We pro-actively used the CNN model to exhaustively screen for novel RBD variants with combinations of up to four single amino acid substitutions and suggested candidates with the highest improvements in RBD-ACE2 binding affinity for human and animal ACE2 receptors. We found that the binding affinity of RBD variants against animal ACE2s follows similar trends as those against human ACE2. White-tailed deer ACE2 binds to RBD almost as tightly as human ACE2 while cattle, pig, and chicken ACE2s bind weakly. The model allows testing whether adaptation of the virus for increased binding with other animals would cause concomitant increases in binding with hACE2 or decreased fitness due to adaptation to other hosts.


title: Suppression of transposon mobilization by m6A-mediated RNA sequestration in stress granules

abstract: Transposon is a mobile and ubiquitous DNA that can vastly causes genomic alterations. In plants, it is well documented that transposon mobilization is strongly repressed by DNA methylation; however, the roles of RNA methylation in transposon control remain unknown. Here we suggest that transposon RNA is marked by m6A RNA methylation and is sequestered in stress granule (SG) in m6A-dependent manner. Intriguingly, a SG-localized AtALKBH9B selectively demethylates a heat-activated retroelement Onsen, and thereby releases from spatial confinement allowing for its mobilization. In addition, we show evidence that m6A RNA methylation contributes to transpositional suppression by inhibiting the virus-like particles assembly and extrachromosomal DNA production. In summary, this study unveils a hidden role for m6A in the suppression of transposon mobility and provides an insight into how transposon counteracts the hosts epitranscriptomic control by hitchhiking RNA demethylase.

category: plant biology


title: Chromatin-associated lncRNA Malat1 regulates Th17 effector program and promotes intestinal inflammation

abstract:
Interleukin IL-17 cytokines are central regulators of mucosal homeostasis and disease. In response to tissue injury, IL-17A promotes epithelial barrier functions and restricts local inflammation. In the resolution phase of colonic inflammation, adaptive Th17 cells are the major source of IL-17A, however, the cell-specific temporal control mechanism(s) underlying the delayed Th17 response remain elusive. Here, we discover a surprising role of the lncRNA Malat1 as a key barrier to achieving a robust and protective Th17 response. Genetic ablation of Malat1 potentiates IL-17A production in Th17 cells and improves disease outcomes in mouse models of colitis. Mechanistically, Malat1 represses Il17a transcription by restricting its bivalent super-enhancer activities. Thus, Malat1 is down-regulated during the resolution phase of inflammation, allowing Th17 to overtake early T{gamma}{delta}17 as the major source of colonic IL-17A. Targeted depletion of Malat1 in T helper cell using anti-sense nucleotides show therapeutic potential in colitis model.

One Sentence SummaryMalat1 regulates super-enhancer transcriptional activities in Th17 cells.


title: Long-read-resolved, ecosystem-wide exploration of nucleotide and structural microdiversity of lake bacterioplankton genomes

abstract: Reconstruction of metagenome-assembled genomes (MAGs) has become a fundamental approach in microbial ecology. However, an MAG is hardly complete and overlooks genomic microdiversity because metagenomic assembly fails to resolve microvariants among closely related genotypes. Aiming at understanding the universal factors that drive or constrain prokaryotic genome diversification, we performed an ecosystem-wide high-resolution metagenomic exploration of microdiversity by combining spatiotemporal (2 depths x 12 samples) sampling from a pelagic freshwater system, MAG reconstruction using long- and short-read metagenomic sequences, and profiling of single nucleotide variants (SNVs) and structural variants (SVs) through mapping of short and long reads to the MAGs, respectively. We reconstructed 575 MAGs, including 29 circular assemblies, providing high-quality reference genomes of freshwater bacterioplankton. Read mapping against these MAGs identified 100-101,781 SNVs/Mb, 0-305 insertions, 0-467 deletions, 0-41 duplications, and 0-6 inversions for each MAG. Nonsynonymous SNVs were accumulated in genes potentially involved in cell surface structural modification to evade phage recognition. Most (80.2%) deletions overlapped with a gene-coding region, and genes of prokaryotic defense systems were most frequently (>8% of the genes) involved in a deletion. Some such deletions exhibited a monthly shift in their allele frequency, suggesting a rapid turnover of genotypes in response to phage predation. MAGs with extremely low microdiversity were either rare or opportunistic bloomers, suggesting that population persistency is key to their genomic diversification. The results lead to the conclusion that prokaryotic genomic diversification is primarily driven by viral load and constrained by a population bottleneck.


title: Kiawah and Seabrook islands are a critical site for the rufa red knot (Calidris canutus rufa)

abstract: The rufa Red Knot (Calidris canutus rufa) is a migratory shorebird that performs one of the longest known migrations of any bird species -- from their breeding grounds in the Canadian Arctic to their nonbreeding grounds as far south as Tierra del Fuego -- and has experienced a population decline of over 85% in recent decades. During migration, knots rest and refuel at stopover sites along the Atlantic Coast, including Kiawah and Seabrook islands in South Carolina. Here, we document the importance of Kiawah and Seabrook islands for knots by providing population and stopover estimates during their spring migration. We conducted on-the-ground surveys between 19 February - 20 May 2021 to record the occurrence of individually marked knots. In addition, we quantified the ratio of marked to unmarked knots and deployed geolocators on knots captured in the area. Using a superpopulation model, we estimated a minimum passage population of 17,247 knots (~41% of the total rufa knot population) and an average stopover duration of 47 days. Our geolocator results also showed that knots using Kiawah and Seabrook islands can bypass Delaware Bay and fly directly to the Canadian Arctic. Finally, our geolocators, combined with resighting data from across the Atlantic Flyway, indicate that a large network of more than 70 coastal sites mostly concentrated along the coasts of Florida, Georgia, South Carolina, and North Carolina provide stopover and overwintering habitat for the knots we observed on Kiawah and Seabrook islands. These findings corroborate that Kiawah and Seabrook islands should be recognized as critical sites in the knot network and, therefore, a conservation priority. As a result, the threats facing the sites -- such as prey management issues, anthropogenic disturbance, and sea level rise -- require immediate attention.

category: ecology


title: Urbanization alters phenology of plant reproduction, foliar infection, and herbivory

abstract:
O_LIUrbanization is associated with numerous environmental changes that may affect the timing of plant reproduction and foliar damage by pathogens, herbivores, and human activities. Yet such effects have not previously been quantified simultaneously in plant populations across levels of urbanization. Thus, it remains unclear how urbanization impacts multiple fitness components of a single plant species. Moreover, it is unclear which effects are consistent between species.
C_LIO_LIWe studied 22 populations of two co-occurring Plantago species in parks and nature areas spanning rural, suburban, and urban sections of the Greater St. Louis metropolitan area in the central United States. In monthly surveys of Plantago lanceolata and P. rugelii during summer- fall in two consecutive years, we quantified plant reproductive development and prevalence of powdery mildew infection. In the second year, we additionally quantified prevalence of insect herbivory and mowing damage and placed potted sentinel plants into the field populations to directly measure rates of infection and herbivory.
C_LIO_LIUrbanization was associated with earlier flowering and more seed production for P. rugelii, but less seed maturation for P. lanceolata. Mildew epidemics on P. rugelii started earlier and achieved greater infection prevalence in more urban sites. Correspondingly, sentinels only became infected in suburban and urban sites. By contrast, there was less mildew infection on P. lanceolata, including on sentinels, suggesting low availability of pathogen genotypes able to infect this species. Urbanization accelerated early-summer herbivory on both plant species, but did not affect herbivory of wild or sentinel plants later in summer. Finally, prevalence of mowing damage on leaves was generally greater in urban and suburban than rural sites.
C_LIO_LISynthesis: We show that urbanization affects the timing of key reproductive events and multiple types of aboveground damage for wild herbaceous plants. The two plant species responded differently to urbanization in terms of their reproductive phenology. However, in months when significant effects of urbanization were detected, they were typically in the direction of greater rates of pathogen infection, herbivory, and mowing damage. Overall, these findings suggest multiple mechanisms by which urbanization may impact plant population growth.
C_LI


title: Measuring plant diversity in a two-stage sampling design by Bayesian updated relative abundances

abstract: The two-stage sampling design provides good local estimates of both the number of plant species and the relative abundances. However, it is a problem to calculate Hill diversity indices at the local scale, because some of the species found in the large plot are not present in the small plot and such species should then incorrectly be weighted with zero relative abundance. A new method for calculating local Hill diversity indices from species richness and relative abundances data is therefore needed. We suggest to replace the local relative abundances with Bayesian updated relative abundance estimates, where the prior probability distribution of the relative abundances are empirically estimated from all plots of the same habitat types. The method is applied on Danish Nardus grasslands.


title: Wavelet characterization of spatial pattern in allele frequency

abstract:
Characterizing spatial patterns in allele frequencies is fundamental to evolutionary biology because such patterns can inform on underlying processes. However, the spatial scales at which changing selection, gene flow, and drift act are often unknown. Many of these processes can operate inconsistently across space (causing non-stationary patterns). We present a wavelet approach to characterize spatial pattern in genotype that helps solve these problems. We show how our approach can characterize spatial patterns in ancestry at multiple spatial scales, i.e. a multi-locus wavelet genetic dissimilarity. We also develop wavelet tests of spatial differentiation in allele frequency and quantitative trait loci (QTL). With simulation we illustrate these methods under a variety of scenarios. We apply our approach to natural populations of Arabidopsis thaliana and traditional varieties of Sorghum bicolor to characterize population structure and locally-adapted loci across scales. We find, for example, that Arabidopsis flowering time QTL show significantly elevated scaled wavelet variance at [~] 300 - 1300 km scales. Wavelet transforms of population genetic data offer a flexible way to reveal geographic patterns and underlying processes.

Author summaryBiologists can learn about evolutionary processes by studying spatial/geographic changes in the genotype of organisms in nature. However, many previous approaches to measure spatial genetic patterns have been limited by forcing individual samples into bins of discrete size and location, hindering our ability to learn about evolution. Here we present a new continuous approach to spatial genetics that allows us to resolve patterns that change in space and opposing patterns that occur at different spatial scales.

category: evolutionary biology


title: Cross-trait assortative mating is widespread and inflates genetic correlation estimates

abstract: The observation of genetic correlations between disparate traits has been interpreted as evidence of widespread pleiotropy, altered theories of human genetic architecture, and spurred considerable research activity across the natural and social sciences. Here, we introduce cross-trait assortative mating (xAM) as an alternative explanation for observed genetic correlations. We observe that xAM is common across a broad array of phenotypes and that phenotypic cross-mate correlation estimates are strongly associated with genetic correlation estimates (R2 = 76%). Then, we present theoretical and simulation-based results demonstrating that, under xAM, genetic correlation estimators yield significant estimates even for traits with entirely distinct genetic bases. We demonstrate that existing xAM plausibly accounts for substantial fractions of genetic correlation estimates in two large samples (N = 827,960). For example, previously reported genetic correlation estimates between many pairs of psychiatric disorders are fully consistent with xAM alone. Finally, we provide evidence for a history of xAM at the genetic level using a novel approach based on cross-trait even/odd chromosome polygenic score correlations. Together, our results demonstrate that previous reports have likely overestimated the true genetic similarity between many phenotypes.

category: genetics


title: Integrative Analysis of Checkpoint Blockade Response in Advanced Non-Small Cell Lung Cancer

abstract: Anti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). While our understanding of the biology underlying immune checkpoint blockade in NSCLC is still incomplete, studies to date have established predictive roles for PD-L1 tumor expression and tumor mutational burden (TMB). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up 2 Cancer - Mark Foundation (SU2C-MARK) Cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including: 1) favorable (e.g., ATM altered), and unfavorable (e.g., TERT amplified) genomic subgroups, 2) distinct immune infiltration signatures associated with wound healing (unfavorable) and immune activation (favorable), and 3) a novel de-differentiated tumor-intrinsic subtype characterized by expression of endodermal lineage genes, immune activation, and enhanced response rate. Taken together, results from this cohort extend our understanding of NSCLC-specific predictors, providing a rich set of molecular and immunologic hypotheses with which to further our understanding of the biology of checkpoint blockade in NSCLC.


title: Targeted delivery of galunisertib attenuates fibrogenesis in an integrated ex vivo renal transplant and fibrosis model

abstract:
Normothermic machine perfusion is an emerging preservation technique for kidney allografts to reduce post-transplant complications, including interstitial fibrosis and tubular atrophy. This technique, however, could be improved by adding antifibrotic molecules to perfusion solutions. We established Machine perfusion and Organ slices as a Platform for Ex vivo Drug delivery (MOPED), to explore fibrogenesis suppression strategies. We perfused porcine kidneys ex vivo with galunisertib--a potent inhibitor of the transforming growth factor beta signaling pathway. To determine whether effects persisted, we also cultured precision-cut tissue slices prepared from the respective kidneys. Galunisertib supplementation improved the general viability, without negatively affecting renal function or elevating levels of injury markers or byproducts of oxidative stress. Galunisertib also reduced inflammation and more importantly, strongly suppressed the onset of fibrosis, especially when the treatment was continued in slices. Our results illustrate the value of targeted drug delivery, using isolated organ perfusion, for reducing post-transplant complications.

One Sentence SummaryGalunisertib supplementation during normothermic machine perfusion attenuates fibrogenesis without compromising renal function.