title: Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq

abstract: Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.


title: Recognition of copy-back defective interfering rabies virus genomes by RIG-I triggers the antiviral response against vaccine strains

abstract:
Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year around the world. A typical feature of RABV infection is the suppression of type I and III interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) to initiate IFN signaling remain elusive. Here, we showed that RABV RNAs are recognized by RIG-I (retinoic acid-inducible gene I) sensor resulting in an IFN response of the infected cells but that this global feature was differently modulated according to the type of RABV used. RNAs from pathogenic RABV strain, THA, were poorly detected in the cytosol by RIG-I and therefore mediated a weak antiviral response. On the opposite, we revealed a strong interferon activity triggered by the RNAs of the attenuated RABV vaccine SAD strain mediated by RIG-I. Using next-generation sequencing (NGS) combined with bioinformatics tools, we characterized two major 5copy-back defective interfering (5cb DI) genomes generated during SAD replication. Furthermore, we identified a specific interaction of 5cb DI genomes and RIG-I that correlated with a high stimulation of the type I IFN signaling. This study indicates that RNAs from a wild-type RABV poorly activate the RIG-I pathway, while the presence of 5cb DIs in vaccine SAD strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection and therefore strongly stimulates the IFN response.

HighlightsO_LIRABV pathogenic strain replication in vitro is characterized by the absence of defective interfering genomes thus induces a weak RLR-mediated innate immunity antiviral response.
C_LIO_LIRABV vaccine attenuated strain shows a high release of 5 copy-back defective interfering genomes during replication in vitro and therefore enhances a strong antiviral response upon infection.
C_LIO_LIRIG-I is the main sensor for RABV RNA detection within cells.
C_LI

category: microbiology


title: The lectin LecB induces patches with basolateral characteristics at the apical membrane to promote Pseudomonas aeruginosa host cell invasion

abstract:
The opportunistic bacterium Pseudomonas aeruginosa can infect mucosal tissues of the human body. To persist at the mucosal barrier, this highly adaptable pathogen has evolved many strategies, including invasion of host cells. Here, we show that the P. aeruginosa lectin LecB binds and cross-links fucosylated receptors at the apical plasma membrane of epithelial cells. This triggers a signaling cascade via Src kinases and PI3K leading to the formation of patches enriched with the basolateral marker PIP3 at the apical plasma membrane. This identifies LecB as causative bacterial factor for activating this well-known host cell response that is elicited upon apical binding of P. aeruginosa. Downstream of PI3K, Rac1 is activated to cause actin rearrangement and the outgrowth of protrusions at the apical plasma membrane. LecB-triggered PI3K activation also results in aberrant recruitment of caveolin-1 to the apical domain. In addition, we reveal a positive feedback loop between PI3K activation and apical caveolin-1 recruitment, which provides a mechanistic explanation for the previously observed implication of caveolin-1 in P. aeruginosa host cell invasion. Interestingly, LecB treatment also reversibly removes primary cilia. To directly prove the role of LecB for bacterial uptake, we coated bacteria-sized beads with LecB, which drastically enhanced their endocytosis. Furthermore, LecB deletion and LecB inhibition with L-fucose diminished the invasion efficiency of P. aeruginosa bacteria. Taken together, our study identifies LecB as missing link that can explain how PI3K signaling and caveolin-1 recruitment are triggered to facilitate invasion of epithelial cells from the apical side by P. aeruginosa.

ImportanceAn intriguing feature of the bacterium P. aeruginosa is its ability to colonize highly diverse niches. P. aeruginosa can, besides biofilm formation, also enter and proliferate within epithelial host cells. Moreover, research during recent years has shown that P. aeruginosa possesses many different mechanisms to invade host cells. In this study we identify LecB as novel invasion factor. In particular, we show that LecB activates PI3K signaling, which is connected via a positive feedback loop to apical caveolin-1 recruitment, and leads to actin rearrangement at the apical plasma membrane. This provides a unifying explanation for the previously reported implication of PI3K and caveolin-1 in P. aeruginosa host cell invasion. In addition, our study adds a further function to the remarkable repertoire of the lectin LecB, which is all brought about by the capability of LecB to recognize fucosylated glycans on many different niche-specific host cell receptors.


title: Discovery of a Novel Inhibitor for Chikungunya Virus

abstract: Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, representing a serious health problem worldwide for which no antivirals are currently available. Despite the efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials and prophylaxis is based on vector control, which has shown limited success in containing the virus. Herein, we screened 36 compounds using a replicon system and ultimately identified 3-methyltoxoflavin with activity against CHIKV using cell assays (EC50 200 nM). We have additionally screened 3-methyltoxoflavin against a panel of viruses and showed that it also inhibits yellow fever virus (IC50 370 nM, SI=3.2 in Huh-7 cells). 3-methyltoxoflavin is a known protein disulfide isomerase (PDI) inhibitor, and inhibitor of alphaviruses which likely depends on this host protein to aid in facilitating disulfide bond formation and isomerization, since alphaviruses require conserved cysteine residues for proper folding and assembly of the E1 and E2 envelope glycoproteins. In summary we demonstrate that 3-methyltoxoflavin has activity against CHIKV and may represent a starting point for optimization to develop inhibitors to this and other viruses.


title: First description of a Yersinia pseudotuberculosis clonal outbreak in France, confirmed using a new core genome multilocus sequence typing method

abstract: Yersinia pseudotuberculosis is an enteric pathogen causing mild enteritis that can lead to mesenteric adenitis and septicemia in elderly patients. Most cases are sporadic, but outbreaks have already been described in different countries. We report for the first time a Y. pseudotuberculosis clonal outbreak in France, that occurred in 2020. An epidemiological investigation pointed towards the consumption of tomatoes as the likely source of contamination. The Yersinia National Reference Laboratory (YNRL) developed a new cgMLST scheme with 1,921 genes specific to Y. pseudotuberculosis that identified the clustering of isolates associated to the outbreak and allowed to perform molecular typing in real time. In addition, this method allowed to retrospectively identify isolates belonging to this cluster from earlier in 2020. This method, which does not require specific bioinformatic skills, is now used systematically at the YNRL and proves to display an excellent discriminatory power and is available to the scientific community.


title: Why is the average collateral effect of synonymous mutations so similar across alternative reading frames?

abstract: The standard genetic code has been shown to have multiple interesting properties which impact on molecular biology and the evolutionary process. One facet of molecular biology where code structure is particularly important is the origin and evolution of overlapping genes. We have previously reported that the structure of the standard genetic code ensures that synonymous mutations in a protein coding gene will lead to a remarkably similar average "collateral" mutation effect size in at least four out of the five alternative reading frames. Here we show that only 0.26% of alternative codes with the block structure of the standard genetic code perform at least as well as the standard code in this property. Considering this finding within a code optimality framework suggests that this consistent effect size across the different frames may be adaptive. Here we give context for this finding and present a simple model where a trade-off between evolvability and robustness leads to an average mutation effect size which maximises population fitness. This supports the intuition that similar mutation effects across the different alternative reading frames may be an adaptive property of the standard genetic code which facilitates evolvability through the use of alternative reading frames.

category: evolutionary biology


title: Using genome scans to identify genes used repeatedly for adaptation

abstract: Adaptation occurring in similar genes or genomic regions in distinct lineages provides evolutionary biologists with a glimpse at the fundamental opportunities for and constraints to diversification. With the widespread availability of high throughput sequencing technologies and the development of population genetic methods to identify the genetic basis of adaptation, studies have begun to compare the evidence for adaptation at the molecular level among distinct lineages. However, methods to study repeated adaptation are often oriented towards genome-wide testing to identify a set of genes with signatures of repeated use, rather than evaluating the significance at the level of an individual gene. In this study, we propose PicMin, a novel statistical method derived from the theory of order statistics that can test for repeated molecular evolution to estimate significance at the level of an individual gene, using the results of genome scans. This method is generalizable to any number of lineages and indeed, statistical power to detect repeated adaptation increases with the number of lineages that have signals of repeated adaptation of a given gene in multiple lineages. An implementation of the method written for R can be downloaded from https://github.com/TBooker/PicMin.


title: Global, regional, and cryptic population structure in a high gene-flow transatlantic fish

abstract: 1Lumpfish (Cyclopterus lumpus) is a transatlantic marine fish displaying large population sizes and a high potential for dispersal and gene-flow. These features are expected to result in weak population structure. Here, we investigated population genetic structure of lumpfish throughout its natural distribution in the North Atlantic using two approaches: I) 4393 genome wide SNPs and 95 individuals from 10 locations, and II) 139 discriminatory SNPs and 1,669 individuals from 40 locations. Both approaches identified extensive population genetic structuring with a major split between the East and West Atlantic and a distinct Baltic Sea population, as well as further differentiation of lumpfish from the English Channel, Iceland, and Greenland. The targeted discriminatory loci displayed [~]2-5 times higher divergence than the genome wide approach, revealing further evidence of local population substructures. Lumpfish from Isfjorden in Svalbard were highly distinct but resembled most fish from Greenland. The Kattegat area in the Baltic transition zone, formed a previously undescribed distinct genetic group. Also, further subdivision was detected within North America, Iceland, West Greenland, Barents Sea, and Norway. Although lumpfish have considerable potential for dispersal and gene-flow, the observed high levels of population structuring throughout the Atlantic suggests that this species displays natal homing and potentially local populations with adaptive differences. This fine-scale population structure calls for consideration when defining management units for lumpfish fishing stocks and in decisions related to sourcing and moving lumpfish for farming and cleaner fish use.


title: Gamete interaction protein evolution evinces Pantherines hybridizing history

abstract: The jaguar (Panthera onca) is the most relevant predator in South America and an endangered species in Argentina. Therefore genomic, evolutionary and functional studies of this species are strategic to gain biological insights and support conservation efforts. With the advent of sequencing advances the study of relevant character species evolution has to be revisited. Recent comparative genomic analysis revealed a long history of hybridization among extant big cats, which indicates an absence of inter-specific isolation barriers. We aimed to uncover the molecular basis of this inter-species crossing and the evolution of the functional capacities of pantherine gamete interaction Zona Pellucida proteins (ZP) and JUNO. Hi-C long-range sequencing and chromosome-resolved jaguar genome assembly was used to resolve ZP genes loci and reconstruct the evolutionary history of these proteins. Exploring sequence variability in more that 30 Carnivora species, Feliforme and Caniforme sister groups show significant differences in average sequence identity for all studied proteins except ZP4, always showing Caniformes as a more diverse group than Feliformes. Molecular evolution of ZP isoforms involved in sperm-recognition, ZP3 and ZP2, showed similar phylogenetic histories, displaying signatures of positive selection among Carnivores and Caniformes, but not among Pantherines or Feliformes. On the other hand, structural cross-linking ZP subunits, ZP1 and ZP4, displayed a different evolutionary history. The fusion protein IZUMO1R was found to be highly conserved in all studied phylogenetic groups. These results strengthen the idea that ZPs are proteins more related to specific-specificity isolation than IZUMO1R, which is specialized in fusion events. Altogether, our findings indicate that sperm-oocyte interaction and fusion proteins lack the degree of diversification necessary to establish a prezygotic reproductive isolation barrier between pantherines, which would explain observed reticulated patterns of evolution in this clade.


title: Full-length transcriptome maps of reef-building coral illuminate the molecular basis of calcification, symbiosis, and circadian genes

abstract:
BackgroundReef-building corals are critical species for sustaining coral reefs and are highly threatened by global climate change. However, relevant transcriptomic data largely rely on short-read sequencing, which severely limits the understanding of coral molecular mechanisms and leaves many important biological questions unresolved.

ResultsWe sequenced the full-length transcriptomes of four common and frequently dominant reef-building corals, including two Robusta clade species, Pocillopora damicornis and Pocillopora verrucosa, and two Complexa clade species, Acropora muricata and Montipora foliosa, using the PacBio Sequel II platform. We obtained information on gene functions, structures and expression profiles. Among them, a comparative analysis of biomineralization-related genes provided insights into the molecular basis of coral skeletal density. The gene expression profiles of the symbiote Symbiodiniaceae were also isolated and annotated from the holobiont sequence data; these profiles showed more highly convergent traits related to gene structure and expression level than those of coral hosts. Interestingly, we observed that intracellular algal cells share some evolutionary convergence between intracellular symbiosis in corals and intracellular digestion in amphioxus. Finally, a phylogenetic analysis of key circadian clock genes among 27 evolutionarily representative species indicated that there are four key members in early metazoans, including cry genes; Clock or Npas2; cyc or Arntl; and tim, while per, as the fifth member, occurs in Bilateria.

ConclusionsOur work overcomes the incompleteness of short-read sequencing and illuminates the molecular basis of calcification, symbiosis, and circadian genes, thus providing a foundation for further work on the manipulation of skeleton production or symbiosis to promote the survival of these important organisms.


title: High resolution analysis of proteolytic substrate processing

abstract: Proteolysis is a key catalytic event in protein and thus cellular homeostasis. Despite the importance and wide implications of proteolytic processing and degradation, methods describing the degradation of folded proteins at high temporal and spatial resolution are not well established. However, this information is required to obtain a deep mechanistic understanding of proteolytic events and their consequences. Here, we describe an integrated method comprising time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics to reveal the sequential degradation and unfolding of the model substrate annexin A1 by the human serine protease HTRA1. This workflow represents a general strategy for obtaining precise molecular insights into protease-substrate interactions that can be conveniently adapted to studying other posttranslational modifications such as phosphorylation in dynamic protein complexes.

category: biochemistry


title: B cell receptor repertoire analysis unveils dynamic antibody response and severity markers in COVID-19 patients

abstract: Humoral and cell mediated immunity are critical against viral infections. The knowledge of composition, diversity, gene usage of the B cell repertoires helps in determining the immune response to SARS-CoV-2 infection. Examining B cell response provides insights on therapeutic antibodies, disease severity markers and aids in predicting vaccine response. We have analyzed public domain immunoglobulin sequencing data from PBMCs of SARS-CoV-2 infected individuals to gain a better understanding of B cell repertoire in patients. Public clonotypes showed increased usage of IGHV3, IGHV4, IGKV1, IGKV3, IGLV3 and IGLV2 family genes during the acute phase infection. Identical CDR3 sequences were identified for heavy (H), kappa (K) and lambda (L) chains across individuals, indicating the convergence of B cell selection during SARS-CoV-2 infection. While the immune repertoire dynamically changed over the course of convalescence, there were persistent clones across early and late timepoints. The diversity of antibody repertoire, measured by Shannon-Weiner diversity index for H and K chains, reduced during the acute phase of infection. In addition, the repertoire diversity was low in severe patients compared to patients with mild or moderate symptoms. Increased usage of IGHV4-59 gene was observed in COVID-19 patients with severe symptoms requiring ventilator support at 2 weeks and 3 weeks post symptom onset. IGHV4-59 is reported to have rheumatoid factor (RF) activity with high affinity for IgG and the elevated level of IGHV4-59 provides a potential mechanism for the increased autoimmune responses in severe patients. Correlation of the clinical features with the B cell receptor repertoire dynamics elucidated public antibody clonotypes and disease severity markers for COVID-19.

category: immunology


title: SARS-CoV-2 harnesses host translational shutoff and autophagy to optimize virus yields: The role of the envelope (E) protein

abstract:
The SARS-CoV-2 virion is composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). E spans the membrane a single time and is the smallest, yet most enigmatic of the structural proteins. E is conserved among coronaviruses and has an essential role in virus-mediated pathogenesis. We found that ectopic expression of E had deleterious effects on the host cell as it activated stress responses, leading to phosphorylation of the translation initiation factor eIF2 and LC3 lipidation that resulted in host translational shutoff. During infection E is highly expressed although only a small fraction is incorporated into virions, suggesting that E activity is regulated and harnessed by the virus to its benefit. In support of this, we found that the {gamma}1 34.5 protein of herpes simplex virus 1 (HSV-1) prevented deleterious effects of E on the host cell and allowed for E protein accumulation. This observation prompted us to investigate whether other SARS-CoV-2 structural proteins regulate E. We found that the N and M proteins enabled E protein accumulation, whereas S prevented E accumulation. While {gamma}1 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. This negative effect of {gamma}1 34.5 was most likely associated with failure of SARS-CoV-2 to divert the translational machinery and with deregulation of autophagy pathways. Overall, our data suggest that SARS-CoV-2 causes stress responses and subjugates these pathways, including host protein synthesis (phosphorylated eIF2) and autophagy, to support optimal virus production.

ImportanceIn 2020, a new {beta}-coronavirus, SARS-CoV-2, entered the human population that has caused a pandemic resulting in 6 million deaths worldwide. Although closely related to SARS-CoV, the mechanisms of SARS-CoV-2 pathogenesis are not fully understood. We found that ectopic expression of the SARS-CoV-2 E protein had detrimental effects on the host cell, causing metabolic alterations including shutoff of protein synthesis and mobilization of cellular resources through autophagy activation. Co-expression of E with viral proteins known to subvert host antiviral responses such as autophagy and translational inhibition, either from SARS-CoV-2 or from heterologous viruses increased cell survival and E protein accumulation. However, such factors were found to negatively impact SARS-CoV-2 infection, as autophagy contributes to formation of viral membrane factories, and translational control offers an advantage for viral gene expression. Overall, SARS-CoV-2 has evolved mechanisms to harness host functions that are essential for virus replication.


title: A Computational Pipeline to Identify Potential Drug Targets and Interacting Chemotypes in SARS-CoV-2

abstract: 1Minimizing the human and economic costs of the COVID-19 pandemic and of future pandemics requires the ability to develop and deploy effective treatments for novel pathogens as soon as possible after they emerge. To this end, we introduce a unique, computational pipeline for the rapid identification and characterization of binding sites in the proteins of novel viruses as well as the core chemical components with which these sites interact. We combine molecular-level structural modeling of proteins with clustering and cheminformatic techniques in a computationally efficient manner. Similarities between our results, experimental data, and other computational studies provide support for the effectiveness of our predictive framework. While we present here a demonstration of our tool on SARS-CoV-2, our process is generalizable and can be applied to any new virus, as long as either experimentally solved structures for its proteins are available or sufficiently accurate homology models can be constructed.

category: bioinformatics


title: Acute manipulation and real-time visualization of membrane trafficking and exocytosis in Drosophila

abstract: Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but essential tools for investigating the dynamics of trafficking have been limited to cultured cells. Here we present a system that enables acute manipulation and real-time visualization of membrane trafficking through reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision using streptavidin "hooks" and biotin-induced release in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and of apical secretion in tracheal tubes and the spatiotemporal dynamics of tricellular junction assembly in epithelia. Furthermore, hook-induced ER retention enables tissue-specific knockdown of secretory protein function. The system is compatible with available protein-traps and is widely applicable to investigate membrane trafficking in diverse cell types in vivo.

category: cell biology


title: Patient-derived parathyroid organoids as tracer and drug-screening application model

abstract: Parathyroid diseases are characterized by dysregulation of calcium homeostasis and alterations in parathyroid hormone (PTH) excretion. The understanding of parathyroid hyperplastic growth and the development of parathyroid-targeted treatment and imaging tracers could benefit from in vitro models. Therefore, we aim to establish stem cell-derived, three-dimensional organoids representing human parathyroid tissue in vitro. Patient-derived hyperplastic parathyroid tissue was dispersed and parathyroid organoids (PTO) were cultured and characterized. PTO-derived cells were shown to exhibit in vitro self-renewal over several passages, indicative of the presence of putative stem cells. Immunofluorescence and RNA-sequencing confirm that PTO phenocopy hyperplastic parathyroid tissue. Exposure of PTO to increasing calcium concentrations and to PTH-lowering drugs resulted in a significantly reduced PTH excretion. Next to this, the PTO showed specific binding of 11C-methionine to the targeted receptor. Additionally, when organoids were incubated with 99mTc-sestamibi, we observed a higher uptake in PTOs from patients with a 99mTc-sestamibi positive scan compared to patients with a negative scan. These data show functionality of PTOs resembling the parathyroid. In conclusion, we present a patient-derived PTO culture, that recapitulates the originating tissue on gene and protein expression and functionality. This PTO model paves the way for future physiology studies and therapeutic target and tracer discovery.


title: A nuclear receptor facilitates differentiation of human PSCs into more mature hepatocytes

abstract: The capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types.

category: developmental biology


title: Forest refuge areas and carbon emissions from tropical deforestation in the 21st century

abstract:
Tropical forests are disappearing at an alarming rate due to human activities. Here, we provide spatial models of deforestation in 92 countries covering all the tropical moist forests in the world. Our models confirm the effectiveness of protected areas in displacing deforestation and the negative impact of roads and landscape fragmentation on forest conservation in the tropics. Using our models, we derive high-resolution pantropical maps of the deforestation risk and future forest cover for the 21st century under a "business-as-usual" scenario based on the deforestation rates observed in the 2010s. Although under this scenario, large areas of tropical moist forest should remain in the heart of the Amazon, in the Congo Basin, and in New Guinea in 2100, 48% (39-56%) of all forest cover is expected to disappear during the course of the 21st century, and many countries will have lost all their forests by 2100. The remaining forests will be highly fragmented and located in remote places. As future deforestation will concern forests with higher aboveground carbon stocks, annual carbon emissions associated with tropical deforestation are expected to increase by +0.161 Pg/yr (+35%) between the 2010s and the 2090s.

Significance StatementUnder a "business-as-usual" scenario of deforestation (i.e. projecting the 2010-2020 deforestation rates at the country level in the future), three quarters of the tropical moist forests that existed in 2000 will have disappeared around years 2120, 2160, and 2220 in Southeast Asia, Africa, and Latin America, respectively. By 2100, 41 tropical countries, plus 14 states in Brazil and one region in India, will lose all their tropical forests. Remaining forests will be highly fragmented and concentrated in remote areas (far from roads and towns), preferentially in protected areas, and at high elevations. Future deforestation will concern forests with higher carbon stocks. In the absence of change in the deforestation rates, annual carbon emissions associated with tropical deforestation will increase from 0.467 Pg/yr in the 2010s to 0.628 Pg/yr in the 2090s (+35%), making tropical forests a major carbon source in the 21st century.

category: ecology


title: Fewer non-native insects in freshwater than in terrestrial habitats across continents

abstract:
AimBiological invasions are a major threat to biodiversity in both aquatic and terrestrial habitats. Insects represent an important group of species in freshwater and terrestrial habitats, and they constitute a large proportion of non-native species. However, while many non-native insects are known from terrestrial ecosystems, it remains unclear how they are represented in freshwater habitats. Comparisons of the richness of invaders relative to the richness of native species between freshwater and terrestrial habitats are scarce, which hinders syntheses of invasion processes. Here, we used data from three regions on different continents to determine whether non-native insects are under- or overrepresented in freshwater compared to terrestrial assemblages.

LocationEurope, North America, New Zealand

MethodsWe compiled a comprehensive inventory of the native and non-native insect species established in freshwater and terrestrial habitats of the three study regions. We then contrasted the richness of non-native and native species among freshwater and terrestrial insects for all insect orders in each region. Using binomial regression, we analysed the proportions of non-native species in freshwater and terrestrial habitats across the three regions.

ResultsIn most insect orders living in freshwater, non-native species were under-represented, while they were over-represented in a number of terrestrial orders. This pattern occurred in purely aquatic orders as well as in orders with both freshwater and terrestrial species. Overall, the proportion of non-native species was significantly lower in freshwater than in terrestrial species.

Main conclusionsDespite the numerical and ecological importance of insects among all non-native species, non-native insect species are surprisingly rare in freshwater habitats, and this pattern is consistent across the three investigated study regions. We briefly review hypotheses concerning species traits and invasion pathways that are most likely to explain these patterns. Our findings contribute to a growing appreciation of drivers and impacts of biological invasions.


title: flashfm-ivis: interactive visualisation for fine-mapping of multiple quantitative traits

abstract:
Summaryflashfm-ivis provides a suite of interactive visualisation plots to view potential causal genetic variants that underlie associations that are shared or distinct between multiple quantitative traits and compares results between single- and multi-trait fine-mapping. Unique features include network diagrams that show joint effects between variants for each trait and regional association plots that integrate fine-mapping results, all with user-controlled zoom features for an interactive exploration of potential causal variants across traits.

Availability and Implementationflashfm-ivis is an open-source software under the MIT license. It is available as an interactive web-based tool (http://shiny.mrc-bsu.cam.ac.uk/apps/flashfm-ivis/) and as an R package format. Code and documentation are available at https://github.com/fz-cambridge/flashfm-ivis and https://zenodo.org/record/6376244#.YjnarC-l2X0. Additional features can be downloaded as standalone R libraries to encourage reuse.

Contactfeng.zhou@mrc-bsu.cam.ac.uk or jennifer.asimit@mrc-bsu.cam.ac.uk

category: genetics


title: High-throughput and high-sensitivity biomarker monitoring in body fluid by FAIMS-enhanced fast LC SureQuant™ IS targeted quantitation

abstract: Targeted proteomics methods have been greatly improved and refined over the last decade and are becoming increasingly the method of choice in protein and peptide quantitative assays. Despite the tremendous progress, targeted proteomics assays still suffer from inadequate sensitivity for lower abundant proteins and throughput, especially in complex biological samples. These attributes are essential for establishing targeted proteomics methods at the forefront of clinical use. Here, we report an assay utilizing the SureQuant internal standard triggered targeted method on a newest generation mass spectrometer coupled with a FAIMS (high-field asymmetric waveform ion mobility spectrometry) interface ion mobility device and an EvoSep One liquid chromatography platform, which displays markedly enhanced sensitivity and a high throughput of 100 samples per day. We demonstrate the robustness of this method by quantifying proteins ranging six orders of magnitude in human wound fluid exudates, a biological fluid that exhibits sample complexity and composition similar to plasma. Among the targets quantified were low-abundance proteins such at TNFA and IL1B, highlighting the value of this method in the quantification of trace amounts of invaluable biomarkers that were until recently hardly accessible by targeted proteomics methods. Taken together, this method extends the toolkit of targeted proteomics assays and will help to drive forward mass spectrometry-based proteomics biomarker quantification.


title: Transfer learning of an in vivo-derived senescence signature identifies conserved and tissue-specific senescence across species and diverse pathologies

abstract: Senescent cells (SnCs) contribute to normal tissue development and repair but accumulate with aging where they are implicated in a number of pathologies and diseases. Despite their pathological role and therapeutic interest, SnC phenotype and function in vivo remains unclear due to the challenges in identifying and isolating these rare cells. Here, we developed an in vivo-derived senescence gene expression signature using a model of the foreign body response (FBR) fibrosis in a p16Ink4a-reporter mouse, a cell cycle inhibitor commonly used to identify SnCs. We identified stromal cells (CD45-CD31- CD29+) as the primary p16Ink4a expressing cell type in the FBR and collected the cells to produce a SnC transcriptomic signature with bulk RNA sequencing. To computationally identify SnCs in bulk and single-cell data sets across species and tissues, we used this signature with transfer learning to generate a SnC signature score (SenSig). We found senescent pericyte and cartilage-like fibroblasts in newly collected single cell RNAseq (scRNASeq) data sets of murine and human FBR suggesting populations associated with angiogenesis and secretion of fibrotic extracellular matrix, respectively. Application of the senescence signature to human scRNAseq data sets from idiopathic pulmonary fibrosis (IPF) and the basal cell carcinoma microenvironment identified both conserved and tissue-specific SnC phenotypes, including epithelial-derived basaloid and endothelial cells. In a wound healing model, ligand-receptor signaling prediction identified putative interactions between SnC SASP and myeloid cells that were validated by immunofluorescent staining and in vitro coculture of SnCs and macrophages. Collectively, we have found that our SenSig transfer learning strategy from an in vivo signature outperforms in vitro-derived signatures and identifies conserved and tissue-specific SnCs and their SASP, independent of p16Ink4a expression, and may be broadly applied to elucidate SnC identity and function in vivo.

category: bioengineering


title: MRSCloud: a Cloud-based MR Spectroscopy Tool for Basis Set Simulation

abstract:
BackgroundAccurate quantification of in vivo proton magnetic resonance spectra involves modeling with a linear combination of known metabolite basis functions. Basis sets can be generated by numerical simulation using the quantum mechanical density-matrix formalism. Accurate simulations for a basis set require correct sequence timings, and pulse shapes and durations.

PurposeTo present a cloud-based spectral simulation tool  MRSCloud. It allows community users of MRS to simulate a vendor- and sequence-specific basis set online in a convenient and timeefficient manner. This tool can simulate basis sets for 3 major MR scanner vendors (GE, Philips, Siemens), including conventional acquisitions and spectral editing schemes (MEGA, HERMES, HERCULES) with PRESS and semi-LASER localization.

Study TypeProspective.

SpecimenN/A

Field Strength/SequenceSimulations of 3T basis sets for conventional and spectral-editing sequences (MEGA, HERMES, HERCULES) with PRESS and sLASER localizations.

AssessmentSimulated metabolite basis functions generated by MRSCloud are compared to those generated by FID-A and MARSS, and a phantom-acquired basis-set from LCModel.

Statistical TestsIntraclass correlation coefficients (ICC) were calculated to measure the agreement between individual metabolite basis functions generated using different packages. Statistical analysis was performed using R in RStudio.

ResultsSimulation time for a full basis set is approximately 1 hour. ICCs between MRSCloud and FID-A were at least 0.98 and ICCs between MRSCloud and MARSS were at least 0.96. ICCs between simulated MRSCloud basis spectra and acquired LCModel basis spectra were lowest for Gln at 0.68 and highest for NAA at 0.96.

Data ConclusionSubstantial reductions in runtime have been achieved by implementing the 1D projection method, coherence-order filtering, and pre-calculation of propagators. High ICC values indicated that the accelerating features are running correctly and produce comparable and accurate basis sets. The generated basis set has been successfully used with LCModel.


title: Inference of B cell clonal families using heavy/light chain pairing information

abstract: AO_SCPLOWBSTRACTC_SCPLOWNext generation sequencing of B cell receptor (BCR) repertoires has become a ubiquitous tool for understanding the antibody-mediated immune response: it is now common to have large volumes of sequence data coding for both the heavy and light chain subunits of the BCR. However, until the recent development of high throughput methods of preserving heavy/light chain pairing information, these samples contained no explicit information on which heavy chain sequence pairs with which light chain sequence. One of the first steps in analyzing such BCR repertoire samples is grouping sequences into clonally related families, where each stems from a single rearrangement event. Many methods of accomplishing this have been developed, however, none so far has taken full advantage of the newly-available pairing information. This information can dramatically improve clustering performance, especially for the case of the light chain; the light chain has traditionally been challenging for clonal family inference because of its low diversity and consequent abundance of non-clonal families with indistinguishable naive rearrangements. Here we present a method of incorporating this pairing information into the clustering process in order to arrive at a more accurate partition of the data into clonally related families. We also demonstrate two methods of fixing imperfect pairing information, which may allow for simplified sample preparation and increased sequencing depth. Finally, we describe several other improvements to the partis software package (https://github.com/psathyrella/partis).


title: Ontology-Aware Biomedical Relation Extraction

abstract:
MotivationAutomatically extracting relationships from biomedical texts among multiple sorts of entities is an essential task in biomedical natural language processing with numerous applications, such as drug development or repurposing, precision medicine, and other biomedical tasks requiring knowledge discovery. Current Relation Extraction (RE) systems mostly use one set of features, either as text, or more recently, as graph structures. The state-of-the-art systems often use resource-intensive hence slow algorithms and largely work for a particular type of relationship. However, a simple yet agile system that learns from different sets of features has the advantage of adaptability over different relationship types without an extra burden required for system re-design.

ResultsWe model RE as a classification task and propose a new multi-channel deep neural network designed to process textual and graph structures in separate input channels. We extend a Recurrent Neural Network (RNN) with a Convolutional Neural Network (CNN) to process three sets of features, namely, tokens, types, and graphs. We demonstrate that entity type and ontology graph structure provide better representations than simple token-based representations for RE. We also experiment with various sources of knowledge, including data resources in the Unified Medical Language System (UMLS) to test our hypothesis. Extensive experiments on four well-studied biomedical benchmarks with different relationship types show that our system outperforms earlier ones. Thus, our system has state-of-the-art performance and allows processing millions of full-text scientific articles in a few days on one typical machine.


title: Crimp: fast and scalable cluster relabeling based on impurity minimization

abstract:
MotivationTo analyze population structure based on multilocus geno-type data, a variety of popular tools perform model-based clustering, as-signing individuals to a prespecified number of ancestral populations. Since such methods often involve stochastic components, it is a common practice to perform multiple replicate analyses based on the same input data and parameter settings. Their results are typically affected by the label-switching phenomenon, which complicates their comparison and summary. Available tools allow to mitigate this problem, but leave room for improvements, in particular, regarding large input datasets.

ResultsIn this work, I present CO_SCPLOWRIMPC_SCPLOW, a lightweight command-line tool, which offers a relatively fast and scalable heuristic to align clusters across multiple replicate clusterings consisting of the same number of clusters. For small problem sizes, an exact algorithm can be used as alternative. Additional features include row-specific weights, input and output files similar to those of CLUMPP (Jakobsson & Rosenberg, 2007), and the evaluation of a given solution in terms of either CLUMPPs and its own objective functions. Benchmark analyses show that CO_SCPLOWRIMPC_SCPLOW, especially when applied to larger datasets, tends to outperform alternative tools considering runtime requirements and various quality measures.

AvailabilityCO_SCPLOWRIMPC_SCPLOWs source code along with precompiled binaries for Linux and Windows, usage guidelines and benchmark code are freely available at https://github.com/ulilautenschlager/crimp.

Contactulrich.lautenschlager@ur.de


title: Competitive membrane wetting of polymer blends in artificial cells initiates phase separation and promotes fractionation

abstract: Biomolecular condensates driven by liquid-liquid phase separation (LLPS) have received attention as novel activity regulators of living organisms. In intracellular LLPS, an important question is what type of biomolecules form condensates under what conditions. In this regard, possible interactions between biomolecules have been investigated. Recently, LLPS condensates have been reported to regulate the membrane structure upon wetting. However, the possibility of membrane wetting, in which the membrane conversely regulates the LLPS, remains unexplored. Using droplets of short polyethylene glycol and long dextran blends encapsulated with a lipid membrane, we demonstrate that membrane wetting regulates LLPS in cell-size spaces and alters the equilibrium state. In smaller droplets, the two-phase region expands beyond the bulk system, and the fractionation degree increases, particularly during the separation between short PEG and long dextran. We explain the space-size dependent LLPS based on the competitive membrane wetting between the polymers. Smaller droplets promote the membrane wetting of short PEG, which enhances the depletion force between long dextran molecules and finally induces LLPS. This shows that competition for membrane wettability among various molecules can regulate LLPS in cell-size spaces, rendering this LLPS principle feasible in living cells.

category: biophysics


title: Long-term cellular cargo tracking reveals intricate trafficking through active cytoskeletal networks

abstract: A eukaryotic cell is a microscopic world within which efficient material transport is essential. To examine cargo transport in a crowded cellular environment, we tracked unlabeled cargos in directional motion in a massively parallel fashion using the interferometric scattering microscopy. Our label-free, cargo-tracing method revealed not only dynamic cargo transportation but also the fine architecture of the actively used cytoskeletal highways and the long-term evolution of the associated traffic at sub-diffraction resolution via a myriad of molecular strokes. Cargos experience a traffic jam, but they have an effective strategy to circumvent it: moving together in tandem or migrating collectively. All taken together, a cell is an incredibly complex and busy space where the principle and practice in transportation intriguingly parallel those of our macroscopic world.


title: Cellular Mechanisms Underlying Central Sensitization in a Mouse Model of Chronic Muscle Pain

abstract: Chronic pain disorders are often associated with psychiatric symptoms. The central nucleus of the amygdala (CeA) has emerged as an integrative hub for nociceptive and affective components during the development of central pain. Although the exact cause for this process remains unknown, prior adverse injuries are precipitating factors and thought to transform nociceptors into a primed state for chronic pain. However, the cellular basis underlying the primed state and the subsequent pain chronification remains unknown. Here, we investigated cellular and synaptic alterations of the CeA in a mouse model of chronic muscle pain. In these mice, local infusion of pregabalin, a clinically approved drug for fibromyalgia and other chronic pain disorders, into the CeA or selective inactivation of somatostatin-expressing CeA (CeA-SST) neurons during the priming phase prevented pain chronification. Further, electrophysiological recording revealed that CeA-SST neurons received increased excitatory transmission and showed enhanced excitability in chronic pain states. In line with the possible role of CeA-SST neurons in central sensitization, chemogenetic inactivation of CeA-SST neurons or pharmacological suppression of nociceptive afferents from the brainstem to CeA-SST neurons by pregabalin after the development of chronic muscle pain alleviated pain and negative emotions.

category: neuroscience


title: Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

abstract: During the SARS-CoV-2 intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-COVID-19 drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the Receptor Binding Domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. When used to immunise mice, the 3CLpro-RBD chimer elicited antibodies mainly to the 3CL-pro portion of the molecule indicating that a different chimeric composition (i.e., RBD/full Spike-3CLpro) or expression system (i.e., mammalian cells), might be required to produce and deliver a RBD with immunogenicity similar to the native protein. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.


title: Changes of urinary proteomic before and after QIV and COVID-19 vaccination

abstract: We first collected a young peoples urine samples cohort of quadrivalent influenza vaccine. Urine protein at 24 hours after vaccination was enriched in immune-related pathways, though the specific pathways varied. Perhaps because different people may be in a previous life encountered some of the viruses in the vaccine, the second immunization was triggered. Or everyone has a different constitution, exposure to the same virus triggering different immunity. We then collected urine samples from several uninfected SARS-CoV-2 young people before and after the first, second, and third doses of the COVID-19 vaccine. We found that the differential protein compared between after the second dose (24h) and before the second dose enriched pathways were involved in regulated exocytosis and immune-related pathways, indicating not first exposure to antigen. Surprisingly, the urine differential protein-enriched pathways before and after the first dose were similar to those before and after the second dose. We assume that although the volunteers have not been infected with SARS-CoV-2, they might have been exposed to other coimmunogenic coronaviruses. 2~4h after the third vaccination, the differentially expressed protein also enriched regulated exocytosis and immune-related pathways, indicating that the body has triggered the immune response in a very short time after vaccination, and urine proteome is a good window to monitor the changes of human immune function.


title: Comparative Analysis of Right Ventricular Metabolic Reprogramming in Pre-clinical Rat Models of Severe Pulmonary Hypertension-induced Right Ventricular Failure

abstract:
BackgroundPulmonary hypertension (PH) leads to right ventricular (RV) hypertrophy and failure (RVF). The precise mechanisms of the metabolic basis of maladaptive PH-induced RVF (PH-RVF) are yet to be fully elucidated. Here we performed a comparative analysis of RV-metabolic reprogramming in MCT and Su/Hx rat models of severe PH-RVF using targeted metabolomics and multi-omics.

MethodsMale Sprague Dawley rats (250-300gm; n=15) were used. Rats received subcutaneous monocrotaline (60mg/kg; MCT; n=5) and followed for [~]30-days or Sugen (20mg/kg; Su/Hx; n=5) followed by hypoxia (10%O2; 3-weeks) and normoxia (2-weeks). Controls received saline (Control; n=5). Serial echocardiography was performed to assess cardiopulmonary hemodynamics. Terminal RV-catheterization was performed to assess PH. Targeted metabolomics was performed on RV tissue using UPLC-MS. RV multi-omics analysis was performed integrating metabolomic and transcriptomic datasets using Joint Pathway Analysis (JPA).

ResultsMCT and Su/Hx rats developed severe PH, RV-hypertrophy and decompensated RVF. Targeted metabolomics of RV of MCT and Su/Hx rats detected 126 and 125 metabolites respectively. There were 28 and 24 metabolites significantly altered in RV of MCT and Su/Hx rats, respectively, including 11 common metabolites. Common significantly upregulated metabolites included aspartate and GSH, whereas downregulated metabolites included phosphate, -ketoglutarate, inositol, glutamine, 5-Oxoproline, hexose phosphate, creatine, pantothenic acid and acetylcarnitine. JPA highlighted common genes and metabolites from key pathways such as glycolysis, fatty acid metabolism, oxidative phosphorylation, TCA cycle etc.

ConclusionsComparative analysis of metabolic reprogramming of RV from MCT and Su/Hx rats reveals common and distinct metabolic signatures which may serve as RV-specific novel therapeutic targets for PH-RVF.

category: physiology


title: A Novel Restraint Device to Improve Safety and Efficacy of Blood Collection During Non-Terminal Sampling of Bats

abstract: There are a variety of blood collection techniques described in the literature for unanesthetized bats, which typically require multiple sharps (e.g., needles, lancets, etc.), competent animal handling for prolonged periods, and usually involve two individuals. With the challenges inherent to non-terminal sampling of blood from bats, as well as the growing need for the use of this technique across multiple disciplines and industries, an improved blood collection method is needed. We report the creation of a bat restraint device specifically designed for a single individual to safely collect blood from anesthetized or non-anesthetized bats. The utility of this restraint device is multifaceted, serving as a safety measure for both animal and handler, as well as increasing the efficiency of blood collection. The restraint device was tested during two laboratory bat studies, Afterwards, the users of the restraint device were provided with a 10-question survey questionnaire to record their opinions on its usage. In total 80% of responses were considered positive, 15% considered neutral, and 5% considered negative. Survey questions that all participants responded to positively when in comparison to the traditional method of blood collection from bats include "easier to perform", "safer to bats", and "safer to the individual". While using the restraint devices during the laboratory studies, no needle sticks, bites, or scratches to laboratorians occurred, and no observable health issues or complication due to blood collection in the bats bled using the restraint devices.

category: zoology


title: Prenatal methadone exposure leads to long-term memory impairments and disruptions of dentate granule cell function in a sex-dependent manner

abstract: Prenatal opioid exposures lead to extensive cognitive and emotion-regulation problems in children, persisting at least through school-age. Methadone, an opioid typically used for the treatment of opioid use disorder, has been approved for use in pregnant women for several decades. Importantly, however, the impacts of prenatal methadone exposure (PME), particularly on offspring as they progress into adulthood, has not been extensively examined. In recent years, children and young animal models have shown cognitive deficits related to PME, including evidence of hippocampal dysfunction. The present work aims to examine the persistent nature of these deficits, as well as determine how they may differ by sex. Pregnant Sprague Dawley rats either received subcutaneous methadone or water injections twice daily from gestational days 3-20 or were left undisturbed. Following postnatal day 70, male and female offspring were behaviorally tested for impairments in recognition memory using the Novel Object Recognition task and working spatial memory through Spontaneous Alternation. Additionally, using whole-cell patch-clamp electrophysiology, hippocampal dentate granule cell function was examined in adult offspring. Results indicate that methadone-exposed females showed decreased excitability and increased inhibition of these dentate granule cells, while males did not. These findings were accompanied by impairments in female working spatial memory and impaired recognition memory of both sexes. Overall, this work supports the continued investigation of the long-term effects of PME on adult male and female learning and memory, as well as promotes further exploration of adult hippocampal function as a neural mechanism impacted by this exposure.


title: Neural activity in the anterior cingulate cortex is required for effort-based decision making

abstract:
Adaptive decision making requires the evaluation of cost-benefit tradeoffs to guide action selection. Effort-based decision making involves weighing predicted gains against effort costs and is disrupted in several neuropsychiatric disorders. The ACC is postulated to control effort-base choice via its role in encoding the value of overcoming effort costs in rodent effort-based decision making assays. However, temporally precise methods of manipulating neural activity have rarely been applied to effort-based decision making. We developed and validated a mouse version of the barrier T-maze, and used optogenetics to inhibit ACC excitatory neurons at specific times during this task. Bilateral inhibition of ACC rapidly and reversibly impaired preference to exert greater effort for a larger reward when a less rewarded, low effort alternative was available. Equalizing effort for potential choice options led mice to choose the high reward arm of the maze regardless of whether the ACC was inhibited or not. The mechanics of choice behavior were altered during trials where the ACC was inhibited, but there were no effects on overall mobility or tendency to exert effort in an unrelated assay. These findings establish causality between ACC neural activity during a choice trial and effortful action selection during cost-benefit decision making.

SIGNIFICANCE STATEMENTDisturbances in evaluating effort-based costs during decision making occur in depression, schizophrenia, addiction and Parkinsons disease. Precisely resolving the function of prefrontal brain regions in mediating these processes will reveal key loci of dysfunction and potential therapeutic intervention in these disorders.


title: Elucidating the role of c-di-AMP in Mycobacterium smegmatis: phenotypic characterization and functional analysis

abstract: Cyclic-di-AMP (c-di-AMP) is a newly discovered secondary messenger molecule that plays a critical role in monitoring several important cellular processes, especially in several Gram-positive bacteria signal transduction pathways. In this study, we seek to unravel the physiological significance of the molecule c-di-AMP in Mycobacterium smegmatis under different conditions, using strains with altered c-di-AMP levels: c-di-AMP null mutant ({Delta}disA) and a c-di-AMP over-expression mutant ({Delta}pde). Our thorough analysis of the mutants revealed that the intracellular concentration of c-di-AMP could determine many basic phenotypes such as colony architecture, cell shape, cell size, membrane permeability etc. Additionally, it was shown to play a significant role in multiple stress adaptation pathways in the case of different DNA and membrane stresses. Our study also revealed how the biofilm phenotype of M. smegmatis cells are dependent on intracellular c-di-AMP concentration. Next, we checked how c-di-AMP contributes to antibiotic tolerance characteristics of M. smegmatis, which was followed by a detailed transcriptome profile analysis to reveal key genes and pathways regulated by c-di-AMP in mycobacteria.


title: Human neutrophils direct epithelial cell extrusion to enhance intestinal epithelial host defense during Salmonella infection

abstract:
Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and then seeded with primary human polymorphonuclear leukocytes (PMN-HIOs), specifically neutrophils and analyzed for bacterial growth and host cell survival. Surprisingly, PMNs did not affect luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed intestinal epithelial cells that could be blocked by Caspase-1 or Caspase-3 inhibition. Cleaved Caspase-3 was present in epithelial cells, but expression of the inflammasome adaptor, ASC, was only detected in PMNs. Caspase inhibition also increased bacterial burden in the epithelium of the PMN-HIO, suggesting PMNs enhance activation of cell death pathways in human intestinal epithelial cells as a protective response to infection. These data support a critical function for neutrophils beyond their antimicrobial role whereby they amplify cell death and extrusion of epithelial cells from the Salmonella-infected intestinal monolayer.

Significance statementNeutrophils are early responders to Salmonella intestinal infection, but how they influence infection progression and outcome is unknown. Here we use a co-culture model of human intestinal organoids and human primary neutrophils to study the contribution of human neutrophils to Salmonella infection of the intestinal epithelium. We found that neutrophils markedly enhanced epithelial defenses, including enhancing cell extrusion to reduce intraepithelial burden of Salmonella and association with the epithelium, rather than directly killing Salmonella in the HIO lumen. These findings reveal a novel role for neutrophils in the gut beyond killing invading pathogens and illuminate how neutrophils can reprogram cells in the gut environment to enhance antimicrobial defenses.


title: Towards a multi-stressor theory for coral reefs in a changing world

abstract:
Coral reefs are facing a constant barrage of human impacts, including eutrophication, overharvesting and climate change. However, research and management are just beginning to depart from a single-dominant-stressor paradigm and a holistic ecosystem-based understanding of these systems is still in its infancy. We expand on a well-known theoretical model to motivate an integrated multi-stressor framework for coral reefs by incorporating empirical evidence of multi-stressor impacts (overfishing, eutrophication and climate-driven mortality) as well as general ecological and theoretical concepts. We show that: i) the geometry of a simple, empirically-motivated model suggests nutrients and harvesting can operate similarly in driving shifts from coral- to algae-dominated reefs; ii) these impacts increase nonlinearly when acting concurrently, resulting in clear context-dependent management implications; and iii) this same geometry suggests climate-driven coral mortality (temperature-stress, cyclonic storms) can drive the presence of long transients and climate-driven alternate states, even in moderately-impacted ecosystems. These results imply that reefs that appear to be in a "safe space" may in fact be in danger of being pushed into a degraded algae-dominated state as storms and bleaching events are increasing in frequency and magnitude. Altogether, we find that responses in benthic composition as "signatures of change" to multi-stressors allows us to develop a predictive multi-stressor framework for coral reefs. In line with this theory, we detail empirical evidence from Barbados that highlights the context-dependent nature of coral reefs in a changing world. Our results present novel and generalizable insights into the functioning of coral reefs, that draw from classic theoretical and ecological concepts such as keystone predation theory, ecological succession and life history theory, as well as the emerging fields of long transients and early warning signals. By bridging coral reef ecology and general ecological concepts, we can better understand ecosystem functioning and resilience in these important yet highly threatened systems.

Open Research Statement: Data used in this manuscript are already published and publicly available. Sources can be found within the manuscript and in Supplement B. Mathematical analyses and simulations were performed in Wolfram Mathematica (version 12.1.0.0); code is available at: https://github.com/carlingbieg/corals


title: Sex and genetic specific effects on behavioral, but not metabolic, responses to a high fat diet in heterogeneous stock rats

abstract: Obesity is a growing epidemic associated with a range of comorbidities, including anxiety and depression. Genetics and environmental factors such as diet contribute to both adiposity and anxiety/depression. Heterogeneous stock (HS) rats are an outbred colony and useful for genetic mapping of complex traits. We have previously shown that HS male rats exhibit worsened metabolic and behavioral health in response to high fat diet (HFD). This study aims to determine if females have similar response to diet and if response to diet interacts with genetic background. We measured multiple metabolic (body weight, fat pad weight, glucose tolerance, fasting glucose and insulin) and behavioral (elevated plus maze, open field test, and forced swim test) outcomes in a large cohort of male and female rats on either HFD or low fat diet (LFD). We estimated overall heritability as well as heritability of response to diet for each outcome. Both sexes showed worsened metabolic measures when fed HFD compared to LFD. In contrast, only males exhibited altered behavioral responses to HFD relative to LFD, with no effect in females. Most metabolic and behavioral measures showed overall heritability in both sexes. In contrast, although there was some evidence for gene by diet (GxD) interactions for behavioral measures in males, GxD interactions were generally not found for the metabolic measures. These data demonstrate an important role of diet, sex and genetics in metabolic and behavioral phenotypes in HS rats, with a potential role of gene by diet interactions for behavioral outcomes only in males.


title: Intrathecal tracer dispersion is accelerated by high volume injection and natural micromixing

abstract:
BackgroundTraditionally, there is a widely held belief that drug dispersion after intrathecal (IT) delivery is confined to a small location near the injection site. We posit that high volume infusions can overcome this perceived limitation of IT administration.

MethodsTo test our hypothesis, subject-specific deformable phantom models of the human central nervous system were manufactured so that tracer infusion could be realistically replicated in vitro over the entire physiological range of pulsating cerebrospinal fluid (CSF) amplitudes and frequencies. Dispersion of IT injected tracers was studied systematically with high-speed optical methods to determine the relative impact of injection parameters including infusion volume, flow rate and catheter configurations and natural CSF oscillations.

ResultsOptical imaging analysis of high-volume infusion experiments showed that tracer disperses quickly throughout the spinal subarachnoid space (SAS) reaching the cervical region in less than ten minutes; this is much faster than suggested by prior theories (Taylor-Aris-Watson dispersion). Our experiments indicate that micro-mixing patterns induced by oscillatory CSF flow around microanatomical features such as nerve roots significantly accelerate solute dispersion. Strong micro mixing effects caused by anatomical features in the spinal subarachnoid space are present in intrathecal drug administration, but were not considered in prior dispersion theories, which explains why prior models developed in the engineering community are poor predictors for IT delivery.

ConclusionOur experiments support the feasibility of targeting large sections of the neuroaxis or brain by means of high-volume injection protocols. The experimental tracer dispersion profiles acquired with an in vitro human CNS analog informed a new predictive model of tracer dispersion as a function of physiological CSF pulsations and adjustable infusion parameters. The ability to predict spatiotemporal dispersion patterns is an essential prerequisite for exploring new indications of IT drug delivery which target specific regions in the central nervous system (CNS) or the brain.


title: Reproducible acquisition, management, and meta-analysis of nucleotide sequence (meta)data using q2-fondue

abstract: The volume of public nucleotide sequence data has blossomed over the past two decades, enabling novel discoveries via re-analysis, meta-analyses, and comparative studies for uncovering general biological trends. However, reproducible re-use and management of sequence datasets remains a challenge. We created the software plugin q2-fondue to enable user-friendly acquisition, re-use, and management of public nucleotide sequence (meta)data while adhering to open data principles. The software allows fully provenance-tracked programmatic access to and management of data from the Sequence Read Archive (SRA). Sequence data and accompanying metadata retrieved with q2-fondue follow a validated format, which is interoperable with the QIIME 2 ecosystem and its multiple user interfaces. To highlight the manifold capabilities of q2-fondue, we present several demonstration analyses using amplicon, whole genome, and shotgun metagenome datasets. These use cases demonstrate how q2-fondue increases analysis reproducibility and transparency from data download to final visualizations by including source details in the integrated provenance graph. We believe q2-fondue will lower existing barriers to comparative analyses of nucleotide sequence data, enabling more transparent, open, and reproducible conduct of meta-analyses. q2-fondue is a Python 3 package released under the BSD 3-clause license at https://github.com/bokulich-lab/q2-fondue.


title: A model for quorum-sensing mediated stochastic biofilm nucleation

abstract: Surface-attached bacterial biofilms cause disease and industrial biofouling, as well as being widespread in the natural environment. Density-dependent quorum sensing is one of the mechanisms implicated in biofilm initiation. Here we present and analyse a model for quorum-sensing triggered biofilm initiation. In our model, individual, planktonic bacteria adhere to a surface, pro-liferate and undergo a collective transition to a biofilm phenotype. This model predicts a stochastic transition between a loosely attached, finite, layer of bacteria near the surface, and a growing biofilm. The transition is governed by two key parameters: the collective transition density relative to the carrying capacity, and the immigration rate relative to the detachment rate. Biofilm initiation is complex, but our model suggests that stochastic nucleation phenomena may be relevant.


title: Structure-based Evolution of G protein-biased μ-opioid Receptor Agonists

abstract:
The -opioid receptor (OR) is the major target for opioid analgesics. Activation of OR initiates signaling through G protein pathways as well as through {beta}-arrestin recruitment. OR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased OR agonist. Here we report the cryoEM structure of PZM21 bound OR in complex with Gi protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced Gi protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to OR in complex with Gi protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce {beta}-arrestin recruitment by the OR, and hold promise for developing next-generation analgesics with fewer adverse effects.

Table of Contents Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=43 SRC="FIGDIR/small/485330v1_ufig1.gif" ALT="Figure 1">
View larger version (14K):
org.highwire.dtl.DTLVardef@125808corg.highwire.dtl.DTLVardef@11aa15corg.highwire.dtl.DTLVardef@157caaaorg.highwire.dtl.DTLVardef@ba837d_HPS_FORMAT_FIGEXP  M_FIG C_FIG We obtained cryoEM structures of the -opioid receptor (OR) bound to the lead compound PZM21 and the newly developed agonist FH210 to understand the mechanism of their biased signaling and to guide the evolution of next-generation analgesics with fewer adverse effects.

category: pharmacology and toxicology


title: An in-silico analysis of experimental designs to study right ventricular function and pulmonary hypertension

abstract:
In-vivo studies of pulmonary hypertension (PH) have provided key insight into the progression of the disease and right ventricular (RV) dysfunction. Additional in-silico experiments using multiscale computational models have provided further details into biventricular mechanics and hemodynamic function in the presence of PH, yet few have assessed whether model parameters are identifiable prior to data collection. Moreover, none have used modeling to devise synergistic experimental designs. To address this knowledge gap, we conduct an identifiability analysis of a multiscale cardiovascular model across four simulated experimental designs. We determine a set of parameters using a combination of Morris screening and local sensitivity analysis, and test for identifiability using profile likelihood based confidence intervals. We employ Markov chain Monte Carlo (MCMC) techniques to quantify parameter and model forecast uncertainty in the presence of noise corrupted data. Our results show that model calibration to only RV pressure suffers from identifiability issues and suffers from large forecast uncertainty in output space. In contrast, parameter and model forecast uncertainty is substantially reduced once additional left ventricular (LV) pressure and volume data is included. A comparison between single point systolic and diastolic LV data and continuous, time-dependent LV pressure volume data reveals that even basic, functional data from the LV remedies identifiability issues and provides substantial insight into biventricular interactions.

Author SummaryComputational models of cardiac dynamics are becoming increasingly useful in understanding the underlying mechanisms of disease. In-silico analyses are especially insightful in understanding PH and eventual RV dysfunction, as these conditions are diagnosed months to years after disease onset. Many researchers couple computational models with in-vivo experimental models of PH, yet few ever assess what data might be necessary or sufficient for parameter inference prior to designing their experiments. Here, we considered a multiscale computational model including sarcomere dynamics, biventricular interactions, and vascular hemodynamics, and assessed whether parameters could be inferred accurately given limited cardiac data. We utilized sensitivity analyses, profile likelihood confidence intervals, and MCMC to quantify parameter influence and uncertainty. We observed that RV pressure alone is not sufficient to infer the influential parameters in the model, whereas combined pressure and volume data in both the RV and LV reduced uncertainty in model parameters and in model forecasts. We conclude that synergistic PH studies utilizing computational modeling include these data to reduce issues with parameter identifiability and minimize uncertainty.


title: The gender gap in academic career achievements and the mediation effect of work-family conflict and partner support

abstract: Parenthood has long been associated with gender disparities in academia. Yet, the underlying mechanism of how parenting is associated with career achievement gaps of academics remains unclear. Using data from a large-scale survey distributed to 7,764 scholars and their publication profiles from the Web of Science database, we analyze the gender differences in parenthood, academic achievements, and the mediation effect of work-family conflict and partner support in these gender gaps. Our results suggest that gender gaps in academic achievements are in fact "parenthood gaps". Specifically, we found significant gender gaps exist in all measures of objective and subjective career achievements of academics in the parent group but not in the non-parent group. Additionally, mothers are more likely than fathers to experience higher levels of work-family conflict, and receive lower levels of partner support, contributing significantly to the gender gaps in objective and subjective career achievements for the parent group. Findings from this study identify the forms and the impact of parenthood on gender disparities in career achievements of academics and shed light on possible interventions and actions for mitigating gender inequalities in academia.

category: scientific communication and education


title: Particle-Swarm Based Modelling Reveals Two Distinct Classes of CRHPVN Neurons

abstract:
Electrophysiological recordings can provide detailed information of single neurons dynamical features and shed light into their response to stimuli. Unfortunately, rapidly modeling electrophysiological data for inferring network-level behaviours remains challenging. Here, we investigate how modeled single neuron dynamics lead to network-level responses in the paraventricular nucleus of the hypothalamus (PVN), a critical nucleus for the mammalian stress response. Recordings of corticotropinreleasing hormone neurons from the PVN (CRHPVN) were performed using whole-cell current-clamp. These, neurons, which initiate the endocrine response to stress, were rapidly and automatically fit to a modified Adaptive Exponential Integrate and Fire model (AdEx) with Particle Swarm Optimization (PSO). All CRHPVN neurons were accurately fit by the AdEx model with PSO. Multiple sets of parameters were found that reliably reproduced current-clamp traces for any single neuron. Despite multiple solutions, the dynamical features of the models such as the rheobase current levels, fixed points, and bifurcations, were shown to be stable across fits. We found that CRHPVN neurons can be divided into two sub-types according to their bifurcation at the onset of firing: saddles (integrators) and sub-critical Hopf (resonators). We constructed networks of these fit CRHPVN model neurons to investigate the network level responses of CRHPVN neurons. We found that CRHPVN-resonators maintain baseline firing in networks even when all inputs are inhibitory. The dynamics of a small subset of CRHPVN neurons may be critical to maintaining a baseline firing tone in the PVN.

Key PointsO_LICorticotropin-releasing hormone neurons (CRHPVN) in the paraventricular nucleus of the hypothalamus act as the final neural controllers of the stress response.
C_LIO_LIWe developed a rapid computational modeling platform that uses Particle-Swarm Optimization to rapidly and accurately fit biophysical neuron models.
C_LIO_LIA model was fit to each patched neuron without the use of dynamic clamping, or other procedures requiring sophisticated inputs and fitting procedures. Any neuron undergoing standard current clamping for a few minutes can be fit by this procedure
C_LIO_LIThe dynamical analysis of the modeled neurons shows thatCRHPVN comes in two specific  flavours: CRHPVN-resonators and CRHPVN-integrators.
C_LIO_LINetwork simulations show thatCRHPVN-resonators are critical to retaining the baseline firing rate of the entire network of CRHPVN neurons as these cells can fire rebound spikes and bursts in the presence of strong inhibitory synaptic input.
C_LI


title: Increased fatty acid metabolism and decreased glycolysis are hallmarks of metabolic reprogramming in the brain during recovery from experimental stroke

abstract: The goal of this study was to evaluate changes in metabolic homeostasis during the first 12 weeks of recovery in a distal middle cerebral artery occlusion mouse model of stroke. To achieve this goal, we compared the brain metabolomes of ipsilateral and contralateral hemispheres from aged male mice up to 12 weeks after stroke to that of age-matched naive and sham operated mice. There were 707 biochemicals detected in each sample by liquid chromatography-mass spectroscopy (LC-MS). Mitochondrial fatty acid {beta}-oxidation, indicated by acyl carnitine levels, was increased in stroked tissue at 1 day and 4 weeks following stroke. Glucose and several glycolytic intermediates were elevated in the ipsilateral hemisphere for 12 weeks compared to the aged naive controls, but pyruvate was decreased. Additionally, itaconate, a glycolysis inhibitor associated with activation of anti-inflammatory mechanisms in myeloid cells, was higher in the same comparisons. These changes correlated with reduced levels of glutamate, dopamine, and adenosine in the ipsilateral hemisphere after stroke. These results indicate that chronic metabolic differences exist between stroked and control tissue, including alterations in fatty acid metabolism and glycolysis for at least 12 weeks after stroke.


title: Two mosquito odorant receptors with reciprocal specificity mediated by a single amino acid residue

abstract: Insects perceive the environment with a repertoire of promiscuous and narrowly tuned odorant receptors (ORs). The southern house mosquito, Culex quinquefasciatus, utilizes two ORs, CquiOR10 and CquiOR2, which are narrowly tuned and well conserved among mosquito species. They detect the oviposition attractants skatole and indole, respectively, with reciprocal specificity. We envisioned that by reverting specificity with chimeric receptors, we could gain a better insight into the specificity determinants of insect ORs. We swapped the seven transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73, although this residue in CquiOR10 did not directly form contacts with skatole or indole. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a higher sensitivity than the wild-type receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10 was insensitive to 3-ethylindole, whereas CquiOR10A73G responded to the three ligands with 3-ethylindole eliciting a slightly higher response than indole. We then concluded that the specificity of these receptors is mediated by a single amino acid substitution leading to dilation or contraction of the binding pocket.


title: Multiple-timescale dynamics, mixed mode oscillations and mixed affective states in a model of Bipolar Disorder

abstract: Mixed affective states in bipolar disorder is a common psychiatric condition that occurs when symptoms of the two opposite poles coexist during an episode of mania or depression. There exist many strategies to attempt to model the mood variations of patients with bipolar disorder (BD) as recently reviewed in [14]. A four-dimensional model by A. Goldbeter [28, 29] rests upon the notion that manic and depressive symptoms are produced by two competing and auto-inhibited neural networks. Some of the rich dynamics that this model can produce, include complex rhythms formed by both small-amplitude (subthreshold) and large-amplitude (suprathreshold) oscillations and could correspond to mixed bipolar states. These rhythms are commonly referred to as mixed mode oscillations (MMOs) and they have already been studied in different contexts ranging from neurons electrical activity [7] to oscillatory chemical reactions [51]. In order to accurately explain these dynamics one has to apply a mathematical apparatus that is making full use of the timescale separation between variables. Here we apply this very framework to the model of BD from [28, 29] in order to understand the mathematical mechanisms underpinning the observed dynamics of changing mood using multiple-timescale dynamics. We show that the observed complex oscillations can be understood as MMOs due to a so-called folded-node singularity. Moreover we explore the bifurcation structure of the system with respect to two meaningful parameters and we provide possible biological interpretations of our findings. Furthermore, we show the possibility of deterministic chaos in this system and the robustness of the MMOs regime to stochastic noise. Finally, we propose a reduction of the original 4D model to a minimal three-dimensional model which, with the addition of noise, exhibits similar yet purely noise-driven dynamics. The broader significance of this work is to introduce mathematical tools that could be used to analyse and potentially control future, more biologically grounded models of BD.


title: Quadripulse Stimulation: A Replication Study with A Newly Developed Stimulator

abstract: Quadripulse stimulation (QPS) is a promising patterned repetitive transcranial magnetic stimulation protocol, which allows the modulation of brain activity for over one hour after the stimulation. Recently, Deymed Diagnostic developed a new stimulator specifically designed to deliver QPS (DuoMAG MP-Quad, https://deymed.com/duomag-qps). The properties of the after-effect with this new stimulator were expected to parallel those obtained with another stimulator, the Magstim stimulator, which is currently used in the psychological and clinical research but not commercially available anymore. Nevertheless, experimental validation of DuoMAG MP-Quad was still warranted. We thus studied the QPS after-effect induced by this stimulator. As a result, motor evoked potentials were found to be bidirectionally modulated by the QPS for more than one hour, consistent with previous studies using the Magstim stimulator. Moreover, the degree of the after-effect was comparable to the after-effect induced by the other stimulator. Taken together, we conclude that the newly developed QPS stimulator is as effective as the Magstim stimulator.


title: Structure and influence in an interconnected world: neurocomputational mechanism of real-time distributed learning on social networks

abstract: Many social species are embedded on social networks, including our own. The structure of social networks shapes our decisions by constraining what information we learn and from whom. But how does the brain incorporate social network structures into learning and decision-making processes, and how does learning in networked environments differ from learning from isolated partners? Combining a real-time distributed learning task with computational modeling, fMRI, and social network analysis, we investigated the process by which humans learn from observing others decisions on 7-node networks with varying topological structures. We show that learning on social networks can be realized by means similar to the well-established reinforcement learning algorithm, supported by an action prediction error encoded in the lateral prefrontal cortex. Importantly, learning is flexibly weighted toward well-connected neighbors, according to activity in the dorsal anterior cingulate cortex, but only insofar as neighbors actions vary in their informativeness. These data suggest a neurocomputational mechanism of network-dependent filtering on the sources of information, which may give rise to biased learning and the spread of misinformation in an interconnected society.


title: FKS1 is required for Cryptococcus neoformans fitness in vivo: application of copper-regulated gene expression to mouse models of cryptococcosis

abstract:
There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used to modulate gene expression in vivo previously. The premise for these experiments is that copper concentrations vary depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied to the gene encoding 1,3-{beta}-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through post-transcriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection.

ImportanceConditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied it to the study of FKS1, the gene encoding for the target of the echinocandin class of 1,3-{beta}-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-{beta}-glucan synthase inhibitors toward C. neoformans.


title: The effects of antibiotic combination treatments on Pseudomonas aeruginosa tolerance evolution and coexistence with Stenotrophomonas maltophilia

abstract: Pseudomonas aeruginosa bacterium is a common pathogen of Cystic Fibrosis (CF) patients due to its ability to evolve resistance to antibiotics during treatments. While P. aeruginosa resistance evolution is well characterised in monocultures, it is less well understood in polymicrobial CF infections. Here, we investigated how exposure to ciprofloxacin, colistin, or tobramycin antibiotics, administered at sub-MIC doses alone and in combination, shaped the tolerance evolution of P. aeruginosa (PAO1 lab and clinical CF LESB58 strains) in the absence and presence of a commonly co-occurring species, Stenotrophomonas maltophilia. Increases in antibiotic tolerances were primarily driven by the presence of that antibiotic in the treatment. We observed a reciprocal cross-tolerance between ciprofloxacin and tobramycin, and when combined these antibiotics selected increased MICs for all antibiotics. Though the presence of S. maltophilia did not affect the tolerance or the MIC evolution, it drove P. aeruginosa into extinction more frequently in the presence of tobramycin due to its relatively greater innate tobramycin tolerance. In contrast, P. aeruginosa dominated and drove S. maltophilia extinct in most other treatments. Together, our findings suggest that besides driving high-level antibiotic tolerance evolution, sub-MIC antibiotic exposure can alter competitive bacterial interactions, leading to target pathogen extinctions in multi-species communities.


title: The genomics and physiology of abiotic stressors associated with global elevation gradients in Arabidopsis thaliana

abstract: Arabidopsis thaliana has a wide elevational range and much of its diversity may be associated with local adaptation to elevation. We took a multi-regional view of the genomics and physiology of elevational adaptation in Arabidopsis, with >200 ecotypes, including 17 newly collected from Africa. We measured plant responses to potential high elevation stressors: low pCO2, high light, and night freezing and conducted genome-wide association studies (GWAS). We found evidence of an adaptive cline in the western Mediterranean with low {delta}13C/early flowering at low elevations to high {delta}13C/late flowering at high elevations. By contrast, central Asian high elevation ecotypes flowered earlier. Antioxidants and pigmentation under high light and freezing showed regional differentiation but not elevational clines and may be associated with maladaptive plasticity. We found natural variation in non-photochemical quenching (NPQ) kinetics in response to chilling and fluctuating light, though with an unclear role in local adaptation. There were several candidate genetic loci mapped, including the ascorbate transporter PHT4;4 (associated with antioxidants) that influences the xanthophyll cycle, and may be involved in local adaptation to Morocco. Our study shows how the ecological strategies and genetic loci causing local adaptation to elevation change across regions and contribute to diversity in Arabidopsis.