210 Citations
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The CRISPR-Cas type V-I is a family of Cas i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA Here we present an engineered Type V-I CRISPR system Cas i ABR- which utilizes a tracr-less guide RNA The compact Cas i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets which facilitates versatile delivery options and multiplexing respectively We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas i The engineered variant ABR- exhibits broad genome editing capability in human cell lines primary T cells and CD hematopoietic stem and ... More
The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy. Less
Understanding macrophage heterogeneity in tissue repair is a major challenge Here we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing We use a modified version of the original Smart-seq protocol to increase speed and accuracy This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation
Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function Recently this motif was suggested to be involved in the catalytic activity of tankyrases In this work we set out to study the effect of the zinc-binding motif on the activity stability and structure of human tankyrases We generated mutants of human tankyrase TNKS and TNKS abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro We further generated a ... More
Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type. Less
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states While mouse models are used extensively to investigate the interplay between microbes and the inflamed state the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships To address this issue we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria CIAMIB The initial release of this collection comprises isolates of unique bacterial species covering phyla and containing previously uncultivated isolates including novel family and novel genera The collection ... More
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states. While mouse models are used extensively to investigate the interplay between microbes and the inflamed state, the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships. To address this issue, we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria (CIAMIB). The initial release of this collection comprises 41 isolates of 39 unique bacterial species, covering 4 phyla and containing 10 previously uncultivated isolates, including 1 novel family and 7 novel genera. The collection significantly expands the number of available Muribaculaceae, Lachnospiraceae, and Coriobacteriaceae isolates and includes microbes from genera associated with inflammation, such as Prevotella and Klebsiella. We characterized the growth of CIAMIB isolates across a diverse range of nutritional conditions and predicted their metabolic potential and anaerobic fermentation capacity based on the genomes of these isolates. We also provide the first metabolic analysis of species within the genus Adlercreutzia, revealing these representatives to be nitrate-reducing and severely restricted in their ability to grow on carbohydrates. CIAMIB isolates are fully sequenced and available to the scientific community as a powerful tool to study host-microbiota interactions. Less
The emergence of several zoonotic viruses in the last twenty years especially the pandemic outbreak of SARS-CoV- has exposed a dearth of antiviral drug therapies for viruses with pandemic potential Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses CoVs and other viruses with pandemic potential Here we focus on the SARS-CoV- conserved macrodomain Mac a small domain of non-structural protein nsp Mac is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose MAR from target proteins protects the virus from the anti-viral effects of host ADP-ribosyltransferases and is critical for the replication and pathogenesis of ... More
The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100µM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors. Less
Chromatin abnormalities are common hallmarks of cancer cells which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes These epigenetic patterns are partly established and maintained by UHRF ubiquitin-like PHD and RING finger domain-containing protein which senses existing methylation states through multiple reader domains and reinforces the modifications through recruitment of DNA methyltransferases Small molecule inhibitors of UHRF would be important tools to illuminate molecular functions yet no compounds capable of blocking UHRF -histone binding in the context of the full-length protein exist Here we report the discovery and mechanism of action of compounds that selectively inhibit ... More
Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition. Less
Tousled-like kinases TLKs are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants A major function of TLKs is to phosphorylate the histone chaperone proteins ASF a and ASF b to facilitate DNA replication-coupled nucleosome assembly but how TLKs selectively target these critical substrates is unknown Here we show that TLK selectivity towards ASF substrates is achieved in two ways First the TLK catalytic domain recognizes consensus phosphorylation site motifs in the ASF C-terminal tail Second a short sequence at the TLK N-terminus docks onto the ASF a globular N-terminal domain in a manner ... More
Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate. Less
Many animal species are susceptible to severe acute respiratory syndrome coronavirus SARS-CoV- infection and could act as reservoirs however transmission in free-living animals has not been documented White-tailed deer the predominant cervid in North America are susceptible to SARS-CoV- infection and experimentally infected fawns can transmit the virus To test the hypothesis that SARS-CoV- is circulating in deer retropharyngeal lymph node RPLN samples collected from free-living and captive deer in Iowa from April through January of were assayed for the presence of SARS-CoV- RNA Ninety-four of the deer samples were positive for SARS-CoV- RNA as assessed by RT-PCR Notably following ... More
Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive “One Health” approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2. Less
Genetic variants of severe acute respiratory syndrome coronavirus SARS-CoV- have repeatedly altered the course of the coronavirus disease COVID- pandemic Delta variants are now the focus of intense international attention because they are causing widespread COVID- globally and are associated with vaccine breakthrough cases We sequenced SARS-CoV- genomes from samples acquired March through September in the Houston Methodist hospital system This sample represents of all Methodist system COVID- patients during the study period Delta variants increased rapidly from late April onward to cause of all COVID- cases and spread throughout the Houston metroplex Compared with all other variants combined Delta ... More
Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly altered the course of the coronavirus disease 2019 (COVID-19) pandemic. Delta variants are now the focus of intense international attention because they are causing widespread COVID-19 globally and are associated with vaccine breakthrough cases. We sequenced 16,965 SARS-CoV-2 genomes from samples acquired March 15, 2021, through September 20, 2021, in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared with all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared with 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold value (a proxy for high virus load). This value was similar to the median cycle threshold value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. The integrated analysis indicates that vaccines used in the United States are highly effective in decreasing severe COVID-19, hospitalizations, and deaths. Less
The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth driving redox reactions that give rise to global biogeochemical cycles Community metabolism emerges from a hierarchy of processes including gene expression ecological interactions and environmental factors In wild communities gene content is correlated with environmental context but predicting metabolite dynamics from genomes remains elusive Here we show for the process of denitrification that metabolite dynamics of a community are predictable from the genes each member of the community possesses A simple linear regression reveals a sparse and generalizable mapping from gene ... More
The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth, driving redox reactions that give rise to global biogeochemical cycles. Community metabolism emerges from a hierarchy of processes, including gene expression, ecological interactions, and environmental factors. In wild communities, gene content is correlated with environmental context, but predicting metabolite dynamics from genomes remains elusive. Here, we show, for the process of denitrification, that metabolite dynamics of a community are predictable from the genes each member of the community possesses. A simple linear regression reveals a sparse and generalizable mapping from gene content to metabolite dynamics for genomically diverse bacteria. A consumer-resource model correctly predicts community metabolite dynamics from single-strain phenotypes. Our results demonstrate that the conserved impacts of metabolic genes can predict community metabolite dynamics, enabling the prediction of metabolite dynamics from metagenomes, designing denitrifying communities, and discovering how genome evolution impacts metabolism. Less
Despite advances in spatial transcriptomics the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge We present a method termed behavioral transcriptomics that allows us to couple physiological behaviors of single cells in an intact tissue to deep molecular profiling of individual cells This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following tissue injury
Here we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP Recombinant PtxS and PARP are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag The protocol does not require specialized compounds or substrates making it accessible and easy to adapt in any laboratory or for other proteins of interest
Squamous cell carcinoma SCC is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses AK Chronic inflammation is a well-known promoter of skin cancer progression AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus S aureus Certain secreted products from S aureus are known to promote cutaneous pro-inflammatory responses however not all S aureus strains produce these As inflammation plays a key role in SCC development we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S aureus Sterile culture supernatants secretomes of S aureus clinical ... More
Squamous cell carcinoma (SCC) is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses (AK). Chronic inflammation is a well-known promoter of skin cancer progression. AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus (S. aureus). Certain secreted products from S. aureus are known to promote cutaneous pro-inflammatory responses; however, not all S. aureus strains produce these. As inflammation plays a key role in SCC development, we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S. aureus. Sterile culture supernatants (“secretomes”) of S. aureus clinical strains isolated from AK and SCC were applied to human keratinocytes in vitro. Some S. aureus secretomes induced keratinocytes to overexpress inflammatory mediators that have been linked to skin carcinogenesis, including IL-6, IL-8, and TNFα. A large phenotypic variation between the tested clinical strains was observed. Strains that are highly pro-inflammatory in vitro also caused more pronounced skin inflammation in mice. Proteomic characterization of S. aureus secretomes using mass spectrometry established that specific S. aureus enzymes and cytolytic toxins, including hemolysins, phenol-soluble modulins, and serine proteases, as well as currently uncharacterized proteins, correlate with the pro-inflammatory S. aureus phenotype. This study is the first to describe the toxin secretion profiles of AK and SCC-associated S. aureus, and their potential to induce a pro-inflammatory environment in the skin. Further studies are needed to establish whether these S. aureus products promote SCC development by mediating chronic inflammation. Less
Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations Although several antibody-based targeted approaches have been readily available for single-cell analysis so far only the mass spectrometry methodology can offer unbiased proteome profiling While this strategy has only recently emerged it has already demonstrated unparalleled analytical power quantifying proteins in single cells Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell ... More
Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells, which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations. Although several antibody-based targeted approaches have been readily available for single-cell analysis, so far only the mass spectrometry methodology can offer unbiased proteome profiling. While this strategy has only recently emerged, it has already demonstrated unparalleled analytical power quantifying >1000 proteins in single cells. Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell types and instrumentation. This chapter provides a typical example of mass spectrometry-based single-cell proteomics workflow with details about the critical steps of analysis and alternative methods useful for optimization purposes. Less
Over the past years single-cell sequencing has become very popular For this reason many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population the choice of the best protocol can be difficult due to the high abundance of available protocols instruments and options For this reason here we describe the Smart-seq protocol for full-length mRNA sequencing of single cell This protocol can be easily optimized in every molecular ... More
Over the past 7 years, single-cell sequencing has become very popular. For this reason, many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique. For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population, the choice of the best protocol can be difficult, due to the high abundance of available protocols, instruments, and options. For this reason, here we describe the Smart-seq2 protocol for full-length mRNA sequencing of single cell. This protocol can be easily optimized in every molecular biology laboratory provided with standard laboratory equipment. The protocol is suitable for many different cell types, and the cost per cell is relatively small, allowing a good balance between costs and transcript coverage. Less
Single cell proteomics is an emerging field of bioanalysis allowing one to capture proteome profiles of isolated single cells which is expected to yield additional biological information in comparison with bulk cell analysis Mass spectrometry-based methods provide unbiased analysis of detectable proteins limited only by technical parameters such as sensitivity which necessitates the development of best-practice workflows Here we describe the entire experimental design of single cell proteome analysis exemplified by cultured A lung adenocarcinoma cells treated with an anti-cancer drug methotrexate and utilizing tandem mass tag TMTpro labeling strategy for mass spectrometric data acquisition
Many biological systems are composed of diverse single cells This diversity necessitates functional and molecular single-cell analysis Single-cell protein analysis has long relied on affinity reagents but emerging mass-spectrometry methods either label-free or multiplexed have enabled quantifying proteins per cell while simultaneously increasing the specificity of protein quantification Here we describe the Single Cell ProtEomics SCoPE protocol which uses an isobaric carrier to enhance peptide sequence identification Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation mPOP and their peptides labeled by isobaric mass tags TMT or TMTpro for multiplexed analysis ... More
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis. Less
Certain genetic variants of severe acute respiratory syndrome coronavirus SARS-CoV- are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients SARS-CoV- genome sequences from patients in the Houston Methodist health care system diagnosed from January through May are reported here Prevalence of the B Alpha variant increased rapidly and caused to of new cases in the latter half of May Eleven B genomes had an E K replacement in spike protein a change also identified in other SARS-CoV- lineages Compared with non B -infected patients individuals with B ... More
Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non–B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. In the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex. Less
Determinants of protective immunity against severe acute respiratory syndrome coronavirus SARS-CoV- infection require the development of well-standardized reproducible antibody assays This need has led to the emergence of a variety of neutralization assays Head-to-head evaluation of different SARS-CoV- neutralization platforms could facilitate comparisons across studies and laboratories Five neutralization assays were compared using plasma samples from convalescent individuals with mild to moderate coronavirus disease COVID- four cell-based systems using either live recombinant SARS-CoV- or pseudotyped viral particles created with lentivirus LV or vesicular stomatitis virus VSV packaging and one surrogate enzyme-linked immunosorbent assay ELISA -based test that measures inhibition of ... More
Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. Less
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids but this technique presents challenges that impede scalable culture expansion experimental manipulation high-throughput screening and diagnostic applications We have developed a low-viscosity matrix LVM suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers We demonstrate the utility of LVM suspension culture for organoid and ... More
Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine. Less
In bacteria trans-translation is the major quality control system for rescuing stalled ribosomes It is mediated by tmRNA a hybrid RNA with properties of both a tRNA and a mRNA and the small protein SmpB Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival it is a promising target for the development of novel antibiotics To facilitate screening of chemical libraries various reliable in vitro and in vivo systems have been created for assessing trans-translational activity However the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from ... More
In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens. Less
A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility
Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing ATAC-seq The method combines up-front bulk Tn tagging of chromatin with flow cytometry to isolate single nuclei or cells Reagents required to generate sequencing libraries are added to the same well in ... More
Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support. Less
The integration of DNA methylation and transcriptional state within single cells is of broad interest Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity including the discovery and in-depth study of rare cell populations Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study Here we present details of the protocol and provide guidance on its utility Our Smart-RRBS reduced representation bisulfite sequencing protocol ... More
The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system. Less
There is increasing attention focussed on the risks associated with mobile phones possibly serving as Trojan Horse fomites for microbial transmission in healthcare settings However little is reported on the presence of microbes on community derived mobile phones which in numbered in the billions in circulation with majority being used on a daily basis Identify viable microbial organisms swabbed from smartphones on a university campus Entire surfaces of mobile phones were swabbed and examined for their microbial content using pre-agar-based growths followed by downstream DNA metagenomic next-generation sequencing analysis All phones were contaminated with viable microbes bacteria fungi protists bacteriophages ... More
There is increasing attention focussed on the risks associated with mobile phones possibly serving as ‘Trojan Horse’ fomites for microbial transmission in healthcare settings. However, little is reported on the presence of microbes on community derived mobile phones which in 2021, numbered in the billions in circulation with majority being used on a daily basis. Identify viable microbial organisms swabbed from smartphones on a university campus. Entire surfaces of 5 mobile phones were swabbed and examined for their microbial content using pre-agar-based growths followed by downstream DNA metagenomic next-generation sequencing analysis. All phones were contaminated with viable microbes. 173 bacteria, 8 fungi, 8 protists, 53 bacteriophages, 317 virulence factor genes and 41 distinct antibiotic resistant genes were identified. While this research represents a pilot study, the snapshot metagenomic analysis of samples collected from the surface of mobile phones has revealed the presence of a large population of viable microbes and an array of antimicrobial resistant factors. With billions of phones in circulation, these devices might be responsible for the rise of community acquired infections. These pilot results highlight the importance of public health authorities considering mobile phones as ‘Trojan Horse’ devices for microbial transmission and ensure appropriate decontamination campaigns are implemented. Less
Aerogels are ultralight porous materials whose matrix structure can be formed by interlinking nm long M phage particles In theory changing the phage properties would alter the aerogel matrix but attempting this using the current production system leads to heterogeneous lengths A phagemid system that yields a narrow length distribution that can be tuned in nm increments from to nm is designed and independently the persistence length varies from to nm by mutating the coat protein A robotic workflow that automates each step from DNA construction to aerogel synthesis is used to build aerogels This is applied to compare Ni ... More
Aerogels are ultralight porous materials whose matrix structure can be formed by interlinking 880 nm long M13 phage particles. In theory, changing the phage properties would alter the aerogel matrix, but attempting this using the current production system leads to heterogeneous lengths. A phagemid system that yields a narrow length distribution that can be tuned in 0.3 nm increments from 50 to 2500 nm is designed and, independently, the persistence length varies from 14 to 68 nm by mutating the coat protein. A robotic workflow that automates each step from DNA construction to aerogel synthesis is used to build 1200 aerogels. This is applied to compare Ni–MnOx cathodes built using different matrixes, revealing a pareto-optimal relationship between performance metrics. This work demonstrates the application of genetic engineering to create “tuning knobs” to sweep through material parameter space; in this case, toward creating a physically strong and high-capacity battery. Less
Blossom-end rot BER is a devastating physiological disorder that affects tomato and other vegetables resulting in significant crop losses To date most studies on BER have focused on the environmental factors that affect calcium translocation to the fruit however the genetic basis of this disorder remains unknown To investigate the genetic basis of BER two F and F populations along with a BC population that segregated for BER occurrence were evaluated in the greenhouse Using the QTL-seq approach quantitative trait loci QTL associated with BER Incidence were identified at the bottom of chromosome ch and ch Additionally linkage-based QTL mapping ... More
Blossom-end rot (BER) is a devastating physiological disorder that affects tomato and other vegetables, resulting in significant crop losses. To date, most studies on BER have focused on the environmental factors that affect calcium translocation to the fruit; however, the genetic basis of this disorder remains unknown. To investigate the genetic basis of BER, two F2 and F3:4 populations along with a BC1 population that segregated for BER occurrence were evaluated in the greenhouse. Using the QTL-seq approach, quantitative trait loci (QTL) associated with BER Incidence were identified at the bottom of chromosome (ch) 3 and ch11. Additionally, linkage-based QTL mapping detected another QTL, BER3.1, on ch3 and BER4.1 on ch4. To fine map the QTLs identified by QTL-seq, recombinant screening was performed. BER3.2, the major BER QTL on ch3, was narrowed down from 5.68 to 1.58 Mbp with a 1.5-LOD support interval (SI) corresponding to 209 candidate genes. BER3.2 colocalizes with the fruit weight gene FW3.2/SlKLUH, an ortholog of cytochrome P450 KLUH in Arabidopsis. Further, BER11.1, the major BER QTL on ch11, was narrowed down from 3.99 to 1.13 Mbp with a 1.5-LOD SI interval comprising of 141 candidate genes. Taken together, our results identified and fine mapped the first loci for BER resistance in tomato that will facilitate marker-assistant breeding not only in tomato but also in many other vegetables suffering for BER. Less
Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information respectively However the mechanisms underlying the assembly of such parallel networks during development remain largely unknown Our study shows that in mice complementary expression of cell surface molecules teneurin- Ten and latrophilin- Lphn in the medial and lateral hippocampal networks respectively guides the precise assembly of CA -to-subiculum connections in both networks In the medial network Ten -expressing Ten CA axons are repelled by target-derived Lphn revealing that Lphn - and Ten -mediated heterophilic repulsion and Ten -mediated homophilic attraction cooperate to control precise target selection of CA ... More
Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3→Ten3, Lphn2→Lphn2" rule instructed by reciprocal repulsions. Less
Background Psoriasis is an inflammatory IL- driven skin disease in which autoantigen-induced CD T cells have been identified as pathogenic drivers Objective Our study focused on comprehensively characterizing the phenotypic variation of CD T cells in psoriatic lesions Methods We used single-cell RNA sequencing to compare CD T-cell transcriptomic heterogeneity between psoriatic and healthy skin Results We identified transcriptionally diverse CD T-cell subsets in psoriatic and healthy skin Among several inflammatory subsets enriched in psoriatic skin we observed Tc cell subsets that were metabolically divergent were developmentally related and expressed CXCL which we found to be a biomarker of psoriasis ... More
Background Psoriasis is an inflammatory, IL-17–driven skin disease in which autoantigen-induced CD8+ T cells have been identified as pathogenic drivers. Objective Our study focused on comprehensively characterizing the phenotypic variation of CD8+ T cells in psoriatic lesions. Methods We used single-cell RNA sequencing to compare CD8+ T-cell transcriptomic heterogeneity between psoriatic and healthy skin. Results We identified 11 transcriptionally diverse CD8+ T-cell subsets in psoriatic and healthy skin. Among several inflammatory subsets enriched in psoriatic skin, we observed 2 Tc17 cell subsets that were metabolically divergent, were developmentally related, and expressed CXCL13, which we found to be a biomarker of psoriasis severity and which achieved comparable or greater accuracy than IL17A in a support vector machine classifier of psoriasis and healthy transcriptomes. Despite high coinhibitory receptor expression in the Tc17 cell clusters, a comparison of these cells with melanoma-infiltrating CD8+ T cells revealed upregulated cytokine, cytolytic, and metabolic transcriptional activity in the psoriatic cells that differed from an exhaustion program. Conclusion Using high-resolution single-cell profiling in tissue, we have uncovered the diverse landscape of CD8+ T cells in psoriatic and healthy skin, including 2 nonexhausted Tc17 cell subsets associated with disease severity. Less
The transcription factor Rora has been shown to be important for the development of ILC and the regulation of ILC macrophages and Treg cells Here we investigate the role of Rora across CD T cells in general but with an emphasis on Th cells both in vitro as well as in the context of several in vivo type infection models We dissect the function of Rora using overexpression and a CD -conditional Rora-knockout mouse as well as a RORA-reporter mouse We establish the importance of Rora in CD T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection and ... More
The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment. Less
Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity thousands of distinct genes with outstanding sensitivity and specificity of transcript quantification These full-length libraries have the advantage of allowing probing of transcript isoforms are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences Since full-length protocols are mostly plate-based at present they are also suited to profiling cell types where cell numbers are limiting such as rare cell types during development A disadvantage of these methods has been the scalability and cost of the experiments which has limited their ... More
Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences. Since full-length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared with droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length single-cell RNA sequencing, including both an in-house automated Smart-seq2 protocol and a commercial kit–based workflow. The protocols take 3–5 d to complete, depending on the number of plates processed in a batch. We discuss these two protocols in terms of ease of use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we have optimized the in-house automated protocol to dramatically reduce its cost. An automated setup can be adopted easily by a competent researcher with basic laboratory skills and no prior automation experience. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org). Less
Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle Despite their significant biogeochemical roles the microbial communities especially in the oxygen depleted compartments are poorly known Here we present a comprehensive dataset including shotgun metagenomes from stratified lakes and ponds mainly located in the boreal and subarctic regions but also including one tropical reservoir and one temperate lake For most lakes and ponds the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer The majority of the samples were collected during ... More
Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle. Despite their significant biogeochemical roles, the microbial communities, especially in the oxygen depleted compartments, are poorly known. Here, we present a comprehensive dataset including 267 shotgun metagenomes from 41 stratified lakes and ponds mainly located in the boreal and subarctic regions, but also including one tropical reservoir and one temperate lake. For most lakes and ponds, the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer. The majority of the samples were collected during the open water period, but also a total of 29 samples were collected from under the ice. In addition to the metagenomic sequences, the dataset includes environmental variables for the samples, such as oxygen, nutrient and organic carbon concentrations. The dataset is ideal for further exploring the microbial taxonomic and functional diversity in freshwater environments and potential climate change impacts on the functioning of these ecosystems. Less
Artemisinin-based combination therapies ACTs the World Health Organization-recommended first-line therapy for uncomplicated falciparum malaria has led to significant decreases in malaria-associated morbidity and mortality in the past two decades Decreased therapeutic efficacy of artemisinins the cornerstone of ACTs is threatening the gains made against this disease As such novel therapeutics with uncompromised mechanisms of action are needed to combat parasite-mediated antimalarial resistance We have previously reported the antimalarial activity of Plasmodium falciparum-specific proteasome inhibitors in conjunction with a variety of antimalarials in clinical use or in preclinical investigations and of proteasome mutants generated in response to these inhibitors Here we ... More
Artemisinin-based combination therapies (ACTs), the World Health Organization-recommended first-line therapy for uncomplicated falciparum malaria, has led to significant decreases in malaria-associated morbidity and mortality in the past two decades. Decreased therapeutic efficacy of artemisinins, the cornerstone of ACTs, is threatening the gains made against this disease. As such, novel therapeutics with uncompromised mechanisms of action are needed to combat parasite-mediated antimalarial resistance. We have previously reported the antimalarial activity of Plasmodium falciparum-specific proteasome inhibitors in conjunction with a variety of antimalarials in clinical use or in preclinical investigations and of proteasome mutants generated in response to these inhibitors. Here, we discover that despite harboring K13C580Y, which has conventionally mediated artemisinin resistance in vitro as measured by increased survival in ring-stage survival assays (RSA), the Cam3.II strain parasites of Cambodian origin that have acquired an additional mutation in the proteasome display increased susceptibility to DHA and OZ439. This discovery implicates the proteasome in peroxide susceptibilities and has favorable implications on the use of peroxide and proteasome inhibitor combination therapy for the treatment of artemisinin-resistant malaria Less
The SpinVessel system provides a methodology using pulsed radial flow to gently mix and uniformly suspend particulates cells magnetic beads silica beads and microcarrier beads for automated assays SpinVessels are well suited for aliquoting on robotic liquid handlers and with robotic reagent dispensers as well as manually The SpinVessel system combines two critical features special internal side fins and projections in the bottom of the vessels and an instrument that quickly spins the vessels and repeatedly reverses the spin direction This rapid reversing motion sends multiple pulses of fluid up the side walls of the SpinVessel creating a circular radial ... More
The SpinVessel system provides a methodology using pulsed radial flow to gently mix and uniformly suspend particulates (cells, magnetic beads, silica beads, and microcarrier beads) for automated assays. SpinVessels are well suited for aliquoting on robotic liquid handlers and with robotic reagent dispensers, as well as manually. The SpinVessel system combines two critical features: (1) special internal side fins and projections in the bottom of the vessels and (2) an instrument that quickly spins the vessels and repeatedly reverses the spin direction. This rapid reversing motion sends multiple pulses of fluid up the side walls of the SpinVessel, creating a circular radial flow pattern. We tested five different particulates and six different SpinVessels with volume capacities varying from 50 mL to 1200 mL. SpinVessels are compatible with either single-, 8-, 12-, 96-, or 384-channel pipettors or with siphon tubing on robotic reagent dispensers. Experiments have demonstrated high viability of cells and undamaged morphology of microcarrier beads even after hours of constant agitation. The uniformity of aliquots collected at various vertical depths and horizontally across the SpinVessels demonstrated that cells, magnetic beads, and silica beads were uniformly suspended throughout the height and breadth of the SpinVessels, and uniformity of samples was consistent from the beginning to the end of the aliquoting procedure. Only 5 min of mixing is required to resuspend settled particulates. This novel mixing methodology has many applications in laboratory automation where particulate aliquot uniformity and/or particulate integrity are important to automating assays. Less
Branching pattern formation is common in many microbes Extensive studies have focused on addressing how such patterns emerge from local cell cell and cell environment interactions However little is known about whether and to what extent these patterns play a physiological role Here we consider the colonization of bacteria as an optimization problem to find the colony patterns that maximize colony growth efficiency under different environmental conditions We demonstrate that Pseudomonas aeruginosa colonies develop branching patterns with characteristics comparable to the prediction of modeling for example colonies form thin branches in a nutrient-poor environment Hence the formation of branching patterns ... More
Branching pattern formation is common in many microbes. Extensive studies have focused on addressing how such patterns emerge from local cell–cell and cell–environment interactions. However, little is known about whether and to what extent these patterns play a physiological role. Here, we consider the colonization of bacteria as an optimization problem to find the colony patterns that maximize colony growth efficiency under different environmental conditions. We demonstrate that Pseudomonas aeruginosa colonies develop branching patterns with characteristics comparable to the prediction of modeling; for example, colonies form thin branches in a nutrient-poor environment. Hence, the formation of branching patterns represents an optimal strategy for the growth of Pseudomonas aeruginosa colonies. The quantitative relationship between colony patterns and growth conditions enables us to develop a coarse-grained model to predict diverse colony patterns under more complex conditions, which we validated experimentally. Our results offer new insights into branching pattern formation as a problem-solving social behavior in microbes and enable fast and accurate predictions of complex spatial patterns in branching colonies. Less
Stem cell dysfunction drives many age-related disorders Identifying mechanisms that initially compromise stem cell behavior represent early targets to promote tissue function later in life Here we pinpoint multiple factors that disrupt neural stem cell NSC behavior in the adult hippocampus Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term NSCs ST-NSCs and long-term NSCs LT-NSCs ST-NSCs divide rapidly to generate neurons and deplete in the young brain Meanwhile multipotent LT-NSCs are maintained for months but are pushed out of homeostasis by lengthening quiescence Single-cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in ... More
Stem cell dysfunction drives many age-related disorders. Identifying mechanisms that initially compromise stem cell behavior represent early targets to promote tissue function later in life. Here, we pinpoint multiple factors that disrupt neural stem cell (NSC) behavior in the adult hippocampus. Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term NSCs (ST-NSCs) and long-term NSCs (LT-NSCs). ST-NSCs divide rapidly to generate neurons and deplete in the young brain. Meanwhile, multipotent LT-NSCs are maintained for months but are pushed out of homeostasis by lengthening quiescence. Single-cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in the mature brain and identified tyrosine-protein kinase Abl1 as an NSC aging factor. Treatment with the Abl inhibitor imatinib increased NSC activation without impairing NSC maintenance in the middle-aged brain. Our study indicates that hippocampal NSCs are particularly vulnerable and adaptable to cellular aging. Less
Understanding antibody responses to SARS-CoV- is indispensable for the development of containment measures to overcome the current COVID- pandemic Recent studies showed that serum from convalescent patients can display variable neutralization capacities Still it remains unclear whether there are specific signatures that can be used to predict neutralization Here we performed a detailed analysis of sera from a cohort of recovered healthcare workers and we addressed their SARS-CoV- antibody response by ELISA against SARS-CoV- Spike receptor binding domain and nucleoprotein Both ELISA methods detected sustained levels of serum IgG against both antigens Yet the majority of individuals from our cohort ... More
Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization. Less
We report the development automation and validation of a D microfluidic liver-on-a-chip for high throughput hepatotoxicity screening the OrganoPlate LiverTox The model is comprised of aggregates of induced pluripotent stem cell iPSC -derived hepatocytes iHep seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP- monoblasts differentiated to macrophages seeded in the vascular channel of the well Mimetas OrganoPlate -lane A key component of high throughput screening is automation and we report a protocol to seed dose collect and replenish media and add assay reagents in the OrganoPlate -lane using a standard laboratory liquid ... More
We report the development, automation and validation of a 3D, microfluidic liver-on-a-chip for high throughput hepatotoxicity screening, the OrganoPlate LiverTox™. The model is comprised of aggregates of induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP-1 monoblasts differentiated to macrophages seeded in the vascular channel of the 96 well Mimetas OrganoPlate 2-lane. A key component of high throughput screening is automation and we report a protocol to seed, dose, collect and replenish media and add assay reagents in the OrganoPlate 2-lane using a standard laboratory liquid handling robot. A combination of secretome measurements and image-based analysis was used to demonstrate stable 15 day cell viability, albumin and urea secretion. Over the same time-period, CYP3A4 activity increased and alpha-fetoprotein secretion decreased suggesting further maturation of the iHeps. Troglitazone, a clinical hepatotoxin, was chosen as a control compound for validation studies. Albumin, urea, hepatocyte nuclear size and viability staining provided Robust Z’factors > 0.2 in plates treated 72 h with 180 μM troglitazone compared with a vehicle control. The viability assay provided the most robust statistic for a Robust Z’ factor = 0.6. A small library of 159 compounds with known liver effects was added to the OrganoPlate LiverTox model for 72 h at 50 μM and the Toxicological Prioritization scores were calculated. A follow up dose-response evaluation of select hits revealed the albumin assay to be the most sensitive in calculating TC50 values. This platform provides a robust, novel model which can be used for high throughput hepatotoxicity screening. Less
Background Huntington's disease HD is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors Using shotgun sequencing we investigated the gut microbiome composition in the R transgenic mouse model of HD from to weeks of age early adolescent through to adult stages Targeted metabolomics was also performed on the blood plasma of these mice n per group at weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile Results ... More
Background Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors. Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors. Using shotgun sequencing, we investigated the gut microbiome composition in the R6/1 transgenic mouse model of HD from 4 to 12 weeks of age (early adolescent through to adult stages). Targeted metabolomics was also performed on the blood plasma of these mice (n = 9 per group) at 12 weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile. Results Modelled time profiles of each species, KEGG Orthologs and bacterial genes, revealed heightened volatility in the R6/1 mice, indicating potential early effects of the HD mutation in the gut. In addition to gut dysbiosis in R6/1 mice at 12 weeks of age, gut microbiome function was perturbed. In particular, the butanoate metabolism pathway was elevated, suggesting increased production of the protective SCFA, butyrate, in the gut. No significant alterations were found in the plasma butyrate and propionate levels in the R6/1 mice at 12 weeks of age. The statistical integration of the metagenomics and metabolomics unraveled several Bacteroides species that were negatively correlated with ATP and pipecolic acid in the plasma. Conclusions The present study revealed the instability of the HD gut microbiome during the pre-motor symptomatic stage of the disease which may have dire consequences on the host's health. Perturbation of the HD gut microbiome function prior to significant cognitive and motor dysfunction suggest the potential role of the gut in modulating the pathogenesis of HD, potentially via specific altered plasma metabolites which mediate gut-brain signaling. Less
Single-cell RNA-sequencing technologies are ideally placed to resolve intratumoral heterogeneity However the lack of coverage across key mutation hotspots has precluded the correlation of genetic and transcriptional readouts from the same single cell To overcome this we developed TARGET-seq a protocol for TARGETed high-sensitivity single-cell mutational analysis with extremely low allelic dropout rates parallel RNA SEQuencing and cell-surface proteomics Here we present a detailed step-by-step protocol for TARGET-seq including troubleshooting tips approaches for automation and methods for high-throughput multiplexing of libraries
How have complex brains evolved from simple circuits Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei the output structures of the cerebellum Using single-nucleus RNA sequencing in mice chickens and humans as well as STARmap spatial transcriptomic analysis and whole central nervous system projection tracing we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in ... More
How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole–central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets. Less
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types Here we examine how specific transcriptomic types of mouse prefrontal cortex PFC projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks We found that most types projected to multiple targets and most targets received projections from multiple types except PFC PAG periaqueductal gray By comparing Ca activity of the molecularly homogeneous PFC PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice ... More
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics. Less
The ongoing severe acute respiratory syndrome coronavirus SARS-CoV- pandemic has devastated the global economy and claimed more than million lives presenting an urgent global health crisis To identify host factors required for infection by SARS-CoV- and seasonal coronaviruses we designed a focused high-coverage CRISPR-Cas library targeting members of a recently published SARS-CoV- protein interactome We leveraged the compact nature of this library to systematically screen SARS-CoV- at two physiologically relevant temperatures along with three related coronaviruses human coronavirus E HCoV- E HCoV-NL and HCoV-OC allowing us to probe this interactome at a much higher resolution than genome-scale studies This approach ... More
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks. Less
The role of gene expression during learning and in short-term memories has been studied extensively but less is known about remote memories which can persist for a lifetime Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning Out of a vast plasticity-coding space we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory Unexpectedly astrocytes and ... More
The role of gene expression during learning and in short-term memories has been studied extensively1,2,3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage. Less
PDGF VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine paracrine and endocrine mechanisms We investigated organ-specific metabolic roles of Drosophila PDGF VEGF-like factors Pvfs We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf signals to the Drosophila hepatocyte-like cells oenocytes to suppress lipid synthesis by activating the Pi K Akt TOR signaling cascade in the oenocytes Functionally this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis We find ... More
PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine, and endocrine mechanisms. We investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells. Less
Lung cancer the leading cause of cancer mortality exhibits heterogeneity that enables adaptability limits therapeutic success and remains incompletely understood Single-cell RNA sequencing scRNA-seq of metastatic lung cancer was performed using clinical biopsies obtained from patients before and during targeted therapy Over cancer and tumor microenvironment TME single-cell profiles exposed a rich and dynamic tumor ecosystem scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically Cancer cells surviving therapy as residual disease RD expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition whereas those present at on-therapy progressive disease PD upregulated kynurenine plasminogen and gap-junction pathways ... More
Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes. Less
In biology it is often critical to determine the identity of an organism and phenotypic traits of interest Whole-genome sequencing can be useful for this but has limited power for trait prediction However we can take advantage of the inherent information content of phenotypes to bypass these limitations We demonstrate in clinical and environmental bacterial isolates that growth dynamics in standardized conditions can differentiate between genotypes even among strains from the same species We find that for pairs of isolates there is little correlation between genetic distance according to phylogenetic analysis and phenotypic distance as determined by growth dynamics This ... More
In biology, it is often critical to determine the identity of an organism and phenotypic traits of interest. Whole-genome sequencing can be useful for this but has limited power for trait prediction. However, we can take advantage of the inherent information content of phenotypes to bypass these limitations. We demonstrate, in clinical and environmental bacterial isolates, that growth dynamics in standardized conditions can differentiate between genotypes, even among strains from the same species. We find that for pairs of isolates, there is little correlation between genetic distance, according to phylogenetic analysis, and phenotypic distance, as determined by growth dynamics. This absence of correlation underscores the challenge in using genomics to infer phenotypes and vice versa. Bypassing this complexity, we show that growth dynamics alone can robustly predict antibiotic responses. These findings are a foundation for a method to identify traits not easily traced to a genetic mechanism. Less
The Mantis microfluidics liquid handler was acquired as a tool for our core facility to improve our workflows and facilitate research projects for others The robot dispensing system can be employed to set up complex dilution patterns which can be advantageous to development projects The Mantis has been utilized in a proof of concept project with our Bio-Rad Droplet DigitalTM PCR ddPCRTM to determine its effectiveness in optimizing reaction primer and template concentrations Protocols were run testing the effectiveness of chip cleaning protocols in removing residual DNA from chips before reusing them in a ddPCR assay Mantis dispensing and dilution ... More
The Mantis microfluidics liquid handler was acquired as a tool for our core facility to improve our workflows and facilitate research projects for others. The robot dispensing system can be employed to set up complex dilution patterns which can be advantageous to development projects. The Mantis has been utilized in a proof of concept project with our Bio-Rad Droplet DigitalTM PCR (ddPCRTM) to determine its effectiveness in optimizing reaction primer and template concentrations. Protocols were run testing the effectiveness of chip cleaning protocols in removing residual DNA from chips before reusing them in a ddPCR assay. Mantis dispensing and dilution series were compared to manual pipetting. In addition, a variable primer setup for ddPCR was run to determine the best conditions for a ddPCR assay. The Mantis has a much speedier delivery than manual setup or our automated pipetting robot. A normalization test of dispensing one reagent with one tip to a plate in variable amounts to each well was completed in less than 2 minutes by the Mantis, while our pipetting robot took over 12 minutes. The cleaning protocols removed DNA below the sensitivity of our ddPCR. The variability of replicate counts of droplets on plate set up with the Mantis has similar spread and deviation compared to a careful manual pipetting setup of a plate. The number of reagents or samples that can be included in a protocol are limited by the number of chips loaded on the Mantis or timeliness between cleanings. Within some limitations the Mantis can be a useful instrument in a core environment. Less
Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes such as mitotic spindle formation telomere homeostasis and Wnt -catenin signalling In recent years tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis Further it has become clear that tankyrases are not only enzymes but also act as scaffolding proteins forming large cellular signalling complexes While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation ... More
Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and cost-effective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein–protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions. Less
Background Immune checkpoint inhibition ICI alone is not efficacious for a large number of patients with melanoma brain metastases We previously established an in situ vaccination ISV regimen combining radiation and immunocytokine to enhance response to ICIs Here we tested whether ISV inhibits the development of brain metastases in a murine melanoma model Methods B GD melanoma primary tumors were engrafted on the right flank of C BL mice After weeks primary tumors were treated with ISV radiation Gy day -GD immunocytokine hu -IL days and ICI -CTLA- days Complete response CR was defined as no residual tumor observed at ... More
Background Immune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model. Methods B78 (GD2+) melanoma ‘primary’ tumors were engrafted on the right flank of C57BL/6 mice. After 3–4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6–10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay. Results ISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8+ and CD4+ T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8+ infiltration and CD8+:FOXP3+ ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or Mhc-1. Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts. Conclusion ISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases. Less