Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated. We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures. We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression. Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally. These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures. We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma. Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.

Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.

The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.

Lysate-based cell-free systems have become a major platform to study gene expression but batch-to-batch variation makes protein production difficult to predict. Here we describe an active learning approach to explore a combinatorial space of ~4,000,000 cell-free buffer compositions, maximizing protein production and identifying critical parameters involved in cell-free productivity. We also provide a one-step-method to achieve high quality predictions for protein production using minimal experimental effort regardless of the lysate quality.

Base editors derived from CRISPR-Cas9 systems and DNA editing enzymes offer an unprecedented opportunity for the precise modification of genes, but have yet to be used at a genome-scale throughput. Here, we test the ability of the Target-AID base editor to systematically modify genes genome-wide by targeting yeast essential genes. We mutate around 17,000 individual sites in parallel across more than 1500 genes. We identify over 700 sites at which mutations have a significant impact on fitness. Using previously determined and preferred Target-AID mutational outcomes, we find that gRNAs with significant effects on fitness are enriched in variants predicted to be deleterious based on residue conservation and predicted protein destabilization. We identify key features influencing effective gRNAs in the context of base editing. Our results show that base editing is a powerful tool to identify key amino acid residues at the scale of proteomes.

Methane is the second most important greenhouse gas after carbon dioxide and approximately 11% of the global anthropogenic methane emissions originate from rice fields. Sulfate amendment is a mitigation strategy to reduce methane emissions from rice fields because sulfate reducers and methanogens compete for the same substrates. Cable bacteria are filamentous bacteria known to increase sulfate levels via electrogenic sulfide oxidation. Here we show that one-time inoculation of rice-vegetated soil pots with cable bacteria increases the sulfate inventory 5-fold, which leads to the reduction of methane emissions by 93%, compared to control pots lacking cable bacteria. Promoting cable bacteria in rice fields by enrichment or sensible management may thus become a strategy to reduce anthropogenic methane emissions.

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

Yield losses caused by fungal pathogens represent a major threat to global food production. One of the most devastating fungal wheat pathogens is Zymoseptoria tritici. Despite the importance of this fungus, the underlying mechanisms of plant-pathogen interactions are poorly understood. Here we present a conceptual framework based on coinfection assays, comparative metabolomics, and microbiome profiling to study the interaction of Z. tritici in susceptible and resistant wheat. We demonstrate that Z. tritici suppresses the production of immune-related metabolites in a susceptible cultivar. Remarkably, this fungus-induced immune suppression spreads within the leaf and even to other leaves, a phenomenon that we term "systemic induced susceptibility". Using a comparative metabolomics approach, we identify defense-related biosynthetic pathways that are suppressed and induced in susceptible and resistant cultivars, respectively. We show that these fungus-induced changes correlate with changes in the wheat leaf microbiome. Our findings suggest that immune suppression by this hemibiotrophic pathogen impacts specialized plant metabolism, alters its associated microbial communities, and renders wheat vulnerable to further infections.

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.

A diagnosis of motor Parkinson's disease (PD) is preceded by a prolonged premotor phase with accumulating neuronal damage. Here we examined the temporal relation between α-synuclein (α-syn) T cell reactivity and PD. A longitudinal case study revealed that elevated α-syn-specific T cell responses were detected prior to the diagnosis of motor PD, and declined after. The relationship between T cell reactivity and early PD in two independent cohorts showed that α-syn-specific T cell responses were highest shortly after diagnosis of motor PD and then decreased. Additional analysis revealed significant association of α-syn-specific T cell responses with age and lower levodopa equivalent dose. These results confirm the presence of α-syn-reactive T cells in PD and show that they are most abundant immediately after diagnosis of motor PD. These cells may be present years before the diagnosis of motor PD, suggesting avenues of investigation into PD pathogenesis and potential early diagnosis.

mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.

Bouts of high frequency activity known as sharp wave ripples (SPW-Rs) facilitate communication between the hippocampus and neocortex. However, the paths and mechanisms by which SPW-Rs broadcast their content are not well understood. Due to its anatomical positioning, the granular retrosplenial cortex (gRSC) may be a bridge for this hippocampo-cortical dialogue. Using silicon probe recordings in awake, head-fixed mice, we show the existence of SPW-R analogues in gRSC and demonstrate their coupling to hippocampal SPW-Rs. gRSC neurons reliably distinguished different subclasses of hippocampal SPW-Rs according to ensemble activity patterns in CA1. We demonstrate that this coupling is brain state-dependent, and delineate a topographically-organized anatomical pathway via VGlut2-expressing, bursty neurons in the subiculum. Optogenetic stimulation or inhibition of bursty subicular cells induced or reduced responses in superficial gRSC, respectively. These results identify a specific path and underlying mechanisms by which the hippocampus can convey neuronal content to the neocortex during SPW-Rs.

Sex determination mechanisms often differ even between related species yet the evolution of sex chromosomes remains poorly understood in all but a few model organisms. Some nematodes such as Caenorhabditis elegans have an XO sex determination system while others, such as the filarial parasite Brugia malayi, have an XY mechanism. We present a complete B. malayi genome assembly and define Nigon elements shared with C. elegans, which we then map to the genomes of other filarial species and more distantly related nematodes. We find a remarkable plasticity in sex chromosome evolution with several distinct cases of neo-X and neo-Y formation, X-added regions, and conversion of autosomes to sex chromosomes from which we propose a model of chromosome evolution across different nematode clades. The phylum Nematoda offers a new and innovative system for gaining a deeper understanding of sex chromosome evolution.

Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 μM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae's surface. These results indicate that bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean.

Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1β is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1β production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities.

Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD+) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2-) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. 15N-labeling experiments revealed that NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH4+ oxidation with electrode as electron acceptor. Complete NH4+ oxidation to N2 without accumulation of NO2- and NO3- was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.

Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100's of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

TENT5C is a non-canonical cytoplasmic poly(A) polymerase highly expressed by activated B cells to suppress their proliferation. Here we measure the global distribution of poly(A) tail lengths in responsive B cells using a Nanopore direct RNA-sequencing approach, showing that TENT5C polyadenylates immunoglobulin mRNAs regulating their half-life and consequently steady-state levels. TENT5C is upregulated in differentiating plasma cells by innate signaling. Compared with wild-type, Tent5c-/- mice produce fewer antibodies and have diminished T-cell-independent immune response despite having more CD138high plasma cells as a consequence of accelerated differentiation. B cells from Tent5c-/- mice also have impaired capacity of the secretory pathway, with reduced ER volume and unfolded protein response. Importantly, these functions of TENT5C are dependent on its enzymatic activity as catalytic mutation knock-in mice display the same defect as Tent5c-/-. These findings define the role of the TENT5C enzyme in the humoral immune response.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.