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Reports on recent research into the operation of neuronal local circuits in higher brain functions. (CP)

Discussed are past work and recent advances in the use of electron microscopes for viewing structures immersed in gas and liquid. Improved environmental chambers make it possible to examine wet specimens easily. (Author/RH)

The Gordon Research Conferences were established to stimulate research in universities, research foundations, and industrial laboratories. The topics for the conferences are listed as well as the locations and scheduled presentations at each conference. (CS)

Included is a list of laboratory instruments and equipment and the names and addresses of their manufacturers. Names of instruments appear in alphabetical order in heavy black type and the names of manufacturers of each instrument appear below each instrument entry in ordinary type. Each instrument is identified by a "category number." Unnumbered cross references are provided for instruments that can be listed in more than one place in the alphabet. Each manufacturer has also been assigned a "manufacturer's number." Complete names, addresses, and telephone numbers of all manufacturers are given in a separate section. Manufacturers are listed in alphabetical and numerical order. (Editor/PS)

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. Here we discover gasdermin homologs encoded in bacteria that execute prokaryotic cell death. Structures of bacterial gasdermins reveal a conserved pore-forming domain that is stabilized in the inactive state with a buried lipid modification. We demonstrate that bacterial gasdermins are activated by dedicated caspase-like proteases that catalyze site-specific cleavage and removal of an inhibitory C-terminal peptide. Release of autoinhibition induces the assembly of >200 Å pores that form a mesh-like structure and disrupt membrane integrity. These results demonstrate that caspase-mediated activation of gasdermins is an ancient form of regulated cell death shared between bacteria and animals.

In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3–pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated A1 of the guide strand and the 1-nt 3′ overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.

A copper-containing antibiotic Bacteria require transition metal ions for biological processes and must also protect themselves against excess metal, which is toxic. Patteson et al . explored how the environmental bacterium Pseudomonas aeruginosa uses a five-enzyme pathway to synthesize a small-molecule complex, fluopsin C, which is built from cysteine and contains a copper ion. The biosynthesis involves unusual enzymatic transformations that convert cysteine to a thiohydroximate, two of which chelate a copper ion in the final natural product. Fluopsin C protects P. aeruginosa from excess copper and also acts as a broad-spectrum antibiotic against other bacteria. —VV

Integrator and protein phosphatase 2A (PP2A) form a complex that dephosphorylates paused RNA polymerase II (Pol II), cleaves the nascent RNA, and terminates transcription. We report the structure of the pretermination complex containing the human Integrator-PP2A complex bound to paused Pol II. Integrator binds Pol II and the pausing factors DSIF and NELF to exclude binding of the elongation factors SPT6 and PAF1 complex. Integrator also binds the C-terminal domain of Pol II and positions PP2A to counteract Pol II phosphorylation and elongation. The Integrator endonuclease docks to the RNA exit site and opens to cleave nascent RNA about 20 nucleotides from the Pol II active site. Integrator does not bind the DNA clamps formed by Pol II and DSIF, enabling release of DNA and transcription termination.

Nodulation regulation Legumes convert atmospheric nitrogen into biologically useful ammonium with the help of symbiotic bacteria housed in root nodules. Much of nodule development is controlled by the transcription factor NODULE INCEPTION (NIN). Feng et al . show that NIN is proteolytically processed to release a fragment that regulates the later stages of nodulation when the nodules acquire nitrogen-fixing capability. In related work, Jiang et al . identified members of the NIN-like protein (NLP) transcription factor family as being regulators of leghemoglobin expression acting through an unusual promotor motif shared across legumes. —PJH

Control by RNA polymerase II Evidence indicates that yeast cells initiate DNA synthesis and transition from the G 1 to the S phase of the cell cycle when cyclin 3 accumulates and causes phosphorylation of Whi5, a functional equivalent of the mammalian Rb (retinoblastoma) protein. Kõivomägi et al . now present evidence for a different cyclin-dependent kinase target (see the Perspective by Fisher). They found that the cyclin 3–cyclin-dependent kinase (Cdk) 1 complex in yeast promoted phosphorylation of RNA polymerase II and thus increased transcription at genes that control entry into the cell cycle. Cdks that regulate the cell cycle can thus act by similar mechanisms to so-called “transcriptional Cdks,” which are known to act as transcriptional regulators but not to function in control of cell division. —LBR

Recent advancements in DNA sequencing technologies and laboratory preparation protocols have rapidly expanded the scope of ancient DNA research over the past decade, both temporally and geographically. Discoveries include interactions between archaic and modern humans as well as modern human population dynamics, including those coinciding with the Last Glacial Maximum and the settlement history of most world regions. This new type of data allows us to examine the deep past of human population dynamics and sharpen the current understanding of our present. The continued development in the ancient DNA field has transformed our understanding of human genetic history and will keep uncovering the further mysteries of our recent evolutionary past.

Silver in the linings The bacterium Shewanella oneidensis is well known to use extracellular electron sinks, metal oxides and ions in nature or electrodes when cultured in a fuel cell, to power the catabolism of organic material. However, the power density of microbial fuel cells has been limited by various factors that are mostly related to connecting the microbes to the anode. Cao et al . found that a reduced graphene oxide–silver nanoparticle anode circumvents some of these issues, providing a substantial increase in current and power density (see the Perspective by Gaffney and Minteer). Electron microscopy revealed silver nanoparticles embedded or attached to the outer cell membrane, possibly facilitating electron transfer from internal electron carriers to the anode. —MAF

Safeguarding protein complex assembly The assembly of multiprotein complexes inside the cell requires each subunit to be produced at a defined level relative to its partners. Imbalances in subunit synthesis are inevitable, necessitating the elimination of unassembled intermediates. Zavodszky et al . found that a ubiquitin ligase called HERC1 is responsible for marking certain assembly intermediates of the proteasome for degradation. HERC1 finds these intermediates by recognizing a proteasome assembly factor that normally dissociates when assembly is complete. A point mutation in HERC1 that impairs its ability to recognize proteasome assembly intermediates causes neurodegeneration in mice, highlighting the importance of this quality control pathway. —SMH

The protective efficacy of neutralizing antibodies (nAbs) elicited during natural infection with SARS-CoV-2 and by vaccination based on its spike protein has been compromised with emergence of the recent SARS-CoV-2 variants. Residues E484 and K417 in the receptor-binding site (RBS) are both mutated in lineages first described in South Africa (B.1.351) and Brazil (B.1.1.28.1). The nAbs isolated from SARS-CoV-2 patients are preferentially encoded by certain heavy-chain germline genes and the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2) can each bind the RBS in two different binding modes. However, their binding and neutralization are abrogated by either the E484K or K417N mutation, whereas nAbs to the cross-reactive CR3022 and S309 sites are largely unaffected. This structural and functional analysis illustrates why mutations at E484 and K417 adversely affect major classes of nAbs to SARS-CoV-2 with consequences for next-generation COVID-19 vaccines.

SARS-CoV-2 from alpha to epsilon As battles to contain the COVID-19 pandemic continue, attention is focused on emerging variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that have been deemed variants of concern because they are resistant to antibodies elicited by infection or vaccination or they increase transmissibility or disease severity. Three papers used functional and structural studies to explore how mutations in the viral spike protein affect its ability to infect host cells and to evade host immunity. Gobeil et al . looked at a variant spike protein involved in transmission between minks and humans, as well as the B1.1.7 (alpha), B.1.351 (beta), and P1 (gamma) spike variants; Cai et al . focused on the alpha and beta variants; and McCallum et al . discuss the properties of the spike protein from the B1.1.427/B.1.429 (epsilon) variant. Together, these papers show a balance among mutations that enhance stability, those that increase binding to the human receptor ACE2, and those that confer resistance to neutralizing antibodies. —VV

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. We demonstrated that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine, musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell (hiPSC)-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive phenylalanine in Pair(−/−) and Pah(R408W/R408W) mice and improved the phenylalanine tolerance of these mice.

Calcium signaling for host cell death In response to microbial pathogens, some plants kill off their own cells to limit further spread of infection. The Toll/Interleukin-1 receptor/Resistance class of nucleotide-binding leucine-rich repeat receptors (known as TNLs) function in plants as immune receptors. These TNLs work together with a dedicated set of helper proteins. Jacob et al. reveal the structure of one of these helpers known as NRG1 (N REQUIREMENT GENE 1). The structure resembles a known animal cation channel. The authors demonstrate that helper NLRs directly control calcium ion influx to initiate host cell death, providing a mechanism for TNL outputs. Science , abg7917, this issue p. 420

Mind your metals Iron–sulfur clusters are important cofactors for proteins involved in metabolism and electron transfer but are also sometimes found in enzymes involved in transcription and replication of DNA. In vitro expression of such enzymes can result in faulty cluster assembly and confusion about the composition of the functional enzyme. Using a careful anoxic purification scheme, Maio et al. found that the severe acute respiratory syndrome coronavirus 2 RNA–dependent RNA polymerase contains two iron–sulfur clusters at two sites previously observed to bind zinc ions. Mutation of the ligating cysteine residues resulted in loss of polymerase activity. A less severe loss of activity was seen in the zinc-containing enzyme. Treatment with the nitroxide drug TEMPOL resulted in degradation of the clusters, enzyme inhibition, and inhibition of viral replication in cell culture. Science , abi5224, this issue p. 236

How Hedgehog gets its lipid tail Phospholipid membranes serve as barriers between different cellular environments but are also crucial platforms for biosynthesis, signaling, and transport. In animals, the developmental signaling protein Hedgehog must be modified with an acyl group by the membrane-embedded enzyme Hedgehog acyltransferase (HHAT) to be recognized by its receptor. Using cryo–electron microscopy, Jiang et al. determined structures of HHAT bound to palmitoyl–coenzyme A or a palmitoylated peptide product. Two cavities connect at the active site, enabling acylation of Hedgehog in the lumen of the endoplasmic reticulum by lipid substrates from the cytosolic face of the membrane. Science , abg4998, this issue p. 1215

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq (1) ) to the spike ectodomain (S-ECD (2) ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å (2) ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning (3,4) our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

Competing signal peptides hold the key When a pollen grain lands on a receptive flower's pistil, a complex dance leading to sexual reproduction begins. Liu et al. show some of the early steps that help to distinguish a compatible pollen grain from a random piece of dust. Normally, a stigmatic gatekeeper, the ANJEA–FERONIA receptor kinase complex, perceives signaling peptides produced by the stigma that drive the production of reactive oxygen species at the stigma papillae. Upon pollination, POLLEN COAT PROTEIN B-class peptides compete with those stigmatic peptides for binding to the stigmatic receptor kinase complex. The subsequent decline of stigmatic reactive oxygen species production allows hydration and opens the gates to pollen germination. Science , this issue p. 171

Ciliary motors locked closed by Shulin Motile cilia and flagella are vital cellular organelles with functions that include setting up the left-right body axis, clearing airways of mucus, and driving single-cell movements. Cilia beating is powered by arrays of dynein motors, the key force generators being the outer dynein arm (ODA) complexes. Using the protozoan Tetrahymena , Mali et al. identified a factor, which they name Shulin, that binds newly synthesized ODAs. Cryo–electron microscopy revealed how Shulin locks the dynein motors together by shutting off motor activity and facilitating delivery of ODAs from the cytoplasm to their final position in the cilia. Science , this issue p. 910

Yeast switches for glucose and galactose Some organisms can switch metabolic pathways depending on their environment. One such example is yeast, which can transition between the sugars glucose and galactose as carbon sources. Boocock et al. show that this ability has undergone selection, resulting in the maintenance of two incompatible metabolic pathways in a select set of yeast strains within a single species. A phylogenetic analysis supports that these different pathways are mediated by three genes that differ between strains within and among yeast species and likely have been maintained over 10 million to 20 million years. Science , this issue p. 415

A tight couple makes messenger RNAs Gene expression in eukaryotes first requires transcription of DNA to an RNA copy and then splicing to form the final, processed messenger RNA (mRNA). Zhang et al. investigated how gene transcription and RNA splicing are physically coupled. Using cryo–electron microscopy, they resolved the molecular structure of a complex of the transcription enzyme RNA polymerase II with part of the splicing machinery, the U1 small nuclear ribonucleoprotein particle. The results provide important details for our understanding of coupled mRNA production. Science , this issue p. 305

PreTCRs use horizontal docking geometry The T cell receptor (TCR) recognizes peptide-bound major histocompatibility complex molecules (pMHCs) and consists of an α chain in association with a β chain. Both chains have hypervariable complementarity-determining regions (CDRs) that inform whether a particular TCR can recognize a given pMHC. To successfully graduate from the thymus, aspiring αβT cells must generate a functional TCR. During one early checkpoint in this process, the β chain is first paired with a preTβ chain to form the preTCR. Li et al. used x-ray crystallography to visualize how preTCRs recognize pMHCs. They report that the CDR3 loop of the preTCR β chain contacts the pMHC with a distinctive lateral topography. This is in contrast to the established binding modality of mature TCRs, whereby all three CDR loops on both α and β chains bind in a vertical orientation. These complexes help solve the mystery of how only functionally rearranged β chains using competent CDR3 loops can properly engage with pMHC at the preTCR stage. Science , this issue p. 181

Nanobodies that neutralize Monoclonal antibodies that bind to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show therapeutic promise but must be produced in mammalian cells and need to be delivered intravenously. By contrast, single-domain antibodies called nanobodies can be produced in bacteria or yeast, and their stability may enable aerosol delivery. Two papers now report nanobodies that bind tightly to spike and efficiently neutralize SARS-CoV-2 in cells. Schoof et al. screened a yeast surface display of synthetic nanobodies and Xiang et al. screened anti-spike nanobodies produced by a llama. Both groups identified highly potent nanobodies that lock the spike protein in an inactive conformation. Multivalent constructs of selected nanobodies achieved even more potent neutralization. Science , this issue p. 1473 , p. 1479

Antibodies predating infection Immunological memory after infection with seasonal human coronaviruses (hCoVs) may potentially contribute to cross-protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Ng et al. report that in a cohort of 350 SARS-CoV-2–uninfected individuals, a small proportion had circulating immunoglobulin G (IgG) antibodies that could cross-react with the S2 subunit of the SARS-CoV-2 spike protein (see the Perspective by Guthmiller and Wilson). By contrast, COVID-19 patients generated IgA, IgG, and IgM antibodies that recognized both the S1 and S2 subunits. The anti-S2 antibodies from SARS-CoV-2–uninfected patients showed specific neutralizing activity against both SARS-CoV-2 and SARS-CoV-2 S pseudotypes. A much higher percentage of SARS-CoV-2–uninfected children and adolescents were positive for these antibodies compared with adults. This pattern may be due to the fact that children and adolescents generally have higher hCoV infection rates and a more diverse antibody repertoire, which may explain the age distribution of COVID-19 susceptibility. Science, this issue p. 1339; see also p. 1272, SARS-CoV-2 neutralizing antibodies can be found in some uninfected individuals—predominantly children and adolescents., Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)–reactive antibodies were detectable using a flow cytometry–based method in SARS-CoV-2–uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S–reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2–uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Spontaneously arising channels transporting the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feed-back between auxin signaling and transport as underlying mechanism but molecular players await discovery. Here we identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related, Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feed-back that coordinates polarization of individual cells during auxin canalization.

Saving a host cell from itself A fundamental mammalian defense mechanism against pathogens and damaged cellular DNA is to recognize DNA fragments in the cytosol and trigger an inflammatory response. The cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) that recognizes cytosolic DNA is also found in the nucleus, but here its activity is suppressed by tethering to chromatin. Two papers now report cryo–electron microscopy structures of cGAS bound to the nucleosome core particle (NCP). Kujirai et al. observed a structure with two cGAS molecules bridging two NCPs, whereas Boyer et al. observed cGAS bound to a single nucleosome. Together, these structures show how cGAS is prevented from autoreactivity toward host DNA. Science , this issue p. 455 , p. 450

Saving a host cell from itself A fundamental mammalian defense mechanism against pathogens and damaged cellular DNA is to recognize DNA fragments in the cytosol and trigger an inflammatory response. The cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) that recognizes cytosolic DNA is also found in the nucleus, but here its activity is suppressed by tethering to chromatin. Two papers now report cryo–electron microscopy structures of cGAS bound to the nucleosome core particle (NCP). Kujirai et al. observed a structure with two cGAS molecules bridging two NCPs, whereas Boyer et al. observed cGAS bound to a single nucleosome. Together, these structures show how cGAS is prevented from autoreactivity toward host DNA. Science , this issue p. 455 , p. 450

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo–electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

Genetic profiles of the bladder Depending on the environment of the individual, the human bladder can be exposed to carcinogens as they are flushed through the body. Lawson et al. and Li et al. examined the genetic composition of laser-dissected microbiopsies from normal and cancer cells collected from the urothelium, a specialized epithelium lining the lower urinary tract (see the Perspective by Rozen). These complementary studies identified the mutational landscape of bladder urothelium through various sequencing strategies and identified high mutational heterogeneity within and between individuals and tumors. Both studies identified mutational profiles related to specific carcinogens such as aristolochic acid and the molecules found in tobacco. These studies present a comprehensive description of the diverse mutational landscape of the human bladder in health and disease, unraveling positive selection for cancer-causing mutations, a diversity of mutational processes, and large differences across individuals. Science , this issue p. 75 , p. 82 ; see also p. 34

Fumarate targets pyroptosis A form of inflammatory cell death called pyroptosis depends on the caspase-mediated cleavage of gasdermin D (GSDMD), the fragments of which assemble into permeability pores that then kill the cell. The mechanisms regulating this important cellular process are not yet fully understood. Humphries et al. now report that the tricarboxylic acid cycle intermediate fumarate can act as an inhibitor of pyroptosis (see the Perspective by Pickering and Bryant). Both endogenous fumarate and exogenously delivered dimethyl fumarate (DMF) convert the cysteines in GSDMD to S-(2-succinyl)-cysteines (a process called succination) to prevent its interaction with caspases and subsequent processing and activation. Administration of DMF to mice alleviated inflammation in models of multiple sclerosis and familial Mediterranean fever. These findings may explain the efficacy of DMF as a treatment for multiple sclerosis and other inflammatory diseases and offer insights into future anti-inflammatory drug design. Science , this issue p. 1633 ; see also p. 1564

How ribosomes are made The formation of eukaryotic ribosomes is a complex process that starts with transcription of a large precursor RNA that assembles into a large 90S preribosome, which matures to finally give the 40S small subunit of the ribosome. Cheng et al. and Du et al. give insight into this process, using cryo–electron microscopy to look at intermediates along the pathway. Together, these studies reveal how a cast of molecular players act to coordinate the compositional and structural changes that transform the 90S preribosome into a pre-40S subunit. Science , this issue p. 1470 , p. 1477

Inosine modulates antitumor immunity Checkpoint blockade immunotherapy harnesses the immune system to kill cancer cells and has been used with great success to treat certain tumors, but not all cancer patients respond. The efficacy of checkpoint blockade immunotherapy has been shown to depend on the presence of distinct, beneficial bacteria residing in the gut of patients, but how the microbiome mediates such beneficial effects is unclear. Mager et al. found that specific bacteria produce a metabolite called inosine that enhances the effect of checkpoint blockade immunotherapy (see the Perspective by Shaikh and Sears). In mouse models, inosine, together with proinflammatory stimuli and immunotherapy, strongly enhanced the antitumor capacities of T cells in multiple tumor types, including colorectal cancer, bladder cancer, and melanoma. Science , this issue p. 1481 ; see also p. 1427

Microbiota protect the kidneys Chronic kidney disease (CKD) afflicts millions of people globally. The first-line treatment for CKD is dietary intervention, so there may be a gut microbiota–associated component. Lobel et al. investigated the mechanistic links between the microbiota and protein intake, because the protein metabolites indole and indoxyl sulfate are known uremic toxins (see the Perspective by Pluznick). The authors used a mouse model of CKD precipitated by a paucity of the dietary sulfur–containing amino acids methionine and cysteine. Bacterial metabolism of sulfur-containing amino acids modulated indole production by sulfide inhibition of the enzyme tryptophanase, thus abrogating uremic toxicity by this metabolite in this model system. Science , this issue p. 1518 ; see also p. 1426

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The pioneering ribosome potentially contacts RNA polymerase (RNAP), forming a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here we present a series of structures representing uncoupled, coupled and collided expressome states determined by electron cryomicroscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, stabilizing an otherwise variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance-channel to the RNAP exit-channel. In this collided complex, NusG-linkage is no longer possible.These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.One Sentence SummaryStructures of the molecular assembly executing gene expression shed light on transcription translation coupling.

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αβ and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αβ T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αβ T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αβ and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.

Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo–electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20.

How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron–specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3–bound c-Fos and importin α3–deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.

Grafting success by cell wall remodeling Plants that produce great fruit may not always have great roots. Grafting of a productive scion onto a resilient rootstock has provided agriculturalists with solutions to this and other challenges. Notaguchi et al. have now studied why some plant grafts work better than others (see the Perspective by McCann). The tobacco relative Nicotiana benthamiana ( Nb ) turns out to be the superhero of grafting, able to form grafts with plants from a wide range of evolutionary families. A bit of Nb , set as a middleman between a tomato scion and an Arabidopsis rootstock, negotiated a successful junction between these two otherwise nonconversant plant species. The expression of β-1,4-glucanases secreted into the extracellular region turns out to be key in facilitating cell wall reconstruction. Science , this issue p. 698 ; see also p. 618

Hitting SARS-CoV-2 in a new spot A key target for therapeutic antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the spike protein, a trimeric protein complex with each monomer comprising an S1 and an S2 domain that mediate binding to host cells and membrane fusion, respectively. In addition to the receptor binding domain (RBD), S1 has an N-terminal domain (NTD). In searching for neutralizing antibodies, there has been a focus on the RBD. Chi et al. isolated antibodies from 10 convalescent patients and identified an antibody that potently neutralizes the virus but does not bind the RBD. Cryo–electron microscopy revealed the epitope as the NTD. This NTD-targeting antibody may be useful to combine with RBD-targeting antibodies in therapeutic cocktails. Science , this issue p. 650