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In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by β-lactams that bind covalently to inhibit transpeptidases and β-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed β-strands of darobactins that target the undruggable β-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

CD4⁺ T cells can either enhance or inhibit tumour immunity. Although regulatory T cells have long been known to impede antitumour responses^1-5, other CD4⁺ T cells have recently been implicated in inhibiting this response^6,7. Yet, the nature and function of the latter remain unclear. Here, using vaccines containing MHC class I (MHC-I) neoantigens (neoAgs) and different doses of tumour-derived MHC-II neoAgs, we discovered that whereas the inclusion of vaccines with low doses of MHC-II-restricted peptides (LDVax) promoted tumour rejection, vaccines containing high doses of the same MHC-II neoAgs (HDVax) inhibited rejection. Characterization of the inhibitory cells induced by HDVax identified them as type 1 regulatory T (Tr1) cells expressing IL-10, granzyme B, perforin, CCL5 and LILRB4. Tumour-specific Tr1 cells suppressed tumour rejection induced by anti-PD1, LDVax or adoptively transferred tumour-specific effector T cells. Mechanistically, HDVax-induced Tr1 cells selectively killed MHC-II tumour antigen-presenting type 1 conventional dendritic cells (cDC1s), leading to low numbers of cDC1s in tumours. We then documented modalities to overcome this inhibition, specifically via anti-LILRB4 blockade, using a CD8-directed IL-2 mutein, or targeted loss of cDC2/monocytes. Collectively, these data show that cytotoxic Tr1 cells, which maintain peripheral tolerance, also inhibit antitumour responses and thereby function to impede immune control of cancer.

Motivations bias our responses to stimuli, producing behavioural outcomes that match our needs and goals. Here we describe a mechanism behind this phenomenon: adjusting the time over which stimulus-derived information is permitted to accumulate towards a decision. As a Drosophila copulation progresses, the male becomes less likely to continue mating through challenges^1-3. We show that a set of copulation decision neurons (CDNs) flexibly integrates information about competing drives to mediate this decision. Early in mating, dopamine signalling restricts CDN integration time by potentiating Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation in response to stimulatory inputs, imposing a high threshold for changing behaviours. Later into mating, the timescale over which the CDNs integrate termination-promoting information expands, increasing the likelihood of switching behaviours. We suggest scalable windows of temporal integration at dedicated circuit nodes as a key but underappreciated variable in state-based decision-making.

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion¹. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation^2-4. At present, it is unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Here, using single-molecule imaging, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA. Notably, sliding D2-I stalls on encountering single-stranded-double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions⁵. Using cryogenic electron microscopy, we determined structures of D2-I on DNA that show that stalled D2-I makes specific interactions with the ss-dsDNA junction that are distinct from those made by sliding D2-I. Thus, D2-I surveys dsDNA and, when it reaches an ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Because ss-dsDNA junctions are found at stalled replication forks, D2-I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2-I in the recognition and protection of stalled replication forks in several DNA repair pathways.

The upper airway is an important site of infection, but immune memory in the human upper airway is poorly understood, with implications for COVID-19 and many other human diseases^1-4. Here we demonstrate that nasal and nasopharyngeal swabs can be used to obtain insights into these challenging problems, and define distinct immune cell populations, including antigen-specific memory B cells and T cells, in two adjacent anatomical sites in the upper airway. Upper airway immune cell populations seemed stable over time in healthy adults undergoing monthly swabs for more than 1 year, and prominent tissue resident memory T (T_RM) cell and B (B_RM) cell populations were defined. Unexpectedly, germinal centre cells were identified consistently in many nasopharyngeal swabs. In subjects with SARS-CoV-2 breakthrough infections, local virus-specific B_RM cells, plasma cells and germinal centre B cells were identified, with evidence of local priming and an enrichment of IgA⁺ memory B cells in upper airway compartments compared with blood. Local plasma cell populations were identified with transcriptional profiles of longevity. Local virus-specific memory CD4⁺ T_RM cells and CD8⁺ T_RM cells were identified, with diverse additional virus-specific T cells. Age-dependent upper airway immunological shifts were observed. These findings provide new understanding of immune memory at a principal mucosal barrier tissue in humans.

During development, motor neurons originating in the brainstem and spinal cord form elaborate synapses with skeletal muscle fibres¹. These neurons release acetylcholine (ACh), which binds to nicotinic ACh receptors (AChRs) on the muscle, initiating contraction. Two types of AChR are present in developing muscle cells, and their differential expression serves as a hallmark of neuromuscular synapse maturation^2-4. The structural principles underlying the switch from fetal to adult muscle receptors are unknown. Here, we present high-resolution structures of both fetal and adult muscle nicotinic AChRs, isolated from bovine skeletal muscle in developmental transition. These structures, obtained in the absence and presence of ACh, provide a structural context for understanding how fetal versus adult receptor isoforms are tuned for synapse development versus the all-or-none signalling required for high-fidelity skeletal muscle contraction. We find that ACh affinity differences are driven by binding site access, channel conductance is tuned by widespread surface electrostatics and open duration changes result from intrasubunit interactions and structural flexibility. The structures further reveal pathogenic mechanisms underlying congenital myasthenic syndromes.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons^1-3. It is a pharmacological target for various antidepressants and analgesic drugs^4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlA^EM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na⁺ and Cl^- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlA^EM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.