Slide-tags enables single-nucleus barcoding for multimodal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

Implementing FCTC Article 17 Through Participatory Research With Bidi Workers in Tamil Nadu, India.

INTRODUCTION: The exploitation, poor conditions, and precarity in the bidi (hand-rolled leaf cigarette) industry in India make it ripe for the application of the FCTC's Article 17, "Provision of support for economically viable alternative activities". "Bottom-up", participatory approaches give scope to explore bidi rollers' own circumstances, experiences, and aspirations. METHODS: A team of six community health volunteers using a participatory research orientation developed a questionnaire-based semi-structured interview tool. Forty-six bidi rolling women were interviewed by pairs of volunteers in two northern Tamil Nadu cities. Two follow-up focus groups were also held. A panel of 11 bidi rollers attended a workshop at which the findings from the interviews and focus groups were presented, further significant points were made and possible alternatives to bidi rolling were discussed. RESULTS: Bidi workers are aware of the adverse impact of their occupation on them and their families, as well as the major risks posed by the product itself for the health of consumers. However, they need alternative livelihoods that offer equivalent remuneration, convenience, and (in some cases) dignity. Alternative livelihoods, and campaigns for better rights for bidi workers while they remain in the industry, serve to undercut industry arguments against tobacco control. Responses need to be diverse and specific to local situations, i.e. "bottom-up" as much as "top-down", which can make the issue of scaling up problematic. CONCLUSION: Participatory approaches involving bidi workers themselves in discussions about their circumstances and aspirations have opened up new possibilities for alternative livelihoods to tobacco. IMPLICATIONS: Progress with the FCTC's Article 17 has generally been slow and has focussed on tobacco cultivation rather than later stages in the production process. The bidi industry in India is ripe for the application of an alternative livelihoods approach. This study is one of the first to use participatory methods to investigate the circumstances, experiences, and aspirations of bidi workers themselves.

Chestnut-crowned babbler calls are composed of meaningless shared building blocks.

A core component of human language is its combinatorial sound system: meaningful signals are built from different combinations of meaningless sounds. Investigating whether nonhuman communication systems are also combinatorial is hampered by difficulties in identifying the extent to which vocalizations are constructed from shared, meaningless building blocks. Here we present an approach to circumvent this difficulty and show that a pair of functionally distinct chestnut-crowned babbler (Pomatostomus ruficeps) vocalizations can be decomposed into perceptibly distinct, meaningless entities that are shared across the 2 calls. Specifically, by focusing on the acoustic distinctiveness of sound elements using a habituation-discrimination paradigm on wild-caught babblers under standardized aviary conditions, we show that 2 multielement calls are composed of perceptibly distinct sounds that are reused in different arrangements across the 2 calls. Furthermore, and critically, we show that none of the 5 constituent elements elicits functionally relevant responses in receivers, indicating that the constituent sounds do not carry the meaning of the call and so are contextually meaningless. Our work, which allows combinatorial systems in animals to be more easily identified, suggests that animals can produce functionally distinct calls that are built in a way superficially reminiscent of the way that humans produce morphemes and words. The results reported lend credence to the recent idea that language's combinatorial system may have been preceded by a superficial stage where signalers neither needed to be cognitively aware of the combinatorial strategy in place, nor of its building blocks.

Single-Cell (Multi)omics Technologies.

Single-cell multiomics technologies typically measure multiple types of molecule from the same individual cell, enabling more profound biological insight than can be inferred by analyzing each molecular layer from separate cells. These single-cell multiomics technologies can reveal cellular heterogeneity at multiple molecular layers within a population of cells and reveal how this variation is coupled or uncoupled between the captured omic layers. The data sets generated by these techniques have the potential to enable a deeper understanding of the key biological processes and mechanisms driving cellular heterogeneity and how they are linked with normal development and aging as well as disease etiology. This review details both established and novel single-cell mono- and multiomics technologies and considers their limitations, applications, and likely future developments.

Temperature-mediated plasticity in incubation schedules is unlikely to evolve to buffer embryos from climatic challenges in a seasonal songbird.

Phenotypic plasticity is hypothesized to facilitate adaptive responses to challenging conditions, such as those resulting from climate change. However, tests of the key predictions of this 'rescue hypothesis', that variation in plasticity exists and can evolve to buffer unfavourable conditions, remain rare. Here, we investigate among-female variation in temperature-mediated plasticity of incubation schedules and consequences for egg temperatures using the chestnut-crowned babbler (Pomatostomus ruficeps) from temperate regions of inland south-eastern Australia. Given recent phenological advances in this seasonal breeder and thermal requirements of developing embryos (>~25°C, optimally ~38°C), support for evolutionary rescue-perhaps paradoxically-requires that plasticity serves to buffer embryos more from sub-optimally low temperatures. We found significant variation in the duration of incubation bouts (mean ± SD = 27 ± 22 min) and foraging bouts (mean ± SD = 17 ± 11 min) in this maternal-only incubator. However, variation in each arose because of variation in the extent to which mothers increased on- and off-bout durations when temperatures (0-36°C) were more favourable rather than unfavourable as required under rescue. In addition, there was a strong positive intercept-slope correlation in on-bout durations, indicating that those with stronger plastic responses incubated more at average temperatures (~19°C). Combined, these effects reduced the functional significance of plastic responses: an individual's plasticity was neither associated with daily contributions to incubation (i.e. attentiveness) nor average egg temperatures. Our results highlight that despite significant among-individual variation in environmental-sensitivity, plasticity in parental care traits need not evolve to facilitate buffering against unfavourable conditions.

Self-Immolative System for Disclosure of Reactive Electrophilic Alkylating Agents: Understanding the Role of the Reporter Group.

The development of stable, efficient chemoselective self-immolative systems, for use in applications such as sensors, requires the optimization of the reactivity and degradation characteristics of the self-immolative unit. In this paper, we describe the effect that the structure of the reporter group has upon the self-immolative efficacy of a prototype system designed for the disclosure of electrophilic alkylating agents. The amine of the reporter group (a nitroaniline unit) was a constituent part of a carbamate that functioned as the self-immolative unit. The number and position of substituents on the nitroaniline unit were found to play a key role in the rate of self-immolative degradation and release of the reporter group. The position of the nitro substituent (meta- vs para-) and the methyl groups in the ortho-position relative to the carbamate exhibited an influence on the rate of elimination and stability of the self-immolative system. The ortho-methyl substituents imparted a twist on the N-C (aromatic) bond leading to increased resonance of the amine nitrogen's lone pair into the carbonyl moiety and a decrease of the leaving character of the carbamate group; concomitantly, this may also make it a less electron-withdrawing group and lead to less acidification of the eliminated ß-hydrogen.

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Exercise-induced hypoxia among emergency department patients admitted for suspected COVID-19.

BACKGROUND: Exercise-induced hypoxia (EIH) has been assessed at ED triage as part of an assessment of COVID-19; however, evidence supporting this practice is incomplete. We assessed the use of a 1-minute sit-to-stand exercise test among ED patients admitted for suspected COVID-19. METHODS: A case note review of all ED patients assessed for suspected COVID-19 between March and May 2020 at Monklands University Hospital was conducted. Demographic characteristics, clinical parameters, baseline blood tests and radiographic findings, hospital length of stay, intensive care and maximum oxygen requirement were obtained for those admitted. Using logistic regression, the association between EIH at admission triage and COVID-19 diagnosis was explored adjusting for confounding clinical parameters. RESULTS: Of 127 ED patients admitted for possible COVID-19, 37 were ultimately diagnosed with COVID-19. 36.4% of patients with COVID-19 and EIH had a normal admission chest radiograph. In multivariate analysis, EIH was an independent predictor of COVID-19 (adjusted OR 3.73 (95% CI (1.25 to 11.15)), as were lymphocyte count, self-reported exertional dyspnoea, C-reactive peptide and radiographic changes. CONCLUSIONS: This observational study demonstrates an association between EIH and a COVID-19 diagnosis. Over one-third of patients with COVID-19 and EIH exhibited no radiographic changes. EIH may represent an additional tool to help predict a COVID-19 diagnosis at initial presentation and may assist in triaging need for admission.

Experimentally induced increases in fecundity lead to greater nestling care in blue tits.

Models on the evolution of bi-parental care typically assume that maternal investment in offspring production is fixed and predict subsequent contributions to offspring care by the pair are stabilized by partial compensation. While experimental tests of this prediction are supportive, exceptions are commonplace. Using wild blue tits ( Cyanistes caeruleus), we provide, to our knowledge, the first investigation into the effects of increasing maternal investment in offspring production for subsequent contributions to nestling provisioning by mothers and male partners. Females that were induced to lay two extra eggs provisioned nestlings 43% more frequently than controls, despite clutch size being made comparable between treatment groups at the onset of incubation. Further, experimental males did not significantly reduce provisioning rates as expected by partial compensation, and if anything contributed slightly (9%) more than controls. Finally, nestlings were significantly heavier in experimental nests compared with controls, suggesting that the 22% average increase in provisioning rates by experimental pairs was beneficial. Our results have potential implications for our understanding of provisioning rules, the maintenance of bi-parental care and the timescale over which current-future life-history trade-offs operate. We recommend greater consideration of female investment at the egg stage to more fully understand the evolutionary dynamics of bi-parental care.

Ring-Opening Polymerisation of Low-Strain Nickelocenophanes: Synthesis and Magnetic Properties of Polynickelocenes with Carbon and Silicon Main Chain Spacers.

Polymetallocenes based on ferrocene, and to a lesser extent cobaltocene, have been well-studied, whereas analogous systems based on nickelocene are virtually unexplored. It has been previously shown that poly(nickelocenylpropylene) [Ni(η5 -C5 H4 )2 (CH2 )3 ]n is formed as a mixture of cyclic (6x ) and linear (7) components by the reversible ring-opening polymerisation (ROP) of tricarba[3]nickelocenophane [Ni(η5 -C5 H4 )2 (CH2 )3 ] (5). Herein the generality of this approach to main-chain polynickelocenes is demonstrated and the ROP of tetracarba[4]nickelocenophane [Ni(η5 -C5 H4 )2 (CH2 )4 ] (8), and disila[2]nickelocenophane [Ni(η5 -C5 H4 )2 (SiMe2 )2 ] (12) is described, to yield predominantly insoluble homopolymers poly(nickelocenylbutylene) [Ni(η5 -C5 H4 )2 (CH2 )4 ]n (13) and poly(tetramethyldisilylnickelocene) [Ni(η5 -C5 H4 )2 (SiMe2 )2 ]n (14), respectively. The ROP of 8 and 12 was also found to be reversible at elevated temperature. To access soluble high molar mass materials, copolymerisations of 5, 8, and 12 were performed. Superconducting quantum interference device (SQUID) magnetometry measurements of 13 and 14 indicated that these homopolymers behave as simple paramagnets at temperatures greater than 50 K, with significant antiferromagnetic coupling that is notably larger in carbon-bridged 6x /7 and 13 compared to the disilyl-bridged 14. However, the behaviour of these polynickelocenes deviates substantially from the Curie-Weiss law at low temperatures due to considerable zero-field splitting.

Chemical regulation of body feather microbiota in a wild bird.

The microbiota has a broad range of impacts on host physiology and behaviour, pointing out the need to improve our comprehension of the drivers of host-microbiota composition. Of particular interest is whether the microbiota is acquired passively, or whether and to what extent hosts themselves shape the acquisition and maintenance of their microbiota. In birds, the uropygial gland produces oily secretions used to coat feathers that have been suggested to act as an antimicrobial defence mechanism regulating body feather microbiota. However, our comprehension of this process is still limited. In this study, we for the first time coupled high-throughput sequencing of the microbiota of both body feathers and the direct environment (i.e., the nest) in great tits with chemical analyses of the composition of uropygial gland secretions to examine whether host chemicals have either specific effects on some bacteria or nonspecific broad-spectrum effects on the body feather microbiota. Using a network approach investigating the patterns of co-occurrence or co-exclusions between chemicals and bacteria within the body feather microbiota, we found no evidence for specific promicrobial or antimicrobial effects of uropygial gland chemicals. However, we found that one group of chemicals was negatively correlated to bacterial richness on body feathers, and a higher production of these chemicals was associated with a poorer body feather bacterial richness compared to the nest microbiota. Our study provides evidence that chemicals produced by the host might function as a nonspecific broad-spectrum antimicrobial defence mechanism limiting colonization and/or maintenance of bacteria on body feathers, providing new insight about the drivers of the host's microbiota composition in wild organisms.

Experimental Evidence for Phonemic Contrasts in a Nonhuman Vocal System.

The ability to generate new meaning by rearranging combinations of meaningless sounds is a fundamental component of language. Although animal vocalizations often comprise combinations of meaningless acoustic elements, evidence that rearranging such combinations generates functionally distinct meaning is lacking. Here, we provide evidence for this basic ability in calls of the chestnut-crowned babbler (Pomatostomus ruficeps), a highly cooperative bird of the Australian arid zone. Using acoustic analyses, natural observations, and a series of controlled playback experiments, we demonstrate that this species uses the same acoustic elements (A and B) in different arrangements (AB or BAB) to create two functionally distinct vocalizations. Specifically, the addition or omission of a contextually meaningless acoustic element at a single position generates a phoneme-like contrast that is sufficient to distinguish the meaning between the two calls. Our results indicate that the capacity to rearrange meaningless sounds in order to create new signals occurs outside of humans. We suggest that phonemic contrasts represent a rudimentary form of phoneme structure and a potential early step towards the generative phonemic system of human language.

Turning risk assessment and adaptation policy priorities into meaningful interventions and governance processes.

The UK is one of the first countries in the world to have set up a statutory system of national climate risk assessments followed by a national adaptation programme. Having this legal framework has been essential for enabling adaptation at the government level in a challenging political environment. However, using this framework to create an improvement in resilience to climate change across the country requires more than publishing a set of documents; it requires careful thought about what interventions work, how they can be enabled and what level of risk acceptability individuals, organizations and the country should be aiming for.This article is part of the theme issue 'Advances in risk assessment for climate change adaptation policy'.

Structural Variability of 4f and 5f Thiocyanate Complexes and Dissociation of Uranium(III)-Thiocyanate Bonds with Increased Ionicity.

A series of complexes [Et4N][Ln(NCS)4(H2O)4] (Ln = Pr, Tb, Dy, Ho, Yb) have been structurally characterized, all showing the same structure, namely a distorted square antiprismatic coordination geometry, and the Ln-O and Ln-N bond lengths following the expected lanthanide contraction. When the counterion is Cs+, a different structural motif is observed and the eight-coordinate complex Cs5[Nd(NCS)8] isolated. The thorium compounds [Me4N]4[Th(NCS)7(NO3)] and [Me4N]4[Th(NCS)6(NO3)2] have been characterized, and high coordination numbers are also observed. Finally, attempts to synthesize a U(III) thiocyanate compound has been unsuccessful; from the reaction mixture, a heterocycle formed by condensation of five MeCN solvent molecules, possibly promoted by U(III), was isolated and structurally characterized. To rationalize the inability to isolate U(III) thiocyanate compounds, thin-layer cyclic voltammetry and IR spectroelectrochemistry have been utilized to explore the cathodic behavior of [Et4N]4[U(NCS)8] and [Et4N][U(NCS)5(bipy)2] along with a related uranyl compound [Et4N]3[UO2(NCS)5]. In all examples, the reduction triggers a rapid dissociation of [NCS]- ions and decomposition. Interestingly, the oxidation chemistry of [Et4N]3[UO2(NCS)5] in the presence of bipy gives the U(IV) compound [Et4N]4[U(NCS)8], an unusual example of a ligand-based oxidation triggering a metal-based reduction. The experimental results have been augmented by a computational investigation, concluding that the U(III)-NCS bond is more ionic than the U(IV)-NCS bond.

Energetics of communal roosting in chestnut-crowned babblers: implications for group dynamics and breeding phenology.

For many endotherms, communal roosting saves energy in cold conditions, but how this might affect social dynamics or breeding phenology is not well understood. Using chestnut-crowned babblers (Pomatostomus ruficeps), we studied the effects of nest use and group size on roosting energy costs. These 50 g cooperatively breeding passerine birds of outback Australia breed from late winter to early summer and roost in huddles of up to 20 in single-chambered nests. We measured babbler metabolism at three ecologically relevant temperatures: 5°C (similar to minimum nighttime temperatures during early breeding), 15°C (similar to nighttime temperatures during late breeding) and 28°C (thermal neutrality). Nest use alone had modest effects: even for solitary babblers at 5°C, it reduced nighttime energy expenditures by <15%. However, group-size effects were substantial, with savings of up to 60% in large groups at low temperatures. Babblers roosting in groups of seven or more at 5°C, and five or more at 15°C, did not need to elevate metabolic rates above basal levels. Furthermore, even at 28°C (thermoneutral for solitary babblers), individuals in groups of four or more had 15% lower basal metabolic rate than single birds, hinting that roosting in small groups is stressful. We suggest that the substantial energy savings of communal roosting at low temperatures help explain why early breeding is initiated in large groups and why breeding females, which roost alone and consequently expend 120% more energy overnight than other group members, suffer relatively higher mortality than communally roosting group mates.

Pseudomonas syringae Effector AvrPphB Suppresses AvrB-Induced Activation of RPM1 but Not AvrRpm1-Induced Activation.

The Pseudomonas syringae effector AvrB triggers a hypersensitive resistance response in Arabidopsis and soybean plants expressing the disease resistance (R) proteins RPM1 and Rpg1b, respectively. In Arabidopsis, AvrB induces RPM1-interacting protein kinase (RIPK) to phosphorylate a disease regulator known as RIN4, which subsequently activates RPM1-mediated defenses. Here, we show that AvrPphB can suppress activation of RPM1 by AvrB and this suppression is correlated with the cleavage of RIPK by AvrPphB. Significantly, AvrPphB does not suppress activation of RPM1 by AvrRpm1, suggesting that RIPK is not required for AvrRpm1-induced modification of RIN4. This observation indicates that AvrB and AvrRpm1 recognition is mediated by different mechanisms in Arabidopsis, despite their recognition being determined by a single R protein. Moreover, AvrB recognition but not AvrRpm1 recognition is suppressed by AvrPphB in soybean, suggesting that AvrB recognition requires a similar molecular mechanism in soybean and Arabidopsis. In support of this, we found that phosphodeficient mutations in the soybean GmRIN4a and GmRIN4b proteins are sufficient to block Rpg1b-mediated hypersensitive response in transient assays in Nicotiana glutinosa. Taken together, our results indicate that AvrB and AvrPphB target a conserved defense signaling pathway in Arabidopsis and soybean that includes RIPK and RIN4.