In situ structure of intestinal apical surface reveals nanobristles on microvilli

Microvilli are actin-bundle-supported membrane protrusions essential for absorption, secretion, and sensation. Microvilli defects cause gastrointestinal disorders; however, mechanisms controlling microvilli formation and organization remain unresolved. Here, we study microvilli by vitrifying the Caenorhabditis elegans larvae and mouse intestinal tissues with high-pressure freezing, thinning them with cryo-focused ion-beam milling, followed by cryo-electron tomography and subtomogram averaging. We find that many radial nanometer bristles referred to as nanobristles project from the lateral surface of nematode and mouse microvilli. The C. elegans nanobristles are 37.5 nm long and 4.5 nm wide. Nanobristle formation requires a protocadherin family protein, CDH-8, in C. elegans. The loss of nanobristles in cdh-8 mutants slows down animal growth and ectopically increases the number of Y-shaped microvilli, the putative intermediate structures if microvilli split from tips. Our results reveal a potential role of nanobristles in separating microvilli and suggest that microvilli division may help generate nascent microvilli with uniformity.

Microvilli are membrane-bound cell-surface protrusions that contain a core bundle of actin filaments enveloped in the plasma membrane (1–3). Many epithelial cells develop microvilli above their apical surface to enhance functional capacity for a range of physiological tasks, including nutrient absorption in the intestine (4), solute uptake in the renal tubules (5), mechanosensation in sensory stereocilia of the inner ear (6), and chemosensation in the gut, lung, and urogenital tracts (7–9). Abnormal microvillar structure and function lead to human disorders, such as life-threatening nutrient malabsorption, osmotic imbalances, and inherited deafness in Usher syndrome (1, 3, 4, 10).An intestinal absorptive cell enterocyte develops up to 1,000 densely packed microvilli in an array known as the brush border. These fingerlike outward projections enhance the functional surface area for nutrient absorption and provide the barrier for host defense against pathogens and toxins (1, 3, 4). Because the gut epithelium undergoes constant regenerative renewal, microvillus assembly is a process that continues throughout our lifetime (1, 3, 4, 11). The long-standing questions regarding microvillus formation are how microvilli are formed with striking uniformity in sizes and how these protrusions are maximally packed in a hexagonal pattern.The tip of microvilli is known to be decorated by additional filamentous structures. The glycoprotein-rich glycocalyx localizes between the apical tip of microvilli and the luminal space (Fig. 1A), provides a barrier for pathogens, and serves as the interface for nutrient digestion (3, 4). The protocadherin-based adhesion tip links localize between adjacent microvilli (Fig. 1A) (12). The mammalian cadherin superfamily members, including CDHR2 and CDHR5, play essential roles in packing microvilli, increasing surface density, and controlling microvilli length (13). Other cadherins, specifically CDH23 and PCDH15, have been implicated in organizing the exaggerated microvilli found on inner-ear hair cells (14, 15).Open in a separate windowFig. 1.In situ cryo-ET of the C. elegans intestinal brush border reveals nanobristles on the lateral surface of microvilli. (A) A schematic diagram of an intestinal epithelial cell (Left) and two microvilli (Right) from the dotted box in Left. The glycocalyx and the protocadherin tip link are the characterized cell-surface structure at microvillar tips. This work shows that numerous nanobristles (magenta) decorate the lateral surface of microvilli. (B, Left and Center) Representative cryo-SEM images of the C. elegans L1 larvae before and after FIB milling. (Scale bars, 10 μm.) (B, Right) Representative FIB image of the ∼200-nm-thick cryo-lamella. (Scale bar, 5 μm.) (C) A 3D rendering of the C. elegans intestinal brush border showing various macromolecules and structures. Magenta, nanobristles; cyan, membrane; yellow, actin; beige, ribosome; green, mitochondria; orange, ER) Nanobristles and ribosomes were mapped back in the tomogram with the computed location and orientation. (D) A selected microvillus from E magnified for visualization. (E and F) Cryo-ET tomogram slices of microvilli (E, top view; F, side view). (Scale bars in C–F, 50 nm.)An individual microvillus is only 0.1 μm in diameter and 1 to ∼2 μm in height (Fig. 1A), the tiny dimension of which, along with their high density and lumen localization, becomes a technical hurdle for in situ structural investigations at high resolution (1–3). Despite our understanding of microvillar tip decorations, it is unclear whether any structure projects from the lateral surface of microvilli. Recent methodology advance of cryo-electron tomography (cryo-ET) makes the platform well suited to address the challenges of studying microvillus structure in animals (16, 17). Here, we used cryo-ET to reveal a previously unrecognized nanobristle structure on the lateral surface of microvilli. We provide evidence that nanobristle formation depends on a protocadherin family protein, CDH-8, and that nanobristles regulate microvilli separation.     

Chlorination of arenes via the degradation of toxic chlorophenols

Aryl chlorides are among the most versatile synthetic precursors, and yet inexpensive and benign chlorination techniques to produce them are underdeveloped. We propose a process to generate aryl chlorides by chloro-group transfer from chlorophenol pollutants to arenes during their mineralization, catalyzed by Cu(NO₃)₂/NaNO₃ under aerobic conditions. A wide range of arene substrates have been chlorinated using this process. Mechanistic studies show that the Cu catalyst acts in cooperation with NO_x species generated from the decomposition of NaNO₃ to regulate the formation of chlorine radicals that mediate the chlorination of arenes together with the mineralization of chlorophenol. The selective formation of aryl chlorides with the concomitant degradation of toxic chlorophenol pollutants represents a new approach in environmental pollutant detoxication. A reduction in the use of traditional chlorination reagents provides another (indirect) benefit of this procedure.

Chlorophenols are widely encountered moieties present in herbicides, drugs, and pesticides (1). Owing to the high dissociation energies of carbon‒chloride bonds, chlorophenols biodegrade very slowly, and their prolonged exposure leads to severe ecological and environmental problems (Fig. 1A) (2–4). Several well-established technologies have been developed for the treating of chlorophenols, including catalytic oxidation (5–11), biodegradation (12–15), solvent extraction (16, 17), and adsorption (18–20) Among these methods, adsorption is the most versatile and widely used method due to its high removal efficiency and simple operation, but the resulting products are of no value, and consequently, these processes are not viable.Open in a separate windowFig. 1.Background and reaction design. (A) Examples of chlorophenol pollutants. (B) Examples of aryl chlorides. (C) The chlorination process reported herein was based on chloro-group transfer from chlorophenol pollutants.With the extensive application of substitution reactions (21, 22), transfunctionalizations (23, 24), and cross-coupling reactions (25, 26), aryl chlorides are regarded as one of the most important building blocks widely used in the manufacture of polymers, pharmaceuticals, and other types of chemicals and materials (Fig. 1B) (27–31). Chlorination of arenes is usually carried out with toxic and corrosive reagents (32–34). Less toxic and more selective chlorination reagents tend to be expensive [e.g., chloroamides (35, 36)] and are not atom economic (37–39). Consequently, from the perspective of sustainability, the ability to transfer a chloro group from unwanted chlorophenols to other substrates would be advantageous.Catalytic isofunctional reactions, including transfer hydrogenation and alkene metathesis, have been widely exploited in organic synthesis. We hypothesized that chlorination of arenes also could be achieved by chloro-group transfer, and since stockpiles of chlorophenols tend to be destroyed by mineralization and chlorophenol pollutants may be concentrated by adsorption (18–20), they could be valorized as chlorination reagents via transfer of the chloro group to arene substrates during their mineralization, thereby adding value to the destruction process (Fig. 1C). Although chlorophenol pollutants are not benign, their application as chlorination reagents, with their concomitant destruction to harmless compounds, may be considered as not only meeting the criteria of green chemistry but also potentially surpassing it. Herein, we describe a robust strategy to realize chloro-group transfer from chlorophenol pollutants to arenes and afford a wide range of value-added aryl chlorides.     

Electrochemical borylation of carboxylic acids

A simple electrochemically mediated method for the conversion of alkyl carboxylic acids to their borylated congeners is presented. This protocol features an undivided cell setup with inexpensive carbon-based electrodes and exhibits a broad substrate scope and scalability in both flow and batch reactors. The use of this method in challenging contexts is exemplified with a modular formal synthesis of jawsamycin, a natural product harboring five cyclopropane rings.

Boronic acids are among the most malleable functional groups in organic chemistry as they can be converted into almost any other functionality (1–3). Aside from these versatile interconversions, their use in the pharmaceutical industry is gaining traction, resulting in approved drugs such as Velcade, Ninlaro, and Vabomere (4). It has been shown that boronic acids can be rapidly installed from simple alkyl halides (5–19) or alkyl carboxylic acids through the intermediacy of redox-active esters (RAEs) (Fig. 1A) (20–24). Our laboratory has shown that both Ni (20) and Cu (21) can facilitate this reaction. Conversely, Aggarwal and coworkers (22) and Li and coworkers (23) demonstrated photochemical variations of the same transformation. While these state-of-the-art approaches provide complementary access to alkyl boronic acids, each one poses certain challenges. For example, the requirement of excess boron source and pyrophoric MeLi under Ni catalysis is not ideal. Although more cost-effective and operationally simple, Cu-catalyzed borylation conditions can be challenging on scale due to the heterogeneity resulting from the large excess of LiOH•H₂O required. In addition to its limited scope, Li and coworkers’ protocol requires 4 equivalence of B₂pin₂ and an expensive Ir photocatalyst. The simplicity of Aggarwal and coworkers’ approach is appealing in this regard and represents an important precedent for the current study.Open in a separate windowFig. 1.(A) Prior approaches to access alkyl boronic esters from activated acids. (B) Inspiration for initiating SET events electrochemically to achieve borylation. (C) Summary of this work.At the heart of each method described above, the underlying mechanism relies on a single electron transfer (SET) event to promote decarboxylation and form an alkyl radical species. In parallel, the related borylation of aryl halides via a highly reactive aryl radical can also be promoted by SET. While numerous methods have demonstrated that light can trigger this mechanism (Fig. 1B) (16, 25–31), simple electrochemical cathodic reduction can elicit the same outcome (32–35). It was postulated that similar electrochemically driven reactivity could be translated to alkyl RAEs. The development of such a transformation would be highly enabling, as synthetic organic electrochemistry allows the direct addition or removal of electrons to a reaction, representing an incredibly efficient way to forge new bonds (36–40). This disclosure reports a mild, scalable, and operationally simple electrochemical decarboxylative borylation (Fig. 1C) not reliant on transition metals or stoichiometric reductants. In addition to mechanistic studies of this interesting transformation, applications to a variety of alkyl RAEs, comparison to known decarboxylative borylation methods, and a formal synthesis of the polycyclopropane natural product jawsamycin [(–)-FR-900848] are presented.     

An image-computable model of how endogenous and exogenous attention differentially alter visual perception

Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: 1) the parafoveal advantage in segmentation, 2) the uniform improvements across eccentricity by endogenous attention, and 3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

Endogenous and exogenous spatial attention prioritize subsets of visual information and facilitate their processing without concurrent eye movements (1–3). Selection by endogenous attention is goal-driven and adapts to task demands, whereas exogenous attention transiently and automatically orients to salient stimuli (1–3). In most visual tasks, both types of attention typically improve visual perception similarly [e.g., acuity (4–6), visual search (7, 8), perceived contrast (9–11)]. Consequently, models of visual attention do not distinguish between endogenous and exogenous attention (e.g., refs. 12–19). However, stark differences also exist. Each attention type differentially modulates neural responses (20, 21) and fundamental properties of visual processing, including temporal resolution (22, 23), texture sensitivity (24), sensory tuning (25), contrast sensitivity (26), and spatial resolution (27–34).The effects of endogenous and exogenous attention are dissociable during texture segmentation, a visual task constrained by spatial resolution [reviews (1–3)]. Whereas endogenous attention optimizes spatial resolution to improve the detection of an attended texture (32–34), exogenous attention reflexively enhances resolution even when detrimental to perception (27–31, 34). Extant models of attention do not explain these well-established effects.Two main hypotheses have been proposed to explain how attention alters spatial resolution. Psychophysical studies ascribe attentional effects to modulations of spatial frequency (SF) sensitivity (30, 33). Neurophysiological (13, 35, 36) and neuroimaging (37, 38) studies bolster the idea that attention modifies spatial profiles of neural receptive fields (RFs) (2). Both hypotheses provide qualitative predictions of attentional effects but do not specify their underlying neural computations.Differences between endogenous and exogenous attention are well established in segmentation tasks and thus provide an ideal model system to uncover their separate roles in altering perception. Texture-based segmentation is a fundamental process of midlevel vision that isolates regions of local structure to extract figures from their background (39–41). Successful segmentation hinges on the overlap between the visual system’s spatial resolution and the levels of detail (i.e., SF) encompassed by the texture (39, 41, 42). Consequently, the ability to distinguish between adjacent textures varies as resolution declines toward the periphery (43–46). Each attention type differentially alters texture segmentation, demonstrating that their effects shape spatial resolution [reviews (1–3)].Current models of texture segmentation do not explain performance across eccentricity and the distinct modulations by attention. Conventional models treat segmentation as a feedforward process that encodes the elementary features of an image (e.g., SF and orientation), transforms them to reflect the local structure (e.g., regions of similarly oriented bars), and then pools across space to emphasize texture-defined contours (39, 41, 47). Few of these models account for variations in resolution across eccentricity (46, 48, 49) or endogenous (but not exogenous) attentional modulations (18, 50). All others postulate that segmentation is a “preattentive” (42) operation whose underlying neural processing is impervious to attention (39, 41, 46–49).Here, we develop a computational model in which feedforward processing and attentional gain contribute to segmentation performance. We augment a conventional model of texture processing (39, 41, 47). Our model varies with eccentricity and includes contextual modulation within local regions in the stimulus via normalization (51), a canonical neural computation (52). The defining characteristic of normalization is that an individual neuron is (divisively) suppressed by the summed activity of neighboring neurons responsive to different aspects of a stimulus. We model attention as multiplicative gains [attentional gain factors (15)] that vary with eccentricity and SF. Attention shifts sensitivity toward fine or coarse spatial scales depending on the range of SFs enhanced.Our model is image-computable, which allowed us to reproduce behavior directly from grayscale images used in psychophysical experiments (6, 26, 27, 29–33). The model explains three signatures of texture segmentation hitherto unexplained within a single computational framework (Fig. 1): 1) the central performance drop (CPD) (27–34, 43–46) (Fig. 1A), that is, the parafoveal advantage of segmentation over the fovea; 2) the improvements in the periphery and impairments at foveal locations induced by exogenous attention (27–32, 34) (Fig. 1B); and 3) the equivalent improvements across eccentricity by endogenous attention (32–34) (Fig. 1C).Open in a separate windowFig. 1.Signatures of texture segmentation. (A) CPD. Shaded region depicts the magnitude of the CPD. Identical axis labels are omitted in B and C. (B) Exogenous attention modulation. Exogenous attention improves segmentation performance in the periphery and impairs it near the fovea. (C) Endogenous attention modulation. Endogenous attention improves segmentation performance across eccentricity.Whereas our analyses focused on texture segmentation, our model is general and can be applied to other visual phenomena. We show that the model predicts the effects of attention on contrast sensitivity and acuity, i.e., in tasks in which both endogenous and exogenous attention have similar or differential effects on performance. To preview our results, model comparisons revealed that normalization is necessary to elicit the CPD and that separate profiles of gain enhancement across SF (26) generate the effects of exogenous and endogenous attention on texture segmentation. A preferential high-SF enhancement reproduces the impairments by exogenous attention due to a shift in visual sensitivity toward details too fine to distinguish the target at foveal locations. The transition from impairments to improvements in the periphery results from exogenous attentional gain gradually shifting to lower SFs that are more amenable for target detection. Improvements by endogenous attention result from a uniform enhancement of SFs that encompass the target, optimizing visual sensitivity for the attended stimulus across eccentricity.     

Microscopic origins of the crystallographically preferred growth in evaporation-induced colloidal crystals

Unlike crystalline atomic and ionic solids, texture development due to crystallographically preferred growth in colloidal crystals is less studied. Here we investigate the underlying mechanisms of the texture evolution in an evaporation-induced colloidal assembly process through experiments, modeling, and theoretical analysis. In this widely used approach to obtain large-area colloidal crystals, the colloidal particles are driven to the meniscus via the evaporation of a solvent or matrix precursor solution where they close-pack to form a face-centered cubic colloidal assembly. Via two-dimensional large-area crystallographic mapping, we show that the initial crystal orientation is dominated by the interaction of particles with the meniscus, resulting in the expected coalignment of the close-packed direction with the local meniscus geometry. By combining with crystal structure analysis at a single-particle level, we further reveal that, at the later stage of self-assembly, however, the colloidal crystal undergoes a gradual rotation facilitated by geometrically necessary dislocations (GNDs) and achieves a large-area uniform crystallographic orientation with the close-packed direction perpendicular to the meniscus and parallel to the growth direction. Classical slip analysis, finite element-based mechanical simulation, computational colloidal assembly modeling, and continuum theory unequivocally show that these GNDs result from the tensile stress field along the meniscus direction due to the constrained shrinkage of the colloidal crystal during drying. The generation of GNDs with specific slip systems within individual grains leads to crystallographic rotation to accommodate the mechanical stress. The mechanistic understanding reported here can be utilized to control crystallographic features of colloidal assemblies, and may provide further insights into crystallographically preferred growth in synthetic, biological, and geological crystals.

As an analogy to atomic crystals, colloidal crystals are highly ordered structures formed by colloidal particles with sizes ranging from 100 nm to several micrometers (1–6). In addition to engineering applications such as photonics, sensing, and catalysis (4, 5, 7, 8), colloidal crystals have also been used as model systems to study some fundamental processes in statistical mechanics and mechanical behavior of crystalline solids (9–14). Depending on the nature of interparticle interactions, many equilibrium and nonequilibrium colloidal self-assembly processes have been explored and developed (1, 4). Among them, the evaporation-induced colloidal self-assembly presents a number of advantages, such as large-size fabrication, versatility, and cost and time efficiency (3–5, 15–18). In a typical synthesis where a substrate is immersed vertically or at an angle into a colloidal suspension, the colloidal particles are driven to the meniscus by the evaporation-induced fluid flow and subsequently self-assemble to form a colloidal crystal with the face-centered cubic (fcc) lattice structure and the close-packed {111} plane parallel to the substrate (2, 3, 19–23) (see Fig. 1A for a schematic diagram of the synthetic setup).Open in a separate windowFig. 1.Evaporation-induced coassembly of colloidal crystals. (A) Schematic diagram of the evaporation-induced colloidal coassembly process. “G”, “M”, and “N” refer to “growth,” “meniscus,” and “normal” directions, respectively. The reaction solution contains silica matrix precursor (tetraethyl orthosilicate, TEOS) in addition to colloids. (B) Schematic diagram of the crystallographic system and orientations used in this work. (C and D) Optical image (Top Left) and scanning electron micrograph (SEM) (Bottom Left) of a typical large-area colloidal crystal film before (C) and after (D) calcination. (Right) SEM images of select areas (yellow rectangles) at different magnifications. Corresponding fast-Fourier transform (see Inset in Middle in C) shows the single-crystalline nature of the assembled structure. (E) The 3D reconstruction of the colloidal crystal (left) based on FIB tomography data and (right) after particle detection. (F) Top-view SEM image of the colloidal crystal with crystallographic orientations indicated.While previous research has focused on utilizing the assembled colloidal structures for different applications (4, 5, 7, 8), considerably less effort is directed to understand the self-assembly mechanism itself in this process (17, 24). In particular, despite using the term “colloidal crystals” to highlight the microstructures’ long-range order, an analogy to atomic crystals, little is known regarding the crystallographic evolution of colloidal crystals in relation to the self-assembly process (3, 22, 25). The underlying mechanisms for the puzzling—yet commonly observed—phenomenon of the preferred growth along the close-packed <110> direction in evaporation-induced colloidal crystals are currently not understood (3, 25–29). The <110> growth direction has been observed in a number of processes with a variety of particle chemistries, evaporation rates, and matrix materials (3, 25–28, 30), hinting at a universal underlying mechanism. This behavior is particularly intriguing as the colloidal particles are expected to close-pack parallel to the meniscus, which should lead to the growth along the <112> direction and perpendicular to the <110> direction (16, 26, 31)^*.Preferred growth along specific crystallographic orientations, also known as texture development, is commonly observed in crystalline atomic solids in synthetic systems, biominerals, and geological crystals. While current knowledge recognizes mechanisms such as the oriented nucleation that defines the future crystallographic orientation of the growing crystals and competitive growth in atomic crystals (32–34), the underlying principles for texture development in colloidal crystals remain elusive. Previous hypotheses based on orientation-dependent growth speed and solvent flow resistance are inadequate to provide a universal explanation for different evaporation-induced colloidal self-assembly processes (3, 25–29). A better understanding of the crystallographically preferred growth in colloidal self-assembly processes may shed new light on the crystal growth in atomic, ionic, and molecular systems (35–37). Moreover, mechanistic understanding of the self-assembly processes will allow more precise control of the lattice types, crystallography, and defects to improve the performance and functionality of colloidal assembly structures (38–40).     

From the Cover: Mammalian face as an evolutionary novelty

The anterior end of the mammalian face is characteristically composed of a semimotile nose, not the upper jaw as in other tetrapods. Thus, the therian nose is covered ventrolaterally by the “premaxilla,” and the osteocranium possesses only a single nasal aperture because of the absence of medial bony elements. This stands in contrast to those in other tetrapods in whom the premaxilla covers the rostral terminus of the snout, providing a key to understanding the evolution of the mammalian face. Here, we show that the premaxilla in therian mammals (placentals and marsupials) is not entirely homologous to those in other amniotes; the therian premaxilla is a composite of the septomaxilla and the palatine remnant of the premaxilla of nontherian amniotes (including monotremes). By comparing topographical relationships of craniofacial primordia and nerve supplies in various tetrapod embryos, we found that the therian premaxilla is predominantly of the maxillary prominence origin and associated with mandibular arch. The rostral-most part of the upper jaw in nonmammalian tetrapods corresponds to the motile nose in therian mammals. During development, experimental inhibition of primordial growth demonstrated that the entire mammalian upper jaw mostly originates from the maxillary prominence, unlike other amniotes. Consistently, cell lineage tracing in transgenic mice revealed a mammalian-specific rostral growth of the maxillary prominence. We conclude that the mammalian-specific face, the muzzle, is an evolutionary novelty obtained by overriding ancestral developmental constraints to establish a novel topographical framework in craniofacial mesenchyme.

In the movie For Whom the Bell Tolls (1943, Paramount), a girl says, “I do not know how to kiss, or I would kiss you. Where do the noses go?” (1) Nothing could reveal more vividly the curious morphological fact that it is the nose, not the tip of the upper jaw, that is the most protruding part of the mammalian face. Therian mammals are thus characterized by a protruding nose, representing a morphologically and functionally semi-independent module for tactile sensory detection and for mammalian olfactory function (Fig. 1A) (2–7). The topographical relationship between the nose and cranial bones also shows an exceptional pattern in mammals: the rostral-most bone of the upper jaw, or premaxilla, is found on the ventrolateral sides of the external nostrils in therian mammals, unlike in other amniotes in whom the premaxilla covers the rostromedial tip of the snout (Fig. 1 A and B) (2–4, 7, 8). However, the evolutionary origin of this therian-specific face (the so-called muzzle) and homology of the therian premaxilla (also known as the incisive bone) have not been examined for a long time (2–4, 7–9).Open in a separate windowFig. 1.Murine “premaxilla” develops differently from premaxillae of other tetrapods. (A) The anatomy of the therian mammal’s face. (B) General scheme of craniofacial development in amniotes (10, 11, 15). (C) Three-dimensional models of tetrapod embryos. The murine premaxilla ossifies in the same topographical position as the septomaxilla (orange) of other species. The infraorbital branch (nerve branch for vibrissae) of V2 was removed in 13.5 dpc mouse. The summary is shown in D. sn, solum nasi; nld, nasolacrimal duct; V1, ophthalmic nerve; V2, maxillary nerve. (Not to scale.)During vertebrate embryogenesis, the upper jaw is primarily formed by growth of the maxillary prominence of the mandibular arch, except for the premaxilla, the rostral midline part of the upper jaw, which develops by the convergence of the premandibular ectomesenchyme (frontonasal prominence) that initially develops rostral to the mandibular arch ectomesenchyme (Fig. 1B) (4, 10–12). This topographical configuration is recognized even in some placoderms; that is, the basic pattern of jaw morphology is thought to be constrained among the jawed vertebrates (12–14). However, the topographical position of the therian premaxilla suggests that this highly conserved pattern is disrupted in mammals in association with the evolution of the mammalian muzzle. Specifically, the innervation pattern of the homonymous “premaxilla” is significantly different in mammals (15), which is also suggestive of fundamental embryological changes.In the present study, we conducted comparative experimental embryological analyses and cell lineage tracing of the facial primordia to investigate the origin of the mammalian face.     

From the Cover: Upslope migration of snow avalanches in a warming climate

Snow is highly sensitive to atmospheric warming. However, because of the lack of sufficiently long snow avalanche time series and statistical techniques capable of accounting for the numerous biases inherent to sparse and incomplete avalanche records, the evolution of process activity in a warming climate remains little known. Filling this gap requires innovative approaches that put avalanche activity into a long-term context. Here, we combine extensive historical records and Bayesian techniques to construct a 240-y chronicle of snow avalanching in the Vosges Mountains (France). We show evidence that the transition from the late Little Ice Age to the early twentieth century (i.e., 1850 to 1920 CE) was not only characterized by local winter warming in the order of +1.35 °C but that this warming also resulted in a more than sevenfold reduction in yearly avalanche numbers, a severe shrinkage of avalanche size, and shorter avalanche seasons as well as in a reduction of the extent of avalanche-prone terrain. Using a substantial corpus of snow and climate proxy sources, we explain this abrupt shift with increasingly scarcer snow conditions with the low-to-medium elevations of the Vosges Mountains (600 to 1,200 m above sea level [a.s.l.]). As a result, avalanches migrated upslope, with only a relict activity persisting at the highest elevations (release areas >1,200 m a.s.l.). This abrupt, unambiguous response of snow avalanche activity to warming provides valuable information to anticipate likely changes in avalanche behavior in higher mountain environments under ongoing and future warming.

Over the last decades, global warming has led to widespread shrinking of the cryosphere (1–4). The reduction of snowfall and amounts of snow on the ground (2) are increasingly well documented, notably in Europe (4–6). Likewise, climate models project further drastic reductions in snow cover extent and duration (1, 4, 7, 8), especially in low-elevation mountain environments (2). Changes in snowfall and snow cover characteristics already have direct consequences on the occurrence and magnitude of natural hazards (1, 2, 4, 9). Among these, snow avalanches not only are a major threat to mountain populations and their assets (10, 11) but also represent the mass-movement process for which climate is considered to exert the largest control through changes in rain–snow partitioning of precipitation and snow cover characteristics (2, 8, 12).Although systematic avalanche records typically extend back to ca. the 1950s, it has so far remained difficult to observe unequivocal trends on climatically driven changes in avalanche numbers, runout distances, or flow types (13–15). Likewise, literature is also critically lacking regarding future avalanche activity in a warmer climate—the only study currently available (16) points to a 20 to 30% reduction in avalanche numbers in the French Alps by the end of the twenty-first century as compared to the reference period 1960 to 1990. Several factors explain this lack of direct evidence: first, homogeneous time series of snow avalanches have remained too short for trend analyses over time scales relevant for the detection of climate change impacts (12, 17). Second, interpretations may have suffered from confounding influences of climatic and socioeconomical drivers or biases in the proxy data (14, 17–20). Eventually, with warming, a temporary increase in extreme snowfall or wet-snow amounts could emerge at higher elevations (2, 21, 22), which in turn could enhance avalanche activity (23). For instance, during the exceptional avalanche episode of January 2018 in the European Alps, and despite extremely high air temperatures, numerous avalanches were triggered across the Alps (24) [in a subregion of Switzerland alone, nearly 19,000 avalanches were observed on satellite images (25)]. Similarly, a drastic increase in avalanche activity has been evidenced lately on high-elevation slopes in the Himalayas and attributed to the increase in increasing air temperatures (2, 25).Yet, the role of climate in controlling snow avalanching at decadal to centennial time scales remains poorly understood and therefore precludes robust anticipation of future changes (1, 2, 8, 12). Closing this gap needs an assessment of avalanche activity in a longer-term context and a focus on areas and/or climate transitions that may have caused significant shifts in avalanche regimes in the past. To this end, we analyzed an extensive compilation of 734 historical records of avalanche events (20) that occurred in the low-to-medium elevation Vosges Mountains (northeast France) over the period 1774 to 2013 (Fig. 1). We developed a statistical framework that 1) accounts for the possible pitfalls affecting historical sources (e.g., biases in observations toward large, destructive snow avalanches; completeness of the record; accuracy of the information) and, 2) quantifies the respective roles of climate and changes in the sources reporting observed snow avalanches. Our analysis reveals a substantial decrease in avalanche frequency between the end of the Little Ice Age (LIA) (26, 27) and the Early Twentieth Century Warming (ETCW) (28, 29), known as one of the most prominent phases of accelerated warming prior to the current anthropogenic warming. Hence, we shed light on an unambiguous relation between climatic changes and snow avalanche activity to demonstrate that the warming-induced shrinkage of snow amounts and snow cover duration led to an upslope migration of snow avalanches. We also evidence that warming resulted in a rapid shift from a widespread to a residual activity—which in addition is restricted to the highest release areas—with a concomitant decrease in avalanche size and a shortening of the avalanche season. Eventually, we discuss how these results allow anticipating likely changes in avalanche behavior in a variety of higher-elevation mountain ranges under future warming.Open in a separate windowFig. 1.Study area and historical archives relating to past snow avalanches. (A) Map from the mid-nineteenth century localizing a house damaged by an avalanche (flow direction is indicated); (B) old photograph of an avalanche that reached a valley in year 1894; (C and D) written sources relating to building destruction and casualties by snow avalanches in 1852 and 1783, respectively; (E) the four avalanches are mapped on the topography of the Vosges Mountains. The three weather stations indicated have long snow series (SI Appendix, Supplementary Material S1-7).     

Emerging predictable features of replicated biological invasion fronts

Biological dispersal shapes species’ distribution and affects their coexistence. The spread of organisms governs the dynamics of invasive species, the spread of pathogens, and the shifts in species ranges due to climate or environmental change. Despite its relevance for fundamental ecological processes, however, replicated experimentation on biological dispersal is lacking, and current assessments point at inherent limitations to predictability, even in the simplest ecological settings. In contrast, we show, by replicated experimentation on the spread of the ciliate Tetrahymena sp. in linear landscapes, that information on local unconstrained movement and reproduction allows us to predict reliably the existence and speed of traveling waves of invasion at the macroscopic scale. Furthermore, a theoretical approach introducing demographic stochasticity in the Fisher–Kolmogorov framework of reaction–diffusion processes captures the observed fluctuations in range expansions. Therefore, predictability of the key features of biological dispersal overcomes the inherent biological stochasticity. Our results establish a causal link from the short-term individual level to the long-term, broad-scale population patterns and may be generalized, possibly providing a general predictive framework for biological invasions in natural environments.What is the source of variance in the spread rates of biological invasions? The search for processes that affect biological dispersal and sources of variability observed in ecological range expansions is fundamental to the study of invasive species dynamics (1–10), shifts in species ranges due to climate or environmental change (11–13), and, in general, the spatial distribution of species (3, 14–16). Dispersal is the key agent that brings favorable genotypes or highly competitive species into new ranges much faster than any other ecological or evolutionary process (1, 17). Understanding the potential and realized dispersal is thus key to ecology in general (18). When organisms’ spread occurs on the timescale of multiple generations, it is the byproduct of processes that take place at finer spatial and temporal scales that are the local movement and reproduction of individuals (5, 10). The main difficulty in causally understanding dispersal is thus to upscale processes that happen at the short-term individual level to long-term and broad-scale population patterns (5, 18–20). Furthermore, the large fluctuations observed in range expansions have been claimed to reflect an intrinsic lack of predictability of the phenomenon (21). Whether the variability observed in nature or in experimental ensembles might be accounted for by systematic differences between landscapes or by demographic stochasticity affecting basic vital rates of the organisms involved is an open research question (10, 18, 21, 22).Modeling of biological dispersal established the theoretical framework of reaction–diffusion processes (1–3, 23–25), which now finds common application in dispersal ecology (5, 14, 22, 26–30) and in other fields (17, 23, 25, 31–36). Reaction–diffusion models have also been applied to model human colonization processes (31), such as the Neolithic transition in Europe (25, 37, 38). The classical prediction of reaction–diffusion models (1, 2, 24, 25) is the propagation of an invading wavefront traveling undeformed at a constant speed (Fig. 1E). Such models have been widely adopted by ecologists to describe the spread of organisms in a variety of comparative studies (5, 10, 26) and to control the dynamics of invasive species (3, 4, 6). The extensive use of these models and the good fit to observational data favored their common endorsement as a paradigm for biological dispersal (6). However, current assessments (21) point at inherent limitations to the predictability of the phenomenon, due to its intrinsic stochasticity. Therefore, single realizations of a dispersal event (as those addressed in comparative studies) might deviate significantly from the mean of the process, making replicated experimentation necessary to allow hypothesis testing, identification of causal relationships, and to potentially falsify the models’ assumptions (39).Open in a separate windowFig. 1.Schematic representation of the experiment. (A) Linear landscape. (B) Individuals of the ciliate Tetrahymena sp. move and reproduce within the landscape. (C) Examples of reconstructed trajectories of individuals (Movie S1). (D) Individuals are introduced at one end of a linear landscape and are observed to reproduce and disperse within the landscape (not to scale). (E) Illustrative representation of density profiles along the landscape at subsequent times. A wavefront is argued to propagate undeformed at a constant speed v according to the Fisher–Kolmogorov equation.Here, we provide replicated and controlled experimental support to the theory of reaction–diffusion processes for modeling biological dispersal (23–25) in a generalized context that reproduces the observed fluctuations. Firstly, we experimentally substantiate the Fisher–Kolmogorov prediction (1, 2) on the existence and the mean speed of traveling wavefronts by measuring the individual components of the process. Secondly, we manipulate the inclusion of demographic stochasticity in the model to reproduce the observed variability in range expansions. We move from the Fisher–Kolmogorov equation (Materials and Methods) to describe the spread of organisms in a linear landscape (1, 2, 24, 25). The equation couples a logistic term describing the reproduction of individuals with growth rate r and carrying capacity K and a diffusion term accounting for local movement, epitomized by the diffusion coefficient D . These species’ traits define the characteristic scales of the dispersal process. In this framework, a population initially located at one end of a linear landscape is predicted to form a wavefront of colonization invading empty space at a constant speed (1, 2, 24, 25), which we measured in our dispersal experiment (Fig. 1D and SI Text).     

Electrophilic aromatic substitution reactions of compounds with Craig-Möbius aromaticity

Electrophilic aromatic substitution (EAS) reactions are widely regarded as characteristic reactions of aromatic species, but no comparable reaction has been reported for molecules with Craig-Möbius aromaticity. Here, we demonstrate successful EAS reactions of Craig-Möbius aromatics, osmapentalenes, and fused osmapentalenes. The highly reactive nature of osmapentalene makes it susceptible to electrophilic attack by halogens, thus osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene can undergo typical EAS reactions. In addition, the selective formation of a series of halogen substituted metalla-aromatics via EAS reactions has revealed an unprecedented approach to otherwise elusive compounds such as the unsaturated cyclic chlorirenium ions. Density functional theory calculations were conducted to study the electronic effect on the regioselectivity of the EAS reactions.

Aromaticity, a core concept in chemistry, was initially introduced to account for the bonding, stability, reactivity, and other properties of many unsaturated organic compounds. There have been many elaborations and extensions of the concept of aromaticity (1, 2). The concepts of Hückel aromaticity and Möbius aromaticity are widely accepted (Fig. 1A). A π-aromatic molecule of the Hückel type is planar and has 4n + 2 conjugated π-electrons (n = 0 or an integer), whereas a Möbius aromatic molecule has one twist of the π-system, similar to that in a Möbius strip, and 4n π-electrons (3, 4). Since the discovery of naphthalene in 1821, aromatic chemistry has developed into a rich field and with a variety of subdisciplines over the course of its 200-y history, and the concept of aromaticity has been extended to other nontraditional structures with “cyclic delocalization of mobile electrons” (5). For example, benzene-like metallacycles—predicted by Hoffmann et al. as metallabenzenes—in which a metal replaces a C–H group in the benzene ring (6), have garnered extensive research interest from both experimentalists and theoreticians (7–12). As paradigms of the metalla-aromatic family, most complexes involving metallabenzene exhibit thermodynamic stability, kinetic persistence, and chemical reactivity associated with the classical aromaticity concept (13–15). Typically, like benzene, metallabenzene can undergo characteristic reactions of aromatics such as electrophilic aromatic substitution (EAS) reactions (16–18) (Fig. 1B, I) and nucleophilic aromatic substitution reactions (19–21).Open in a separate windowFig. 1.Schematic representations of aromaticity classification (A) and EAS reactions (B) of benzene, metallabenzene, and polycyclic metallacycles with Craig-Möbius aromaticity.The incorporation of transition metals has also led to an increase in the variety of the aromatic families (22–25). We have reported that stable and highly unusual bicyclic systems, metallapentalenes (osmapentalenes), benefit from Craig-Möbius aromaticity (26–30). In contrast to other reported Möbius aromatic compounds with twisted topologies, which are known as Heilbronner-Möbius aromatics (31–34), the involvement of transition metal d orbitals in π-conjugation switches the Hückel anti-aromaticity of pentalene into the planar Craig-Möbius aromaticity of metallapentalene (35–38) (Fig. 1A, III). Both the twisted topology and the planar Craig-Möbius aromaticity are well established and have been accepted as reasonable extensions of aromaticity (39–43). There has been no experimental evidence, however, as to whether these Möbius aromatic molecules can undergo classical aromatic substitution reactions, such as EAS reactions, instead of addition reactions. Given the key role of EAS in aromatic chemistry to obtain various derivatives, we sought to extend the understanding of the reactivity paradigm in the metalla-aromatic family.Our recent synthetic efforts associated with the metallapentalene system prompted us to investigate whether typical EAS reactions could proceed in these Craig-Möbius aromatics. If so, how could substitution be achieved in the same way that it is with traditional Hückel aromatics such as benzenes? In this paper, we present EAS reactions, mainly the halogenation of osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene, which follow the classic EAS mechanistic scheme (Fig. 1B). With the aid of density functional theory (DFT) calculations, we characterized the effects on EAS reactivity and regioselectivity.     

The embryonic node behaves as an instructive stem cell niche for axial elongation

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen’s node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle–related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

In higher vertebrate embryos the body axis forms in head-to-tail direction from a growth zone at the tail end, which is present from gastrula stages through to the end of axis elongation, several days later. Hensen’s node is part of this growth zone. Rather than defining a distinct cell population arising very early in development, the node represents a dynamic region at the tip of the primitive streak, which appears as a morphological “node” from HH4 (1) in chick. The initial cells that make up this region are derived from two distinct cell populations, which meet at the tip of the elongating primitive streak (HH3 to 3+ in chick) (2–4). These are then joined by cells from the epiblast lateral to the anterior streak and node (5) (at stages HH3+ to HH4) and from the primitive streak immediately caudal to the node during regression (from stage HH5) (6, 7). Although ingression of cells from adjacent epiblast along most of the length of the streak continues later into development (6), this ceases at the level of the node by HH4+ (5, 8, 9). After stage 5, the node begins to regress caudally (7), while cells exit the node to lay down the midline of the developing head–tail axis, contributing to axial (notochord) and paraxial (medial somite) mesoderm, definitive endoderm, and neural midline (floorplate) tissues (Fig. 1 A–C) (5, 10–12).Open in a separate windowFig. 1.The node confers resident behavior. (A–C) Node replacement using a GFP donor showing normal node axial fates. (D and E) Epiblast lateral to the HH3+/4 node ingresses into it and gives rise to the axis and to regressing node as resident cells. (F and G) Anterior epiblast not normally fated to enter the node behaves as lateral epiblast when forced to do so. (H and I) Anterior epiblast normally gives rise to head structures. (J and K) Lateral epiblast no longer gives rise to node-derived axial structures when prevented from entering the node. (L) Quantifying tissue contribution of lateral (D, green) versus anterior (F, blue) epiblast grafts to the host. E, endoderm; F, floorplate; MS, medial-somite; N, notochord; RC, resident cell. Transverse dashed lines show levels of accompanying sections. The field of view of the wholemount images (C, E, G, I, K) is approximately 2 mm x 5 mm.Therefore, most cells pass transiently though the node, temporarily gaining a node-like gene-expression signature, which they lose upon leaving the node (5). However, transplantation of cell groups and fate-mapping experiments in chick (10, 13–15) and mouse (16–20) during early development have suggested that the node may also contain a few resident self-renewing cells that persist within the node during axial elongation, while other cells leave (Fig. 1C, “RC”). In particular, labeling of single cells in the node has provided a few examples of cells that contribute to midline structures and appear to self-renew because one or more cells remain at the site of labeling after some progeny have left (10, 17, 21, 22). At a cell-population level, grafts of groups of cells transplanted repeatedly between older and younger tailbud regions can contribute to midline structures over two or more hosts, while again some cells remain in the tailbud (14, 19). These findings have led to the idea that some cells in the node (most likely a very small subset) may have the ability to self-renew, perhaps indefinitely, thus displaying stem cell behavior.Are the self-renewing cells a special population that arose in earlier development, or might the node act as an environment (niche) (23–25) that captures a subset of the cells that enter it and instructs them to become resident and acquire self-renewal behavior and act as stem cells (26–28)? To demonstrate self-renewal and to test whether the node is an instructive stem cell niche, it is critical to test whether an individual cell can acquire this behavior when introduced to the node environment; this has not yet been attempted. Here we address this question using transplantation of groups of cells and of single cells in vivo and single-cell RNA sequencing (scRNA-seq). We find that the tip of the primitive streak is able to impart notochord and somite identity to most or all cells that enter it, while capturing a small subset to become resident and acquire self-renewal behavior. Cells from epiblast that would never have entered the node region during normal development are able to read these cues. We also define the developmental stage at which epiblast cells lose competence to respond to node signals. Long-term resident cells are preferentially located in the posterior part of the node, and display enriched expression of G2/M cell cycle markers.     

Opacity and conductivity measurements in noble gases at conditions of planetary and stellar interiors

The noble gases are elements of broad importance across science and technology and are primary constituents of planetary and stellar atmospheres, where they segregate into droplets or layers that affect the thermal, chemical, and structural evolution of their host body. We have measured the optical properties of noble gases at relevant high pressures and temperatures in the laser-heated diamond anvil cell, observing insulator-to-conductor transformations in dense helium, neon, argon, and xenon at 4,000–15,000 K and pressures of 15–52 GPa. The thermal activation and frequency dependence of conduction reveal an optical character dominated by electrons of low mobility, as in an amorphous semiconductor or poor metal, rather than free electrons as is often assumed for such wide band gap insulators at high temperatures. White dwarf stars having helium outer atmospheres cool slower and may have different color than if atmospheric opacity were controlled by free electrons. Helium rain in Jupiter and Saturn becomes conducting at conditions well correlated with its increased solubility in metallic hydrogen, whereas a deep layer of insulating neon may inhibit core erosion in Saturn.Noble gases play important roles in the evolution and dynamics of planets and stars, especially where they appear in a condensed, purified state. In gas giant planets, helium and neon can precipitate as rain in metallic hydrogen envelopes, leading to planetary warming and specifically the anomalously slow cooling of Saturn (1–8). In white dwarf stars cooling can be especially fast due to the predicted low opacity of dense helium atmospheres, affecting the calibration of these objects as cosmological timekeepers (9–12). In these systems, the transformation of dense noble gases (particularly He) from optically transparent insulators to opaque electrical conductors is of special importance (2, 9, 11, 12).Dense noble gases are expected to show systematic similarities in their properties at extreme conditions (13–17); however, a general understanding of their insulator–conductor transformation remains to be established. Xe is observed to metallize near room temperature under pressures similar to those at Earth’s core–mantle boundary (18, 19). Ar and He are observed to conduct only at combined high pressure and temperature (12, 13, 17). Ne is predicted to have the highest metallization pressure of all known materials—10³ times that of Xe and 10 times that of He (14, 18, 20, 21)—and has never been documented outside of its insulating state. Experimental probes of extreme densities and temperatures in noble gases have previously relied on dynamic compression by shock waves (12, 13, 17, 22–24). However, in such adiabatic experiments, light and compressible noble gases heat up significantly and can ultimately reach density maxima (12, 13, 17, 21, 24, 25), so that conditions created often lie far from those deep within planets (7, 8) and stars (9).Here we report experiments in the laser-heated diamond anvil cell (15, 16, 26–29) on high-density and high-temperature states of the noble gases Xe, Ar, Ne, and He (Fig. 1). Rapid heating and cooling of compressed samples using pulsed laser heating (26, 27) is coupled with time domain spectroscopy of thermal emission (26) to determine sample temperature and transient absorption to establish corresponding sample optical properties (Figs. S1 and S2). A sequence of heat cycles to increasing temperature documents optical changes in these initially transparent insulators.Open in a separate windowFig. 1.Creating and probing extreme states of noble gases. (A) Configuration of laser heating and transient absorption probing of the diamond anvil cell, with probe beams transmitted through the cell into the detection system. (B) Microscopic view of the diamond cell cavity, which contains a noble gas sample and a metal foil (Ir) which converts laser radiation to heat and has small hole at the heated region through which probe beams are transmitted to test optical character of samples. (C) Finite element model (26) (Fig. S3) of the temperature distribution in heated Ar at 51 GPa (Fig. 2), with solid–melt (16) and insulator–conductor (α = 0.1 μm⁻¹) boundaries in the sample marked dashed and dotted, respectively. (D) Schematic of time domain probing during transient heating. Temperature is determined from thermal emission (red) and absorption from transmitted probe beams: a continuous laser (cw; green) and pulsed supercontinuum broadband (bb; blue).     

Conjugated polymers based on metalla-aromatic building blocks

Conjugated polymers usually require strategies to expand the range of wavelengths absorbed and increase solubility. Developing effective strategies to enhance both properties remains challenging. Herein, we report syntheses of conjugated polymers based on a family of metalla-aromatic building blocks via a polymerization method involving consecutive carbyne shuttling processes. The involvement of metal d orbitals in aromatic systems efficiently reduces band gaps and enriches the electron transition pathways of the chromogenic repeat unit. These enable metalla-aromatic conjugated polymers to exhibit broad and strong ultraviolet–visible (UV–Vis) absorption bands. Bulky ligands on the metal suppress π–π stacking of polymer chains and thus increase solubility. These conjugated polymers show robust stability toward light, heat, water, and air. Kinetic studies using NMR experiments and UV–Vis spectroscopy, coupled with the isolation of well-defined model oligomers, revealed the polymerization mechanism.

Conjugated polymers are macromolecules usually featuring a backbone chain with alternating double and single bonds (1–3). These characteristics allow the overlapping p-orbitals to form a system with highly delocalized π-electrons, thereby giving rise to intriguing chemical and physical properties (4–6). They have exhibited many applications in organic light-emitting diodes, organic thin film transistors, organic photovoltaic cells, chemical sensors, bioimaging and therapies, photocatalysis, and other technologies (7–10). To facilitate the use of solar energy, tremendous efforts have been devoted in recent decades to developing previously unidentified conjugated polymers exhibiting broad and strong absorption bands (11–13). The common strategies for increasing absorption involve extending π-conjugation by incorporating conjugated cyclic moieties, especially fused rings; modulating the strength of intramolecular charge transfer between donor and acceptor units (D–A effect); increasing the coplanarity of π conjugation through weak intramolecular interactions (e.g., hydrogen bonds); and introducing heteroatoms or heavy atoms into the repeat units of conjugated polymers (11–16). Additionally, appropriate solubility is a prerequisite for processing and using polymers and is usually achieved with the aid of long alkyl or alkoxy side chains (12, 17).Aromatic rings are among the most important building blocks for conjugated polymers. In addition to aromatic hydrocarbons, a variety of aromatic heterocycles composed of main-group elements have been used as fundamental components. These heteroatom-containing conjugated polymers show unique optical and electronic properties (4–10). However, while metalla-aromatic systems bearing a transition metal have been known since 1979 due to the pioneering work by Thorn and Hoffmann (18), none of them have been used as building blocks for conjugated polymers. The HOMO–LUMO gaps (E_g) of metalla-aromatics are generally narrower (Fig. 1) than those of their organic counterparts (19–22). We reasoned that this feature should broaden the absorption window if polymers stemming from metalla-aromatics are achievable.Open in a separate windowFig. 1.Comparison of traditional organic skeletons with metalla-aromatic building blocks (the computed energies are in eV). (A) HOMO–LUMO gaps of classic aromatic skeletons. (B) Carbolong frameworks as potential building blocks for novel conjugated polymers with broad absorption bands and improved solubility.In recent years, we have reported a series of readily accessible metal-bridged bicyclic/polycyclic aromatics, namely carbolong complexes, which are stable in air and moisture (23–25). The addition of osmium carbynes (in carbolong complexes) and alkynes gave rise to an intriguing family of d_π–p_π conjugated systems, which function as excellent electron transport layer materials in organic solar cells (26, 27). These observations raised the following question: Can this efficient addition reaction be used to access metalla-aromatic conjugated polymers? It is noteworthy that incorporation of metalla-aromatic units into conjugated polymers is hitherto unknown. In this contribution, we disclose a polymerization reaction involving M≡C analogs of C≡C bonds, which involves a unique carbyne shuttling strategy (Fig. 2A). This led to examples of metalla-aromatic conjugated polymers (polycarbolongs) featuring metal carbyne units in the main chain. On the other hand, the development of polymerization reactions plays a crucial role in involving certain building blocks in conjugated polymers (28–32). These efficient, specific, and feasible polymerizations could open an avenue for the synthesis of conjugated polymers.Open in a separate windowFig. 2.Design of polymers and synthesis of monomers. (A) Schematic illustration of the polymerization strategy. (B) Preparation of carbolong monomers. Insert: X-ray molecular structure for the cations of complex 3. Ellipsoids are shown at the 50% probability level; phenyl groups in PPh₃ are omitted for clarity.     

A noncanonical cytochrome c stimulates calcium binding by PilY1 for type IVa pili formation

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope–spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many several other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on calcium and a noncanonical cytochrome c (TfcP) with an unusual His/Cys heme ligation. We provide evidence that TfcP is unlikely to participate in electron transport and instead stimulates calcium binding by PilY1.1 at low-calcium concentrations, thereby stabilizing PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results not only identify a previously undescribed function of cytochromes c but also illustrate how incorporation of an accessory factor expands the environmental range under which the T4aP system functions.

In bacteria, motility is important for virulence, promotes colonization of habitats of diverse composition, and stimulates biofilm formation (1). Type IVa pili (T4aP) are filamentous cell surface structures that enable cell translocation across surfaces and also have critical functions in surface adhesion, surface sensing, host cell interaction, biofilm formation, predation, virulence, and DNA uptake (2–4). The versatility of T4aP is based on their ability to undergo cycles of extension, surface adhesion, and retraction (5, 6). Retractions generate a force up to 150 pN per pilus, pulling cells across surfaces (7).In Gram-negative bacteria, the extension/retraction cycles of T4aP are driven by the T4aP machine (T4aPM), which consists of 15 conserved proteins that form a complex that spans from the outer membrane (OM) across the periplasm and inner membrane (IM) to the cytoplasm (8–10) (Fig. 1A). Pilus extension and retraction are powered by the PilB and PilT ATPases, respectively, that bind in a mutually exclusive manner to the cytoplasmic base of the T4aPM (8, 11–13). All 15 proteins are essential for T4aP extension except for PilT, which is only important for retraction (4). The so-called priming complex is an integral part of the T4aPM, composed of the major pilin, four minor pilins and the PilY1 protein, and incorporated into the machine independently of the PilB ATPase (10, 14) (Fig. 1A). The five pilins interact directly to form a short pilus that is capped by PilY1, which interacts directly with the minor pilins (10). Pilus extension is initiated by the incorporation of additional major pilin subunits from a reservoir in the IM to the base of the priming complex in a process stimulated by PilB (6, 10, 14). Conversely, during retractions, major pilin subunits are removed from the base of the pilus and reinserted into the IM in a process stimulated by PilT (12, 15). Because the major pilin is added to the priming complex during the initiation of the extension process, the priming complex remains at the tip of the extended pilus (10, 14, 16). Consistently, PilY1 is involved in surface adhesion, surface sensing, specificity in host cell recognition during infections, and virulence (14, 16–19).Open in a separate windowFig. 1.TfcP is a noncanonical cytochrome c. (A) Architectural model of nonpiliated and piliated T4aPM. PilB and PilT associate with PilC in a mutually exclusive manner during extension and retraction, respectively. Bent arrows, incorporation and removal of the major pilin PilA from the pilus base during extension and retraction, respectively. Proteins labeled with single letters have the Pil prefix. Y1_N and Y1_C indicate the N- and C-terminal domains of PilY1, respectively. The color code for the four minor pilins is as in B. (B) Genetic organization of cluster_1 encoding minor pilins, PilY1.1 and TfcP. Locus tags are included Above and gene names within genes. Distances between start and stop codons are shown Above. (C) Domain architecture of TfcP and homologs. Pink, type I signal peptide; red, cytochrome c domain; and brown, C-terminal extension. The cytochrome c signature motif CxxCH and the distal Cys⁹¹ residue are indicated. Numbering of amino acids is according to the unprocessed, full-length TfcP. (D) Sequence alignment of TfcP homologs. Residues are highlighted based on >80% similarity. Domains are indicated using the color code from C. The cytochrome c signature motif CxxCH and the distal Cys⁹¹ residue are indicated. Numbering of amino acids is according to the unprocessed, full-length proteins.Among the 15 proteins of the T4aPM, 9 are generally encoded by single-copy genes (20). Some species contain multiple PilT paralogs that enable retractions with different characteristics (21). The genes for the four minor pilins and PilY1 are also often present in multiple copies (10, 22–24). The multiplicity of minor pilins and PilY1 proteins has been suggested to allow individual species to assemble priming complexes and tip complexes of different composition and with different properties, thereby allowing the formation of T4aP that can function in a variety of different habitats (10, 14, 25). Minor pilins are low-abundance proteins that share overall structure and sequence homology with the major pilin and have a prepilin signal peptide, a hydrophobic N-terminal α-helix, and a C-terminal globular domain, which is less conserved (26). PilY1 proteins have a type I signal peptide, are secreted to the periplasm, and are composed of two domains. The conserved C-terminal PilY1 domain adopts a beta-propeller fold (27) that interacts with the minor pilins in the priming and tip complex (10) (Fig. 1A). The N-terminal domain is much less conserved and is the domain that mediates host cell recognition, adhesion, and surface sensing (10, 17, 28).The soil-dwelling δ-proteobacterium Myxococcus xanthus uses T4aP-dependent motility (T4aPdM) and gliding motility to move on surfaces to generate spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients (29, 30). The M. xanthus genome contains three gene clusters (from here on cluster_1, _2, and _3; proteins labeled with suffixes 1, 2, and 3), each encoding four minor pilins and a PilY1 protein (8, 10). Cluster_1 alone and cluster_3 alone support T4aPdM under standard conditions (10). While the four respective minor pilins share overall sequence homology, the three PilY1 proteins are highly divergent in their N-terminal domains (10). Thus, M. xanthus has the potential to generate at least two, and possibly three, different T4aPM and T4aP that differ in their priming and tip complexes.To understand the functional range of the three minor pilin/PilY1 protein sets, we focused on the proteins of cluster_1. Here, we provide evidence that these proteins form priming and tip complexes in a calcium-dependent manner. We identify the TfcP protein and show that it is a noncanonical cytochrome c with an unusual His/Cys heme ligation that is important for PilY1.1 stability under low-calcium conditions; PilY1.1, in turn, is important for the stability of the cluster_1 minor pilins. The effect of TfcP on PilY1.1 stability depends on calcium binding by PilY1.1 and is bypassed at high-calcium concentrations. Our data support a model whereby TfcP promotes calcium binding by PilY1.1 at low-calcium concentrations, thereby, allowing cluster_1 to support T4aP function in a broader range of environmental conditions.     

Volatile-consuming reactions fracture rocks and self-accelerate fluid flow in the lithosphere

Hydration and carbonation reactions within the Earth cause an increase in solid volume by up to several tens of vol%, which can induce stress and rock fracture. Observations of naturally hydrated and carbonated peridotite suggest that permeability and fluid flow are enhanced by reaction-induced fracturing. However, permeability enhancement during solid-volume–increasing reactions has not been achieved in the laboratory, and the mechanisms of reaction-accelerated fluid flow remain largely unknown. Here, we present experimental evidence of significant permeability enhancement by volume-increasing reactions under confining pressure. The hydromechanical behavior of hydration of sintered periclase [MgO + H₂O → Mg(OH)₂] depends mainly on the initial pore-fluid connectivity. Permeability increased by three orders of magnitude for low-connectivity samples, whereas it decreased by two orders of magnitude for high-connectivity samples. Permeability enhancement was caused by hierarchical fracturing of the reacting materials, whereas a decrease was associated with homogeneous pore clogging by the reaction products. These behaviors suggest that the fluid flow rate, relative to reaction rate, is the main control on hydromechanical evolution during volume-increasing reactions. We suggest that an extremely high reaction rate and low pore-fluid connectivity lead to local stress perturbations and are essential for reaction-induced fracturing and accelerated fluid flow during hydration/carbonation.

Hydration and carbonation reactions in the crust and mantle transport H₂O and CO₂ from Earth’s surface to the interior and control volatile budgets within the Earth (1–6). These reactions are characterized by solid-volume increase, by up to several tens of vol%, which induces stress that may lead to fracturing (7–10). The driving force of such stress generation is the thermodynamic free energy released when metastable anhydrous/noncarbonate minerals react with fluids (7). The stress generated by the reaction has the potential to cause rock fracture and fragmentation (7, 11–13), thereby increasing the reactive surface area and fluid flow and further accelerating the reactions (7, 8, 14). Such chemical breaking of rocks, or reaction-induced fracturing, appears to be important in driving hydration and carbonation reactions to completion (8, 15, 16) in an otherwise self-limiting process where reaction products can clog pores and suppress fluid flow, thereby hindering the reaction (15, 17).Observations of naturally serpentinized and fractured ultramafic rocks indicate a volume increase of 20 to 60% during hydration reactions (13, 18–20), providing evidence of an accelerated supply of fluids during hydration (Fig. 1 A and B). Natural carbonation of ultramafic rocks is also associated with extensive fracture networks, and reaction-induced fracturing is considered a key process in mineral carbonation (Fig. 1C) (7, 8, 21). Numerical simulations indicate a positive feedback between volume-increasing reaction, fracturing, and fluid flow (10, 22–32). Laboratory experiments partially reproduce fracturing during peridotite carbonation, serpentinization, and periclase hydration (29, 33–36); however, hydrothermal flow-through experiments of peridotite serpentinization and carbonation show a decrease in permeability and deceleration of fluid flow and reaction rate (37–42). Observations of the natural carbonation of serpentinized peridotite indicate the decrease in permeability and reduced fluid flow and reaction rate are a consequence of pore clogging related to carbonation (43). Until now, no experimental studies have shown a clear increase in permeability during expansive fluid–rock reactions under confining pressure. As such, despite their geological and environmental importance, the evolution of expansive fluid–rock reactions remains difficult to predict, owing to the complex hydraulic–chemical–mechanical feedbacks underlying these reactions (15, 16, 44). The processes controlling the self-acceleration or deceleration of these reactions remain largely unknown.Open in a separate windowFig. 1.Reaction-induced fractures related to natural hydration/carbonation. (A) Polygonal block of serpentinite cut by planar lizardite veins, extracted from a serpentinite body, San Andreas Lake, California. (B) Photomicrograph of mesh structure in partly serpentinized peridotite, Redwood City serpentinite, California [crossed-polarized light (61)]. (C) Quartz veins in silica–carbonate rocks (i.e., listvenite, a carbonated ultramafic rock) that occur along the boundaries of serpentinite bodies, San Jose, California. ol, olivine; serp, serpentine (lizardite ± antigorite mixture); br, brucite.Here, we use the hydration of periclase to brucite [MgO + H₂O → Mg(OH)₂] as an analog for solid-volume–increasing reactions in the Earth. This reaction produces an extreme solid-volume increase of 119%, with a high reaction rate at 100 to 600 °C (45). Previous experimental studies on periclase hydration have revealed that extensive fracturing occurs under certain conditions (29, 33, 35), yet the links between fracturing experiments (periclase hydration), nonfracturing experiments (peridotite hydration/carbonation), and natural observations are unknown. On the basis of in situ observations of fluid flow during the reactions, we clearly show that fluid flow and associated permeability are strongly enhanced by solid-volume–increasing reactions under confining pressure (i.e., at simulated depth). Based on the experimental results and nondimensional parameterization, we propose that the ratio of the initial fluid flow rate to the reaction rate has a primary control on the self-acceleration and deceleration of fluid flow and reactions during hydration and carbonation within the Earth.