Mesoscale networks and corresponding transitions from self-assembly of block copolymers

A series of cubic network phases was obtained from the self-assembly of a single-composition lamellae (L)-forming block copolymer (BCP) polystyrene-block-polydimethylsiloxane (PS-b-PDMS) through solution casting using a PS-selective solvent. An unusual network phase in diblock copolymers, double-primitive phase (DP) with space group of Im3¯m, can be observed. With the reduction of solvent evaporation rate for solution casting, a double-diamond phase (DD) with space group of Pn3¯m can be formed. By taking advantage of thermal annealing, order–order transitions from the DP and DD phases to a double-gyroid phase (DG) with space group of Ia3¯d can be identified. The order–order transitions from DP (hexapod network) to DD (tetrapod network), and finally to DG (trigonal planar network) are attributed to the reduction of the degree of packing frustration within the junction (node), different from the predicted Bonnet transformation from DD to DG, and finally to DP based on enthalpic consideration only. This discovery suggests a new methodology to acquire various network phases from a simple diblock system by kinetically controlling self-assembling process.

From constituted molecules to polymers, finally ordered hierarchical superstructures, self-assembled solids cover a vast area of nanostructures where the characters of building blocks direct the progress of self-assembly (1, 2). In nature, fascinating periodic network structures and morphologies from different species are appealing in nanoscience and nanotechnology due to their superior properties, especially for photonic crystal structures (3–7). For gyroid, trigonal planar network with chirality demonstrates its potential as chiropitc metamaterial (8–10). Beyond the splendid colors, networks either in macroscale or mesoscale mechanically strengthen their skeletons and protect those fragile but vital organs from impact (11, 12). Inspired by nature, biomimicking materials with mesoscale network may exceed the limitation of the intrinsic properties (13). The topology of networks could further improve their adaptability, allowing extreme deformation for energy dissipation (14). Moreover, network materials from hybridization of self-assembled block copolymers (BCPs) have been exploited to the design of mesoscale quantum metamaterials (15, 16). With the desire to acquire network textures for biomimicking nanomaterials, BCPs with immiscible constituted segments covalently joined together give the accessibility to the formation of nanonetwork morphologies via balancing enthalpic penalty from the repulsive interaction of constituted blocks and entropic penalty from the stretching of polymer chains (17–21). By taking advantage of precise synthesis procedures, it is feasible to obtain the aimed network phases from the self-assembly of BCPs such as Fddd (O⁷⁰) (22–24), gyroid (Q²¹⁴, Q²³⁰) (20, 21, 25–27), and diamond (Q²²⁴, Q²²⁷) (28–31) experimentally and theoretically. On the basis of theoretical prediction, the junction points (nodes) in the network phases could be coordinated with three, four, or six neighbors in three-dimensional space, resulting in the enhancement of packing frustration (31). Topologically, all these phases match the coordination number to neighbors (n = 3, 4, 6), showing no special case of quasicrystal. Accordingly, an order–order transition from double-diamond phase (DD, tetrapod) to double-gyroid phase (DG, trigonal planar network) has been observed (29). Yet, there is no DP phase being found in simple diblock systems except for liquid crystals (32, 33) or organic–inorganic nanocomposites from the mixtures of BCP with inorganic precursors (34, 35). Searching the rare occurrence of network phases and the corresponding phase transitions among phases will be essential to the demands for application by considering the deliberate structuring effects on aimed properties but the approaches remain challenging (8, 36–40). For instance, viewing the narrow window for network morphologies in diblock copolymer phase diagram, it demands harsh requirements for syntheses (2, 41). Recently, by taking advantage of using selective solvent for solution casting, it is feasible to acquire DG phase and even inverted DG phase from the self-assembly of lamellae (L)-forming polystyrene-block-polydimethylsiloxane (PS-b-PDMS) (42). Apart from that, a triclinic DG phase was recently discovered from the PS-b-PDMS which is commonly believed nonexisting in the conventional phase diagram (43). As a result, the phase diagram of BCPs with high interaction parameter is worthy of study for searching the metastable phases with unique network textures (44). Herein, we aim to acquire network phases from a simple diblock system by kinetically controlling the transformation mechanisms of self-assembly. As exemplified by using the PS-b-PDMS for solution casting, with the use of a PS-selective solvent (chloroform), a DP phase and a DD phase could be formed through controlled self-assembly, giving unique network phases simply from solution casting. Moreover, a DG phase can be also acquired from phase transformation. Consequently, a series of network phases with hexapod, tetrapod, and trigonal planar building units could be successfully obtained by using a single-composition L-forming PS-b-PDMS for self-assembly. The corresponding order–order transitions among these network phases examined by temperature-resolved in situ small-angle X-ray scattering (SAXS) combining with electron tomography results provide insights of network phase formation and the corresponding phase transformation mechanisms in the self-assembly of BCPs.     

Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism

Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine–isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae. We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.

The cellular surface of many bacteria is elaborated with thin appendages called pili (also called fimbriae) which have a range of roles including twitching motility, conjugation, immunomodulation, biofilm formation, and adherence (1, 2). These long proteinaceous fibers are key virulence factors that mediate initial host–pathogen interactions, which are subsequently strengthened by more intimate contacts from shorter pili and cell wall–attached adhesins (1–13 ). As the infection progresses, pili also facilitate biofilm formation, protecting invading microbes from host immune clearance and exogenous antibiotics (1, 5, 11, 14). Gram-positive bacteria display very thin (1 to 5 nm) (15) hair-like pili that nevertheless possess enormous tensile strength because their protein components are crosslinked together by lysine–isopeptide bonds. These crosslinked fibers are displayed by a wide range of pathogenic and commensal Gram-positive bacteria, but their structures and mechanism of assembly remain poorly understood (2, 5, 7–10).Pili in Gram-positive bacteria are assembled by pilus-specific sortase enzymes that crosslink the pilus subunits (called pilins) together via lysine–isopeptide bonds. Our current understanding of this process has been significantly advanced by studies of the SpaA pilus in Corynebacterium diphtheriae, a pathogen that causes pharyngeal diphtheria (5, 8, 10, 16). The SpaA pilus mediates adherence to the pharyngeal epithelium and consists of three types of pilins: the pilus shaft is formed by SpaA, and the tip and base are formed by SpaC and SpaB, respectively (17). The C. diphtheriae pilus-specific sortase (^CdSrtA) assembles the pilus by catalyzing a repetitive, irreversible transpeptidation reaction that covalently links pilin subunits together via an isopeptide bond. The shaft of the pilus is formed by ∼100 to 250 crosslinked SpaA pilins (16). ^CdSrtA-catalyzed SpaA polymerization begins when SpaA prepilin proteins containing an N-terminal signal peptide sequence are exported via the Sec pathway and retained on the extracellular surface via a C-terminal cell wall sorting signal (CWSS) (18). ^CdSrtA then crosslinks SpaA proteins together via a two-step process. First, an LPLTG sorting signal sequence within the CWSS is cleaved between Thr and Gly residues by the sortase, generating a thioacyl-linked ^CdSrtA-SpaA intermediate in which the enzyme’s active site cysteine residue is covalently linked to the carbonyl atom of the sorting signal threonine. In the second step, a lysine ε-amine group originating from another SpaA pilin attacks the thioacyl linkage in the ^CdSrtA-SpaA intermediate, thereby joining distinct SpaA proteins together via a K190-T494 isopeptide bond (Fig. 1A). The reactive lysine in SpaA is housed within the N-terminal domain and is part of a highly conserved WxxxVxVYPK sequence motif that is found in many pilin proteins (17). The shaft of the pilus is constructed by repeating this two-step process, and a similar ^CdSrtA-catalyzed reaction is used to add the SpaC tip pilin to SpaA. Pilus assembly is completed by incorporating the SpaB basal pilin, which promotes pilus attachment to the cell wall using a distinct housekeeping sortase ^CdSrtF (3). Pilus biogenesis is thought to occur within “pilusosomes” on the cell surface, at which pilin substrates and pilus-specific sortases colocalize to facilitate rapid polymerization (18).Open in a separate windowFig. 1.Structure of the ^NSpaA-signal peptide complex. (A) Schematic of pilus polymerization with full-length SpaA molecules. An expanded view of the two portions of the crosslinked SpaA polymer investigated in this study, ^CSpaA-^NSpaA complex and ^NSpaA-signal, are boxed in gray dashed lines and solid black lines, respectively. (B) The ^NSpaA-signal peptide complex is represented in surface representation with relative conservation of each residue indicated by a color gradient ranging from highly variable positions (blue) to highly conserved residues (yellow). The peptide (magenta sticks) is docked into a highly conserved, nonpolar binding groove on SpaA. (C) A bundle of the 40 lowest energy structures of the SpaA-signal complex is displayed. The backbone of the ^NSpaA domain is represented by blue ribbons. The last five residues of the sorting signal peptide are depicted as red sticks, and Lys190 is shown as green sticks. (D) Secondary structural elements of the NMR structure are highlighted. (E) An expanded view of the peptide binding interface shows how the peptide is bound in the cleft of ^NSpaA. Residues on SpaA exhibiting intermolecular NOEs to the peptide are shown as sticks. Interacting residues in the core of the domain and within the AB loop are colored yellow and pink, respectively.Despite their importance in bacterial physiology and pathogenesis, only structures of isolated, noncrosslinked pilins have been determined at atomic-level resolution (19). This is because it has been challenging to obtain homogenous crosslinked pili that are suitable for biophysical analyses and because Gram-positive pili are thin and flexible, making them difficult to study using cryogenic electron microscopy and X-ray crystallography. Crystal structures of isolated pilins have revealed that they contain IgG-like Cna-type domains and frequently one or more spontaneously forming intradomain isopeptide bonds that impart significant resistance to mechanical forces (19–21). Internal isopeptide bond linkages exist as either D- or E-type and are extremely stabilizing, allowing pilin domains to withstand the highest unfolding forces yet reported for a globular protein (20). Atomic-level structures of sortase crosslinked pilins have yet to be visualized, but a transmission electron microscopy study of the Streptococcus pneumoniae RrgB pilus enabled the periodicity and polarity of individual subunits within the pilus fiber to be determined (22). This work revealed that the subunits in the pilus are arranged in a head-to-tail manner, enabling sortase-catalyzed isopeptide crosslinking between the lysine and LPxTG motifs located at the N- and C-terminal ends of the pilin, respectively. In crystals, similar head-to-tail packing arrangements are observed, but whether these lattice interactions are also present in the intact pilus is not known.In this study, we used a recently developed in vitro pilus assembly system and hybrid structural biology methods to gain insight into the structure and biogenesis mechanism of the SpaA pilus from C. diphtheriae. We first determined the NMR structure of the N-terminal domain of SpaA crosslinked to the sorting signal peptide and then used small angle X-ray scattering (SAXS), NMR, and crystallographic data to model the structure of the isopeptide-linked SpaA-SpaA building block that is repeated to construct the pilus shaft. We show that crosslinking is accompanied by a large disordered-to-ordered structural change in the SpaA pilin, which forms an interpilin interface that differs markedly from packing interactions observed in crystals of the isolated SpaA. Quantitative measurements of kinetics of the sortase-catalyzed transpeptidation reaction suggest that the enzyme uses a latch mechanism to select the appropriate lysine residue on SpaA for interpilin crosslinking.     

Trends of Stroke Incidence and 28-Day All-Cause Mortality after a Stroke in Malaysia: A Linkage of National Data Sources

Background:Data on nationwide trends for stroke metrics are crucial to understand the extent of the disease burden to a country’s health system. Yet, this information remains scarce in low- and middle-income countries.Objectives:This study investigated trends of stroke incidence and 28-day all-cause mortality after a stroke from 2008 to 2016 in Malaysia, through linkage across national data sources.Methods:Hospital admissions with a principal diagnosis of stroke or transient ischemic attack were included. Cases with first stroke were identified through linkage of hospital admission registers where age and sex-standardized trends of stroke incidence and its subtypes were calculated. By linking hospital registers to the National Death Register, the 28-day all-cause mortality rates after a stroke were estimated. Mann-Kendall’s test was used for trend evaluation.Results:From 243,765 records, the trend of stroke incidence showed an increase of 4.9% in men and a drop of 3.8% among women. Incidences were higher in men, at 99.1 per 100,000 population in 2008 and 103.9 per 100,000 in 2016 than women (80.3 per 100,000 in 2008 and 77.2 per 100,000 in 2016). There was a substantial increase in stroke incidence among those below 65 years old, with the largest increase of 53.3% in men aged between 35–39 years and 50.4% in women of similar age group. The trend for 28-day all-cause mortality showed a decline for men at –13.1% and women, –10.6%. Women had higher mortality from stroke (22.0% in 2008 and 19.7% in 2016) than men (19.4% in 2008 to 17.2% in 2016).Conclusion:This first empirical study on stroke trends in Malaysia revealed a worrying increase in stroke incidence among the younger population. Despite a declining trend, mortality rates remained moderately high especially in women. Comprehensive strategies to strengthen the prevention and management of stroke care are warranted.     

Risk factors and adverse maternal and perinatal outcomes for women with dichorionic twin pregnancies complicated by gestational diabetes mellitus: A retrospective cross-sectional study

Aims/IntroductionThe association between gestational diabetes mellitus (GDM) and adverse maternal and perinatal outcomes in twin pregnancies remains unclear. This study was undertaken to highlight risk factors for GDM in women with dichorionic (DC) twins, and to determine the association between GDM DC twins and adverse maternal and perinatal outcomes in a large homogeneous Taiwanese population.Materials and MethodsA retrospective cross‐sectional study was carried out on 645 women with DC twins, excluding pregnancies complicated by one or both fetuses with demise (n = 22) or congenital anomalies (n = 9), who gave birth after 28 complete gestational weeks between 1 January 2001 and 31 December 2018. Univariable and multiple logistic regression analyses were carried out.ResultsMaternal age >34 years (adjusted odds ratio 2.52; 95% confidence interval 1.25–5.07) and pre‐pregnancy body mass index >24.9 kg/m² (adjusted odds ratio 2.83, 95% confidence interval 1.47–5.46) were independent risk factors for GDM in women with DC twins. Newborns from women with GDM DC twins were more likely to be admitted to the neonatal intensive care unit (adjusted odds ratio 1.70, 95% confidence interval 1.06–2.72) than newborns from women with non‐GDM DC twins. Other pregnancy and neonatal outcomes were similar between the two groups.ConclusionsAdvanced maternal age and pre‐pregnancy overweight or obesity are risk factors for GDM in women with DC twins. Except for a nearly twofold increased risk of neonatal intensive care unit admission of newborns, the pregnancy and neonatal outcomes for women with GDM DC twins are similar to those for women with non‐GDM DC twins.     

Role of modern radiotherapy in managing patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Several treatment options are available for managing HCC patients, classified roughly as local, local-regional, and systemic therapies. The high post-monotherapy recurrence rate of HCC urges the need for the use of combined modalities to increase tumor control and patient survival. Different international guidelines offer treatment recommendations based on different points of view and classification systems. Radiotherapy (RT) is a well-known local-regional treatment modality for managing many types of cancers, including HCC. However, only some of these treatment guidelines include RT, and the role of combined modalities is rarely mentioned. Hence, the present study reviewed clinical evidence for the use of different combined modalities in managing HCC, focusing on modern RT's role. Modern RT has an increased utility in managing HCC patients, mainly due to two driving forces. First, technological advancement (e.g., stereotactic body radiotherapy and advanced proton-beam therapy) enables precise delivery of radiation to increase tumor control and reduce side effects in the surrounding normal tissue. Second, the boom in developing target therapies and checkpoint-blockade immunotherapy prolongs overall survival in HCC patients, re-emphasizing the importance of local tumor control. Remarkably, RT combines with systemic therapies to generate the systemic therapy augmented by radiotherapy effect, a benefit now being actively investigated.     

The Distribution and Composition of Vector Abundance in Hanoi City,Vietnam: Association with Livestock Keeping and Flavivirus Detection

Background: Dengue virus and Japanese encephalitis virus are two common flaviviruses that are spread widely by Aedes and Culex mosquitoes. Livestock keeping is vital for cities; however, it can pose the risk of increasing the mosquito population. Our study explored how livestock keeping in and around a large city is associated with the presence of mosquitoes and the risk of them spreading flaviviruses. Methods: An entomological study was conducted in 6 districts with 233 households with livestock, and 280 households without livestock, in Hanoi city. BG-Sentinel traps and CDC light traps were used to collect mosquitoes close to animal farms and human habitats. Adult mosquitoes were counted, identified to species level, and grouped into 385 pools, which were screened for flaviviruses using a pan-flavivirus qPCR protocol and sequencing. Results: A total of 12,861 adult mosquitoes were collected at the 513 households, with 5 different genera collected, of which the Culex genus was the most abundant. Our study found that there was a positive association between livestock keeping and the size of the mosquito population—most predominantly between pig rearing and Culex species (p < 0.001). One pool of Cx. tritaeniorhynchus, collected in a peri-urban district, was found to be positive for Japanese encephalitis virus. Conclusions: The risk of flavivirus transmission in urban areas of Hanoi city due to the spread of Culex and Aedes mosquitoes could be facilitated by livestock keeping.     

Density estimation using deep generative neural networks

Density estimation is one of the fundamental problems in both statistics and machine learning. In this study, we propose Roundtrip, a computational framework for general-purpose density estimation based on deep generative neural networks. Roundtrip retains the generative power of deep generative models, such as generative adversarial networks (GANs) while it also provides estimates of density values, thus supporting both data generation and density estimation. Unlike previous neural density estimators that put stringent conditions on the transformation from the latent space to the data space, Roundtrip enables the use of much more general mappings where target density is modeled by learning a manifold induced from a base density (e.g., Gaussian distribution). Roundtrip provides a statistical framework for GAN models where an explicit evaluation of density values is feasible. In numerical experiments, Roundtrip exceeds state-of-the-art performance in a diverse range of density estimation tasks.

Let p(·) be a density on a n-dimensional Euclidean space χ. The task of density estimation is to estimate p(·) based on a set of independently and identically distributed data points {xi}i=1Ndrawn from this density.Traditional density estimators such as histograms (1, 2) and kernel density estimators (KDEs) (3, 4) typically perform well only in low dimension. Recently, neural network-based approaches were proposed for density estimation and yielded promising results in problems with high-dimensional data points such as images. There are mainly two families of such neural density estimators: autoregressive models (5–7) and normalizing flows (8–11). Autoregression-based neural density estimators decompose the density into the product of conditional densities based on probability chain rule p(x)=∏ip(xi|x1:i−1). Each conditional probability p(xi|x1:i−1) is modeled by a parametric density (e.g., Gaussian or mixture of Gaussian), of which the parameters are learned by neural networks. Density estimators based on normalizing flows represent x as an invertible transformation of a latent variable z with known density, where the invertible transformation is a composition of a series of simple functions whose Jacobian is easy to compute. The parameters of these component functions are then learned by neural networks.As suggested in ref. 12, both of these are special cases of the following general framework. Given a differentiable and invertible mapping G:Rn→Rn and a base density pzz, the density of x=G(z) can be represented using the change of variable rule as follows:pxx=pzz|detJz|−1,[1]where Jz=∂Gz/∂zT is the Jacobian matrix of function G(·) at point z. Density estimation at x can be solved if the base density pzz is known and the determinant of Jacobian matrix is feasible to calculate. To achieve this, previous neural density estimators have to impose heavy constraints on the model architecture. For example, refs. 7, 10, and 12 require the Jacobian to be triangular, ref. 13 constructed low rank perturbations of a diagonal matrix as the Jacobian, and ref. 14 proposed a circular convolution where the Jacobian is a circulant matrix. These strong constraints diminish the expressiveness of neural networks, which may lead to poor performance. For example, autoregressive neural density estimators based on learning p(xi|x1:i−1) are naturally sensitive to the order of the features. Moreover, the change of variable rule is not applicable when the domain dimension in base density differs from target density. However, experiences from deep generative models [e.g., GAN (15) and VAE (16)] suggested that it is often desirable to use a latent space of smaller dimension than the data space.To overcome the limitations above, we propose a neural density estimator called Roundtrip. Our approach is motivated by recent advances in deep generative neural networks (15, 17, 18). Roundtrip differs from previous neural density estimators in two ways. 1) It allows the direct use of a deep generative network to model the transformation from the latent variable space to the data space, while previous neural density estimators use neural networks only to learn the parameters in the component functions that are used for building up an invertible transformation. 2) It can efficiently model data densities that are concentrated near learned manifolds, which is difficult to achieve by previous approaches as they require the latent space to have the same dimension as the data space. Importantly, we also provide methods, based on either importance sampling and Laplace approximation, for the pointwise evaluation of the density estimate. We summarize our major contributions in this study as follows: 1) We propose a general-purpose neural density estimator based on deep generative models, which requires less restrictive model assumptions compared to previous neural density estimators. 2) We show that the principle in previous neural density estimators can be regarded as a special case in our Roundtrip framework. 3) We demonstrate state-of-the-art performance of Roundtrip model through a series of experiments, including density estimation tasks in simulations as well as in real data applications ranging from image generation to outlier detection.     

Dogs as Sentinels for Flavivirus Exposure in Urban,Peri-Urban and Rural Hanoi,Vietnam

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.