Characterization of the primary structure of the major silk gene, h-fibroin, across caddisfly (Trichoptera) suborders.

Larvae of caddisflies (Trichoptera) produce silk to build various underwater structures allowing them to exploit a wide range of aquatic environments. The silk adheres to various substrates underwater and has high tensile strength, extensibility, and toughness and is of interest as a model for biomimetic adhesives. As a step toward understanding how the properties of underwater silk evolved in Trichoptera, we used genomic data to identify full-length sequences and characterize the primary structure of the major silk protein, h-fibroin, across the order. The h-fibroins have conserved termini and basic motif structure with high variation in repeating modules and variation in the percentage of amino acids, mainly proline. This finding might be linked to differences in mechanical properties related to the different silk usage and sets a starting point for future studies to screen and correlate amino acid motifs and other sequence features with quantifiable silk properties.

Allelic resolution of insect and spider silk genes reveals hidden genetic diversity.

Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation-both in terms of length and repeat motif order-between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 Mya: the insect clade containing caddisflies and butterflies and spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests common mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation.

Highly accurate long reads are crucial for realizing the potential of biodiversity genomics.

BACKGROUND: Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. RESULTS: HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. CONCLUSIONS: Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

Material characterization of GPX®: A versatile in situ solidifying embolic platform technology.

Endovascular embolization is a minimally invasive procedure during which blood flow to targeted tissues is selectively occluded. The list of clinical indications for embolization continues to expand. Liquid embolic agents are injectable compositions that transition into a solid or semi-solid form when introduced into blood vessels. The mechanism that triggers the liquid-to-solid transition is a key distinguishing feature of liquid embolic agents. GPX is a waterborne liquid embolic agent comprising oppositely charged polyelectrolytes: polyguanidinum and inorganic polyphoshate. In situ solidification is driven by electrostatic condensation of the polyelectrolytes, triggered by ionic strength differentials. We report in vitro characterization of the material properties of GPX, it is in vivo effectiveness in acute animal studies, and its potential for chemoembolization. The viscosity of GPX can be varied over a wide range by adjusting the polyguanidinium MW and/or concentration. Formulation of GPX with either tantalum microparticles (30 wt%) or iodinated radiocontrast agents (300 mgI ml-1) did not significantly change the flow behavior of GPX; the viscosity was independent of shear rate and remained within a clinically practical range (80-160 cP). Formulation of GPX with doxorubicin substantially increased viscosity at low shear rates and resulted in a power law dependence on shear rate. High contrast and effective vascular occlusion were demonstrated in both swine kidneys and rete mirabile. Contrast from iodinated compounds was temporary, dissipating within hours. The doxorubicin in vitro release profile was linear over 90 days. The results demonstrate that GPX is a versatile liquid embolic platform that can be formulated with a wide range of viscosities injectable at clinically practical flow rates, with either transient or permanent contrast, and that can provide prolonged zero-order delivery of doxorubicin to embolized tissues.

Conservation of Three-Dimensional Structure of Lepidoptera and Trichoptera L-Fibroins for 290 Million Years.

The divergence of sister orders Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) from a silk-spinning ancestor occurred around 290 million years ago. Trichoptera larvae are mainly aquatic, and Lepidoptera larvae are almost entirely terrestrial-distinct habitats that required molecular adaptation of their silk for deployment in water and air, respectively. The major protein components of their silks are heavy chain and light chain fibroins. In an effort to identify molecular changes in L-fibroins that may have contributed to the divergent use of silk in water and air, we used the ColabFold implementation of AlphaFold2 to predict three-dimensional structures of L-fibroins from both orders. A comparison of the structures revealed that despite the ancient divergence, profoundly different habitats, and low sequence conservation, a novel 10-helix core structure was strongly conserved in L-fibroins from both orders. Previously known intra- and intermolecular disulfide linkages were accurately predicted. Structural variations outside of the core may represent molecular changes that contributed to the evolution of insect silks adapted to water or air. The distributions of electrostatic potential, for example, were not conserved and present distinct order-specific surfaces for potential interactions with or modulation by external factors. Additionally, the interactions of L-fibroins with the H-fibroin C-termini are different for these orders; lepidopteran L-fibroins have N-terminal insertions that are not present in trichopteran L-fibroins, which form an unstructured ribbon in isolation but become part of an intermolecular ß-sheet when folded with their corresponding H-fibroin C-termini. The results are an example of protein structure prediction from deep sequence data of understudied proteins made possible by AlphaFold2.

fastMRI+, Clinical pathology annotations for knee and brain fully sampled magnetic resonance imaging data.

Improving speed and image quality of Magnetic Resonance Imaging (MRI) using deep learning reconstruction is an active area of research. The fastMRI dataset contains large volumes of raw MRI data, which has enabled significant advances in this field. While the impact of the fastMRI dataset is unquestioned, the dataset currently lacks clinical expert pathology annotations, critical to addressing clinically relevant reconstruction frameworks and exploring important questions regarding rendering of specific pathology using such novel approaches. This work introduces fastMRI+, which consists of 16154 subspecialist expert bounding box annotations and 13 study-level labels for 22 different pathology categories on the fastMRI knee dataset, and 7570 subspecialist expert bounding box annotations and 643 study-level labels for 30 different pathology categories for the fastMRI brain dataset. The fastMRI+ dataset is open access and aims to support further research and advancement of medical imaging in MRI reconstruction and beyond.

Genome size evolution in the diverse insect order Trichoptera.

BACKGROUND: Genome size is implicated in the form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: polyploidy (i.e., whole-genome duplication) or smaller duplication events and bursts in the activity of repetitive elements. Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages. RESULTS: We detect a ∼14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single-copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments). CONCLUSION: Our findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20 Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50  = 9.7 Mbp/32.4 Mbp, L50  = 13/11) and complete (BUSCO complete  = 99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

Perceptions of quality of communication in family interactions in neurocritical care.

OBJECTIVE: Given the challenges of patient-provider communication in neurocritical care lacking robust decision-making tools on prognostication, we investigated concordance in perceptions of communication among participants in family discussions and assess the different domains of communication that affect these perceptions. METHODS: Prospective observational study conducted over 4 months in a tertiary-level academic medical center neurocritical care unit. Our study involved family discussions regarding plan of care for admitted patients observed by a neutral observer. All participants completed a survey. The first four questions rated the understanding of the discussion and general satisfaction; the remaining questions were open-ended to assess the quality of communication by the physician leading the discussion. Responses were scored and compared among participants using a Likert scale. A difference of < 1 in scores among participants was rated as concordance, whereas that of > 1 was designated as discordance. All open-ended responses were classified into six domains. RESULTS: We observed 35 family discussions. Questions 1 to 3 inquiring on general satisfaction, impact, and understanding of treatment options yielded 99 cross-comparisons per question (297 compared responses). Most responses were either "Strongly Agree" or "Agree," with "Neutral" or "Disagree" responses being more prevalent in Question 2 regarding the impact of the conversation. Overall concordance of responses between participants was 88% with a lower rate of concordance (72%) on Q2. Further open-ended questions queried observers on specific physician-spoken content, and answers were analyzed to identify domains that affected the perception of quality of communication. Education was the most frequently cited domain of communication in response to open-ended questions. Among family and neutral observers, empathy was frequently listed, whereas providers more often listed family engagement. CONCLUSION: Overall, satisfaction was high among providers, families, and the observer regarding the quality of communication during family discussions in the unit. Perceptual differences emerged over whether this communication impacted healthcare decision-making during that encounter.

Rapid Entrapment of Phenazine Ethosulfate within a Polyelectrolyte Complex on Electrodes for Efficient NAD+ Regeneration in Mediated NAD+-Dependent Bioelectrocatalysis.

Over the past two decades, the designs of redox polymers have become critical to the field of mediated bioelectrocatalysis and are used in commercial glucose biosensors, as well as other bioelectrochemical applications (e.g., energy harvesting). These polymers are specifically used to immobilize redox mediators on electrode surfaces, allowing for self-exchange-based conduction of electrons from enzymes far from the electrode to the electrode surface. However, the synthesis of redox polymers is challenging and results in large batch-to-batch variability. Herein, we report a rapid entrapment of mediators for NAD+-dependent bioelectrocatalysis within reverse ionically condensed polyelectrolytes. A high ionic strength aqueous solution of oppositely charged polyelectrolytes, composed of cationic polyguanidinium (PG) chloride and anionic sodium hexametaphosphate (P6), undergoes phase inversion into a solid microporous polyelectrolyte complex (PEC) when introduced into a low ionic strength aqueous solution. The ionic strength-triggered phase inversion of PGP6 solutions was investigated as a means to entrap mediators on the surface of electrodes for mediated bioelectrocatalysis. Compared to the traditional cross-linked immobilizations using redox polymers, this phase inversion takes place within seconds and requires up to 60 min for complete stabilization. In this work, redox mediator phenazine ethosulfate (PES) was entrapped within PGP6 on electrode surfaces for nicotinamide adenine dinucleotide (NAD+)-dependent bioelectrocatalysis. In the bulk solution, NAD+-dependent dehydrogenase enzymes catalyze the oxidation of the substrate while reducing NAD to reduced nicotinamide adenine dinucleotide (NADH). The resulting NADH is reoxidized to NAD+ by the entrapped PES that gets reduced on the electrode, completing the NAD+-regeneration-based bioelectrocatalysis. To show the use of these new materials in an application, biofuel cells were evaluated using four different anodic enzyme systems (alcohol dehydrogenase, lactate hydrogenase, glycerol dehydrogenase, and glucose dehydrogenase).

Draft Genome Assemblies and Annotations of Agrypnia vestita Walker, and Hesperophylax magnus Banks Reveal Substantial Repetitive Element Expansion in Tube Case-Making Caddisflies (Insecta: Trichoptera).

Trichoptera (caddisflies) play an essential role in freshwater ecosystems; for instance, larvae process organic material from the water and are food for a variety of predators. Knowledge on the genomic diversity of caddisflies can facilitate comparative and phylogenetic studies thereby allowing scientists to better understand the evolutionary history of caddisflies. Although Trichoptera are the most diverse aquatic insect order, they remain poorly represented in terms of genomic resources. To date, all long-read based genomes have been sequenced from individuals in the retreat-making suborder, Annulipalpia, leaving ∼275 Ma of evolution without high-quality genomic resources. Here, we report the first long-read based de novo genome assemblies of two tube case-making Trichoptera from the suborder Integripalpia, Agrypnia vestita Walker and Hesperophylax magnus Banks. We find that these tube case-making caddisflies have genome sizes that are at least 3-fold larger than those of currently sequenced annulipalpian genomes and that this pattern is at least partly driven by major expansion of repetitive elements. In H. magnus, long interspersed nuclear elements alone exceed the entire genome size of some annulipalpian counterparts suggesting that caddisflies have high potential as a model for understanding genome size evolution in diverse insect lineages.

Capsular thinning on magnetic resonance arthrography is associated with intra-operative hip joint laxity in women.

Hip microinstability is a recognized cause of hip pain in young patients. Intra-operative evaluation is used to confirm the diagnosis, but limited data exist associating magnetic resonance arthrography (MRA) findings with hip microinstability. To determine if a difference exists in the thickness of the anterior joint capsule and/or the width of the anterior joint recess on MRA in hip arthroscopy patients with and without an intra-operative diagnosis of hip laxity. Sixty-two hip arthroscopy patients were included in the study. Two musculoskeletal radiologists blinded to surgical results reviewed the MRAs for two previously described findings: (i) anterior joint capsule thinning; (ii) widening of the anterior joint recess distal to the zona orbicularis. Operative reports were reviewed for the diagnosis of joint laxity. In all patients with and without intra-operative laxity, there were no significant differences with either MRA measurement. However, twenty-six of 27 patients with intra-operative laxity were women compared with 11 of 35 patients without laxity (P < 0.001). In subgroup analysis of women, the intra-operative laxity group had a higher rate of capsular thinning compared with the non-laxity group (85% versus 45%; P = 0.01). A 82% of women with capsular thinning also had intra-operative laxity, compared with 40% without capsular thinning (P = 0.01). There were no differences regarding the width of the anterior joint recess. In this study, there was an association between capsular thinning and intra-operative laxity in female patients. Measuring anterior capsule thickness on a pre-operative MRA may be useful for the diagnosis of hip microinstability.

Direct bioelectrocatalysis by redox enzymes immobilized in electrostatically condensed oppositely charged polyelectrolyte electrode coatings.

The immobilization of enzymes on an electrode surface is critical in preserving enzyme activity and providing a sufficient electron transfer pathway for bioelectrocatalysis. Here, we present a novel single-step, cross-linker free immobilization for direct bioelectrocatalysis using an ionic strength induced phase inversion of oppositely charged polyelectrolytes. Cationic poly-guanidinyl-propyl-methacrylate (pGPMA, PG) and anionic inorganic polyphosphate, sodium hexametaphosphate (P6) were used to make an electrostatically condensed phase (PGP6). A mixture of PGP6 and laccase (LAC) from Tramates versicolor or HRP (HRP) from Armoracia rusticana were deposited on the electrode surface and were submerged in DI water to form white porous electrode coatings. Each electrode showed a current generation corresponding to the respective substrates via direct bioelectrocatalysis.

Annotated Draft Genomes of Two Caddisfly Species Plectrocnemia conspersa CURTIS and Hydropsyche tenuis NAVAS (Insecta: Trichoptera).

Members of the speciose insect order Trichoptera (caddisflies) provide important ecosystem services, for example, nutrient cycling through breaking down of organic matter. They are also of industrial interest due to their larval silk secretions. These form the basis for their diverse case-making behavior that allows them to exploit a wide range of ecological niches. Only five genomes of this order have been published thus far, with variable qualities regarding contiguity and completeness. A low-cost sequencing strategy, that is, using a single Oxford Nanopore flow cell per individual along with Illumina sequence reads was successfully used to generate high-quality genomes of two Trichoptera species, Plectrocnemia conspersa and Hydropsyche tenuis. Of the de novo assembly methods compared, assembly of low coverage Nanopore reads (∼18×) and subsequent polishing with long reads followed by Illumina short reads (∼80-170× coverage) yielded the highest genome quality both in terms of contiguity and BUSCO completeness. The presented genomes are the shortest to date and extend our knowledge of genome size across caddisfly families. The genomic region that encodes for light (L)-chain fibroin, a protein component of larval caddisfly silk was identified and compared with existing L-fibroin gene clusters. The new genomic resources presented in this paper are among the highest quality Trichoptera genomes and will increase the knowledge of this important insect order by serving as the basis for phylogenomic and comparative genomic studies.

Outcomes of Subacromial Balloon Spacer Implantation for Massive and Irreparable Rotator Cuff Tears: A Systematic Review.

BACKGROUND: Selection of optimal treatment for massive to irreparable rotator cuff tears (RCTs) entails a challenging decision-making process in which surgeons must consider several factors, including duration of symptoms, tear pattern, tear size, and muscle quality, as well as patient characteristics such as age, comorbidities, shoulder dominance, and activity level. Unfortunately, no clear consensus has been reached regarding optimal management. PURPOSE: To systematically review the published literature assessing outcomes after subacromial balloon spacer implantation for treatment of massive and irreparable RCTs. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: A comprehensive literature search was performed in September 2018 through use of MEDLINE and the Cochrane Library electronic databases. Studies were assessed for multiple outcomes of interest including Constant score, Oxford Shoulder Score (OSS), University of California Los Angeles (UCLA) Shoulder Score, complications, and patient satisfaction. RESULTS: After applying the selection criteria, 12 clinical studies were included for data extraction and analysis. In total, 291 shoulders (in 284 patients) treated with subacromial balloon spacer implantation were pooled for evaluation, with a mean follow-up of 22.9 ± 14.9 months (range, 6-60 months). Constant scores were used as an outcome metric for 267 shoulders (91.7%; 11 studies), with improvements in mean Constant score ranging from 18.5 to 49.6 points. Patient satisfaction was assessed in 105 patients (37.0%; 5 studies), with rates of patients indicating they were satisfied or very satisfied with their treatment outcome ranging from 45.8% to 100%. A total of 6 patients (2.1%) experienced complications related to balloon spacer implantation, including transient neurapraxia of the lateral antebrachial cutaneous nerve, superficial wound infection, deep wound infection, and balloon migration. Of these, 3 patients (2 balloon migration, 1 deep wound infection) required subsequent surgeries for balloon removal. CONCLUSION: Placement of the subacromial balloon spacer is a minimally invasive, technically simple procedure with favorable patient-reported outcomes at limited short-term follow-up. However, inherent methodological limitations and patient heterogeneity between studies may impair our ability to fully characterize the longer term efficacy, particularly relative to other potential surgical options. Further prospective randomized or comparative studies are warranted to ascertain clinical outcomes of subacromial balloon spacer in the management of massive and irreparable RCTs.

Exploring the underwater silken architectures of caddisworms: comparative silkomics across two caddisfly suborders.

Caddisfly (Trichoptera) larvae assemble a variety of underwater structures using bioadhesive silk. The order is divided into two primary sub-orders distinguished by how the larvae deploy their silk. Foraging Integripalpia larvae construct portable tube cases. Annulipalpia larvae construct stationary retreats, some with suspended nets to capture food. To identify silk molecular adaptations that may have contributed to caddisfly diversification, we report initial characterization of silk from a net-spinner genus, Parapsyche, for comparison with the silk of a tube case-maker genus, Hesperophylax. Overall, general features of silk structure and processing are conserved across the sub-orders despite approximately 200 Ma of divergence: the H-fibroin proteins comprise repeating phosphoserine-rich motifs, naturally spun silk fibres contain approximately 1 : 1 molar ratios of divalent metal ions to phosphate, silk fibre precursors are stored as complex fluids of at least two types of complexes, and silk gland proteins contain only traces of divalent metal ions, suggesting metal ions that solidify the fibres are absorbed from the aqueous environment after silk extrusion. However, the number and arrangement of the repeating phosphoserine blocks differ between genera, suggesting molecular adaptation of H-fibroin through duplication and shuffling of conserved structural modules may correspond with the radiation of caddisflies into diverse environments. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Aqueous Liquid-Liquid Phase Separation of Natural and Synthetic Polyguanidiniums.

Protamines are natural polyguanidiniums, arginine(R)-rich proteins involved in the compaction of chromatin during vertebrate spermatogenesis. Salmine, a protamine isolated from salmon sperm, contains 65 mol% R residues, with positively charged guanidino (Gdm⁺) sidechains, and no other amino acids with ionizable or aromatic sidechains. Salmine sulfate solutions undergo liquid-liquid phase separation (LLPS) with a concentration-dependent upper critical solution temperature (UCST). The condensed liquid phase comprises 50 wt % water and >600 mg·ml-¹ salmine with a constant 1:2 ratio of sulfate (SO4²-) to Gdm⁺. Isothermal titration calorimetry, titrating Na2SO4 into salmine chloride above and below the UCST, allowed isolation of exothermic sulfate binding to salmine chloride from subsequent endothermic condensation and exothermic phase separation events. Synthetic random polyacrylate analogs of salmine, with 3-guanidinopropyl sidechains, displayed similar counterion dependent phase behavior, demonstrating that the LLPS of polyguanidiniums does not depend upon subunit sequence or polymer backbone chirality, and was due entirely to Gdm⁺ sidechain interactions. The results provide experimental evidence for like-charge pairing of Gdm⁺ sidechains, and an experimental approach for further characterizing these interactions.

Aquatic caddisworm silk is solidified by environmental metal ions during the natural fiber-spinning process.

Aquatic caddisfly larvae (caddisworms) wet-spin fibers to construct composite cases of silk and stone. The silk emerges from labial ducts as a nanofibrous fluid gel, flowing over the stone substrate and making intimate interfacial adhesive contacts before being drawn into tough fibers that rapidly solidify underwater to span gaps in the construction. Divalent metal ions are responsible for the unique mechanical properties of naturally spun silk fibers; however, when and where divalent metal ions are incorporated into the metallofibers and other aspects of the fiber solidification mechanism are poorly understood. To investigate, the elemental composition and secondary structure of silk precursors stored in the silk gland lumen were compared with naturally spun fibers by inductively coupled plasma optical emission spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy. Naturally spun fibers contained near equimolar ratios of Ca2+ to P. In contrast, silk precursors stored in the silk gland lumen contained only traces of Ca2+ and other multivalent metal ions. Ca2+ was also undetectable in anterior lumenal silk using the histochemical Ca2+ indicator, alizarin S red. Addition of Ca2+ to isolated lumenal silk resulted in Ca2+ complexation by H-fibroin phosphoserines (pSs) and a shift in secondary structure from random coils to ß-structures, creating infrared spectra in the phosphate and amide I regions nearly equivalent to those found in naturally spun Ca2+-containing silk fibers. Light and electron microscopy within distinct regions of the silk gland suggested that posterior gland silk colloidal complexes transition into a nanofibrous morphology as they pass into the chitin-lined anterior lumen. Altogether, the results suggest that environmental Ca2+ absorbed from natural water triggers silk fiber solidification postdraw by complexing H-fibroin pSs, creating Ca2+-stabilized crystalline ß-nanodomains that cross-link and toughen the freshly drawn silk fibers.-Ashton, N. N., Stewart, R. J. Aquatic caddisworm silk is solidified by environmental metal ions during the natural fiber-spinning process.

The genome of an underwater architect, the caddisfly Stenopsyche tienmushanensis Hwang (Insecta: Trichoptera).

Background: Caddisflies (Insecta: Trichoptera) are a highly adapted freshwater group of insects split from a common ancestor with Lepidoptera. They are the most diverse (>16,000 species) of the strictly aquatic insect orders and are widely employed as bio-indicators in water quality assessment and monitoring. Among the numerous adaptations to aquatic habitats, caddisfly larvae use silk and materials from the environment (e.g., stones, sticks, leaf matter) to build composite structures such as fixed retreats and portable cases. Understanding how caddisflies have adapted to aquatic habitats will help explain the evolution and subsequent diversification of the group. Findings: We sequenced a retreat-builder caddisfly Stenopsyche tienmushanensis Hwang and assembled a high-quality genome from both Illumina and Pacific Biosciences (PacBio) sequencing. In total, 601.2 M Illumina reads (90.2 Gb) and 16.9 M PacBio subreads (89.0 Gb) were generated. The 451.5 Mb assembled genome has a contig N50 of 1.29 M, has a longest contig of 4.76 Mb, and covers 97.65% of the 1,658 insect single-copy genes as assessed by Benchmarking Universal Single-Copy Orthologs. The genome comprises 36.76% repetitive elements. A total of 14,672 predicted protein-coding genes were identified. The genome revealed gene expansions in specific groups of the cytochrome P450 family and olfactory binding proteins, suggesting potential genomic features associated with pollutant tolerance and mate finding. In addition, the complete gene complex of the highly repetitive H-fibroin, the major protein component of caddisfly larval silk, was assembled. Conclusions: We report the draft genome of Stenopsyche tienmushanensis, the highest-quality caddisfly genome so far. The genome information will be an important resource for the study of caddisflies and may shed light on the evolution of aquatic insects.

Deep-learning-assisted diagnosis for knee magnetic resonance imaging: Development and retrospective validation of MRNet.

BACKGROUND: Magnetic resonance imaging (MRI) of the knee is the preferred method for diagnosing knee injuries. However, interpretation of knee MRI is time-intensive and subject to diagnostic error and variability. An automated system for interpreting knee MRI could prioritize high-risk patients and assist clinicians in making diagnoses. Deep learning methods, in being able to automatically learn layers of features, are well suited for modeling the complex relationships between medical images and their interpretations. In this study we developed a deep learning model for detecting general abnormalities and specific diagnoses (anterior cruciate ligament [ACL] tears and meniscal tears) on knee MRI exams. We then measured the effect of providing the model's predictions to clinical experts during interpretation. METHODS AND FINDINGS: Our dataset consisted of 1,370 knee MRI exams performed at Stanford University Medical Center between January 1, 2001, and December 31, 2012 (mean age 38.0 years; 569 [41.5%] female patients). The majority vote of 3 musculoskeletal radiologists established reference standard labels on an internal validation set of 120 exams. We developed MRNet, a convolutional neural network for classifying MRI series and combined predictions from 3 series per exam using logistic regression. In detecting abnormalities, ACL tears, and meniscal tears, this model achieved area under the receiver operating characteristic curve (AUC) values of 0.937 (95% CI 0.895, 0.980), 0.965 (95% CI 0.938, 0.993), and 0.847 (95% CI 0.780, 0.914), respectively, on the internal validation set. We also obtained a public dataset of 917 exams with sagittal T1-weighted series and labels for ACL injury from Clinical Hospital Centre Rijeka, Croatia. On the external validation set of 183 exams, the MRNet trained on Stanford sagittal T2-weighted series achieved an AUC of 0.824 (95% CI 0.757, 0.892) in the detection of ACL injuries with no additional training, while an MRNet trained on the rest of the external data achieved an AUC of 0.911 (95% CI 0.864, 0.958). We additionally measured the specificity, sensitivity, and accuracy of 9 clinical experts (7 board-certified general radiologists and 2 orthopedic surgeons) on the internal validation set both with and without model assistance. Using a 2-sided Pearson's chi-squared test with adjustment for multiple comparisons, we found no significant differences between the performance of the model and that of unassisted general radiologists in detecting abnormalities. General radiologists achieved significantly higher sensitivity in detecting ACL tears (p-value = 0.002; q-value = 0.019) and significantly higher specificity in detecting meniscal tears (p-value = 0.003; q-value = 0.019). Using a 1-tailed t test on the change in performance metrics, we found that providing model predictions significantly increased clinical experts' specificity in identifying ACL tears (p-value < 0.001; q-value = 0.006). The primary limitations of our study include lack of surgical ground truth and the small size of the panel of clinical experts. CONCLUSIONS: Our deep learning model can rapidly generate accurate clinical pathology classifications of knee MRI exams from both internal and external datasets. Moreover, our results support the assertion that deep learning models can improve the performance of clinical experts during medical imaging interpretation. Further research is needed to validate the model prospectively and to determine its utility in the clinical setting.