Degradation profiles of the poly(ε-caprolactone)/silk fibroin electrospinning membranes and their potential applications in tissue engineering.

Degradation profiles are critical for the optimal application of electrospun polymer nanofibers in tissue regeneration, wound healing, and drug delivery systems. In this study, natural and synthetic polymers and their composites were subjected to in vivo transplantation and in vitro treatment with lipases, macrophages, and acetic acid to evaluate their degradation patterns. The effects of environmental stimulation, surface wettability, and polymer components on the degradation profiles of the electrospinning poly(ε-caprolactone)/silk fibroin (PCL/SF) nanofibers were first evaluated. In vivo degradation study demonstrated that bulk degradation, characterized by the transition from microfibers to nanofibers, and surface erosion, characterized by fusion between the microfibers or direct erosion from both ends of the microfibers, occurred in the electrospun membranes; however, bulk degradation dominated their overall degradation. Furthermore, the degradation rates of the electrospun PCL/SF membranes varied according to the composition, morphology, and surface wettability of the composite membranes. After the incorporation of silk fibroin (SF), the degradation rate of the SF/PCL composite membranes was faster, accompanied by larger values of weight loss and molecular weight (Mw) loss when compared with that of the pure poly(ε-caprolactone) (PCL) membrane, indicating a close relationship between degradation rate and hydrophilicity of the electrospinning membranes. The in vitro experimental results demonstrated that enzymes and oxidation partially resulted in the surface erosion of the PCL/SF microfibers. Consequently, bulk degradation and surface erosion coordinated with each other to enhance the hydrophilicity of the electrospinning membranes and accelerate the in vivo degradation.

Structural basis of peptide secretion for Quorum sensing by ComA.

Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA's peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg2+ triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.

Practices for running a research-oriented shared cryo-EM facility.

The Harvard Cryo-Electron Microscopy Center for Structural Biology, which was formed as a consortium between Harvard Medical School, Boston Children's Hospital, Dana-Farber Cancer Institute, and Massachusetts General Hospital, serves both academic and commercial users in the greater Harvard community. The facility strives to optimize research productivity while training users to become expert electron microscopists. These two tasks may be at odds and require careful balance to keep research projects moving forward while still allowing trainees to develop independence and expertise. This article presents the model developed at Harvard Medical School for running a research-oriented cryo-EM facility. Being a research-oriented facility begins with training in cryo-sample preparation on a trainee's own sample, ideally producing grids that can be screened and optimized on the Talos Arctica via multiple established pipelines. The first option, staff assisted screening, requires no user experience and a staff member provides instant feedback about the suitability of the sample for cryo-EM investigation and discusses potential strategies for sample optimization. Another option, rapid access, allows users short sessions to screen samples and introductory training for basic microscope operation. Once a sample reaches the stage where data collection is warranted, new users are trained on setting up data collection for themselves on either the Talos Arctica or Titan Krios microscope until independence is established. By providing incremental training and screening pipelines, the bottleneck of sample preparation can be overcome in parallel with developing skills as an electron microscopist. This approach allows for the development of expertise without hindering breakthroughs in key research areas.

Rotavirus VP4 Epitope of a Broadly Neutralizing Human Antibody Defined by Its Structure Bound with an Attenuated-Strain Virion.

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

Early detection of COPD based on graph convolutional network and small and weakly labeled data.

Chronic obstructive pulmonary disease (COPD) is a common disease with high morbidity and mortality, where early detection benefits the population. However, the early diagnosis rate of COPD is low due to the absence or slight early symptoms. In this paper, a novel method based on graph convolution network (GCN) for early detection of COPD is proposed, which uses small and weakly labeled chest computed tomography image data from the publicly available Danish Lung Cancer Screening Trial database. The key idea is to construct a graph using regions of interest randomly selected from the segmented lung parenchyma and then input it into the GCN model for COPD detection. In this way, the model can not only extract the feature information of each region of interest but also the topological structure information between regions of interest, that is, graph structure information. The proposed GCN model achieves an acceptable performance with an accuracy of 0.77 and an area under a curve of 0.81, which is higher than the previous studies on the same dataset. GCN model also outperforms several state-of-the-art methods trained at the same time. As far as we know, it is also the first time using the GCN model on this dataset for COPD detection.

Akt-GSK3ß-mPTP pathway regulates the mitochondrial dysfunction contributing to odontoblasts apoptosis induced by glucose oxidative stress.

Diabetes Mellitus can cause dental pulp cells apoptosis by oxidative stress, and affect the integrity and function of dental pulp tissue. Mitochondria are the main attack targets of oxidative stress and have a critical role in apoptosis. However, whether mitochondria are involved in dental pulp damage caused by diabetes mellitus remains unclear. This study aimed to investigate the role of mitochondria in the apoptosis of odontoblast-like cell line (mDPC6T) induced by glucose oxidative stress, and to explore its possible mechanism. We established an oxidative stress model in vitro using glucose oxidase/glucose to simulate the pathological state under diabetic conditions. We found that the opening of mitochondrial permeability transition pore (mPTP) contributed to the apoptosis of mDPC6T treated with glucose oxidase, as evidenced by enhanced mitochondrial reactive oxygen species (mtROS) and intracellular Ca2+ disorder, significantly reduced mitochondrial membrane potential (MMP) and ATP production. Antioxidant N-acetylcysteine (NAC) or Cyclosporine A (mPTP inhibitor) blocked the mPTP opening, which significantly attenuated mitochondrial dysfunction and apoptosis induced by glucose oxidative stress. In addition, we found that glucose oxidative stress stimulated mPTP opening may through inhibition of Akt-GSK3ß pathway. This study provides a new insight into the mitochondrial mechanism underlying diabetes-associated odontoblast-like cell apoptosis, laying a foundation for the prevention and treatment of diabetes-associated pulp injury.

A Novel CT-Based Radiomics Features Analysis for Identification and Severity Staging of COPD.

RATIONALE AND OBJECTIVES: To evaluate the role of radiomics based on Chest Computed Tomography (CT) in the identification and severity staging of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: This retrospective analysis included 322 participants (249 COPD patients and 73 control subjects). In total, 1395 chest CT-based radiomics features were extracted from each participant's CT images. Three feature selection methods, including variance threshold, Select K Best method, and least absolute shrinkage and selection operator (LASSO), and two classification methods, including support vector machine (SVM) and logistic regression (LR), were used as identification and severity classification of COPD. Performance was compared by AUC, accuracy, sensitivity, specificity, precision, and F1-score. RESULTS: 38 and 10 features were selected to construct radiomics models to detect and stage COPD, respectively. For COPD identification, SVM classifier achieved AUCs of 0.992 and 0.970, while LR classifier achieved AUCs of 0.993 and 0.972 in the training set and test set, respectively. For the severity staging of COPD, the mentioned two machine learning classifiers can better differentiate less severity (GOLD1 + GOLD2) group from greater severity (GOLD3 + GOLD4) group. The AUCs of SVM and LR is 0.907 and 0.903 in the training set, and that of 0.799 and 0.797 in the test set. CONCLUSION: The present study showed that the novel radiomics approach based on chest CT images that can be used for COPD identification and severity classification, and the constructed radiomics model demonstrated acceptable performance.

TRPC4 ion channel regulations by small-molecular inhibitors and calmodulin.

TRPC4 ion channel was reported to be regulated by small molecular inhibitors and calmodulin. We discuss these findings in the context of other members of TRPC subfamily modulated by different stimulants.

PFAT5 stability assessment of Lipovenoes MCT in total nutrient admixtures.

BACKGROUND: The purpose of this study was to evaluate the effects of monovalent and divalent cations on the stability of a fat emulsion (Lipovenoes MCT) in total nutrient admixtures (TNAs) by testing the percentage of fat residing in globules >5 µm (PFAT5) values. METHODS: TNAs with different combinations of glucose (5% and 10%), amino acids (3.35 and 4.5 g/100 mL), Na+/K+ (100/39 mmol/L), Mg2+ (3.4 and 2.7 mmol/L), and fat emulsion (2.4%) were tested in triplicate at room temperature. The pH, mean droplet size (MDS), and PFAT5 were assessed at 0, 6, 12, 24, 36, and 48 h. RESULTS: In all seven groups, the TNA globule distribution was uniform, the pH value fluctuated in the range of 5.93-6.06, and the MDS met the limit of the United States Pharmacopeia (USP) within 48 h. The PFAT5 value of the control group 0 without electrolytes was the lowest; group 1 added monovalent ions of 139 mmol/L was significantly higher (P<0.05) but without exceed the USP limit after 48 h. Groups 2 and 3 added Mg2+ 3.4 and 2.7 mmol/L respectively, based on group 1. Group 4 increased the amino acid concentration from 3.35% to 4.5% based on group 2, and group 5 reduced the glucose concentration from 10% to 5% based on group 4. Group 6 removed monovalent ions and retained only Mg2+ based on group 5. The PFAT5 values of group 2, 3, 4, and 5 exceeded the limit after 6 h and group 6 after 12 h. There was no statistical difference between group 2 and 4 (P>0.05) or between group 4 and 5 (P>0.05). CONCLUSIONS: When the concentration of glucose is 10-25% and the amino acid is 2.5-4.5%, The addition of monovalent ions affects the stability of fat emulsion in TNAs, however when the concentrations of Na+ ≤100 mmol/L and K+ ≤39 mmol/L, the PFAT5 value will not exceed the USP limit within 24 h. Mg2+ has a significant effect, the PFAT5 value will exceed the USP limit after 6 h when the concentration ≥2.7 mmol/L, which may cause potential safety hazards.

Structure of a nascent membrane protein as it folds on the BAM complex.

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a ß-barrel structure1,2. ß-Barrels are sheets of ß-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3-5. One subunit of the machines is itself a ß-barrel protein that has a central role in folding other ß-barrels. In Gram-negative bacteria, the ß-barrel assembly machine (BAM) consists of the ß-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid ß-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.

Analogous Diamondene Nanotube Structure Prediction Based on Molecular Dynamics and First-Principle Calculations.

A concentric twin tube (CTT) can be built by placing a carbon nanotube (CNT) in another identical CNT. Different from diamondene nanotubes, a stable CTT has no inter-shell covalent bond. As a prestressed double-walled nanotube, CTT has a lower structural stability at a finite temperature. According to the molecular dynamics and first-principle calculations, (a) CTTs have three types of relaxed configurations. In a type III CTT, the inner tube buckles to produce a V-shaped cross-section, and the outer tube may be convex or concave. (b) The minimal radii of relaxed zigzag and armchair CTTs with concave outer tubes were found. (c) After relaxation, the circumferences and areas of the two tubes in a type III CTT are different from those of the corresponding ideal CNT. The area change rate (A-CR) and circumference change rate (C-CR) of the outer tube are the first-order Gaussian function of the radius of the ideal CNT (which forms the CTT), and tends to be 73.3% of A-CR or 95.3% of C-CR, respectively. For the inner tube of a CTT, the A-CR is between 29.3% and 37.0%, and the C-CR is close to 95.8%. (d) The temperature slightly influences the findings given above.

Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function.

The transient receptor potential canonical subfamily member 5 (TRPC5), one of seven mammalian TRPC members, is a nonselective calcium-permeant cation channel. TRPC5 is of considerable interest as a drug target in the treatment of progressive kidney disease, depression, and anxiety. Here, we present the 2.8-Å resolution cryo-electron microscopy (cryo-EM) structure of the mouse TRPC5 (mTRPC5) homotetramer. Comparison of the TRPC5 structure to previously determined structures of other TRPC and TRP channels reveals differences in the extracellular pore domain and in the length of the S3 helix. The disulfide bond at the extracellular side of the pore and a preceding small loop are essential elements for its proper function. This high-resolution structure of mTRPC5, combined with electrophysiology and mutagenesis, provides insight into the lipid modulation and gating mechanisms of the TRPC family of ion channels.

In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase.

Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each 5-fold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual 5-fold positions. We have analyzed intact virions ("triple-layer particles"), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosylmethionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, double-strand RNA genome.

Comparison of IDW, cokriging and ARMA for predicting spatiotemporal variability of soil salinity in a gravel-sand mulched jujube orchard.

Information about the spatiotemporal variability of soil salinity is important for managing salinization in gravel-sand mulched fields. We used inverse distance weighting (IDW) and cokriging to model the spatial variability of soil salinity from 2013 to 2016 and used an autoregressive moving-average (ARMA) model time series to analyze the temporal variability. The objectives of this paper are (a) to compare IDW and cokriging for predicting salinity in deep soil layers from surface data, thus finding a more appropriate method to model the spatial variability of soil salinity, and, using ARMA time series, (b) to identify one or a few sampling points, where soil salt content is the most temporally stable, to increase sampling efficiency or decrease cost and to estimate the overall soil salt content of a field. The IDW interpolation was more accurate than cokriging when using surface salt content to estimate the content in deep layers; so, we used IDW to interpolate the data and draw spatial distribution maps of salt content. Salinity in the 0-10 cm layer gradually decreased with the amount of gravel-sand mulching, from 1.02 to 0.7 g/kg over four years, and increased with depth. ARMA was accurate when using sample dates to predict soil salinity in the time series, and the model was more stable. The stability of the salt spatial patterns over time and along the soil profile allowed us to identify a location representative of the field-mean salt content, with mean relative error ranging between 0.56 and 2.19%. The monitoring of soil salt from a few observations is thus a valuable tool for practitioners and will aid the management of soil salt in gravel-sand-mulched fields in arid regions, with a range of potential applications beyond the framework of monitoring salinity.

The structure of TRPC ion channels.

Briefly review the recent structural work of transient receptor potential canonical (TRPC) ion channels by using electron cryo-microscopy (cryo-EM). The high resolution structures of TRPC3, TRPC4, TRPC5 and TRPC6 are discussed.

Structural basis of RIP2 activation and signaling.

Signals arising from bacterial infections are detected by pathogen recognition receptors (PRRs) and are transduced by specialized adapter proteins in mammalian cells. The Receptor-interacting-serine/threonine-protein kinase 2 (RIPK2 or RIP2) is such an adapter protein that is critical for signal propagation of the Nucleotide-binding-oligomerization-domain-containing proteins 1/2 (NOD1 and NOD2). Dysregulation of this signaling pathway leads to defects in bacterial detection and in some cases autoimmune diseases. Here, we show that the Caspase-activation-and-recruitment-domain (CARD) of RIP2 (RIP2-CARD) forms oligomeric structures upon stimulation by either NOD1-CARD or NOD2-2CARD. We reconstitute this complex, termed the RIPosome in vitro and solve the cryo-EM filament structure of the active RIP2-CARD complex at 4.1 Å resolution. The structure suggests potential mechanisms by which CARD domains from NOD1 and NOD2 initiate the oligomerization process of RIP2-CARD. Together with structure guided mutagenesis experiments at the CARD-CARD interfaces, we demonstrate molecular mechanisms how RIP2 is activated and self-propagating such signal.

Structure of the mammalian TRPM7, a magnesium channel required during embryonic development.

The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg2+ and Zn2+ Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg2+ ions occupying the center of the conduction pore. In high [Mg2+], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.

Structure of the mouse TRPC4 ion channel.

Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels.

Cryo-EM structure of the polycystin 2-l1 ion channel.

We report the near atomic resolution (3.3 Å) of the human polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1.