A minimum functional form of the Escherichia coli BAM complex constituted by BamADE assembles outer membrane proteins in vitro.

The biogenesis of outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in Escherichia coli. Despite great progress in the BAM structural analysis, the molecular details of BAM-mediated processes as well as the exact function of each BAM component during OMP assembly are still not fully understood. To enable a distinguishment of the function of each BAM component, it is the aim of the present work to examine and identify the effective minimum form of the E. coli BAM complex by use of a well-defined reconstitution strategy based on a previously developed versatile assay. Our data demonstrate that BamADE is the core BAM component and constitutes a minimum functional form for OMP assembly in E. coli, which can be stimulated by BamB and BamC. While BamB and BamC have a redundant function based on the minimum form, both together seem to cooperate with each other to substitute for the function of the missing BamD or BamE. Moreover, the BamAE470K mutant also requires the function of BamD and BamE to assemble OMPs in vitro, which vice verse suggests that BamADE are the effective minimum functional form of the E. coli BAM complex.

Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Chemoproteomic mapping of the glycolytic targetome in cancer cells.

Hyperactivated glycolysis is a metabolic hallmark of most cancer cells. Although sporadic information has revealed that glycolytic metabolites possess nonmetabolic functions as signaling molecules, how these metabolites interact with and functionally regulate their binding targets remains largely elusive. Here, we introduce a target-responsive accessibility profiling (TRAP) approach that measures changes in ligand binding-induced accessibility for target identification by globally labeling reactive proteinaceous lysines. With TRAP, we mapped 913 responsive target candidates and 2,487 interactions for 10 major glycolytic metabolites in a model cancer cell line. The wide targetome depicted by TRAP unveils diverse regulatory modalities of glycolytic metabolites, and these modalities involve direct perturbation of enzymes in carbohydrate metabolism, intervention of an orphan transcriptional protein's activity and modulation of targetome-level acetylation. These results further our knowledge of how glycolysis orchestrates signaling pathways in cancer cells to support their survival, and inspire exploitation of the glycolytic targetome for cancer therapy.

Chondroitin/dermatan sulfate glycosyltransferase genes are essential for craniofacial development.

Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.

Signal peptide replacement of Ag43 enables an efficient bacterial cell surface display of receptor-binding domain of coronavirus.

To enable an efficient bacterial cell surface display with effective protein expression and cell surface loading ability via autotransporter for potential vaccine development applications, the inner membrane protein translocation efficiency was investigated via a trial-and-error strategy by replacing the original unusual long signal peptide of E. coli Ag43 with 11 different signal peptides. The receptor-binding domain (RBD) of coronavirus was used as a neutral display substrate to optimize the expression conditions, and the results showed that signal peptides from PelB, OmpC, OmpF, and PhoA protein enhance the bacterial cell surface display efficiency of RBD. In addition, the temperature has also a significant effect on the autodisplay efficiency of RBD. Our data provide further technical basis for the biotechnological application of Ag43 as a bacterial surface display carrier system and further potential application in vaccine development.

An association between elevated telomerase reverse transcriptase expression and the immune tolerance disruption of dendritic cells.

BACKGROUND: To elucidate the mechanism of dysfunction of tolerogenic dendritic cells (DCs) is of significance. Telomerase involves the regulation of the cell fate and activities. The objective of this study is to investigate the role of telomerase reverse transcriptase (TERT) in regulating the tolerogenic feature of DCs. METHODS: The telomerase was assessed in DCs, which were collected from patients with allergic rhinitis (AR), healthy control (HC) subjects, and mice. RNAs were extracted from DCs, and analyzed by RNA sequencing (RNAseq), real-time quantitative RT-PCR, and Western blotting. RESULTS: The results showed that expression of TERT was higher in peripheral DCs of AR patients. The expression of IL10 in DCs was negatively correlated with the levels of TERT expression. Importantly, the levels of TERT mRNA in DCs were associated with the AR response in patients with AR. Endoplasmic reticulum (ER) stress promoted the expression of Tert in DCs. Sensitization with the ovalbumin-aluminum hydroxide protocol increased the expression of Tert in DCs by exacerbating ER stress. TERT interacting with c-Maf (the transcription factor of IL-10) inducing protein (CMIP) in DCs resulted in CMIP ubiquitination and degradation, and thus, suppressed the production of IL-10. Inhibition of Tert in DCs mitigated experimental AR. CONCLUSIONS: Elevated amounts of TERT were detected in DCs of patients with AR. The tolerogenic feature of DCs was impacted by TERT. Inhibited TERT attenuated experimental AR.

Effects of edaravone dexborneol on functional outcome and inflammatory response in patients with acute ischemic stroke.

BACKGROUND: Edaravone dexborneol has been reported as an effective neuroprotective agent in the treatment of acute ischemic stroke (AIS). This study aimed at investigating the impact of edaravone dexborneol on functional outcomes and systematic inflammatory response in AIS patient. METHODS: All participants were recruited from the AISRNA study (registered 21/11/2019, NCT04175691 [ClinicalTrials.gov]) between January 2022 and December 2022. The AIS patients were divided into two groups based on whether they received the treatment of edaravone dexborneol (37.5 mg/12 hours, IV) within 48 h after stroke onset. Inflammatory response was determined by detecting levels of cytokines (interleukin-2 [IL-2], IL-4, IL-5, IL-8, IL-6, IL-10, IL-12p70, IL-17, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], IFN-α, and IL-1ß) within 14 days after stroke onset. RESULTS: Eighty-five AIS patients were included from the AISRNA study. Patients treated with edaravone dexborneol showed a significantly higher proportion of modified Rankin Scale score < 2 compared to those who did not receive this treatment (70.7% versus 47.8%; P = 0.031). Furthermore, individuals receiving edaravone dexborneol injection exhibited lower expression levels of interleukin (IL)-1ß, IL-6, and IL-17, along with higher levels of IL-4 and IL-10 expression during the acute phase of ischemic stroke (P < 0.05). These trends were not observed for IL-2, IL-5, IL-8, IL-12p70, tumor necrosis factor-α, interferon-γ [IFN-γ], and IFN-α (P > 0.05). CONCLUSIONS: Treatment with edaravone dexborneol resulted in a favorable functional outcome at 90 days post-stroke onset when compared to patients without this intervention; it also suppressed proinflammatory factors expression while increasing anti-inflammatory factors levels. TRIAL REGISTRATION: ClinicalTrials.gov NCT04175691. Registered November 21, 2019, https://www. CLINICALTRIALS: gov/ct2/show/NCT04175691 .

Dynamic network reorganization underlying neuroplasticity: the deficits-severity-related language network dynamics in patients with left hemispheric gliomas involving language network.

Brain network dynamics not only endow the brain with flexible coordination for various cognitive processes but also with a huge potential of neuroplasticity for development, skill learning, and after cerebral injury. Diffusive and progressive glioma infiltration triggers the neuroplasticity for functional compensation, which is an outstanding pathophysiological model for the investigation of network reorganization underlying neuroplasticity. In this study, we employed dynamic conditional correlation to construct framewise language networks and investigated dynamic reorganizations in 83 patients with left hemispheric gliomas involving language networks (40 patients without aphasia and 43 patients with aphasia). We found that, in healthy controls (HCs) and patients, the language network dynamics in resting state clustered into 4 temporal-reoccurring states. Language deficits-severity-dependent topological abnormalities of dFCs were observed. Compared with HCs, suboptimal language network dynamics were observed for those patients without aphasia, while more severe network disruptions were observed for those patients with aphasia. Machine learning-based dFC-linguistics prediction analyses showed that dFCs of the 4 states significantly predicted individual patients' language scores. These findings shed light on our understanding of metaplasticity in glioma. Glioma-induced language network reorganizations were investigated under a dynamic "meta-networking" (network of networks) framework. In healthy controls and patients with glioma, the framewise language network dynamics in resting-state robustly clustered into 4 temporal-reoccurring states. The spatial but not temporal language deficits-severity-dependent abnormalities of dFCs were observed in patients with left hemispheric gliomas involving language network. Language network dynamics significantly predicted individual patients' language scores.

The domain-separation language network dynamics in resting state support its flexible functional segregation and integration during language and speech processing.

Modern linguistic theories and network science propose that language and speech processing are organized into hierarchical, segregated large-scale subnetworks, with a core of dorsal (phonological) stream and ventral (semantic) stream. The two streams are asymmetrically recruited in receptive and expressive language or speech tasks, which showed flexible functional segregation and integration. We hypothesized that the functional segregation of the two streams was supported by the underlying network segregation. A dynamic conditional correlation approach was employed to construct framewise time-varying language networks and k-means clustering was employed to investigate the temporal-reoccurring patterns. We found that the framewise language network dynamics in resting state were robustly clustered into four states, which dynamically reconfigured following a domain-separation manner. Spatially, the hub distributions of the first three states highly resembled the neurobiology of speech perception and lexical-phonological processing, speech production, and semantic processing, respectively. The fourth state was characterized by the weakest functional connectivity and was regarded as a baseline state. Temporally, the first three states appeared exclusively in limited time bins (∼15%), and most of the time (> 55%), state 4 was dominant. Machine learning-based dFC-linguistics prediction analyses showed that dFCs of the four states significantly predicted individual linguistic performance. These findings suggest a domain-separation manner of language network dynamics in resting state, which forms a dynamic "meta-network" framework to support flexible functional segregation and integration during language and speech processing.

Continuous suture technique increases the complete closure rate of colorectal mucosal defects after endoscopic resection: a single-blind, randomized controlled trial.

BACKGROUND: Complete closure of mucosal defects after colorectal endoscopic submucosal dissection (ESD)/piecemeal endoscopic mucosal resection (p-EMR) procedures reduces postoperative adverse events, but the complete closure rate of the traditional method using only hemostatic clips is not satisfactory. Therefore, we invented a continuous suture technique using a barbed suture and clips to increase the complete closure rate of colorectal mucosal defects. METHODS: Patients with a single large (≥ 2 cm) colorectal lesion were recruited. After completion of the ESD/p-EMR procedures, they were randomly allocated to the treatment group or control group. The mucosal defects of the treatment group were closed using barbed suture and clips, while the control group was closed using only clips. RESULTS: From January 18, 2022 to April 13, 2022, a total of 62 patients with colorectal lesions were enrolled, with 31 patients in each group. Complete closure was achieved in 29 patients (93.5%) in the treatment group and 18 patients (58.1%) in the control group (P = 0.001). The median closure time was 13 min in the treatment group and 19 min in the control group (P < 0.001). The median closure speed was 6.4 cm2/10 min in the treatment group and 3.5 cm2/10 min in the control group (P = 0.008). CONCLUSIONS: This study provided a clinically feasible continuous suture technique that was safe and effective for the complete closure of colorectal mucosal defects after endoscopic resection.

GLP-1R Signaling and Functional Molecules in Incretin Therapy.

Glucagon-like peptide-1 receptor (GLP-1R) is a critical therapeutic target for type 2 diabetes mellitus (T2DM). The GLP-1R cellular signaling mechanism relevant to insulin secretion and blood glucose regulation has been extensively studied. Numerous drugs targeting GLP-1R have entered clinical treatment. However, novel functional molecules with reduced side effects and enhanced therapeutic efficacy are still in high demand. In this review, we summarize the basis of GLP-1R cellular signaling, and how it is involved in the treatment of T2DM. We review the functional molecules of incretin therapy in various stages of clinical trials. We also outline the current strategies and emerging techniques that are furthering the development of novel therapeutic drugs for T2DM and other metabolic diseases.

The role of Gdf5 in the development of the zebrafish fin endoskeleton.

BACKGROUND: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. RESULTS: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. CONCLUSIONS: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

Living Cell-Target Responsive Accessibility Profiling Reveals Silibinin Targeting ACSL4 for Combating Ferroptosis.

We report a living cell-target responsive accessibility profiling (LC-TRAP) approach to identify the targetome of silibinin (SIL), a well-established hepatoprotective natural product (NP), in HepG2 cells. Proteins showing accessibility changes, probed by covalent lysine labeling reagents and leveraged by multiplexed quantitative proteomics, following the administration of SIL to the living cells were assigned as potential targets. Among the assigned targetome, ACSL4, an enzyme essential for ferroptosis induction, might be involved in the hepatoprotective effects of SIL and hence was intensively validated. We first demonstrated that SIL protected HepG2 cells from ferroptosis dependent on ACSL4. Then, we used biophysical assays and a SIL-derivatized chemical probe to corroborate that SIL can bind to ACSL4. The ensuing enzymatic assays showed that SIL inhibited ACSL4 enzymatic activity, thereby mitigating the ACSL4-mediated ferroptosis. As such, we revealed that ACSL4 inhibition, using SIL as a model compound, represents a promising hepatoprotective strategy. Further, since TRAP probes the accessibility changes of reactive proteinaceous lysines, it can pinpoint the proximal regions where the ligand engagement may occur. Thus, the LC-TRAP analysis of SIL, the newly discovered ligand of ACSL4, and arachidonic acid (AA), the substrate, intriguingly showed that SIL and AA both affected the conformation of the K536-proximal region of ACSL4, albeit through distinct binding patterns. Collectively, we describe a straightforward LC-TRAP workflow that does not involve ligand-derived probe synthesis and is widely applicable to target discovery of NPs.

Drynaria fortunei improves lipid profiles of elderly patients with postmenopausal osteoporosis via regulation of Notch1-NLRP3 inflammasome-mediated inflammation.

BACKGROUND: Dyslipidemia is a common comorbidity in elderly patients with postmenopausal osteoporosis (PMOP). Drynaria fortunei (Rhizoma drynariae) is well-known in traditional Chinese medicine for its ability to improve bone mineral density (BMD). However, whether and how Drynaria fortunei improves plasma lipid profiles in elderly PMOP patients remains unclear. METHODS: Eighty elderly female patients with concurrent PMOP and hyperlipemia were randomly assigned to Drynaria fortunei 2(n = 40) or control (n = 40) groups. The clinical efficacies of Drynaria fortunei were evaluated. At 0, 3-, 6-, 9-, and 12-month of follow-up, plasma levels of IL-1ß, IL-18, TNF-α, IL-6, IL-8, and IL-10 were measured using ELISA, whereas PBMC levels of NLRP3, ASC, caspase-1, NF-κB, SIRT1, and Notch1 were measured using RT-qPCR. PBMC isolated from PMOP patients were cultured and treated with Drynaria fortunei to determine its influence on NLRP3 inflammasome and associated cytokines. RESULTS: Drynaria fortunei effectively improved patients' BMD and lipid profiles. IL-1ß, IL-18, TNF-α, IL-6, IL-8 levels, as well as inflammasome-molecules of NLRP3, ASC, caspase-1, and NF-κB increased over time in the control group, but were significantly attenuated with Drynaria fortunei administration. In vitro, Drynaria fortunei suppressed NLRP3 inflammasome and associated cytokines by increasing SIRT1 or decreasing Notch1. Drynaria fortunei had inhibitory effects on NLRP3 inflammasome and Notch1 even when SIRT1 expression was suppressed. CONCLUSIONS: Drynaria fortunei has been demonstrated to significantly improve lipid profiles for elderly PMOP patients. Drynaria fortunei may down-regulate Notch1 independently of SIRT1 to suppress NLRP3 inflammasome-mediated inflammation, thus improving plasma lipid profile.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

p53 mediates hydroxyurea resistance in aneuploid cells of colon cancer.

Aneuploidy refers to aberrancies in cellular chromosome count, which is prevalent in most human cancers. Chemotherapy is an effective cancer treatment; however, the development of drug resistance is a major concern of conventional chemotherapy. The chemotherapy agent hydroxyurea (HU) targets proliferating cells and has long been applied to treat various human cancers. It remains elusive whether aneuploidy affects the drug sensitivity of hydroxyurea. By generating an inducible aneuploidy model, we found that aneuploid colon cancer cells were resistant to HU treatment compared to euploid controls. Surprisingly, further analyses showed that the HU resistance was dependent on the expression of wild type p53. Activation of the p53 pathway in aneuploidy cells reduced cell proliferation but generated resistance of tumor cells to HU treatment. HU resistance was abrogated in aneuploid cells if p53 was absent but re-gained when inducing proliferation repression in cells by serum deprivation. Our results demonstrate that the HU resistance developed in aneuploid colon cancer cells is mediated by wild type p53 and indicates the prognostic value of combining karyotypic and p53 status in clinical cancer treatment.

Systemic immune-inflammation index acts as a novel diagnostic biomarker for postmenopausal osteoporosis and could predict the risk of osteoporotic fracture.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a bone metabolism disorder involving systematic inflammation activation. Blood routine examination is easily available in clinical practice and contains abundant information reflecting the systematic inflammation level. Thus, it is attractive to achieve early diagnosis of PMOP and predict osteoporotic fracture risk just based on the biomarkers in blood routine examination. METHODS: A multi-centric prospective cohort study was designed and enrolled postmenopausal women from two independent institutions. All participants underwent the dual-energy X-ray absorptiometry (DEXA) scanning for diagnosing PMOP. Blood routine examination was conducted, and the key inflammatory biomarkers such as neutrophil-to-lymphocyte ratio (NLR) and systemic immune-inflammation index (SII) were calculated. PMOP patients were followed up to observe osteoporotic fracture and identify the related risk predictors. RESULTS: A total of 92 participants out of 238 enrolled postmenopausal women were diagnosed with PMOP, with a prevalence of 38.66%. The main risk factors identified for PMOP included older age (OR = 2.06, 95% CI = 1.14-3.72), longer menopause duration (OR = 3.14, 95% CI = 2.06-4.79), higher NLR (OR = 2.11, 95% CI = 1.37-3.25), and higher SII (OR = 3.02, 95% CI = 1.98-4.61). Besides age and menopause duration, SII ≥834.89 was newly identified as a prominent risk factor for discriminating osteoporotic fracture risk in PMOP patients (HR = 3.66, 95% CI = 1.249-10.71). CONCLUSION: As an easy and economical biomarker calculated from blood routine examination, SII not only acts as a good risk predictor for PMOP diagnosis but also well discriminates the osteoporotic fracture risk, which deserves further investigation and application in clinical practice.

Integrated automated particle tracking microfluidic enables high-throughput cell deformability cytometry for red cell disorders.

Investigating individual red blood cells (RBCs) is critical to understanding hematologic diseases, as pathology often originates at the single-cell level. Many RBC disorders manifest in altered biophysical properties, such as deformability of RBCs. Due to limitations in current biophysical assays, there exists a need for high-throughput analysis of RBC deformability with single-cell resolution. To that end, we present a method that pairs a simple in vitro artificial microvasculature network system with an innovative MATLAB-based automated particle tracking program, allowing for high-throughput, single-cell deformability index (sDI) measurements of entire RBC populations. We apply our technology to quantify the sDI of RBCs from healthy volunteers, Sickle cell disease (SCD) patients, a transfusion-dependent beta thalassemia major patient, and in stored packed RBCs (pRBCs) that undergo storage lesion over 4 weeks. Moreover, our system can also measure cell size for each RBC, thereby enabling 2D analysis of cell deformability vs cell size with single cell resolution akin to flow cytometry. Our results demonstrate the clear existence of distinct biophysical RBC subpopulations with high interpatient variability in SCD as indicated by large magnitude skewness and kurtosis values of distribution, the "shifting" of sDI vs RBC size curves over transfusion cycles in beta thalassemia, and the appearance of low sDI RBC subpopulations within 4 days of pRBC storage. Overall, our system offers an inexpensive, convenient, and high-throughput method to gauge single RBC deformability and size for any RBC population and has the potential to aid in disease monitoring and transfusion guidelines for various RBC disorders.

Upregulation of lncRNA ADAMTS9-AS2 Promotes Salivary Adenoid Cystic Carcinoma Metastasis via PI3K/Akt and MEK/Erk Signaling.

Neurotropic infiltrative growth and distant metastasis are the main causes of death in salivary adenoid cystic carcinoma (SACC) patients. Long noncoding RNAs (lncRNAs) are involved in many human neoplasms, however, their potential roles in SACC are unclear. In our study, we found that ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9) antisense RNA 2 (ADAMTS9-AS2) was significantly upregulated in SACC patients with metastasis and SACC-lung metastasis (LM) cells. Moreover, ADAMTS9-AS2 expression was closely associated with the prognosis and distant metastasis in SACC patients. Next, we found that c-myc could specifically bind to the promoter of ADAMTS9-AS2 and activated its transcription. Knockdown of ADAMTS9-AS2 significantly inhibited migration and invasion of SACC cells in vitro and distant lung metastasis in vivo. Furthermore, ADAMTS9-AS2, which mainly expressed in the cytoplasm, shared microRNA (miRNA) response elements with Integrin α6 (ITGA6). Overexpression of ADAMTS9-AS2 competitively bound to miR-143-3p that inhibited ITGA6 from miRNA-mediated degradation, and thus it activated the activity of PI3K/Akt and MEK/Erk signaling and facilitated SACC metastasis. In summary, ADAMTS9-AS2 promotes migration and invasion in SACC by competing with miR-143-3p. This sheds a new insight into the regulation mechanism of ADAMTS9-AS2, and it provides a possible application for the SACC treatment.

DSeg: A Dynamic Image Segmentation Program to Extract Backbone Patterns for Filamentous Bacteria and Hyphae Structures.

Analysis of numerous filamentous structures in an image is often limited by the ability of algorithms to accurately segment complex structures or structures within a dense population. It is even more problematic if these structures continuously grow when recording a time-series of images. To overcome these issues we present DSeg; an image analysis program designed to process time-series image data, as well as single images, to segment filamentous structures. The program includes a robust binary level-set algorithm modified to use size constraints, edge intensity, and past information. We verify our algorithms using synthetic data, differential interference contrast images of filamentous prokaryotes, and transmission electron microscopy images of bacterial adhesion fimbriae. DSeg includes automatic segmentation, tools for analysis, and drift correction, and outputs statistical data such as persistence length, growth rate, and growth direction. The program is available at Sourceforge.