Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart.

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

iRice-MS: An integrated XGBoost model for detecting multitype post-translational modification sites in rice.

Post-translational modification (PTM) refers to the covalent and enzymatic modification of proteins after protein biosynthesis, which orchestrates a variety of biological processes. Detecting PTM sites in proteome scale is one of the key steps to in-depth understanding their regulation mechanisms. In this study, we presented an integrated method based on eXtreme Gradient Boosting (XGBoost), called iRice-MS, to identify 2-hydroxyisobutyrylation, crotonylation, malonylation, ubiquitination, succinylation and acetylation in rice. For each PTM-specific model, we adopted eight feature encoding schemes, including sequence-based features, physicochemical property-based features and spatial mapping information-based features. The optimal feature set was identified from each encoding, and their respective models were established. Extensive experimental results show that iRice-MS always display excellent performance on 5-fold cross-validation and independent dataset test. In addition, our novel approach provides the superiority to other existing tools in terms of AUC value. Based on the proposed model, a web server named iRice-MS was established and is freely accessible at http://lin-group.cn/server/iRice-MS.

Hemispheric epilepsy surgery for hemimegalencephaly: The UCLA experience.

OBJECTIVES: Hemimegalencephaly (HME) is a rare congenital brain malformation presenting predominantly with drug-resistant epilepsy. Hemispheric disconnective surgery is the mainstay of treatment; however, little is known about how postoperative outcomes compare across techniques. Thus we present the largest single-center cohort of patients with HME who underwent epilepsy surgery and characterize outcomes. METHODS: This observational study included patients with HME at University of California Los Angeles (UCLA) from 1984 to 2021. Patients were stratified by surgical intervention: anatomic hemispherectomy (AH), functional hemispherectomy (FH), or less-than-hemispheric resection (LTH). Seizure freedom, functional outcomes, and operative complications were compared across surgical approaches. Regression analysis identified clinical and intraoperative variables that predict seizure outcomes. RESULTS: Of 56 patients, 43 (77%) underwent FH, 8 (14%) underwent AH, 2 (4%) underwent LTH, 1 (2%) underwent unknown hemispherectomy type, and 2 (4%) were managed non-operatively. At median last follow-up of 55 months (interquartile range [IQR] 20-92 months), 24 patients (49%) were seizure-free, 17 (30%) required cerebrospinal fluid (CSF) shunting for hydrocephalus, 9 of 43 (21%) had severe developmental delay, 8 of 38 (21%) were non-verbal, and 15 of 38 (39%) were non-ambulatory. There was one (2%) intraoperative mortality due to exsanguination earlier in this cohort. Of 12 patients (29%) requiring revision surgery, 6 (50%) were seizure-free postoperatively. AH, compared to FH, was not associated with statistically significant improved seizure freedom (hazard ratio [HR] = .48, p = .328), although initial AH trended toward greater odds of seizure freedom (75% vs 46%, p = .272). Younger age at seizure onset (HR = .29, p = .029), lack of epilepsia partialis continua (EPC) (HR = .30, p = .022), and no contralateral seizures on electroencephalography (EEG) (HR = .33, p = .039) independently predicted longer duration of seizure freedom. SIGNIFICANCE: This study helps inform physicians and parents of children who are undergoing surgery for HME by demonstrating that earlier age at seizure onset, absence of EPC, and no contralateral EEG seizures were associated with longer postoperative seizure freedom. At our center, initial AH for HME may provide greater odds of seizure freedom with complications and functional outcomes comparable to those of FH.

Development of an online calculator for the prediction of seizure freedom following pediatric hemispherectomy using the Hemispherectomy Outcome Prediction Scale (HOPS).

OBJECTIVES: Although hemispheric surgeries are among the most effective procedures for drug-resistant epilepsy (DRE) in the pediatric population, there is a large variability in seizure outcomes at the group level. A recently developed HOPS score provides individualized estimation of likelihood of seizure freedom to complement clinical judgement. The objective of this study was to develop a freely accessible online calculator that accurately predicts the probability of seizure freedom for any patient at 1-, 2-, and 5-years post-hemispherectomy. METHODS: Retrospective data of all pediatric patients with DRE and seizure outcome data from the original Hemispherectomy Outcome Prediction Scale (HOPS) study were included. The primary outcome of interest was time-to-seizure recurrence. A multivariate Cox proportional-hazards regression model was developed to predict the likelihood of post-hemispheric surgery seizure freedom at three time points (1-, 2- and 5- years) based on a combination of variables identified by clinical judgment and inferential statistics predictive of the primary outcome. The final model from this study was encoded in a publicly accessible online calculator on the International Network for Epilepsy Surgery and Treatment (iNEST) website (https://hops-calculator.com/). RESULTS: The selected variables for inclusion in the final model included the five original HOPS variables (age at seizure onset, etiologic substrate, seizure semiology, prior non-hemispheric resective surgery, and contralateral fluorodeoxyglucose-positron emission tomography [FDG-PET] hypometabolism) and three additional variables (age at surgery, history of infantile spasms, and magnetic resonance imaging [MRI] lesion). Predictors of shorter time-to-seizure recurrence included younger age at seizure onset, prior resective surgery, generalized seizure semiology, FDG-PET hypometabolism contralateral to the side of surgery, contralateral MRI lesion, non-lesional MRI, non-stroke etiologies, and a history of infantile spasms. The area under the curve (AUC) of the final model was 73.0%. SIGNIFICANCE: Online calculators are useful, cost-free tools that can assist physicians in risk estimation and inform joint decision-making processes with patients and families, potentially leading to greater satisfaction. Although the HOPS data was validated in the original analysis, the authors encourage external validation of this new calculator.

MI-DenseCFNet: deep learning-based multimodal diagnosis models for Aureus and Aspergillus pneumonia.

OBJECTIVE: To build and merge a diagnostic model called multi-input DenseNet fused with clinical features (MI-DenseCFNet) for discriminating between Staphylococcus aureus pneumonia (SAP) and Aspergillus pneumonia (ASP) and to evaluate the significant correlation of each clinical feature in determining these two types of pneumonia using a random forest dichotomous diagnosis model. This will enhance diagnostic accuracy and efficiency in distinguishing between SAP and ASP. METHODS: In this study, 60 patients with clinically confirmed SAP and ASP, who were admitted to four large tertiary hospitals in Kunming, China, were included. Thoracic high-resolution CT lung windows of all patients were extracted from the picture archiving and communication system, and the corresponding clinical data of each patient were collected. RESULTS: The MI-DenseCFNet diagnosis model demonstrates an internal validation set with an area under the curve (AUC) of 0.92. Its external validation set demonstrates an AUC of 0.83. The model requires only 10.24s to generate a categorical diagnosis and produce results from 20 cases of data. Compared with high-, mid-, and low-ranking radiologists, the model achieves accuracies of 78% vs. 75% vs. 60% vs. 40%. Eleven significant clinical features were screened by the random forest dichotomous diagnosis model. CONCLUSION: The MI-DenseCFNet multimodal diagnosis model can effectively diagnose SAP and ASP, and its diagnostic performance significantly exceeds that of junior radiologists. The 11 important clinical features were screened in the constructed random forest dichotomous diagnostic model, providing a reference for clinicians. CLINICAL RELEVANCE STATEMENT: MI-DenseCFNet could provide diagnostic assistance for primary hospitals that do not have advanced radiologists, enabling patients with suspected infections like Staphylococcus aureus pneumonia or Aspergillus pneumonia to receive a quicker diagnosis and cut down on the abuse of antibiotics. KEY POINTS: • MI-DenseCFNet combines deep learning neural networks with crucial clinical features to discern between Staphylococcus aureus pneumonia and Aspergillus pneumonia. • The comprehensive group had an area under the curve of 0.92, surpassing the proficiency of junior radiologists. • This model can enhance a primary radiologist's diagnostic capacity.

GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis.

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.

miRNA-mediated loss of m6A increases nascent translation in glioblastoma.

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition.

Concussions in ice hockey: mixed methods study including assessment of concussions on games missed and cap hit among National Hockey League players, systematic review, and concussion protocol analysis.

OBJECTIVE: Concussions can occur at any level of ice hockey. Incidence estimates of concussions in ice hockey vary, and optimal prevention strategies and return-to-play (RTP) considerations have remained in evolution. The authors performed a mixed-methods study with the aim of elucidating the landscape of concussion in ice hockey and catalyzing initiatives to standardize preventative mechanisms and RTP considerations. METHODS: The authors performed a five-part mixed-methods study that includes: 1) an analysis of the impact of concussions on games missed and income for National Hockey League (NHL) players using a publicly available database, 2) a systematic review of the incidence of concussion in ice hockey, 3) a systematic review of preventative strategies, 4) a systematic review of RTP, and 5) a policy review of documents from major governing bodies related to concussions in sports with a focus on ice hockey. The PubMed, Embase, and Scopus databases were used for the systematic reviews and focused on any level of hockey. RESULTS: In the NHL, 689 players had 1054 concussions from the 2000-2001 to 2022-2023 seasons. A concussion led to a mean of 13.77 ± 19.23 (range 1-82) games missed during the same season. After cap hit per game data became available in 2008-2009, players missed 10,024 games due to 668 concussions (mean 15.13 ± 3.81 per concussion, range 8.81-22.60 per concussion), with a cap hit per game missed of $35,880.85 ± $25,010.48 (range $5792.68-$134,146.30). The total cap hit of all missed games was $385,960,790.00, equating to $577,635.91 per concussion and $25,724,052.70 per NHL season. On systematic review, the incidence of concussions was 0.54-1.18 per 1000 athlete-exposures. Prevention mechanisms involved education, behavioral and cognitive interventions, protective equipment, biomechanical studies, and policy/rule changes. Rules prohibiting body checking in youth players were most effective. Determination of RTP was variable. Concussion protocols from both North American governing bodies and two leagues mandated that a player suspected of having a concussion be removed from play and undergo a six-step RTP strategy. The 6th International Conference on Concussion in Sport recommended the use of mouthguards for children and adolescents and disallowing body checking for all children and most levels of adolescents. CONCLUSIONS: Concussions in ice hockey lead to substantial missed time from play. The authors strongly encourage all hockey leagues to adopt and adhere to age-appropriate rules to limit hits to the head, increase compliance in wearing protective equipment, and utilize high-quality concussion protocols.

Novel chromosomes and genomes provide new insights into evolution and adaptation of the whole genome duplicated yeast-like fungus TN3-1 isolated from natural honey.

Aureobasidium melanogenum TN3-1 strain and A. melanogenum P16 strain were isolated from the natural honey and the mangrove ecosystem, respectively. The former can produce much higher pullulan from high concentration of glucose than the latter. In order to know what happened to their genomes, the PacBio sequencing and Hi-C technologies were used to create the first high-quality chromosome-level reference genome assembly of A. melanogenum TN3-1 (51.61 Mb) and A. melanogenum P16 (25.82 Mb) with the contig N50 of 2.19 Mb and 2.26 Mb, respectively. Based on the Hi-C results, a total of 93.33% contigs in the TN3-1 strain and 92.31% contigs in the P16 strain were anchored onto 24 and 12 haploid chromosomes, respectively. The genomes of the TN3-1 strain had two subgenomes A and B. Synteny analysis showed that the genomic contents of the two subgenomes were asymmetric with many structural variations. Intriguingly, the TN3-1 strain was revealed as a recent hybrid/fusion between the ancestor of A. melanogenum CBS105.22/CBS110374 and the ancestor of another unidentified strain of A. melanogenum similar to P16 strain. We estimated that the two ancient progenitors diverged around 18.38 Mya and merged around 10.66-9.98 Mya. It was found that in the TN3-1 strain, telomeres of each chromosome contained high level of long interspersed nuclear elements (LINEs), but had low level of the telomerase encoding gene. Meanwhile, there were high level of transposable elements (TEs) inserted in the chromosomes of the TN3-1 strain. In addition, the positively selected genes of the TN3-1 strain were mainly enriched in the metabolic processes related to harsh environmental adaptability. Most of the stress-related genes were found to be related to the adjacent LTRs, and the glucose derepression was caused by the mutation of the Glc7-2 in the Snf-Mig1 system. All of these could contribute to its genetic instability, genome evolution, high stress resistance, and high pullulan production from glucose.

The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

Three-dimensional multifocal scanning microscopy for super-resolution cell and tissue imaging.

Recent advancements in image-scanning microscopy have significantly enriched super-resolution biological research, providing deeper insights into cellular structures and processes. However, current image-scanning techniques often require complex instrumentation and alignment, constraining their broader applicability in cell biological discovery and convenient, cost-effective integration into commonly used frameworks like epi-fluorescence microscopes. Here, we introduce three-dimensional multifocal scanning microscopy (3D-MSM) for super-resolution imaging of cells and tissue with substantially reduced instrumental complexity. This method harnesses the inherent 3D movement of specimens to achieve stationary, multi-focal excitation and super-resolution microscopy through a standard epi-fluorescence platform. We validated the system using a range of phantom, single-cell, and tissue specimens. The combined strengths of structured illumination, confocal detection, and epi-fluorescence setup result in two-fold resolution improvement in all three dimensions, effective optical sectioning, scalable volume acquisition, and compatibility with general imaging and sample protocols. We anticipate that 3D-MSM will pave a promising path for future super-resolution investigations in cell and tissue biology.

Stanniocalcin-1 secreted by human umbilical mesenchymal stem cells regulates interleukin-10 expression via the PI3K/AKT/mTOR pathway in alveolar macrophages.

Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure caused by infection, trauma, shock, aspiration or drug reaction. The pathogenesis of ARDS is characterized as an unregulated inflammatory storm, which causes endothelial and epithelial layer damage, leading to alveolar fluid accumulation and pulmonary edema. Previous studies have shown the potential role of mesenchymal stem cells (MSC) in combating the inflammatory cascade by increasing the anti-inflammatory mediator interleukin-10 (IL-10). However, the involved mechanisms are unclear. Here we investigated whether a key immunomodulatory regulator, stanniocalcin-1 (STC-1), was secreted by MSC to activate phosphoinositide 3-kinase/protein kinase B (PI3K/AKT)/ mammalian target of rapamycin (mTOR) signaling pathway to increase IL-10 expression in alveolar macrophages. Lipopolysaccharide (LPS)-stimulated alveolar macrophages co-cultured with human umbilical mesenchymal stem cells (HUMSC) secreted high levels of IL-10. HUMSC co-cultured with alveolar macrophages expressed high STC-1 levels and increased PI3K, AKT and mTOR phosphorylation after LPS activation in alveolar macrophages. STC-1 knockdown in HUMSC decreased the phosphorylation of PI3K, AKT and mTOR and suppressed IL-10 expression in alveolar macrophages. Rapamycin (an mTOR inhibitor) reduced IL-10 secretion in alveolar macrophages. These results, together with our previous study and others, indicate that the PI3K/AKT/mTOR pathway is involved in the regulation of IL-10 production by STC-1 secreted by HUMSC in alveolar macrophages.

Clinical utility of intraoperative electrocorticography for epilepsy surgery: A systematic review and meta-analysis.

Despite the widespread use of intraoperative electrocorticography (iECoG) during resective epilepsy surgery, there are conflicting data on its overall efficacy and inability to predict benefit per pathology. Given the heterogeneity of iECoG use in resective epilepsy surgery, it is important to assess the utility of interictal-based iECoG. This individual patient data (IPD) meta-analysis seeks to identify the benefit of iECoG during resective epilepsy surgery in achieving seizure freedom for various pathologies. Embase, Scopus, and PubMed were searched from inception to January 31, 2021 using the following terms: "ecog", "electrocorticography", and "epilepsy". Articles were included if they reported seizure freedom at ≥12-month follow-up in cohorts with and without iECoG for epilepsy surgery. Non-English articles, noncomparative iECoG cohorts, and studies with <10% iECoG use were excluded. This meta-analysis followed the PRISMA 2020 guidelines. The primary outcome was seizure freedom at last follow-up and time to seizure recurrence, if applicable. Forest plots with random effects modeling assessed the relationship between iECoG use and seizure freedom. Cox regression of IPD was performed to identify predictors of longer duration of seizure freedom. Kaplan-Meier curves with log-rank test were created to visualize differences in time to seizure recurrence. Of 7504 articles identified, 18 were included for study-level analysis. iECoG was not associated with higher seizure freedom at the study level (relative risk = 1.09, 95% confidence interval [CI] = 0.96-1.23, p = .19, I2  = 64%), but on IPD (n = 7 studies, 231 patients) iECoG use was independently associated with more favorable seizure outcomes (hazard ratio = 0.47, 95% CI = .23-.95, p = .037). In Kaplan-Meier analysis of specific pathologies, iECoG use was significantly associated with longer seizure freedom only for focal cortical dysplasia (FCD; p < .001) etiology. Number needed to treat for iECoG was 8.8, and for iECoG in FCD it was 4.7. We show iECoG seizure freedom is not achieved uniformly across centers. iECoG is particularly beneficial for FCD etiology in improving seizure freedom.

Magnetic resonance-guided laser interstitial thermal therapy for drug-resistant epilepsy: A systematic review and individual participant data meta-analysis.

Magnetic resonance-guided laser interstitial thermal therapy (MRgLITT) has emerged as a popular minimally invasive alternative to open resective surgery for drug-resistant epilepsy (DRE). We sought to perform a systematic review and individual participant data meta-analysis to identify independent predictors of seizure outcome and complications following MRgLITT for DRE. Eleven databases were searched from January 1, 2010 to February 6, 2021 using the terms "MR-guided ablation therapy" and "epilepsy". Multivariable mixed-effects Cox and logistic regression identified predictors of time to seizure recurrence, seizure freedom, operative complications, and postoperative neurological deficits. From 8705 citations, 46 studies reporting on 450 MRgLITT DRE patients (mean age = 29.5 ± 18.1 years, 49.6% female) were included. Median postoperative seizure freedom and follow-up duration were 15.5 and 19.0 months, respectively. Overall, 240 (57.8%) of 415 patients (excluding palliative corpus callosotomy) were seizure-free at last follow-up. Generalized seizure semiology (hazard ratio [HR] = 1.78, p = .020) and nonlesional magnetic resonance imaging (MRI) findings (HR = 1.50, p = .032) independently predicted shorter time to seizure recurrence. Cerebral cavernous malformation (CCM; odds ratio [OR] = 7.97, p < .001) and mesial temporal sclerosis/atrophy (MTS/A; OR = 2.21, p = .011) were independently associated with greater odds of seizure freedom at last follow-up. Operative complications occurred in 28 (8.5%) of 330 patients and were independently associated with extratemporal ablations (OR = 5.40, p = .012) and nonlesional MRI studies (OR = 3.25, p = .017). Postoperative neurological deficits were observed in 53 (15.1%) of 352 patients and were independently predicted by hypothalamic hamartoma etiology (OR = 5.93, p = .006) and invasive electroencephalographic monitoring (OR = 4.83, p = .003). Overall, MRgLITT is particularly effective in treating patients with well-circumscribed lesional DRE, such as CCM and MTS/A, but less effective in nonlesional cases or lesional cases with a more diffuse epileptogenic network associated with generalized seizures. This study identifies independent predictors of seizure freedom and complications following MRgLITT that may help further guide patient selection.

Fangchinoline derivatives inhibits PI3K signaling in vitro and in vivo in non-small cell lung cancer.

Fangchinoline (Fan) are extracted from the traditional Chinese medicine Stephania tetrandra S., which is a bis-benzyl isoquinoline alkaloids with anti-tumor activity. Therefore, 25 novel Fan derivatives have been synthesized and evaluated for their anti-cancer activity. In CCK-8 assay, these fangchinoline derivatives displayed higher proliferation inhibitory activity on six tumor cell lines than the parental compound. Compared to the parent Fan, compound 2h presented the anticancer activity against most cancer cells, especially A549 cells, with an IC50 value of 0.26 µM, which was 36.38-fold, and 10.61-fold more active than Fan and HCPT, respectively. Encouragingly, compound 2h showed low biotoxicity to the human normal epithelial cell BEAS-2b with an IC50 value of 27.05 µM. The results indicated compound 2h remarkably inhibited the cell migration by decreasing MMP-2 and MMP-9 expression and inhibited the proliferation of A549 cells by arresting the G2/M cell cycle. Meanwhile, compound 2h could also induce A549 cell apoptosis by promoting endogenous pathways of mitochondrial regulation. In nude mice presented that the growth of tumor tissues was markedly inhibited by the consumption of compound 2h in a dose-dependent manner, and it was found that compound 2h could inhibit the mTOR/PI3K/AKT pathway in vivo. In docking analysis, high affinity interaction between 2h and PI3K was responsible for drastic kinase inhibition by the compound. To conclude, this derivative compound may be useful as a potent anti-cancer agent for treatment of NSCLC.

Dl-3-n-butylphthalide promotes angiogenesis in ischemic stroke mice through upregulating autocrine and paracrine sonic hedgehog.

Dl-3-n-butylphthalide (NBP) is a small-molecule drug used in the treatment of ischemic stroke in China, which is proven to ameliorate the symptoms of ischemic stroke and improve the prognosis of patients. Previous studies have shown that NBP accelerates recovery after stroke by promoting angiogenesis. In this study, we investigated the mechanisms underlying the angiogenesis-promoting effects of NBP in ischemic stroke models in vitro and in vivo. OGD/R model was established in human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), while the tMCAO model was established in mice. The cells were pretreated with NBP (10, 50, 100 µM); the mice were administered NBP (4, 8 mg/kg, i.v.) twice after tMCAO. We showed that NBP treatment significantly stimulated angiogenesis by inducing massive production of angiogenic growth factors VEGFA and CD31 in both in vitro and in vivo models of ischemic stroke. NBP also increased the tubule formation rate and migration capability of HUVECs in vitro. By conducting the weighted gene co-expression network analysis, we found that these effects were achieved by upregulating the expression of a hedgehog signaling pathway. We demonstrated that NBP treatment not only changed the levels of regulators of the hedgehog signaling pathway but also activated the transcription factor Gli1. The pro-angiogenesis effect of NBP was abolished when the hedgehog signaling pathway was inhibited by GDC-0449 in HUVECs, by Sonic Hedgehog(Shh) knockdown in HUVECs, or by intracerebroventricular injection of AAV-shRNA(shh)-CMV in tMCAO mice. Furthermore, we found that HUVECs produced a pro-angiogenic response not only to autocrine Shh, but also to paracrine Shh secreted by astrocytes. Together, we demonstrate that NBP promotes angiogenesis via upregulating the hedgehog signaling pathway. Our results provide an experimental basis for the clinical use of NBP.

Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging.

Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, because current live-cell imaging systems are limited in their adaptability, portable cell imaging systems have been adapted by a variety of strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15c m×15c m×3c m) and provides in situ cell imaging inside an incubator with a subcellular lateral resolution (∼3µm). We demonstrated the improved stability of the MAM system with fluorescent targets and live HeLa cells, enabling long-term imaging for 12 h without the need for external support or post-processing. We believe the protocol could guide scientists to construct a compact portable fluorescence imaging system and perform time-lapse in situ single-cell imaging and analysis.

Association of spinopelvic index with proximal junctional failure developing in adult spinal deformity after surgical treatment: an observational study.

BACKGROUND: Those pelvic parameters of sacral slope (SS) and pelvic tilt (PT) correlated significantly to lumbar spine and hip joints respectively. We proposed the match between SS and PT, namely spinopelvic index (SPI), in order to investigate whether the SPI correlated to proximal junctional failure (PJF) in adult spinal deformity (ASD) after correction surgery. METHODS: Ninety-nine ASD patients who had undergone long-fusion (≥ 5 vertebras) surgeries were reviewed retrospectively in two medical institutions from January 2018 to December 2019. Those SPI were calculated with the equation: SPI = SS/PT, and analyzed using the receiver operating characteristic curve (ROC) analysis. All participants were subdivided into the observational and control group. Comparisons of demographics, surgical and radiographic data between the two groups were performed. A Kaplan-Meier curve and log-rank test was used to analyze the differences in PJF-free survival time, and the 95% confidence intervals (CI) were recorded respectively. RESULTS: Nineteen patients suffering from PJF had much smaller postoperative SPI (P = 0.015), but much larger TK postoperatively (P < 0.001). ROC analysis determined the best cutoff value of 0.82 for SPI (sensitivity = 88.5%, specificity = 57.9%; AUC = 0.719, 95%CI: 0.612-0.864; P = 0.003). There were 19 and 80 cases in the observational (SPI ≤ 0.82) and control group (SPI > 0.82) respectively. The incidence of PJF in the observational group was much higher (11/19 VS 8/80, P < 0.001); further logistic regression analysis showed that SPI ≤ 0.82 was associated with increased odds of PJF (odds ratio: 12.375; 95%CI: 3.851-39.771). PJF-free survival time in the observational group decreased significantly (P < 0.001, log-rank test), moreover, multivariate analysis demonstrated that a value of SPI ≤ 0.82 (HR 6.626, 95%CI: 1.981-12.165) was significantly associated with PJF. CONCLUSIONS: For ASD patients underwent long-fusion surgeries, the SPI should be over 0.82. The incidence of PJF may increase by 12-fold in such individuals with the immediate SPI ≤ 0.82 postoperatively.

Health disparities attributed to Medicare-Medicaid dual-eligible status in patients with peripheral arterial disease.

OBJECTIVE: Peripheral arterial disease (PAD) is a prevalent and debilitating disease that can be effectively treated by surgical revascularization. However, Medicare-Medicaid dual-eligible patients have experienced worse long-term outcomes, notably higher rates of amputation and mortality, relative to other insurance groups. In the present study, we investigated how insurance status can perpetuate health disparities in PAD outcomes. METHODS: The National Inpatient Sample was queried from 2000 to 2011 for patients aged ≥18 years with PAD who had undergone surgical revascularization with hospitalization. Patients were stratified by insurance status, and dual-eligible patients were compared with Medicare-only, Medicaid-only, private insurance, and self-pay patients. Multivariable regression analysis was performed to assess the effect of dual-eligible status on postoperative outcomes such as inpatient mortality, complications, and favorable discharge (home or home with services). RESULTS: A total of 771,790 hospitalizations were included in the present analysis and stratified by insurance type. Dual-eligible patients had the highest rates of major (32%) and extreme (11%) severity of illness and the highest rates of major (19%) and extreme (6%) risk of mortality among all insurance groups (P < .001). Dual-eligibility status was independently associated with reduced odds of favorable discharge relative to all patients (P < .001) and increased length of stay relative to Medicare-only (P = .002) and private-payor groups (P < .001). Although dual-eligible patients had increased mortality odds relative to the Medicaid-only and self-pay groups, they did not have significantly different odds of perioperative complications relative to all other insurance groups. CONCLUSIONS: Medicare-Medicaid dual-eligible patients with PAD had had more severe clinical presentations, a greater risk of extended hospitalizations, and a lower likelihood of discharge to home, relative to patients without dual eligibility. Further studies are needed to examine the link between discharge disposition and disparities in healthcare outcomes and to investigate the interventions that effectively address the increased severity of PAD in dual-eligible patients.