Three-Dimensional Manipulation of Micromodules Using Twin Optothermally Actuated Bubble Robots.

A 3D manipulation technique based on two optothermally generated and actuated surface-bubble robots is proposed. A single laser beam can be divided into two parallel beams and used for the generation and motion control of twin bubbles. The movement and spacing control of the lasers and bubbles can be varied directly and rapidly. Both 2D and 3D operations of micromodules were carried out successfully using twin bubble robots. The cooperative manipulation of twin bubble robots is superior to that of a single robot in terms of stability, speed, and efficiency. The operational technique proposed in this study is expected to play an important role in tissue engineering, drug screening, and other fields.

Fast estimation of generalized linear latent variable models for performance and process data with ordinal, continuous, and count observed variables.

Different data types often occur in psychological and educational measurement such as computer-based assessments that record performance and process data (e.g., response times and the number of actions). Modelling such data requires specific models for each data type and accommodating complex dependencies between multiple variables. Generalized linear latent variable models are suitable for modelling mixed data simultaneously, but estimation can be computationally demanding. A fast solution is to use Laplace approximations, but existing implementations of joint modelling of mixed data types are limited to ordinal and continuous data. To address this limitation, we derive an efficient estimation method that uses first- or second-order Laplace approximations to simultaneously model ordinal data, continuous data, and count data. We illustrate the approach with an example and conduct simulations to evaluate the performance of the method in terms of estimation efficiency, convergence, and parameter recovery. The results suggest that the second-order Laplace approximation achieves a higher convergence rate and produces accurate yet fast parameter estimates compared to the first-order Laplace approximation, while the time cost increases with higher model complexity. Additionally, models that consider the dependence of variables from the same stimulus fit the empirical data substantially better than models that disregarded the dependence.

Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study, we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C150 mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF1-5 repeats for a total of 4 months resulted in a marked reduction (38-48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients.

Insulator Defect Detection Based on ML-YOLOv5 Algorithm.

To address the challenges of balancing accuracy and speed, as well as the parameters and FLOPs in current insulator defect detection, we propose an enhanced insulator defect detection algorithm, ML-YOLOv5, based on the YOLOv5 network. The backbone module incorporates depthwise separable convolution, and the feature fusion C3 module is replaced with the improved C2f_DG module. Furthermore, we enhance the feature pyramid network (MFPN) and employ knowledge distillation using YOLOv5m as the teacher model. Experimental results demonstrate that this approach achieved a 46.9% reduction in parameter count and a 43.0% reduction in FLOPs, while maintaining an FPS of 63.6. It exhibited good accuracy and detection speed on both the CPLID and IDID datasets, making it suitable for real-time inspection of high-altitude insulator defects.

Acoustics-Controlled Microdroplet and Microbubble Fusion and Its Application in the Synthesis of Hydrogel Microspheres.

Droplet fusion technology is a key technology for many droplet-based biochemical medical applications. By integrating a symmetrical flow channel structure, we demonstrate an acoustics-controlled fusion method of microdroplets using surface acoustic waves. Different kinds of microdroplets can be staggered and ordered in the symmetrical flow channel, proving the good arrangement effect of the microfluidic chip. This method can realize not only the effective fusion of microbubbles but also the effective fusion of microdroplets of different sizes without any modification. Further, we investigate the influence of the input frequency and peak-to-peak value of the driving voltage on microdroplets fusion, giving the effective fusion parameter conditions of microdroplets. Finally, this method is successfully used in the preparation of hydrogel microspheres, offering a new platform for the synthesis of hydrogel microspheres.

Frequentist Model Averaging in Structure Equation Model With Ordinal Data.

In practice, it is common that a best fitting structural equation model (SEM) is selected from a set of candidate SEMs and inference is conducted conditional on the selected model. Such post-selection inference ignores the model selection uncertainty and yields too optimistic inference. Using the largest candidate model avoids model selection uncertainty but introduces a large variation. Jin and Ankargren (Psychometrika 84:84-104, 2019) proposed to use frequentist model averaging in SEM with continuous data as a compromise between model selection and the full model. They assumed that the true values of the parameters depend on [Formula: see text] with n being the sample size, which is known as a local asymptotic framework. This paper shows that their results are not directly applicable to SEM with ordinal data. To address this issue, we prove consistency and asymptotic normality of the polychoric correlation estimators under the local asymptotic framework. Then, we propose a new frequentist model averaging estimator and a valid confidence interval that are suitable for ordinal data. Goodness-of-fit test statistics for the model averaging estimator are also derived.

Estimating and Testing Random Intercept Multilevel Structural Equation Models with Model Implied Instrumental Variables.

This study develops a new limited information estimator for random intercept Multilevel Structural Equation Models (MSEM). It is based on the Model Implied Instrumental Variable Two-Stage Least Squares (MIIV-2SLS) estimator, which has been shown to be an excellent alternative or supplement to maximum likelihood (ML) in SEMs (Bollen, 1996). We also develop a multilevel overidentification test statistic that applies to equations at the within or between levels. Our Monte Carlo simulation analysis suggests that MIIV-2SLS is more robust than ML to misspecification at within or between levels, performs well given fewer that 100 clusters, and shows that our multilevel overidentification test for equations performs well at both levels of the model.

γ-Secretase modulators show selectivity for γ-secretase-mediated amyloid precursor protein intramembrane processing.

The aggregation of ß-amyloid peptide 42 results in the formation of toxic oligomers and plaques, which plays a pivotal role in Alzheimer's disease pathogenesis. Aß42 is one of several Aß peptides, all of Aß30 to Aß43 that are produced as a result of γ-secretase-mediated regulated intramembrane proteolysis of the amyloid precursor protein. γ-Secretase modulators (GSMs) represent a promising class of Aß42-lowering anti-amyloidogenic compounds for the treatment of AD. Gamma-secretase modulators change the relative proportion of secreted Aß peptides, while sparing the γ-secretase-mediated processing event resulting in the release of the cytoplasmic APP intracellular domain. In this study, we have characterized how GSMs affect the γ-secretase cleavage of three γ-secretase substrates, E-cadherin, ephrin type A receptor 4 (EphA4) and ephrin type B receptor 2 (EphB2), which all are implicated in important contexts of cell signalling. By using a reporter gene assay, we demonstrate that the γ-secretase-dependent generation of EphA4 and EphB2 intracellular domains is unaffected by GSMs. We also show that γ-secretase processing of EphA4 and EphB2 results in the release of several Aß-like peptides, but that only the production of Aß-like proteins from EphA4 is modulated by GSMs, but with an order of magnitude lower potency as compared to Aß modulation. Collectively, these results suggest that GSMs are selective for γ-secretase-mediated Aß production.

The Molecular Assembly State of Drp1 Controls its Association With the Mitochondrial Recruitment Receptors Mff and MIEF1/2.

Drp1 is a central player in mitochondrial fission and is recruited to mitochondria by Mff and MIEFs (MIEF1 and MIEF2), but little is known about how its assembly state affects Drp1 mitochondrial recruitment and fission. Here, we used in vivo chemical crosslinking to explore the self-assembly state of Drp1 and how it regulates the association of Drp1 with MIEFs and Mff. We show that in intact mammalian cells Drp1 exists as a mixture of multiple self-assembly forms ranging from the minimal, probably tetrameric, self-assembly subunit to several higher order oligomers. Precluding mitochondria-bound Drp1 in Mff/MIEF1/2-deficient cells does not affect the oligomerization state of Drp1, while conversely forced recruitment of Drp1 to mitochondria by MIEFs or Mff facilitates Drp1 oligomerization. Mff preferentially binds to higher order oligomers of Drp1, whereas MIEFs bind to a wider-range of Drp1 assembly subunits, including both lower and higher oligomeric states. Mff only recruits active forms of Drp1, while MIEFs are less selective and recruit both active and inactive Drp1 as well as oligomerization- or GTPase-deficient Drp1 mutants to mitochondria. Moreover, all the fission-incompetent Drp1 mutants tested (except the monomeric mutant K668E) affect Drp1-driven mitochondrial dynamics via incorporation of the mutants into the native oligomers to form function-deficient Drp1 assemblies. We here confirm that MIEFs also serve as a platform facilitating the binding of Drp1 to Mff and loss of MIEFs severely impairs the interaction between Drp1 and Mff. Collectively, our findings suggest that Mff and MIEFs respond differently to the molecular assembly state of Drp1 and that the extent of Drp1 oligomerization regulates mitochondrial dynamics.

MIEF1/2 orchestrate mitochondrial dynamics through direct engagement with both the fission and fusion machineries.

BACKGROUND: Mitochondrial dynamics is the result of a dynamic balance between fusion and fission events, which are driven via a set of mitochondria-shaping proteins. These proteins are generally considered to be binary components of either the fission or fusion machinery, but potential crosstalk between the fission and fusion machineries remains less explored. In the present work, we analyzed the roles of mitochondrial elongation factors 1 and 2 (MIEF1/2), core components of the fission machinery in mammals. RESULTS: We show that MIEFs (MIEF1/2), besides their action in the fission machinery, regulate mitochondrial fusion through direct interaction with the fusion proteins Mfn1 and Mfn2, suggesting that MIEFs participate in not only fission but also fusion. Elevated levels of MIEFs enhance mitochondrial fusion in an Mfn1/2- and OPA1-dependent but Drp1-independent manner. Moreover, mitochondrial localization and self-association of MIEFs are crucial for their fusion-promoting ability. In addition, we show that MIEF1/2 can competitively decrease the interaction of hFis1 with Mfn1 and Mfn2, alleviating hFis1-induced mitochondrial fragmentation and contributing to mitochondrial fusion. CONCLUSIONS: Our study suggests that MIEFs serve as a central hub that interacts with and regulates both the fission and fusion machineries, which uncovers a novel mechanism for balancing these opposing forces of mitochondrial dynamics in mammals.

A unified model-implied instrumental variable approach for structural equation modeling with mixed variables.

The model-implied instrumental variable (MIIV) estimator is an equation-by-equation estimator of structural equation models that is more robust to structural misspecifications than full information estimators. Previous studies have concentrated on endogenous variables that are all continuous (MIIV-2SLS) or all ordinal . We develop a unified MIIV approach that applies to a mixture of binary, ordinal, censored, or continuous endogenous observed variables. We include estimates of factor loadings, regression coefficients, variances, and covariances along with their asymptotic standard errors. In addition, we create new goodness of fit tests of the model and overidentification tests of single equations. Our simulation study shows that the proposed MIIV approach is more robust to structural misspecifications than diagonally weighted least squares (DWLS) and that both the goodness of fit model tests and the overidentification equations tests can detect structural misspecifications. We also find that the bias in asymptotic standard errors for the MIIV estimators of factor loadings and regression coefficients are often lower than the DWLS ones, though the differences are small in large samples. Our analysis shows that scaling indicators with low reliability can adversely affect the MIIV estimators. Also, using a small subset of MIIVs reduces small sample bias of coefficient estimates, but can lower the power of overidentification tests of equations.

Novel Cysteine-Sparing Hypomorphic NOTCH3 A1604T Mutation Observed in a Family With Migraine and White Matter Lesions.

OBJECTIVE: To conduct a clinical study of a family with neurologic symptoms and findings carrying a novel NOTCH3 mutation and to analyze the molecular consequences of the mutation. METHODS: We analyzed a family with complex neurologic symptoms by MRI and neurologic examinations. Exome sequencing of the NOTCH3 locus was conducted, and whole-genome sequencing was performed to identify COL4A1, COL4A2, and HTRA1 mutations. Cell lines expressing the normal or NOTCH3A1604T receptors were analyzed to assess proteolytic processing, cell morphology, receptor routing, and receptor signaling. RESULTS: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary form of cerebral small vessel disease (SVD) and caused by mutations in the NOTCH3 gene. Most CADASIL mutations alter the number of cysteine residues in the extracellular domain of the NOTCH3 receptor, but in this article, we describe a family in which some members carry a novel cysteine-sparing NOTCH3 mutation (c.4810 G>A, p.Ala1604Thr). Two of 3 siblings heterozygous for the NOTCH3A1604T mutation presented with migraine and white matter lesions (WMLs), the latter of a type related to but distinct from what is normally observed in CADASIL. Two other members instead carried a novel COL4A1 missense mutation (c.4795 G>A; p.(Ala1599Thr)). The NOTCH3A1604T receptor was aberrantly processed, showed reduced presence at the cell surface, and less efficiently activated Notch downstream target genes. CONCLUSIONS: We identify a family with migraine and WML in which some members carry a cysteine-sparing hypomorphic NOTCH3 mutation. Although a causal relationship is not established, we believe that the observations contribute to the discussion on dysregulated Notch signaling in cerebral SVDs.

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion.

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression.

Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this report, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between the NOTCH and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3-PDGFRB axis in IMF, where an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.

Construction of core-shell microcapsules via focused surface acoustic wave microfluidics.

The ability to construct core-shell microcapsules has the potential to shift the paradigm in the development of new delivery systems for nutrients, cosmetics, and drugs. In this work, we demonstrate an application of focused surface acoustic wave (FSAW) microfluidics to produce microcapsules with a core-shell structure using one or two focused interdigital transducers (FIDTs) on the microfluidic device. Solid particles or liquid microdroplets without any special modification in multiphase laminar flow are driven by the acoustic radiation force arising from the FSAW, and cross the oil/water interface back and forth, which is not only suitable for generation of core-shell microcapsules with solid cores but also used for coating an aqueous microdroplet core with an oil shell. On this basis, more FIDTs can be added to the device to manufacture more layers of microcapsules if needed. Single-layer, two-layer, or even multi-layer microcapsules can be selectively fabricated. This work provides a promising and robust platform to construct core-shell microcapsules via FSAW microfluidics, which are suitable for a wide range of applications.

Acoustic-Controlled Bubble Generation and Fabrication of 3D Polymer Porous Materials.

Porous materials have a variety of applications such as catalysis, gas separation, sensing, tissue engineering, sewage treatment, and so on. However, there are still challenges in the synthesis of porous materials with light weight, high porosity, and superhydrophobicity. Herein, we demonstrate one acoustic-controlled microbubble generation method, which is used to synthesize 3D polymer porous materials. The acoustic-controlled microbubble generation based on focused surface acoustic wave (FSAW) is suitable for not only the generation of gas-in-oil microbubbles but also the gas-in-water microbubbles. The size of microbubbles can be real-time controlled by adjusting the frequency or the driving voltage of the FSAW. The as-prepared poly(vinyl alcohol) (PVA) foams composed of microbubbles can be used as a template to fabricate the PVA-based porous gel materials through freezing-thawing cyclic processing, and the various sized bubbles result in different porosity of the PVA-based porous gel materials. Moreover, excellent properties like oleophilicity and superhydrophobicity of the PVA-based porous gel materials can be obtained through a further hydrophobic modification treatment. The oil/water separation experiments have been done to demonstrate the good absorption and reliability of the modified porous gel materials, which are capable of multiple uses.

Are degenerative spondylolisthesis and further slippage postoperatively really issues in spinal stenosis surgery?

Background and aims Opinions diverge concerning the prognostic importance of preoperative degenerative spondylolisthesis in patients with lumbar spinal stenosis, as well as the significance of further slippage post-operatively following decompression alone. However, a slip is only one among several factors related to the topic, e.g. duration and intensity of back and leg pain, pre-operative walking ability, number of levels operated and not least the experience of the surgeon. Our aim was to take all of the above-mentioned factors into consideration when analysing the patients' clinical outcome, reported as Change in back pain, Change in leg pain, Overall satisfaction and Change in walking ability, with special emphasis on the possible importance of pre- and/or post-operative degenerative spondylolisthesis. Methods We studied 200 consecutive patients, mean follow-up time 81 months (range 62-108). Before treatment and on the follow-up occasion all patients answered the SF-36 questionnaire and assessed their back and leg pain on a visual analogue scale (VAS). At follow-up the patients were asked about possible changes in back and leg pain (completely free, much better, somewhat better, unchanged, somewhat worse, much worse) and whether they were; satisfied with the outcome, in doubt or not satisfied. Before treatment and at follow-up the presence or not of degenerative spondylolisthesis was determined in the lateral view on a plain X-ray or MRI. By use of a microsurgical technique decompression was achieved in all patients by bilateral laminotomy not sparing the midline ligaments, irrespective of a degenerative spondylolisthesis or not. Eight surgeons with different surgical experience performed the operations. Four separate multivariate analyses were conducted, one for each clinical outcome. The Lasso method was used for variable selection and multiple imputation was applied to handle missing values. Results At follow-up 78.5% of the patients were completely satisfied with the outcome. Minimal clinical important difference (MCID) was achieved for 69% of the patients. Before surgery 28 patients were able to walk more than 1 km compared to 111 at follow-up. The reoperation rate at 6.8 years was 12% further decompressions and 2.5% fusions at the index level. Post-operative slippage was equally common in patients with and without a preoperative slip (around 30%). There were no notable differences in outcome in patients with and without a preoperative slip and no effect of further slippage at the index or another level post-operatively. Nor could the statistical analysis show any of the other covariates (age, gender, duration and intensity of back and leg pain, pre-operative walking ability or number of levels operated) to be of statistically significant importance for predicting the outcome. In the univariate statistical analysis differences were found between the patients of individual surgeons regarding satisfaction, pain improvement, and reoperation rates in favour of surgical experience, which were, however, not statistically significant in the multivariate analysis. Conclusions None of the covariates, including pre-operative spondylolisthesis and further slippage post-operatively, were statistically significant for predicting the clinical outcome. Implication Our results provide no evidence for adding fusion to the decompression.

The phosphorylation status of Ser-637 in dynamin-related protein 1 (Drp1) does not determine Drp1 recruitment to mitochondria.

Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a central step required for mitochondrial fission. Reversible Drp1 phosphorylation has been implicated in the regulation of this process, but whether Drp1 phosphorylation at Ser-637 determines its subcellular localization and fission activity remains to be fully elucidated. Here, using HEK 293T cells and immunofluorescence, immunoblotting, RNAi, subcellular fractionation, co-immunoprecipitation assays, and CRISPR/Cas9 genome editing, we show that Drp1 phosphorylated at Ser-637 (Drp1pS637) resides both in the cytosol and on mitochondria. We found that the receptors mitochondrial fission factor (Mff) and mitochondrial elongation factor 1/2 (MIEF1/2) interact with and recruit Drp1pS637 to mitochondria and that elevated Mff or MIEF levels promote Drp1pS637 accumulation on mitochondria. We also noted that protein kinase A (PKA), which mediates phosphorylation of Drp1 on Ser-637, is partially present on mitochondria and interacts with both MIEFs and Mff. PKA knockdown did not affect the Drp1-Mff interaction, but slightly enhanced the interaction between Drp1 and MIEFs. In Drp1-deficient HEK 293T cells, both phosphomimetic Drp1-S637D and phospho-deficient Drp1-S637A variants, like wild-type Drp1, located to the cytosol and to mitochondria and rescued a Drp1 deficiency-induced mitochondrial hyperfusion phenotype. However, Drp1-S637D was less efficient than Drp1-WT and Drp1-S637A in inducing mitochondrial fission. In conclusion, the Ser-637 phosphorylation status in Drp1 is not a determinant that controls Drp1 recruitment to mitochondria.

Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery.

Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin-related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1-mediated mitochondrial fragmentation occurs in the absence of Drp1 and Dyn2, suggesting that they are dispensable for hFis1 function. hFis1 instead binds to Mfn1, Mfn2, and OPA1 and inhibits their GTPase activity, thus blocking the fusion machinery. Consistent with this, disruption of the fusion machinery in Drp1-/- cells phenocopies the fragmentation phenotype induced by hFis1 overexpression. In sum, our data suggest a novel role for hFis1 as an inhibitor of the fusion machinery, revealing an important functional evolutionary divergence between yeast and mammalian Fis1 proteins.