Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Pluripotency state of mouse ES cells determines their contribution to self-organized layer formation by mesh closure on microstructured adhesion-limiting substrates.

Assembly of pluripotent stem cells to initiate self-organized tissue formation on engineered scaffolds is an important process in stem cell engineering. Pluripotent stem cells are known to exist in diverse pluripotency states, with heterogeneous subpopulations exhibiting differential gene expression levels, but how such diverse pluripotency states orchestrate tissue formation is still an unrevealed question. In this study, using microstructured adhesion-limiting substrates, we aimed to clarify the contribution to self-organized layer formation by mouse embryonic stem cells in different pluripotency states: ground and naïve state. We found that while ground state cells as well as sorted REX1-high expression cells formed discontinuous cell layers with limited lateral spread, naïve state cells could successfully self-organize to form a continuous layer by progressive mesh closure within 3 days. Using sequential immunofluorescence microscopy to examine the mesh closure process, we found that KRT8+ cells were particularly localized around unfilled holes, occasionally bridging the holes in a manner suggestive of their role in the closure process. These results highlight that compared with ground state cells, naïve state cells possess a higher capability to contribute to self-organized layer formation by mesh closure. Thus, this study provides insights with implications for the application of stem cells in scaffold-based tissue engineering.

Creative Destruction: A Basic Computational Model of Cortical Layer Formation.

One of the most characteristic properties of many vertebrate neural systems is the layered organization of different cell types. This cytoarchitecture exists in the cortex, the retina, the hippocampus, and many other parts of the central nervous system. The developmental mechanisms of neural layer formation have been subject to substantial experimental efforts. Here, we provide a general computational model for cortical layer formation in 3D physical space. We show that this multiscale, agent-based model, comprising two distinct stages of apoptosis, can account for the wide range of neuronal numbers encountered in different cortical areas and species. Our results demonstrate the phenotypic richness of a basic state diagram structure. Importantly, apoptosis allows for changing the thickness of one layer without automatically affecting other layers. Therefore, apoptosis increases the flexibility for evolutionary change in layer architecture. Notably, slightly changed gene regulatory dynamics recapitulate the characteristic properties observed in neurodevelopmental diseases. Overall, we propose a novel computational model using gene-type rules, exhibiting many characteristics of normal and pathological cortical development.

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.

Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination.

To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

The Pace of Neurogenesis Is Regulated by the Transient Retention of the Apical Endfeet of Differentiating Cells.

The development of the mammalian cerebral cortex involves a variety of temporally organized events such as successive waves of neuronal production and the transition of progenitor competence for each neuronal subtype generated. The number of neurons generated in a certain time period, that is, the rate of neuron production, varies across the regions of the brain and the specific developmental stage; however, the underlying mechanism of this process is poorly understood. We have recently found that nascent neurons communicate with undifferentiated progenitors and thereby regulate neurogenesis, through a transiently retained apical endfoot that signals via the Notch pathway. Here, we report that the retention time length of the neuronal apical endfoot correlates with the rate of neuronal production in the developing mouse cerebral cortex. We further demonstrate that a forced reduction or extension of the retention period through the disruption or stabilization of adherens junction, respectively, resulted in the acceleration or deceleration of neurogenesis, respectively. Our results suggest that the apical endfeet of differentiating cells serve as a pace controller for neurogenesis, thereby assuring the well-proportioned laminar organization of the neocortex.

Process optimization and filtration performance of an anaerobic dynamic membrane bioreactor treating textile wastewaters.

The study aimed at investigating the performance of anaerobic dynamic MBR (AnDMBR) for the treatment of synthetic textile wastewater. A laboratory scale anaerobic bioreactor was operated to test nylon mesh support materials with different pore sizes (20 µm, 53 µm and 100 µm). The performances of the AnDMBR were evaluated with a stimulated wastewater containing 1,000 mg.L-1 COD and 100 mg.L-1 dye (Remazol Brilliant Violet 5R). To develop an effective dynamic cake layer on the support material, different operational strategies, i.e. high flux, continuous and intermittently biogas recycle were studied for process optimization and increase the filtration performances. Initially, the bioreactor was operated under continuous biogas recycle. Under this operation strategy, the cake layer was not formed, then intermittent biogas recycle was applied to improve the development of dynamic layer. Effluent SS decreased below 20 mg-SS.L-1 for all the tested different pore sized supports after the development of the cake layer. Almost complete color (>99%) and high COD removal efficiencies (95-97%) were observed. For all the three supports, the bioreactor was operated at fluxes of 5-15 L.(m2.h)-1 (LMH), which was quite high compared to conventional AnMBRs equipped with micro/ultra-filtration membranes. In order to better understand the formation and its structure, detailed cake layer characterization analyses were conducted with scanning electron microscopy (SEM), SEM coupled Energy Dispersive X-ray Spectroscopy (EDS) and inductively coupled plasma-optical emission spectrometer (ICP). Provided the formation of the cake layer, the comparable flux and removal performances with AnMBRs for all three tested support materials were possible.

Antagonism between ß-catenin and Gata.a sequentially segregates the germ layers of ascidian embryos.

Many animal embryos use nuclear ß-catenin (nß-catenin) during the segregation of endomesoderm (or endoderm) from ectoderm. This mechanism is thus likely to be evolutionarily ancient. In the ascidian embryo, nß-catenin reiteratively drives binary fate decisions between ectoderm and endomesoderm at the 16-cell stage, and then between endoderm and margin (mesoderm and caudal neural) at the 32-cell stage. At the 16-cell stage, nß-catenin activates endomesoderm genes in the vegetal hemisphere. At the same time, nß-catenin suppresses the DNA-binding activity of a maternal transcription factor, Gata.a, through a physical interaction, and Gata.a thereby activates its target genes only in the ectodermal lineage. In the present study, we found that this antagonism between nß-catenin and Gata.a also operates during the binary fate switch at the 32-cell stage. Namely, in marginal cells where nß-catenin is absent, Gata.a directly activates its target, Zic-r.b (ZicL), to specify the marginal cell lineages. Thus, the antagonistic action between nß-catenin and Gata.a is involved in two consecutive stages of germ layer segregation in ascidian embryos.

Mutually repressive interaction between Brn1/2 and Rorb contributes to the establishment of neocortical layer 2/3 and layer 4.

Although several molecules have been shown to play important roles in subtype specification of neocortical neurons, the entire mechanism involved in the specification, in particular, of upper cortical plate (UCP) neurons still remains unclear. The UCP, which is responsible for intracortical connections in the neocortex, comprises histologically, functionally, and molecularly different layer 2/3 (L2/3) and L4. Here, we report the essential interactions between two types of transcription factors, Rorb (RAR-related orphan receptor beta) and Brn1/2 (Brain-1/Brain-2), for UCP specification. We found that Brn2 expression was detected in all upper layers in the immature UCP, but was subsequently restricted to L2/3, accompanied by up-regulation of Rorb in L4, suggesting demarcation of L2/3 and L4 during cortical maturation. Rorb indeed inhibited Brn2 expression and the expression of other L2/3 characteristics, revealed by ectopic expression and knockdown studies. Moreover, this inhibition occurred through direct binding of Rorb to the Brn2 locus. Conversely, Brn1/2 also inhibited Rorb expression and the expression of several L4 characteristics. Together, these results suggest that a mutually repressive mechanism exists between Brn1/2 and Rorb expression and that the established expression of Brn1/2 and Rorb further specifies those neurons into L2/3 and L4, respectively, during UCP maturation.

FIP4/Arfophilin-2 plays overlapping but distinct roles from FIP3/Arfophilin-1 in neuronal migration during cortical layer formation.

The class II Rab11 family-interacting proteins, FIP3 and FIP4, also termed Arfophilin-1 and Arfophilin-2, respectively, are endosomal proteins that function as dual effector proteins for Rab11 and ADP ribosylation factor (Arf) small GTPases. In the present study, we examined the expression and role of FIP4 in neuronal migration during cerebral layer formation. FIP4 mRNA was first weakly detected in post-mitotic migrating neurons in the upper intermediate zone, and expression was markedly increased in the cortical layer. Exogenously expressed FIP4 protein was localized to subpopulations of EEA1- and syntaxin 12-positive endosomes in migrating neurons, and was partially colocalized with FIP3. Knockdown of FIP4 by in utero electroporation significantly stalled transfected neurons in the lower cortical layer and decreased the speed of neuronal migration in the upper intermediate zone and in the cortical plate compared with control small hairpin RNA (shRNA)-transfected neurons. Furthermore, co-transfection of shRNA-resistant wild-type FIP4, but not wild type FIP3 or FIP4 mutants lacking the binding region for Rab11 or Arf, significantly improved the disturbed cortical layer formation caused by FIP4 knockdown. Collectively, our findings suggest that FIP4 and FIP3 play overlapping but distinct roles in neuronal migration downstream of Arf and Rab11 during cortical layer formation.

Tangential cell migration during layer formation of chick optic tectum.

The laminated structure of the optic tectum is formed by radial and tangential cell migration during development. Studies of developing chick optic tectum have revealed two streams of tangential cell migration in the middle and superficial layers, which have distinctive origins, migratory paths, modes of migration, and destinations. We will review the process of the two types of tangential migrations, in order to elucidate their roles in the formation of the optic tectum layers.

How variable progenitor clones construct a largely invariant neocortex.

The neocortex contains a vast collection of diverse neurons organized into distinct layers. While nearly all neocortical neurons are generated by radial glial progenitors (RGPs), it remains largely unclear how a complex yet organized neocortex is constructed reliably and robustly. Here, we show that the division behavior and neuronal output of RGPs are highly constrained with patterned variabilities to support the reliable and robust construction of the mouse neocortex. The neurogenic process of RGPs can be well-approximated by a consistent Poisson-like process unfolding over time, producing deep to superficial layer neurons progressively. The exact neuronal outputs regarding layer occupation are variable; yet, this variability is constrained systematically to support all layer formation, largely reflecting the variable intermediate progenitor generation and RGP neurogenic entry and exit timing differences. Together, these results define the fundamental features of neocortical neurogenesis with a balanced reliability and variability for the construction of the complex neocortex.

A Careful Insight into DDI-Type Receptor Layers on the Way to Improvement of Click-Biology-Based Immunosensors.

Protein-based microarrays are important tools for high-throughput medical diagnostics, offering versatile platforms for multiplex immunodetection. However, challenges arise in protein microarrays due to the heterogeneous nature of proteins and, thus, differences in their immobilization conditions. This article advocates DNA-directed immobilization (DDI) as a solution, emphasizing its rapid and cost-effective fabrication of biosensing platforms. Thiolated single-stranded DNA and its analogues, such as ZNA® and PNA probes, were used to immobilize model proteins (anti-CRP antibodies and SARS-CoV nucleoprotein). The study explores factors influencing DDI-based immunosensor performance, including the purity of protein-DNA conjugates and the stability of their duplexes with DNA and analogues. It also provides insight into backfilling agent type and probe surface density. The research reveals that single-component monolayers lack protection against protein adsorption, while mixing the probes with long-chain ligands may hinder DNA-protein conjugate anchoring. Conventional DNA probes offer slightly higher surface density, while ZNA® probes exhibit better binding efficiency. Despite no enhanced stability in different ionic strength media, the cost-effectiveness of DNA probes led to their preference. The findings contribute to advancing microarray technology, paving the way for new generations of DDI-based multiplex platforms for rapid and robust diagnostics.

Effect of fiber layer formation on mechanical and wear properties of natural fiber filled epoxy hybrid composites.

Natural fiber-reinforced polymer matrix composites are gathering significance in future trend applications such as automotive, aerospace, sport, and other engineering applications due to their superior enhanced mechanical, wear, and thermal properties. Compared to synthetic fiber, natural fiber is low adhesive and flexural strength properties. The research aims to synthesize the epoxy hybrid composites by utilizing the silane (pH = 4) treated Kenaf (KF) and sisal fiber (SF) as layering by uni, bi, and multi-unidirectional via hand layup techniques. Thirteen composite samples have been prepared by three-layer formation adopted with different weight ratios of E/KF/SF such as 100E/0KF/0SF, 70E/30KF/0SF, 70E/0KF/30SF, 70E/20KF/10SF, and 70E/10KF/20SF respectively. The effect of layer formation on the tensile, flexural, and impact strength of composites is studied by ASTM D638, D790, and D256 standards. The unidirectional fiber layer formed (sample 5) 70E/10KF/20SF composite is found maximum tensile and flexural strength of 57.9 ± 1.2 MPa and 78.65 ± 1.8 MPa. This composite is subjected to wear studies by pin-on-disc wear apparatus configured with a hardened grey cast-iron plate under an applied load of 10, 20, 30, and 40 N at different sliding velocities of 0.1, 0.3, 0.5, and 0.7 m/s. The wear rate of the sample progressively increases with increasing load and sliding speed of the composite. The minimum wear rate of 0.012 mg/min (sample 4) is found on 7.6 N frictional force at 0.1 m/s sliding speed. Moreover, sample 4 at a high velocity of 0.7 m/s with a low load (10 N) shows a wear rate of 0.034 mg/min. The wear-worn surface is examined and found adhesive and abrasive wear on a high frictional force of 18.54 N at 0.7 m/s. The enhanced mechanical and wear behavior of sample 5 is recommended for automotive seat frame applications.

Simulation-Assisted Process Design and Experimental Verification of Laterally Confined Oxide Areas Generated with Continuous Electrolytic Free Jet on EN AW-7075 Aluminum Alloy.

Local anodization with a free electrolyte jet is a suitable solution for locally confined surface functionalization without additionally required preparation of the parts. However, the geometrical formation of the anodic oxide layer in jet-based anodization is not yet sufficiently understood. In this study, numerical calculations based on physical descriptions are used to describe the lateral and vertical oxide formation on aluminum alloy EN AW-7075. The required electrical resistance and capacitance were determined by immersion-based anodization and implemented into the numerical simulation model to evaluate the electrical conductivity of the porous layer. The simulation results showed an electrical conductivity of 2.6 × 10-6 S/m for the porous layer. Subsequently, a model for jet-based anodization was developed and the previous results were implemented to calculate the oxide formation. The simulation results showed decreasing oxide layer thickness at increasing radial distance from the center of the jet, which corresponds to experimental results. The simulation model was validated by varying the current efficiency from 5% to 90%, where similar developments of the anodic oxide layer thickness compared with experimental results were determined at 5%.

YAP1 is essential for self-organized differentiation of pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) form aggregates that recapitulate aspects of the self-organization in early embryogenesis. Within few days, cells undergo a transition from epithelial-like structures to organized three-dimensional embryoid bodies (EBs) with upregulation of germ layer-specific genes. However, it is largely unclear, which signaling cascades regulate self-organized differentiation. The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. YAP1 has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human iPSCs remains to be elucidated. To gain insights into the function of YAP1 in early cell-fate decisions, we generated YAP1 knockout (YAP-/-) iPSC lines with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. YAP-/- iPSCs showed increased expression of several YAP1 targets and of NODAL, an important regulator of cell differentiation. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies towards endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in undirected self-organized YAP-/- EBs germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the undirected self-organization of EBs, and this is at least partly attributed to activation of the NODAL signaling.

Impact of polysaccharide and protein interactions on membrane fouling: Particle deposition and layer formation.

Membrane fouling, which limits the application of membrane bioreactors, has received considerable research attention in recent years. In this work, filtration modeling was performed in combination with surface plasmon resonance (SPR) analysis to investigate the membrane fouling mechanism. Sodium alginate (SA) and bovine serum albumin (BSA) were used to perform dead-end filtration on hydrophilic and hydrophobic poly (vinylidene fluoride) (PVDF) membranes. The initial foulant deposition and layer formation on membranes as well as the interaction between the BSA and SA were comprehensively analyzed. Results indicated that during SA filtration, initial fouling on hydrophilic membranes were primarily attributed to the particle-membrane interactions, while the fouling on the hydrophobic membrane were dominantly caused by the interactions among SA particles. The interaction between BSA and SA led to more severe membrane fouling and hydrophobic membrane was more sensitive to it, especially in the initial filtration process. The SPR results helped clarify the in-situ deposition behavior of BSA and SA particles on the PVDF surface. Compared to SA, BSA adsorbed faster on the PVDF membrane, and specific interactions played an essential role in BSA adsorption, whereas the deposition of SA on PVDF could be easily removed by shear force. Interactions between BSA and SA could alleviate the bonding between BSA and the PVDF membrane.

Determination of Compressibility and Relaxation Behavior of Yeast Cell Sediments by Analytical Centrifugation and Comparison with Deposit Formation on Membrane Surfaces.

Separation of cells from produced biomolecules is a challenging task in many biotechnological downstream operations due to deposit formation of the retained cells, affecting permeation of the target product. Compression and relaxation behavior of cell deposits formed during filtration are important factors affecting operational performance. The determination of these factors by flux or pressure stepping experiments is time- and labor-intensive. In this work, we propose a screening method by analytical centrifugation, which is capable of detecting small differences in compression and relaxation behavior induced by milieu changes, using a model system comprised of washed and unwashed yeast cells in the presence or absence of bovine serum albumin as a model target protein. The main effects observed were firstly the impact of pH value, affecting interaction of bovine serum albumin and yeast cells especially close to the isoelectric point, and secondly the effect of washing the yeast cells prior to analysis, where the presence of extracellular polymeric substances led to higher compressibility of the deposited cells. By comparing and validating the obtained results with dead-end filtration trials, the stabilizing role of bovine serum albumin in deposits formed at low pH values due to interactions with the yeast cells was underlined.

An Optimalization Study on the Surface Texture and Machining Parameters of 60CrMoV18-5 Steel by EDM.

As a non-conventional machining technology, EDM is used extensively in modern industry, particularly in machining difficult-to-cut materials. CALMAX is a chromium-molybdenum-vanadium tool steel with exceptional toughness, ductility, and wear resistance that has a wide range of applications. Despite the fact that EDM is routinely used in CALMAX machining, the related published research is brief and limited. The current research gives a complete experimental study of CALMAX machining using EDM. A Taguchi Design of Experiment (DOE) was used, using pulse-on current, pulse-on time, and open-circuit voltage as control parameters. Material Removal Rate (MRR), Tool Material Removal Rate (TMRR), and Tool Wear Ratio (TWR) were used to evaluate machining performance, while Ra and Rz were used to estimate Surface Quality (SQ). The produced White Layer (WL) parameters were determined using optical and SEM microscopy, as well as EDX measurements and micro-hardness studies. Finally, for each of the aforementioned indexes, Analysis of Variance (ANOVA) was performed, and multi-objective optimization was based on Grey Relational Analysis (GRA). The results show that higher open-circuit voltage produces lower WL thickness, although by increasing the pulse-on time, the TWR is increased. The average hardness of the WL is increased about 400% compared to the micro-hardness of the bulk material.

Dab1-deficient deep layer neurons prevent Dab1-deficient superficial layer neurons from entering the cortical plate.

The mammalian neocortex has a 6-layered cytoarchitecture, where early- and late-born neurons are positioned deeply and superficially, respectively. Inverted lamination has been observed in mice defective in the Reelin/Disabled-1 (Dab1) pathway. Considering that Dab1-deficient superficial layer neurons can migrate into the Dab1 +/+ cortical plate and that Dab1 is thought to function cell-autonomously, it is unclear why superficial layer neurons are positioned below deep layer neurons in Reelin/Dab1-deficient mice. Here, we reconfirmed that Dab1 -/- superficial layer neurons enter the cortical plate using in utero electroporation on embryonic day (E) 14.5 Dab1-floxed mice. Electroporation in E12.5 Dab1-floxed mice reconfirmed that many deep layer neurons were mispositioned below the subplate. We also found an accumulation of Dab1-deficient superficial layer neurons below the cortical plate in many of these brains, in which deep layer neurons below the subplate showed high cell density. These phenotypes were rescued by decreasing the knockout probability and by expressing Dab1 in deep layer neurons. These observations suggest that cell-dense Dab1 -/- deep layer neurons prevent Dab1 -/- superficial layer neurons from entering the cortical plate. This reflects a non-cell-autonomous function of Dab1 and may explain the preplate splitting failure and outside-in lamination observed in Reelin/Dab1-deficient mice.