Neuroligin-3 and neuroligin-4X form nanoscopic clusters and regulate growth cone organization and size.

The cell-adhesion proteins neuroligin-3 and neuroligin-4X (NLGN3/4X) have well described roles in synapse formation. NLGN3/4X are also expressed highly during neurodevelopment. However, the role these proteins play during this period is unknown. Here we show that NLGN3/4X localized to the leading edge of growth cones where it promoted neuritogenesis in immature human neurons. Super-resolution microscopy revealed that NLGN3/4X clustering induced growth cone enlargement and influenced actin filament organization. Critically, these morphological effects were not induced by autism spectrum disorder (ASD)-associated NLGN3/4X variants. Finally, actin regulators p21-activated kinase 1 and cofilin were found to be activated by NLGN3/4X and involved in mediating the effects of these adhesion proteins on actin filaments, growth cones and neuritogenesis. These data reveal a novel role for NLGN3 and NLGN4X in the development of neuronal architecture, which may be altered in the presence of ASD-associated variants.

Schizophrenia risk proteins ZNF804A and NT5C2 interact in cortical neurons.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.

Enabling the characterization of the nonlinear electrokinetic properties of particles using low voltage.

Insulator-based electrokinetically driven microfluidic devices stimulated with direct current (DC) voltages are an attractive solution for particle separation, concentration, or isolation. However, to design successful particle manipulation protocols, it is mandatory to know the mobilities of electroosmosis, and linear and nonlinear electrophoresis of the microchannel/liquid/particle system. Several techniques exist to characterize the mobilities of electroosmosis and linear electrophoresis. However, only one method to characterize the mobility of nonlinear electrophoresis has been thoroughly assessed, which generally requires DC voltages larger than 1000 V and measuring particle velocity in a straight microchannel. Under such conditions, Joule heating, electrolysis, and the DC power source cost become a concern. Also, measuring particle velocity at high voltages is noisy, limiting characterization quality. Here we present a protocol-tested on 2 µm polystyrene particles-for characterizing the mobility of nonlinear electrophoresis of the liquid/particle system using a DC voltage of only 30 V and visual inspection of particle dynamics in a microchannel featuring insulating obstacles. Multiphysics numerical modelling was used to guide microchannel design and to correlate particle location during an experiment with electric field intensity. The method was validated against the conventional characterization protocol, exhibiting excellent agreement while significantly reducing measurement noise and experimental complexity.

Molecular diversity and polyparasitism of avian trypanosomes in the Brazilian Atlantic Rainforest.

The current study proposes to investigate the diversity and phylogeny of trypanosomes parasitizing wild birds from the Brazilian Atlantic Forest. Cytological examination was carried out by light microscopy of blood smears and positive birds were selected for amplification of the 18S rDNA sequence through PCR. The resulting amplicons were subjected to purification, cloning, and sequencing analysis. Phylogenetic reconstruction was conducted, including all avian trypanosomes representative's lineages. A total of ten bird samples from species of Turdus flavipes (N=1/12), T. albicollis (N=1/8), Tachyphonus coronatus (N=6/121), Thamnophilus caerulescens (N=1/22) and Synallaxis spixi (N=1/8) were positive for Trypanosoma spp. In the six specimens of T. coronatus, five distinct lineages of Trypanosoma spp. 18S-rRNA were observed in ninety sequences obtained, and using the strategy of cloning independent PCR, it was possible to observe that two of them were related to T. avium (JB01/JB02), and three were closed related to T. bennetti (JB03/ JB04/JB05). Addionaly, all fifteen sequences obtained from T. caerulescens/ S. spixi/T. flavipes/T. albicollis were identical. The present research is the first study to access molecular diversity and polyparasitism by avian trypanosomes in Brazil. The current research exhibits the wide genetic variability in avian trypanosomes and its non-specific relationship with its avian hosts.

Acute IL-6 exposure triggers canonical IL6Ra signaling in hiPSC microglia, but not neural progenitor cells.

BACKGROUND: Prenatal exposure to elevated interleukin (IL)-6 levels is associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ), autism spectrum condition (ASC) and bipolar disorder (BD). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterized the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. RESULTS: We observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both protein and transcript levels due to the absence of IL6R expression and soluble (s)IL6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL6, JMJD3 and IL10 expression in MGL, confirming activation of canonical IL6Ra signaling. Bulk RNAseq identified 156 up-regulated genes (FDR < 0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an up-regulated gene set from human post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility, consistent with gene ontology pathways highlighted from the RNAseq data and replicating rodent model indications that IRF8 regulates microglial motility. Finally, IL-6 induces MGLs to secrete CCL1, CXCL1, MIP-1α/ß, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 after 3 h and 24 h. CONCLUSION: Our data provide evidence for cell specific effects of acute IL-6 exposure in a human model system, ultimately suggesting that microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.

A critical review on the fabrication techniques that can enable higher throughput in dielectrophoresis devices.

The sorting of targeted cells in a sample is a cornerstone of healthcare diagnostics and therapeutics. This work focuses on the use of dielectrophoresis for the selective sorting of targeted bioparticles in a sample and how the lack of throughput has been one important practical challenge to its widespread practical implementation. Increasing the cross-sectional area of a channel can lead to higher flow rates and thus the capability to process a larger sample volume per unit of time. However, the required electric field gradient that is generated by polarized electrodes drastically decreases as one moves away from the electrodes. Hence, the scaling up of the channel cross section must be done asymmetrically. One desires a channel aspect ratio AR = height/width that is much smaller or much larger than 1. Since reducing footprint of the DEP device is important to ensure affordability, the use of channels with AR ≫ 1 is desired. This creates the challenge to fabricate electrodes on the sidewalls of multiple channels with AR ≫ 1, or a channel embedding an array of electrodes with a gap in between them with AR ≫ 1. This critical review first details the motivation for using three-dimensional (3D) DEP devices to improve throughput and then describes selected techniques that have been used to fabricate them. Techniques include electrodeposition, deep etching, thick-film photolithography, and co-fabrication. Electrode materials addressed include metals, silicon, carbon, PDMS-based composites as well as conductive polymers and fluids.

Electrically driven microfluidic platforms for exosome manipulation and characterization.

Exosomes are small extracellular vesicles that can be obtained from several body fluids such as blood and urine. Since these vesicles can carry biomarkers and other cargo, they have application in healthcare diagnostics and therapeutics, such as liquid biopsies and drug delivery. Yet, their identification and separation from a sample remain challenging due to their high degree of heterogeneity and their co-existence with other bioparticles. In this contribution, we review the state-of-the-art on electrical techniques and methods to displace, selectively trap/isolate, and detect/characterize exosomes in microfluidic devices. Although there are many reviews focused on exosome separation using benchtop equipment, such as ultracentrifugation, there are limited reviews focusing on the use of electrical phenomena in microfluidic devices for exosome manipulation and detection. Here, we highlight contributions published during the past decade and present perspectives for this research field for the near future, outlining challenges to address in years to come.

Attenuated transcriptional response to pro-inflammatory cytokines in schizophrenia hiPSC-derived neural progenitor cells.

Maternal immune activation (MIA) during prenatal development is an environmental risk factor for psychiatric disorders including schizophrenia (SZ). Converging lines of evidence from human and animal model studies suggest that elevated cytokine levels in the maternal and fetal compartments are an important indication of the mechanisms driving this association. However, there is variability in susceptibility to the psychiatric risk conferred by MIA, likely influenced by genetic factors. How MIA interacts with a genetic profile susceptible to SZ is challenging to test in animal models. To address this gap, we examined whether differential gene expression responses occur in forebrain-lineage neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC) generated from three individuals with a diagnosis of schizophrenia and three healthy controls. Following acute (24 h) treatment with either interferon-gamma (IFNγ; 25 ng/µl) or interleukin (IL)-1ß (10 ng/µl), we identified, by RNA sequencing, 3380 differentially expressed genes (DEGs) in the IFNγ-treated control lines (compared to untreated controls), and 1980 DEGs in IFNγ-treated SZ lines (compared to untreated SZ lines). Out of 4137 genes that responded significantly to IFNγ across all lines, 1223 were common to both SZ and control lines. The 2914 genes that appeared to respond differentially to IFNγ treatment in SZ lines were subjected to a further test of significance (multiple testing correction applied to the interaction effect between IFNγ treatment and SZ diagnosis), yielding 359 genes that passed the significance threshold. There were no differentially expressed genes in the IL-1ß-treatment conditions after Benjamini-Hochberg correction. Gene set enrichment analysis however showed that IL-1ß impacts immune function and neuronal differentiation. Overall, our data suggest that a) SZ NPCs show an attenuated transcriptional response to IFNγ treatment compared to controls; b) Due to low IL-1ß receptor expression in NPCs, NPC cultures appear to be less responsive to IL-1ß than IFNγ; and c) the genes differentially regulated in SZ lines - in the face of a cytokine challenge - are primarily associated with mitochondrial, "loss-of-function", pre- and post-synaptic gene sets. Our findings particularly highlight the role of early synaptic development in the association between maternal immune activation and schizophrenia risk.

Identifying FDA-approved drugs with multimodal properties against COVID-19 using a data-driven approach and a lung organoid model of SARS-CoV-2 entry.

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Caudal Extension Graft of the Lower Lateral Cartilage: Technique and Aesthetic and Functional Results.

Here we describe a new technique to deal with alar retraction, a highly undesirable imperfection of the nose. The procedure involves placing a caudal extension graft below the vestibular portion of the lower lateral cartilage (LLC) after its detachment from the vestibular skin. The graft is fixed to the cartilage and, subsequently, to the vestibular tissue. The present retrospective study included 20 patients, 11 females and 9 males, with a mean age of 28.90 years. Follow-up ranged from 1 to 18 months. Surgery improved alar notching to a smoother dome shape and nostril exposure was reduced in every patient. The caudal extension graft of the LLC contributed to rise in overall patient satisfaction, as revealed by the postoperative increase of the Rhinoplasty Outcomes Evaluation (ROE) mean score from 40.0 to 79.17 (p < 0.0001). It also contributed to and improved functional outcomes, as indicated by the decrease of the Nasal Obstruction Symptom Evaluation (NOSE) mean score from 52.75 to 13.25 (p = 0.0001). Sex did not affect the mean ROE and NOSE scores. Thus, increased patient satisfaction measured by the ROE is present in both sexes and at both age groups but it is better detected in the first year after surgery. Functional improvements analyzed with NOSE are best detected in patients aged ≥ 30 years and in follow-ups of 11 months. The caudal extension graft of the LLC technique described herein effectively and safely corrects alar retraction and the collapse of the nasal valve while filling the soft triangle.

Nondimensional Streaming Dielectrophoresis Number for a System of Continuous Particle Separation.

Cell sorting methods are required in numerous healthcare assays. Although flow cytometry and magnetically actuated sorting are widespread techniques for cell sorting, there is intense research on label-free techniques to reduce the cost and complexity of the process. Among label-free techniques, dielectrophoresis (DEP) offers the capability to separate cells not only on the basis of size but also on their membrane capacitance. This is important because it enables cell discrimination on the basis of specific traits such as viability, identity, fate, and age. StreamingDEP refers to the continuous sorting of cells thanks to the generation of streams of targeted particles by equilibrating the drag and DEP forces acting on targeted particles. In this work, we provide an analytical expression for a streamingDEP number toward enabling the a priori design of DEP devices to agglomerate targeted particles into streams. The nondimensional streamingDEP number (SDN) obtained in this analysis is applied to experiments with 1 µm polystyrene particles and Candida cells. On the basis of these experiments, three characteristic zones are mapped to different values of the SDN: (1) physical capture thanks to DEP for 0 < SDN < 0.6; (2) streaming due to DEP for 0.6 < SDN < 1; (3) elution without experiencing DEP for SDN > 1.

Dielectrophoretic characterization and separation of monocytes and macrophages using 3D carbon-electrodes.

Monocyte heterogeneity and its prevalence are revealed as indicator of several human diseases ranking from cardiovascular diseases to rheumatoid arthritis, chronic kidney diseases, autoimmune multiple sclerosis, and stroke injuries. When monocytes and macrophages are characterized and isolated with preserved genetic, phenotypic and functional properties, they can be used as label-free biomarkers for precise diagnostics and treatment of various diseases. Here, the dielectrophoretic responses of the monocytes and macrophages were examined. We present 3D carbon-electrode dielectrophoresis (carbon-DEP) as a separation tool for U937 monocytes and U937 monocyte-differentiated macrophages. The carbon-electrodes advanced the usability and throughput of DEP separation, presented wider electrochemical stability. Using the 3D carbon-DEP chip, we first identified the selective positive and negative DEP responses and specific crossover frequencies of monocytes and macrophages as their signatures for separation. The crossover frequency of monocytes and macrophages was 17 and 30 kHz, respectively. Next, we separated monocyte and macrophage subpopulations using their specific dielectrophoretic responses. Afterward, we used a fluorescence-activated cell sorter to confirm our results. Finally, we enriched 70% of monocyte cells from the mixed cell population, in other words, concentration of monocyte cells to macrophage cells was five times increased, using the 30-kHz, 10-Vpp electric field and 1 µL/min flow rate.

Experimental and theoretical investigation of long-wavelength fluorescence emission in push-pull benzazoles: intramolecular proton transfer or charge transfer in the excited state?

This study presents the synthesis, characterisation and theoretical calculations of compounds that contain electron donor and withdrawing groups connected through a π-conjugated benzazolic structure. The compounds in solution show an absorption maximum in the UV-visible spectrum (380-390 nm) due to spin and symmetry allowed electronic 1ππ* transitions with no clear evidence for charge transfer in either compound in the ground state. A fluorescence emission located in the violet-blue-green region, tailored by solvent polarity, with a large Stokes shift was observed. Taking the long-wavelength emission into account, the Lippert-Mataga plot indicates a positive solvatochromism in the solvent polarity function (Δf) range 0.02-0.20, related to the occurrence of an ICT mechanism in the excited state. At Δf greater than 0.20, the polarity of the medium seems no longer to increase the stabilization of the compounds, reaching a plateau. Time-dependent density functional theory (TD-DFT) and resolution-of-identity second-order approximate coupled-cluster (RI-CC2) calculations were also used to better understand the excited state of these compounds. The results indicated that ESIPT was disfavoured in the compounds, mainly in polar solvents, and the emission wavelengths were primarily associated with ICT. In summary, in these push-pull compounds, the electron donating and withdrawing groups do not favour the ESIPT process.

Dielectrophoretic Separation of Live and Dead Monocytes Using 3D Carbon-Electrodes.

Blood has been the most reliable body fluid commonly used for the diagnosis of diseases. Although there have been promising investigations for the development of novel lab-on-a-chip devices to utilize other body fluids such as urine and sweat samples in diagnosis, their stability remains a problem that limits the reliability and accuracy of readouts. Hence, accurate and quantitative separation and characterization of blood cells are still crucial. The first step in achieving high-resolution characteristics for specific cell subpopulations from the whole blood is the isolation of pure cell populations from a mixture of cell suspensions. Second, live cells need to be purified from dead cells; otherwise, dead cells might introduce biases in the measurements. In addition, the separation and characterization methods being used must preserve the genetic and phenotypic properties of the cells. Among the characterization and separation approaches, dielectrophoresis (DEP) is one of the oldest and most efficient label-free quantification methods, which directly purifies and characterizes cells using their intrinsic, physical properties. In this study, we present the dielectrophoretic separation and characterization of live and dead monocytes using 3D carbon-electrodes. Our approach successfully removed the dead monocytes while preserving the viability of the live monocytes. Therefore, when blood analyses and disease diagnosis are performed with enriched, live monocyte populations, this approach will reduce the dead-cell contamination risk and achieve more reliable and accurate test results.

Genome-wide significant schizophrenia risk variation on chromosome 10q24 is associated with altered cis-regulation of BORCS7, AS3MT, and NT5C2 in the human brain.

Chromosome 10q24.32-q24.33 is one of the most robustly supported risk loci to emerge from genome-wide association studies (GWAS) of schizophrenia. However, extensive linkage disequilibrium makes it difficult to distinguish the actual susceptibility gene(s) at the locus, limiting its value for improving biological understanding of the condition. In the absence of coding changes that can account for the association, risk is likely conferred by altered regulation of one or more genes in the region. We, therefore, used highly sensitive measures of allele-specific expression to assess cis-regulatory effects associated with the two best-supported schizophrenia risk variants (SNP rs11191419 and indel ch10_104957618_I/rs202213518) on the primary positional candidates BORCS7, AS3MT, CNNM2, and NT5C2 in the human brain. Heterozygosity at rs11191419 was associated with increased allelic expression of BORCS7 and AS3MT in the fetal and adult brain, and with reduced allelic expression of NT5C2 in the adult brain. Heterozygosity at ch10_104957618_I was associated with reduced allelic expression of NT5C2 in both the fetal and adult brain. Comparisons between cDNA ratios in heterozygotes and homozygotes for the risk alleles indicated that cis-effects on NT5C2 expression in the adult dorsolateral prefrontal cortex could be largely accounted for by genotype at these two risk variants. While not excluding effects on other genes in the region, this study implicates altered neural expression of BORCS7, AS3MT, and NT5C2 in susceptibility to schizophrenia arising from genetic variation at the chromosome 10q24 locus. © 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc.

Massive Pilomatrixoma of the Scalp: A Case Report.

Pilomatrixoma, also called epithelioma of Malherbe, is a benign neoplasm derived from hair follicle matrix cells. It usually presents as a solitary mass in the head and neck region and is more frequent in children and young adults, females, and the Caucasian population. Lesions equal to or greater than 5 cm are categorized as giant pilomatrixomas. We present a case of a 75-year-old female, with no known medical history, who was brought to the emergency department (ED) after falling on the street. She had a giant soft head tissue tumor, severe anemia due to intralesional chronic small hemorrhages and folates and cobalamin deficiencies, and delirant speech. The anatomopathological result of the biopsy of the tumor revealed to be a pilomatrixoma. The patient was then referred to plastic surgery, with complete excision of the tumor. After surgery, she was transferred to the psychiatric team, who assumed the delirant speech to be in the context of schizophrenia. She was discharged four months after admission.

The Effect of Different System Parameters on the Movement of Microbial Cells Using Light-Induced Dielectrophoresis.

The manipulation of single particles remains a topic of interest with many applications. Here we characterize the impact of selected parameters on the motion of single particles thanks to dielectrophoresis (DEP) induced by visible light, in a technique called Light-induced Dielectrophoresis, or LiDEP, also known as optoelectronic tweezers, optically induced DEP, and image-based DEP. Baker's yeast and Candida cells are exposed to an electric field gradient enabled by shining a photoconductive material with a specific pattern of visible light, and their response is measured in terms of the average cell velocity towards the gradient. The impact on cell velocity when varying the shape and color of the light pattern, as well as the distance from the cell to the pattern, is presented. The experimental setup featured a commercial light projector featuring digital light processing (DLP) technology but mechanically modified to accommodate a 40× microscope objective lens. The minimal resolution achieved on the light pattern was 8 µm. Experimental results show the capability for single cell manipulation and the possibility of using different shapes, colors, and distances to determine the average cell velocity.

Integrating human endogenous retroviruses into transcriptome-wide association studies highlights novel risk factors for major psychiatric conditions.

Human endogenous retroviruses (HERVs) are repetitive elements previously implicated in major psychiatric conditions, but their role in aetiology remains unclear. Here, we perform specialised transcriptome-wide association studies that consider HERV expression quantified to precise genomic locations, using RNA sequencing and genetic data from 792 post-mortem brain samples. In Europeans, we identify 1238 HERVs with expression regulated in cis, of which 26 represent expression signals associated with psychiatric disorders, with ten being conditionally independent from neighbouring expression signals. Of these, five are additionally significant in fine-mapping analyses and thus are considered high confidence risk HERVs. These include two HERV expression signatures specific to schizophrenia risk, one shared between schizophrenia and bipolar disorder, and one specific to major depressive disorder. No robust signatures are identified for autism spectrum conditions or attention deficit hyperactivity disorder in Europeans, or for any psychiatric trait in other ancestries, although this is likely a result of relatively limited statistical power. Ultimately, our study highlights extensive HERV expression and regulation in the adult cortex, including in association with psychiatric disorder risk, therefore providing a rationale for exploring neurological HERV expression in complex neuropsychiatric traits.

Dielectrophoresis of lambda-DNA using 3D carbon electrodes.

Carbon electrodes have recently been introduced as an alternative to metal electrodes and insulator structures for dielectrophoretic applications. Here, an experimental and theoretical study employing an array of 3D carbon electrodes contained in a microfluidic channel for the dielectrophoretic manipulation of DNA is presented. First evidence that carbon-electrode DEP can be used for the manipulation and trapping of biomolecules such as DNA is reported. In particular, the dielectrophoretic response of λ-DNA (48.5 kbp) under various frequencies and flow conditions necessary for retention of λ-DNA are studied. Negative DEP is observed at frequencies above 75 kHz and positive DEP is present in the range below 75 kHz and down to 5 kHz. We further implement a theoretical model to capture the experimental findings in sufficient detail. Our theoretical considerations based on reported scaling laws for linear and supercoiled DNA further suggest that carbon-electrode DEP devices could be employed in future analytical applications such as DNA preconcentration and fractionation.