Monitoring of Chlorpyrifos Residues in Corn Oil Based on Raman Spectral Deep-Learning Model.

This study presents a novel method for the quantitative detection of residual chlorpyrifos in corn oil through Raman spectroscopy using a combined long short-term memory network (LSTM) and convolutional neural network (CNN) architecture. The QE Pro Raman+ spectrometer was employed to collect Raman spectra of corn oil samples with varying concentrations of chlorpyrifos residues. A deep-learning model based on LSTM combined with a CNN structure was designed to realize feature self-learning and model training of Raman spectra of corn oil samples. In the study, it was discovered that the LSTM-CNN model has superior generalization performance compared to both the LSTM and CNN models. The root-mean-square error of prediction (RMSEP) of the LSTM-CNN model is 12.3 mg·kg-1, the coefficient of determination (RP2) is 0.90, and the calculation of the relative prediction deviation (RPD) results in a value of 3.2. The study demonstrates that the deep-learning network based on an LSTM-CNN structure can achieve feature self-learning and multivariate model calibration for Raman spectra without preprocessing. The results of this study present an innovative approach for chemometric analysis using Raman spectroscopy.

Commensal microbiome promotes hair follicle regeneration by inducing keratinocyte HIF-1α signaling and glutamine metabolism.

Tissue injury induces metabolic changes in stem cells, which likely modulate regeneration. Using a model of organ regeneration called wound-induced hair follicle neogenesis (WIHN), we identified skin-resident bacteria as key modulators of keratinocyte metabolism, demonstrating a positive correlation between bacterial load, glutamine metabolism, and regeneration. Specifically, through comprehensive multiomic analysis and single-cell RNA sequencing in murine skin, we show that bacterially induced hypoxia drives increased glutamine metabolism in keratinocytes with attendant enhancement of skin and hair follicle regeneration. In human skin wounds, topical broad-spectrum antibiotics inhibit glutamine production and are partially responsible for reduced healing. These findings reveal a conserved and coherent physiologic context in which bacterially induced metabolic changes improve the tolerance of stem cells to damage and enhance regenerative capacity. This unexpected proregenerative modulation of metabolism by the skin microbiome in both mice and humans suggests important methods for enhancing regeneration after injury.

Wound-Induced Hair Neogenesis Model.

Skin wounds in adult mammals typically heal with a fibrotic scar and fail to restore ectodermal appendages, such as hair follicles or adipose tissue. Intriguingly, new hair follicles regenerate in the center of large full-thickness wounds of mice in a process called wound-induced hair neogenesis (WIHN). WIHN is followed by neogenesis of dermal adipose tissue. Both neogenic events reactivate embryonic-like cellular and molecular programs. The WIHN model provides a platform for studying mammalian regeneration, and findings from this model could instruct future regenerative medicine interventions for treating wounds and alopecia. Since Ito et al. rediscovered WIHN 15 years ago, numerous investigators have worked on the WIHN model using varying wounding protocols and model interpretations. Because a variety of factors, including environmental variables and choice of mouse strains, can affect the outcomes of a WIHN study, the purpose of this article is to provide an overview of the experimental variables that impact WIHN so that experiments between laboratories can be compared in a meaningful manner.

Toward Understanding Wound Immunology for High-Fidelity Skin Regeneration.

Effective tissue repair is vital for the survival of organisms. Yet, how the immune system coordinates with tissue stem cells (SCs) to effect postnatal tissue restoration remains elusive. This review presents current knowledge surrounding wound-induced SC and immune signaling that favors tissue repair, including wound healing and regeneration. We discuss factors that affect regenerative capacities among organisms and the dynamics of local immune cells and SCs during reepithelialization. We also present recent insights into how immune niches communicate with SCs or other body systems to restore the epithelial architecture. Additionally, we summarize our findings on functional wound regeneration, specifically how alarmin (double-stranded RNA [dsRNA])-activated Toll-like receptor signaling and host-microbe interaction-related immune pathways alter the regenerative property of skin SCs. Last, we touch on mechanisms by which known immunologic cellular and molecular signaling might boost the skin's regenerative property. Overall, this review will provide insights into how therapeutically modulating immune signaling could enhance postnatal tissue regeneration.

Mechanical tension mobilizes Lgr6+ epidermal stem cells to drive skin growth.

Uniquely among mammalian organs, skin is capable of marked size change in adults, yet the mechanisms underlying this notable capacity are unclear. Here, we use a system of controlled tissue expansion in mice to uncover cellular and molecular determinants of skin growth. Through machine learning-guided three-dimensional tissue reconstruction, we capture morphometric changes in growing skin. We find that most growth is driven by the proliferation of the epidermis in response to mechanical tension, with more limited changes in dermal and subdermal compartments. Epidermal growth is achieved through preferential activation and differentiation of Lgr6+ stem cells of the epidermis, driven in part by the Hippo pathway. By single-cell RNA sequencing, we uncover further changes in mechanosensitive and metabolic pathways underlying growth control in the skin. These studies point to therapeutic strategies to enhance skin growth and establish a platform for understanding organ size dynamics in adult mammals.

Edaravone activates the GDNF/RET neurotrophic signaling pathway and protects mRNA-induced motor neurons from iPS cells.

BACKGROUND: Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. METHODS: We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. RESULTS: We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. CONCLUSIONS: This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.

Bacteria induce skin regeneration via IL-1ß signaling.

Environmental factors that enhance regeneration are largely unknown. The immune system and microbiome are attributed roles in repairing and regenerating structure but their precise interplay is unclear. Here, we assessed the function of skin bacteria in wound healing and wound-induced hair follicle neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers correlate with bacterial counts, being lowest in germ-free (GF), intermediate in conventional specific pathogen-free (SPF), and highest in wild-type mice, even those infected with pathogenic Staphylococcus aureus. Reducing skin microbiota via cage changes or topical antibiotics decreased WIHN. Inflammatory cytokine IL-1ß and keratinocyte-dependent IL-1R-MyD88 signaling are necessary and sufficient for bacteria to promote regeneration. Finally, in a small trial, a topical broad-spectrum antibiotic also slowed skin wound healing in adult volunteers. These results demonstrate a role for IL-1ß to control morphogenesis and support the need to reconsider routine applications of topical prophylactic antibiotics.

Frondoside A Inhibits an MYC-Driven Medulloblastoma Model Derived from Human-Induced Pluripotent Stem Cells.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the MYC oncogene combined with p53 loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non-small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.

Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage-specific neurons. However, current strategies mainly rely on genome-integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons. mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications led to higher and more stable protein expression, and induced more efficient neuron conversion, as compared to mRNAs coding wild-type proteins. Using these two modified mRNAs with morphogens, we established a 5-day protocol that can rapidly generate mDA neurons with >90% purity from normal and Parkinson's disease iPSCs. After in vitro maturation, these mRNA-induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high-content neuron cultures for drug discovery. Proteomic analysis of Atoh1-binding proteins identified the nonmuscle myosin II (NM-II) complex as a new binding partner of nuclear Atoh1. The NM-II complex, commonly known as an ATP-dependent molecular motor, binds more strongly to phosphosite-modified Atoh1 than the wild type. Blebbistatin, an NM-II complex antagonist, and bradykinin, an NM-II complex agonist, inhibited and promoted, respectively, the transcriptional activity of Atoh1 and the efficiency of miDA neuron generation. These findings established the first mRNA-driven strategy for efficient iPSC differentiation to mDA neurons. We further identified the NM-II complex as a positive modulator of Atoh1-driven neuron differentiation. The methodology described here will facilitate the development of mRNA-driven differentiation strategies for generating iPSC-derived progenies widely applicable to disease modeling and cell replacement therapy. Stem Cells Translational Medicine 2019;8:112&12.

The Effects of Quinine on Neurophysiological Properties of Dopaminergic Neurons.

Quinine is an antimalarial drug that is toxic to the auditory system by commonly inducing hearing loss and tinnitus, presumably due to its ototoxic effects on disruption of cochlear hair cells and blockade of ion channels of neurons in the auditory system. To a lesser extent, quinine also causes ataxia, tremor, and dystonic reactions. As dopaminergic neurons are implicated to play a role in all of these diseases, we tested the toxicity of quinine on induced dopaminergic (iDA) neurons derived from human pluripotent stem cells (iPSCs) and primary dopaminergic (DA) neurons of substantia nigra from mice brain slices. Patch clamp recordings and combined drug treatments were performed to examine key physiological properties of the DA neurons. We found that quinine (12.5-200 µM) depolarized the resting membrane potential and attenuated the amplitudes of rebound spikes induced by hyperpolarization. Action potentials were also broadened in spontaneously spiking neurons. In addition to quinine attenuating hyperpolarization-dependent conductance, the tail currents following withdrawal of hyperpolarizing currents were also attenuated. Taken together, we found that iPSC-derived DA neurons recapitulated all the tested physiological properties of human DA neurons, and quinine had distinct effects on the physiology of both iDA and primary DA neurons. This toxicity of quinine may be the underlying mechanism for the movement disorders of cinchonism or quinism and may play a role in tinnitus modulation.

Genome-wide prediction of DNase I hypersensitivity using gene expression.

We evaluate the feasibility of using a biological sample's transcriptome to predict its genome-wide regulatory element activities measured by DNase I hypersensitivity (DH). We develop BIRD, Big Data Regression for predicting DH, to handle this high-dimensional problem. Applying BIRD to the Encyclopedia of DNA Elements (ENCODE) data, we found that to a large extent gene expression predicts DH, and information useful for prediction is contained in the whole transcriptome rather than limited to a regulatory element's neighboring genes. We show applications of BIRD-predicted DH in predicting transcription factor-binding sites (TFBSs), turning publicly available gene expression samples in Gene Expression Omnibus (GEO) into a regulome database, predicting differential regulatory element activities, and facilitating regulome data analyses by serving as pseudo-replicates. Besides improving our understanding of the regulome-transcriptome relationship, this study suggests that transcriptome-based prediction can provide a useful new approach for regulome mapping.

Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile.

Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel's Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj's findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis.