Neural Gene Expression Research Articles

Impact of cryopreservation on viability, gene expression and function of enteric nervous system derived neurospheres.

Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children's quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality. Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function. Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol (M1, slow freezing in fetal calf serum plus 10% DMSO) were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing. Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues.

Artificial neural networks vs. gene expression programming for predicting emission & engine efficiency of SI operated on blends of gasoline-methanol-hydrogen fuel

While retaining environmental friendliness, robust modelling and enhancing spark ignition engine efficacy can be done using improved innovative fuel and unconventional robust hybrid tools. This study is the first to employ Al techniques such as artificial neural networks (ANN) and gene expression programming (GEP) to predict the performance and emissions of a gasohol/hydrogen-powered SI engine. The ANN was adopted to correlate the engine variables viz. engine speed and gasohol/hydrogen mix vs. responses namely brake thermal efficiency (BTE), brake specific energy consumption (BSEC), carbon monoxide (CO), hydrocarbon (HC), oxides of nitrogen (NOx) and carbon dioxide (CO2). GEP model was further employed to predict BTE, BSEC, CO, HC, NOx and CO2. To examine the prediction efficacy of both AI techniques, a set of advanced statistical approaches was used. A set of advanced statistical techniques were employed to test the prediction efficiency of both AI techniques. It was revealed that ANN outperformed the GEP since the values for R in the case of ANN were 0.9864–0.9998 whereas the values for R in the case of GEP were 0.9864–0.9994. Similarly, in the instance of R2, ANN outperformed GEP. Furthermore, Kling-Gupta efficiency was greater in the case of ANN (0.9684–0.9999) than in GEP (0.8912–0.9991). Both AI approaches, however, displayed great prognostic effectiveness in forecasting engine performance and emissions.

Forecasting the seepage loss for lined and un-lined canals using artificial neural network and gene expression programming

Canal lining is customarily used to raise water-use effectiveness and reduce seepage loss. The major water losses in an irrigation channel are due to leakage and evaporation. The Egyptian General Integrated Management for Water Resources and Irrigation introduced a proposal for lining the Al-Hagar canal based on these losses. This study investigates the effect of lining in the Al-Hagar canal on flow characteristics, and compares the canal before and after introducing the lining. Additionally, it discusses the most common type of water loss, namely, losses due to seepage. Fieldwork was conducted on the Al-Hagar canal, Al-Saff Center, South of Helwan city, Egypt. The result revealed that the discharge of the canal after the lining is approximately 1.362–1.573 times greater than that of the un-lined section. Water losses in the Al-Hagar canal were 38.736% when un-lined but decreased to 29.253% when lined. The conveyance effectiveness in the un-lined canal, which is approximately 61.26%, increased to 70.75% when the entire canal is lined, which means a 9.483% improvement of conveyance. New relations were introduced using Artificial Neural Network and Gene Expression Programming to forecast the seepage loss in the lined and the un-lined canal as a function of Manning’s coefficient, Froude number and hydraulic radius. The consequences were better using the GEP program than using ANN for the lined and the un-lined canals. The value of the determination coefficient was 0.98, Correlation factor was 0.99, and the RMSE was 0.0017 for lined canals and the value of determination coefficient was 1, Correlation factor was 1, and the RMSE was 0.0003 for un-lined canals.

Carbon nanofibers fabrication, surface modifications, and application as the innovative substrate for electrical stimulation of neural cell differentiation

Engineered nanostructures are innovative and precisely designed, synthesized, and tailored with outstanding physicochemical properties that can be applied as the game-changer in neural tissue engineering. The present study aimed to develop an innovative approach based on electrical stimulation through a conductive scaffold to differentiate neural cells from human adipose mesenchymal stem cells without the use of a specific environment for neural differentiation. Electrospun carbon nanofibers (CNFs) were obtained using heat treatment of polyacrylonitrile nanofibers and treated by nitric acid, ethylenediamine, and oxygen Plasma. SEM imaging revealed that the treated nanofibers have s diameter in the range of 120–200 nm and the treatment did not significantly change the CNFs diameter. The FTIR results showed that the treatments were able to introduce COOH, OH, and NH2 functional groups on the CNFS surface. The XRD and Raman analysis showed that the plasma treatment induced the lowest structural changes in the CNFs microstructure. The biocompatibility assessments showed that the pristine and treated CNFs were nontoxic induced proliferative effect on human adipose-derived mesenchymal stem cells. The electrical stimulation (1.5 mA current with a frequency of 500 Hz and CMOS waveform for 7 days 10 min each day) induced the expression of neural genes and proteins by the cells cultured on the treated CNFs. The Plasma-treated CNFs mediated the highest differentiation outcome. These results indicate that electrospun CNFs can be applied as the innovative interface applicable for neural tissue regeneration under electrical stimulation.

Application of Human Umbilical Cord Mesenchymal Stem Cells in Rat Spinal Cord Injury Model.

The treatment of spinal cord injury (SCI) is a hot topic in clinic. In this study, female rats were selected and randomly divided into four groups (normal, sham, SCI, and mesenchymal stem cells [MSCs] groups). Hemostatic forceps were used to clamp the spinal cord for 1 min to establish the SCI animal model in rats. The levels of proinflammatory factors in the blood of each group were compared 4 h after operation. The motor function of hind limb was estimated by Basso, Beattie & Bresnahan Locomotor rating scale (BBB scale) at 3 months after surgery, the spinal cord tissue from the experimental area was obtained and stained histologically and immunohistochemically. Basso, Beattie & Bresnahan Locomotor rating scale results indicated that human umbilical cord (HUC) MSCs transplantation could improve the walking ability in rats with the SCI. Human umbilical cord mesenchymal stem cells substantially upregulated the secretion of anti-inflammatory factors and downregulated the secretion of proinflammatory factors, and promoted the repair of the SCI and inhibited the increase of glial cells induced by the SCI. Human umbilical cord mesenchymal stem cells transplantation can partially recovered the motor ability of rats with the SCI through promoting the regeneration of nerve cell and the expression of neural related genes, and inhibiting inflammatory reaction.

Analysis of local head losses in microirrigation lateral connectors based on machine learning approaches

The presence of emitters along the lateral, as well as of connectors along the manifold, causes additional local head losses other than friction losses. An accurate estimation of local losses is of crucial importance for a correct design of microirrigation systems. This paper presents a procedure to assess local head losses caused by 6 lateral start connectors of 32- and 40-mm nominal diameter each under actual hydraulic working conditions based on artificial neural networks (ANN) and gene expression programming (GEP) modelling approaches. Different input–output combinations and data partitions were assessed to analyse the hydraulic performance of the system and the optimum training strategy of the models, respectively. The range of the head losses in the manifold (hsM) is considerable lower than in the lateral (hsL). hsM increases with the protrusion ratio (s/S). hsL does not decrease for a decreasing s/S. There is a correlation between hsL and the Reynolds number in the lateral (ReL). However, this correlation might also be dependent on the flow conditions in the manifold before the derivation. The value of the head loss component due to the protrusion might be influenced by the flow derivation. DN32 connectors and hsM present more accurate estimates. Crucial input parameters are flow velocity and protrusion ratio. The inclusion of friction head loss as input also improves the estimating accuracy of the models. The range of the indicators is considerably worse for DN40 than for DN32. The models trained with all patterns lead to more accurate estimations in connectors 7 to 12 than the models trained exclusively with DN40 patterns. On the other hand, including DN40 patterns in the training process did not involve any improvement for estimating the head losses of DN32 connectors. ANN were more accurate than GEP in DN32. In DN40 ANN were less accurate than GEP for hsM, but they were more accurate than GEP for hsL, while both presented a similar performance for hscombined. Different equations were obtained using GEP to easily estimate the two components of the local loss. The equation that should be used in practice depends on the availability of inputs.

Combined exposure to titanium dioxide and tetracycline induces neurotoxicity in zebrafish

In aquatic environment, engineered materials may inevitably interact with the coexisted organic pollutants, which affect their bioavailability and toxicity. In this contribution, the combined impacts of tetracycline (TC) and titanium dioxide nanoparticles (TiO2 NPs) on the neurodevelopment of zebrafish larvae were investigated, and the underlying mechanisms were further elucidated. Firstly, it was confirmed that the co-existence of TC would increase the size and decrease the zeta potential of TiO2 NPs. Following, developmental indicators and motor behaviors were investigated. Our results indicated that co-exposure to TC and TiO2 NPs exhibited enhanced embryonic malformation rates and abnormal nervous system development in zebrafish embryos. Meanwhile, the locomotor behavior was increased upon treatment of TC and TiO2 NP. Further, pathway enrichment analyses of transcriptomic sequencing provided detailed information that either lipid metabolism or PPAR signaling pathway were significantly affected in the co-exposure group. Also, TC + TiO2 NP exposure significantly changed the mRNA expression of neural development-related genes and up-regulated the expression levels of neurotransmitters like 5-hydroxytryptamine, dopamine, acetylcholinesterase, and γ-aminobutyric acid. Taken together, our results demonstrated that the co-exposure of TC and TiO2 NPs had the potential to cause neurotoxicity in zebrafish embryos.

NuRD‐independent Mi‐2 activity represses ectopic gene expression during neuronal maturation

During neuronal development, extensive changes to chromatin states occur to regulate lineage‐specific gene expression. The molecular factors underlying the repression of non‐neuronal genes in differentiated neurons are poorly characterised. The Mi2/NuRD complex is a multiprotein complex with nucleosome remodelling and histone deacetylase activity. Whilst NuRD has previously been implicated in the development of nervous system tissues, the precise nature of the gene expression programmes that it coordinates is ill‐defined. Furthermore, evidence from several species suggests that Mi‐2 may be incorporated into multiple complexes that may not possess histone deacetylase activity. We show that Mi‐2 activity is required for suppressing ectopic expression of germline genes in neurons independently of HDAC1/NuRD, whilst components of NuRD, including Mi‐2, regulate neural gene expression to ensure proper development of the larval nervous system. We find that Mi‐2 binding in the genome is dynamic during neuronal maturation, and Mi‐2‐mediated repression of ectopic gene expression is restricted to the early stages of neuronal development, indicating that Mi‐2/NuRD is required for establishing stable neuronal transcriptomes during the early stages of neuronal differentiation.

Comparing machine-learning-based black box techniques and white box models to predict rainfall-runoff in a northern area of Iraq, the Little Khabur River.

The rainfall-runoff process is one of the most complex hydrological phenomena. Estimating runoff in the basin is one of the main conditions for planning and optimal use of rainfall. Using machine learning models in various sciences to investigate phenomena for which statistical information is available is a helpful tool. This study investigates and compares the abilities of HEC-HMS and TOPMODEL as white box models and adaptive neural fuzzy inference system (ANFIS) and gene expression programming (GEP) as black box models in rainfall-runoff simulation using 5-year statistical data. Using the inputs of rainfall and temperature of the previous day and discharge in the steps of the previous 2 days reduced the prediction error of both models. Examining the role of different parameters in improving the accuracy of simulations showed that the temperature as an effective parameter in cold months reduces the amount of prediction error. A comparison of R2, RMSE, and MBE showed that black box models are more effective forecasting tools. Among the black box models, the ANFIS model with R2 = 0.82 has performed better than the GEP model with R2 = 0.76. For white box models, the HEC-HMS and TOPMODEL had R2 equal to 0.3 and 0.25, respectively.

Abstract A009: NR2F1 inhibits Gaq/11-YAP-TAZ signaling and limits reactivation of uveal melanoma disseminated cancer cells via induction of dormancy

Abstract Introduction: At the time of diagnosis, less than 5% of uveal melanoma (UM) patients have evidence of metastasis. However, up to 50% of UM patients will succumb to advanced metastatic in liver. UM cancer cells can persist undetected in liver for prolonged periods mainly and often take years-to-decades to grow out and do so without recurrence at the primary site. However, the mechanisms underlying the prolonged dormancy and why the liver is a preferred site for survival and eventual regrowth are a long-standing medical mystery. Results: GNAQ/11 are the main driving mutations in UM that activate downstream oncogenic YAP signaling. Using Gaq/11mut /BAPwt human uveal melanoma lines, we reveal that NR2F1, a neural cell commitment regulator is the main driver of regulating dormancy program. RNA-seq of rare UM disseminated cancer cells (DCCs) in livers identified that dormant DCCs upregulate the expression of neural differentiation genes, including NR2F1, while silencing Gaq downstream YAP/TAZ and cell cycle gene expression compared to proliferative cell clusters. Targeted pharmacological inhibition of Gaq/11mut signaling using YM-254890, an inhibitor of Gaq/11 resulted in NR2F1 upregulation and robust UM growth arrest. In contrast, inhibition of NR2F1 either by siRNA or CRISPR/cas9 upregulated YAP1 and its downstream targets at both transcript and protein levels along with increased colony growth in 3D culture. Importantly, UM solitary DCCs in nude mouse upregulated NR2F1 and in vivo CRISPR KO of NR2F1 results in dormant UM DCC awakening and massive metastatic growth in the murine liver. Immunofluorescence of UM patients’ liver metastatic lesions showed high expression of NR2F1 in solitary DCCs compared to the tumor mass. Chromatin-immunoprecipitation assays revealed that NR2F1 binds to the YAP1 promoter and regulates its gene expression. OMM1.3 CRISPR/Cas9 KO of NR2F1 cells showed increased recruitment of H3K4me3 and H3K27ac histone marks onto the YAP1 promoter while overexpression of NR2F1 decreased the recruitment of histone activating marks. Co-immunoprecipitation assay showed increased YAP1-TEAD1 complex formation in NR2F1 KO OMM1.3 cells while overexpression of NR2F1 reduced the abundance of this complex. Summary: Our work suggests that NR2F1 is required to maintain the single cell state of UM cells in the liver by inhibiting the YAP signally pathway. This study provides insight into the previously unrecognized mechanism of uveal melanoma dormancy opening a new space to design anti-metastatic therapies for UM patients. Citation Format: Rama Kadamb, Melisa Lopez Anton, Vivian Chua, Timothy Purwin, Maria Angelo Nieto Toledano, Andrew Aplin, Julio Aguirre-Ghiso. NR2F1 inhibits Gaq/11-YAP-TAZ signaling and limits reactivation of uveal melanoma disseminated cancer cells via induction of dormancy [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A009.

TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

Long bones are generated by mesoderm-derived skeletal progenitor/stem cells (SSCs) through endochondral ossification, a process of sequential chondrogenic and osteogenic differentiation tightly controlled by the synergy between intrinsic and microenvironment cues. Here, we report that loss of TRIM28, a transcriptional corepressor, in mesoderm-derived cells expands the SSC pool, weakens SSC osteochondrogenic potential, and endows SSCs with properties of ectoderm-derived neural crest cells (NCCs), leading to severe defects of skeletogenesis. TRIM28 preferentially enhances H3K9 trimethylation and DNA methylation on chromatin regions more accessible in NCCs; loss of this silencing upregulates neural gene expression and enhances neurogenic potential. Moreover, TRIM28 loss causes hyperexpression of GREM1, which is an extracellular signaling factor promoting SSC self-renewal and SSC neurogenic potential by activating AKT/mTORC1 signaling. Our results suggest that TRIM28-mediated chromatin silencing establishes a barrier for maintaining the SSC lineage trajectory and preventing a transition to ectodermal fate by regulating both intrinsic and microenvironment cues.

KLF4 inhibits early neural differentiation of ESCs by coordinating specific 3D chromatin structure.

Neural differentiation of embryonic stem cells (ESCs) requires precisely orchestrated gene regulation, a process governed in part by changes in 3D chromatin structure. How these changes regulate gene expression in this context remains unclear. In this study, we observed enrichment of the transcription factor KLF4 at some poised or closed enhancers at TSS-linked regions of genes associated with neural differentiation. Combination analysis of ChIP, HiChIP and RNA-seq data indicated that KLF4 loss in ESCs induced changes in 3D chromatin structure, including increased chromatin interaction loops between neural differentiation-associated genes and active enhancers, leading to upregulated expression of neural differentiation-associated genes and therefore early neural differentiation. This study suggests KLF4 inhibits early neural differentiation by regulation of 3D chromatin structure, which is a new mechanism of early neural differentiation.

Data-driven models for prediction of peak strength of R-CFST circular columns subjected to axial loading

Reinforced concrete-filled steel tubular (R-CFST) columns are a relatively new member of the CFST family. They have been developed with the purpose of reducing the local buckling of steel tubes and enhancing both the load-carrying capacity and ductility of conventional CFST by configuring reinforcing bars in the concrete core. The study herein aims to develop consistent, functional, and robust analytical design models to predict the peak strength of the circular R-CFST columns. To this, an extensive database has been created by gathering up the 115 experimentally tested axially loaded R-CFST circular columns from 15 different studies existing in the literature. In this context, two famous and commonly used soft-computing methods known as artificial neural network (ANN) and gene expression programming (GEP) were employed to derive an analytical design model by means of the compiled experimental-based dataset. The analytical design models developed in this study were proposed based on their prediction performance that was determined by verifying and validating them with a testing dataset, statistically analyzing and comparing them with the existing design codes (Eurocode 4, AISC, ACI, and GB) and formulations proposed by some researchers, and performing sensitivity analysis. The results indicated that the developed ANN and GEP-based analytical design models have a superior prediction performance and considerably lower mean absolute error occurrence of 3.6 and 6.8%, respectively in comparison to the existing models.

Soft surfaces induce neural differentiation via the neuron restrictive silencer factor

The physical and mechanical environment influence the differentiation and culture of stem cells. Early research in this field showed that mesenchymal stem cells (MSCs) adopt preferential lineages when cultured on surfaces of varying stiffness. Culture on very soft surfaces promoted neural differentiation. This effect has been observed in mesenchymal and embryonic stem cells and was shown to increase development of neural stem cells. However, providing further information on the molecular characterization of this phenomenon could help to guide the development of materials that enhance neural differentiation. Previously, our laboratory showed that chemical cues, forskolin and IBMX, induced neural differentiation of MSCs by downregulating the Neuron Restrictive Silencer Factor, leading to de-repression of neural gene expression. We sought to determine if the mechanism whereby soft surfaces induce neural differentiation could also involve NRSF function. We show that MSCs cultured on soft polydimethylsiloxane (PDMS) surfaces have reduced expression of NRSF as well as altered localization of NRSF. This suggests the modulation of transcription factors by surface substrate could be capitalized upon in designing materials for neural cell culture.

Neural progenitor cell-derived extracellular matrix as a new platform for neural differentiation of human induced pluripotent stem cells

The culture microenvironment has been demonstrated to regulate stem cell fate and to be a crucial aspect for quality-controlled stem cell maintenance and differentiation to a specific lineage. In this context, extracellular matrix (ECM) proteins are particularly important to mediate the interactions between the cells and the culture substrate. Human induced pluripotent stem cells (hiPSCs) are usually cultured as anchorage-dependent cells and require adhesion to an ECM substrate to support their survival and proliferation in vitro. Matrigel, a common substrate for hiPSC culture is a complex and undefined mixture of ECM proteins which are expensive and not well suited to clinical application. Decellularized cell-derived ECM has been shown to be a promising alternative to the common protein coatings used in stem cell culture. However, very few studies have used this approach as a niche for neural differentiation of hiPSCs.Here, we developed a new stem cell culture system based on decellularized cell-derived ECM from neural progenitor cells (NPCs) for expansion and neural differentiation of hiPSCs, as an alternative to Matrigel and poly-l-ornithine/laminin-coated well plates. Interestingly, hiPSCs were able to grow and maintain their pluripotency when cultured on decellularized ECM from NPCs (NPC ECM). Furthermore, NPC ECM enhanced the neural differentiation of hiPSCs compared to poly-l-ornithine/laminin-coated wells, which are used in most neural differentiation protocols, presenting a statistically significant enhancement of neural gene expression markers, such as βIII-Tubulin and MAP2.Taken together, our results demonstrate that NPC ECM provides a functional microenvironment, mimicking the neural niche, which may have interesting future applications for the development of new strategies in neural stem cell research.

Application of Soft Computing for Estimation of Pavement Condition Indicators and Predictive Modeling

The pavement management system is recognized as an assertive discipline that works on pavement indices to predict the pavement performance condition. This study used soft computing methods such as genetic algorithms and artificial intelligence to propose a modern generation of pavement indices for road networks in Jordan. The datasets used in this study were collected from multiple roads in Jordan, and 128 data points were used in this study. The input variables are the pavement condition index (PCI) and the international roughness index (IRI) in the artificial neural network (ANN) and gene expression programming (GEP) models. The output variable is the pavement serviceability rate (PSR). The results show an efficient performance benefit of using these techniques. In addition, the ANN and GEP models were able to predict the output variable with a reasonable accuracy, where the ANN model has an R2 value of 0.95, 0.87, and 0.98 for the PCI, IRI, and PSR, respectively. The (R2) values of the GEP model are 0.94, 0.89, and 0.99 for PCI, IRI, and PSR, respectively.

Interaction between the hypothalamo-pituitary-adrenal and thyroid axes during immobilization stress.

The location of corticotropin-releasing hormone receptor 2 (CRH-R2) on thyrotropes within the avian anterior pituitary (APit) and its activation by different stressors indicate a possible communication between hypothalamo-pituitary-adrenal (HPA) and thyroid (HPT) axes. Therefore, an experiment was designed to 1) compare the timing of major components of the HPT axis to those of the HPA axis; 2) address whether stressors activating the HPA axis may simultaneously upregulate components of the HPT axis. Blood, brain, and APit were sampled from chicks prior to stress (control) and 15, 30, 60, 90, and 120min following immobilization (IM) stress. The nucleus of the hippocampal commissure (NHpC) and paraventricular nucleus (PVN) were cryo-dissected from brains for RT-qPCR. Gene expression of thyrotropin-releasing hormone (TRH) and its receptors (TRH-R1 and TRH-R3), urocortin3 (UCN3), deiodinase 2 (D2), and the second type of corticotropin-releasing hormone (CRH2) within the NHpC and PVN was measured. Additionally, gene expression of TRH receptors, thyroid stimulating hormone subunit beta (TSHβ), and D2 was determined in the APit and corticosterone assayed in blood. In brains, a significant upregulation in examined genes occurred at different times of IM. Specifically, UCN3 and CRH2 which have a high affinity to CRH-R2 showed a rapid increase in their mRNA levels that were accompanied by an early upregulation of TRHR1 in the NHpC. In the APit, a significant increase in gene expression of TSHβ and TRH receptors was observed. Therefore, results supported concurrent activation of major brain and APit genes associated with the HPA and HPT axes following IM. The initial neural gene expression originating within the NHpC resulted in the increase of TSHβ mRNA in the APit. Specifically, the rapid upregulation of UCN3 in the NHpC appeared responsible for the early activation of TSHβ in the APit. While sustaining TSHβ activation appeared to be due to both CRH2 and TRH. Therefore, data indicate that CRH-producing neurons and corticotropes as well as CRH- and TRH-producing neurons and thyrotropes are activated to produce the necessary energy required to maintain homeostasis in birds undergoing stress. Overall, data support the inclusion of the NHpC in the classical avian HPA axis and for the first time show the concurrent activation of the HPA axis and components of the HPT axis following a psychogenic stressor.

A Bone Morphogenetic Protein Signaling Inhibitor, LDN193189, Converts Ischemia-Induced Multipotent Stem Cells into Neural Stem/Progenitor Cell-Like Cells.

Stem cell therapy is used to restore neurological function in stroke patients. We have previously reported that ischemia-induced multipotent stem cells (iSCs), which are likely derived from brain pericytes, develop in poststroke human and mouse brains. Although we have demonstrated that iSCs can differentiate into neural lineage cells, the factors responsible for inducing this differentiation remain unclear. In this study, we found that LDN193189, a bone morphogenetic protein (BMP) inhibitor, caused irreversible changes in the shape of iSCs. In addition, compared with iSCs incubated without LDN193189, the iSCs incubated with LDN193189 (LDN-iSCs) showed upregulated expression of neural lineage-related genes and proteins, including those expressed in neural stem/progenitor cells (NSPCs), and downregulated expression of mesenchymal and pericytic-related genes and proteins. Moreover, microarray analysis revealed that LDN-iSCs and NSPCs had similar gene expression profiles. Furthermore, LDN-iSCs differentiated into electrophysiologically functional neurons. These results indicate that LDN193189 induces NSPC-like cells from iSCs, suggesting that bioactive molecules regulating BMP signaling are potential targets for promoting neurogenesis from iSCs in the pathological brain, such as during ischemic stroke. We believe that our findings will bring us one step closer to the clinical application of iSCs.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body’s immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

Ash2l, an obligatory component of H3K4 methylation complexes, regulates neural crest development

The vertebrate nervous system develops from embryonic neural plate and neural crest. Although genetic mechanisms governing vertebrate neural development have been investigated in depth, epigenetic regulation of this process remains less understood. Redundancy of epigenetic factors and early lethality of animals deficient in critical epigenetic components pose major challenges in characterization of epigenetic factors in vertebrate neural development. In this study, we use the amphibian model Xenopus laevis to investigate the roles of non-redundant, obligatory components of all histone H3K4 activating methylation complexes (COMPASS, also known as SET1/MLL complexes) in early neural development. The two genes that we focus on, Ash2l and Dpy30, regulate mesendodermal differentiation in mouse embryonic stem cells and cause early embryonic lethality when removed from mouse embryos. Using targeted knockdown of the genes in dorsal ectoderm of Xenopus that gives rise to future nervous system, we show here that ash2l and dpy30 are required for neural and neural crest marker expression in Xenopus late neurula embryos but are dispensable for early neural and neural plate border gene expression. Co-immunoprecipitation assays reveal that Dpy30 and Ash2L associate with the neural plate border transcription factors, such as Msx1 and Tfap2a. Chromatin immunoprecipitation (ChIP) assay further demonstrates that Ash2L and the H3K4me3 active histone mark accumulate at the promoter regions of the neural crest gene sox10 in a Tfap2a-dependent manner. Collectively, our data suggest that Ash2l and Dpy30 interact with specific transcription factors to recruit COMPASS complexes to the regulatory regions of neural crest specification genes to control their expression and influence development of the nervous system during vertebrate embryogenesis.