Comparative epigenomic annotation of regulatory DNA.

Despite the explosive growth of genomic data, functional annotation of regulatory sequences remains difficult. Here, we introduce "comparative epigenomics"-interspecies comparison of DNA and histone modifications-as an approach for annotation of the regulatory genome. We measured in human, mouse, and pig pluripotent stem cells the genomic distributions of cytosine methylation, H2A.Z, H3K4me1/2/3, H3K9me3, H3K27me3, H3K27ac, H3K36me3, transcribed RNAs, and P300, TAF1, OCT4, and NANOG binding. We observed that epigenomic conservation was strong in both rapidly evolving and slowly evolving DNA sequences, but not in neutrally evolving sequences. In contrast, evolutionary changes of the epigenome and the transcriptome exhibited a linear correlation. We suggest that the conserved colocalization of different epigenomic marks can be used to discover regulatory sequences. Indeed, seven pairs of epigenomic marks identified exhibited regulatory functions during differentiation of embryonic stem cells into mesendoderm cells. Thus, comparative epigenomics reveals regulatory features of the genome that cannot be discerned from sequence comparisons alone.

Nutritional supplement induced modulations in the functional connectivity of a porcine brain.

BACKGROUND: Functional connectivity (FC) measures statistical dependence between cortical brain regions. Studies of FC facilitate understanding of the brain's function and architecture that underpin normal cognition, behavior, and changes associated with various factors (e.g. nutritional supplements) at a large scale. OBJECTIVE: We aimed to identify modifications in FC patterns and targeted brain anatomies in piglets following perinatal intake of different nutritional diets using a graph theory based approach. METHODS: Forty-four piglets from four groups of pregnant sows, who were treated with nutritional supplements, including control diet, docosahexaenoic acid (DHA), egg yolk (EGG), and DHA + EGG, went through resting-state functional magnetic resonance imaging (rs-fMRI). We introduced the use of differential degree test (DDT) to identify differentially connected edges (DCEs). Simulation studies were first conducted to compare the DDT with permutation test, using three network structures at different noise levels. DDT was then applied to rs-fMRI data acquired from piglets. RESULTS: In simulations, the DDT showed a greater accuracy in detecting DCEs when compared with the permutation test. For empirical data, we found that the strength of internodal connectivity is significantly increased for more than 6% of edges in the EGG group and more than 8% of edges in the DHA and DHA + EGG groups, all compared to the control group. Moreover, differential wiring diagrams between group comparisons provided means to pinpoint brain hubs affected by nutritional supplements. CONCLUSION: DDT showed a greater accuracy of detection of DCEs and demonstrated EGG, DHA, and DHA + EGG supplemented diets lead to an improved internodal connectivity in the developing piglet brain.

Sparse multiway canonical correlation analysis for multimodal stroke recovery data.

Conventional canonical correlation analysis (CCA) measures the association between two datasets and identifies relevant contributors. However, it encounters issues with execution and interpretation when the sample size is smaller than the number of variables or there are more than two datasets. Our motivating example is a stroke-related clinical study on pigs. The data are multimodal and consist of measurements taken at multiple time points and have many more variables than observations. This study aims to uncover important biomarkers and stroke recovery patterns based on physiological changes. To address the issues in the data, we develop two sparse CCA methods for multiple datasets. Various simulated examples are used to illustrate and contrast the performance of the proposed methods with that of the existing methods. In analyzing the pig stroke data, we apply the proposed sparse CCA methods along with dimension reduction techniques, interpret the recovery patterns, and identify influential variables in recovery.

An Adolescent Porcine Model of Voluntary Alcohol Consumption Exhibits Binge Drinking and Motor Deficits in a Two Bottle Choice Test.

AIMS: Alcohol is the most commonly abused substance leading to significant economic and medical burdens. Pigs are an attractive model for studying alcohol abuse disorder due to the comparable alcohol metabolism and consumption behavior, which are in stark contrast to rodent models. This study investigates the usage of a porcine model for voluntary binge drinking (BD) and a detailed analysis of gait changes due to motor function deficits during alcohol intoxication. METHODS: Adolescent pigs were trained to drink increasing concentration (0-8%) of alcohol mixed in a 0.2% saccharin solution for 1 h in a two bottle choice test for 2 weeks. The training period was followed by a 3-week alcohol testing period, where animals were given free access to 8% alcohol in 0.2% saccharin solution and 0.2% saccharin water solution. Blood alcohol levels were tested and gait analysis was performed pre-alcohol consumption, last day of training, and Day 5 of each testing period. RESULTS: Pigs voluntarily consumed alcohol to intoxication at all timepoints with blood alcohol concentration (BAL) ≥80 mg/dl. Spatiotemporal gait parameters including velocity, cadence, cycle time, swing time, stance time, step time, and stride length were perturbed as a result of intoxication. The stratification of the gait data based on BAL revealed that the gait parameters were affected in a dose-dependent manner. CONCLUSION: This novel adolescent BD porcine model with inherent anatomical and physiological similarities to humans display similar consumption and intoxication behavior that is likely to yield results that are translatable to human patients.

Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia.

BACKGROUND: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. RESULTS: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. CONCLUSION: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Human Neural Stem Cell Extracellular Vesicles Improve Recovery in a Porcine Model of Ischemic Stroke.

BACKGROUND AND PURPOSE: Recent work from our group suggests that human neural stem cell-derived extracellular vesicle (NSC EV) treatment improves both tissue and sensorimotor function in a preclinical thromboembolic mouse model of stroke. In this study, NSC EVs were evaluated in a pig ischemic stroke model, where clinically relevant end points were used to assess recovery in a more translational large animal model. METHODS: Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO), and either NSC EV or PBS treatment was administered intravenously at 2, 14, and 24 hours post-MCAO. NSC EV effects on tissue level recovery were evaluated via magnetic resonance imaging at 1 and 84 days post-MCAO. Effects on functional recovery were also assessed through longitudinal behavior and gait analysis testing. RESULTS: NSC EV treatment was neuroprotective and led to significant improvements at the tissue and functional levels in stroked pigs. NSC EV treatment eliminated intracranial hemorrhage in ischemic lesions in NSC EV pigs (0 of 7) versus control pigs (7 of 8). NSC EV-treated pigs exhibited a significant decrease in cerebral lesion volume and decreased brain swelling relative to control pigs 1-day post-MCAO. NSC EVs significantly reduced edema in treated pigs relative to control pigs, as assessed by improved diffusivity through apparent diffusion coefficient maps. NSC EVs preserved white matter integrity with increased corpus callosum fractional anisotropy values 84 days post-MCAO. Behavior and mobility improvements paralleled structural changes as NSC EV-treated pigs exhibited improved outcomes, including increased exploratory behavior and faster restoration of spatiotemporal gait parameters. CONCLUSIONS: This study demonstrated for the first time that in a large animal model novel NSC EVs significantly improved neural tissue preservation and functional levels post-MCAO, suggesting NSC EVs may be a paradigm changing stroke therapeutic.

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection.

BACKGROUND: Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a devastating disease of poultry and wild birds. ND is prevented by rigorous biocontainment and vaccination. One potential approach to prevent spread of the virus is production of birds that show innate resistance to NDV-caused disease. Induced pluripotent stem cell (iPSC) technology allows adult cells to be reprogrammed into an embryonic stem cell-like state capable of contributing to live offspring and passing on unique traits in a number of species. Recently, iPSC approaches have been successfully applied to avian cells. If chicken induced pluripotent stem cells (ciPSCs) are genetically or epigenetically modified to resist NDV infection, it may be possible to generate ND resistant poultry. There is limited information on the potential of ciPSCs to be infected by NDV, or the capacity of these cells to become resistant to infection. The aim of the present work was to assess the characteristics of the interaction between NDV and ciPSCs, and to develop a selection method that would increase tolerance of these cells to NDV-induced cellular damage. RESULTS: Results showed that ciPSCs were permissive to infection with NDV, and susceptible to virus-mediated cell death. Since ciPSCs that survived infection demonstrated the ability to recover quickly, we devised a system to select surviving cells through multiple infection rounds with NDV. ciPSCs that sustained 9 consecutive infections had a statistically significant increase in survival (up to 36 times) compared to never-infected ciPSCs upon NDV infection (tolerant cells). Increased survival was not caused by a loss of permissiveness to NDV replication. RNA sequencing followed by enrichment pathway analysis showed that numerous metabolic pathways where differentially regulated between tolerant and never-infected ciPSCs. CONCLUSIONS: Results demonstrate that ciPSCs are permissive to NDV infection and become increasingly tolerant to NDV under selective pressure, indicating that this system could be applied to study mechanisms of cellular tolerance to NDV.

Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium.

Delayed Newcastle disease virus replication using RNA interference to target the nucleoprotein.

Each year millions of chickens die from Newcastle disease virus (NDV) worldwide leading to severe economic and food losses. Current vaccination campaigns have limitations especially in developing countries, due to elevated costs, need of trained personnel for effective vaccine administration, and functional cold chain network to maintain vaccine viability. These problems have led to heightened interest in producing new antiviral strategies, such as RNA interference (RNAi). RNAi methodology is capable of substantially decreasing viral replication at a cellular level, both in vitro and in vivo. In this study, we utilize microRNA (miRNA)-expressing constructs (a type of RNA interference) in an attempt to target and knockdown five NDV structural RNAs for nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), and large (L) protein genes. Immortalized chicken embryo fibroblast cells (DF-1) that transiently expressed miRNA targeting NP mRNA, showed increased resistance to NDV-induced cytopathic effects, as determined by cell count, relative to the same cells expressing miRNA against alternative NDV proteins. Upon infection with NDV, DF-1 cells constitutively expressing the NP miRNA construct had improved cell survival up to 48 h post infection (h.p.i) and decreased viral yield up to 24 h.p.i. These results suggest that overexpression of the NP miRNA in cells and perhaps live animal may provide resistance to NDV.

Affinity of structural white matter tracts between infant and adult pig.

BACKGROUND: The piglet brain has been increasingly used as an excellent surrogate for investigation of pediatric neurodevelopment, nutrition, and traumatic brain injuries. This study intends to establish a piglet brain's structural connectivity model and compare it with the adult pig, enhancing its application for structurally guided functional analysis. METHODS: In this study, diffusion-weighted (DW)-MRI data from piglets (n=11, 3-week-old) was used to establish piglet model and compare with adult pigs. We employed a data-driven independent component analysis (ICA) method to derive piglet-specific tracts. Pearson correlations and Kullback-Leibler (KL) divergences was employed to identify common tracts and unique tracts for piglet. Common tracts were then used in a blueprint connectome study to highlight differences in regions of interest (ROI). RESULTS: The data-driven approach applied to piglet brains revealed 17 common tracts, showing high similarity with adult pigs' white matter (WM) tracts, and identified 3 tracts unique to piglets and 10 negative marker tracts. Additionally, the study highlighted notable differences in 3 ROIs associated with blueprint connectome. COMPARING WITH EXISTING METHODS: This study marks a significant shift from surface-based to voxel-based methodologies in analyzing pig brain structural connectivity and generating connectome blueprints. Additionally, it sheds light on the use of the piglet model for developmental studies, offering new perspectives in this area. CONCLUSION: This study established a piglet brain tract model and conducts a comparative analysis of adult pig's and piglet's structural connectivity. These findings underscore the potential use of the piglet brain model in employing piglet model for developmental studies.

Evaluation of Brain Function Recovery After Traumatic Brain Injury Treatment in a Porcine Model by Cross-Group Temporal-Spatial Correlation Analysis.

Traumatic brain injury (TBI), a significant global health issue, is affecting ∼69 million annually. To better understand TBI's impact on brain function and assess the efficacy of treatments, this study uses a novel temporal-spatial cross-group approach with a porcine model, integrating resting-state functional magnetic resonance imaging (rs-fMRI) for temporal and arterial spin labeling for spatial information. Our research used 18 four-week-old pigs divided into three groups: TBI treated with saline (SLN, n = 6), TBI treated with fecal microbial transplant (FMT, n = 6), and a sham group (sham, n = 6) with only craniectomy surgery as the baseline. By applying machine learning techniques-specifically, independent component analysis and sparse dictionary learning-across seven identified resting-state networks, we assessed the temporal and spatial correlations indicative of treatment efficacy. Both temporal and spatial analyses revealed a consistent increase of correlation between the FMT and sham groups in the executive control and salience networks. Our results are further evidenced by a simulation study designed to mimic the progression of TBI severity through the introduction of variable Gaussian noise to an independent rs-fMRI dataset. The results demonstrate a decreasing temporal correlation between the sham and TBI groups with increasing injury severity, consistent with the experimental results. This study underscores the effectiveness of the methodology in evaluating post-TBI treatments such as the FMT. By presenting comprehensive experimental and simulated data, our research contributes significantly to the field and opens new paths for future investigations into TBI treatment evaluations.

White Matter Integrity and Motor Function Disruption Due to Traumatic Brain Injury in Piglets: Impacts on Motor-Related Brain Fibers.

Pediatric traumatic brain injury (TBI) often induces significant disability in patients, including long-term motor deficits. Early detection of injury severity is key in determining a prognosis and creating appropriate intervention and rehabilitation plans. However, conventional magnetic resonance imaging (MRI) scans, such as T2 Weighted (T2W) sequences, do not reliably assess the extent of microstructural white matter injury. Diffusion tensor imaging (DTI) tractography enables three-dimensional reconstruction of specific white matter tracts throughout the brain in order to detect white matter injury based on anisotropic diffusion. The objective of this study was to employ DTI tractography to detect acute changes to white matter integrity within the intersecting fibers of key motor-related brain regions following TBI. Piglets were assigned to either the sham craniectomy group (sham; n = 6) or the controlled cortical impact TBI group (TBI; n = 6). Gait and MRI were collected at seven days post-surgery (DPS). T2W sequences confirmed a localized injury predominately in the ipsilateral hemisphere in TBI animals. TBI animals, relative to sham animals, showed an increased apparent diffusion coefficient (ADC) and decreased fractional anisotropy (FA) in fiber bundles associated with key brain regions involved in motor function. TBI animals exhibited gait deficits, including stride and step length, compared to sham animals. Together these data demonstrate acute reductions in the white matter integrity, measured by DTI tractography, of fibers intersecting key brain regions that strongly corresponded with acute motor deficits in a pediatric piglet TBI model. These results provide the foundation for the further development of DTI-based biomarkers to evaluate motor outcomes following TBI.

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential.

BACKGROUND: Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype. METHODS: hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer. RESULTS: Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential. CONCLUSION: Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

Fecal microbial transplantation limits neural injury severity and functional deficits in a pediatric piglet traumatic brain injury model.

Pediatric traumatic brain injury (TBI) is a leading cause of death and disability in children. Due to bidirectional communication between the brain and gut microbial population, introduction of key gut bacteria may mitigate critical TBI-induced secondary injury cascades, thus lessening neural damage and improving functional outcomes. The objective of this study was to determine the efficacy of a daily fecal microbial transplant (FMT) to alleviate neural injury severity, prevent gut dysbiosis, and improve functional recovery post TBI in a translational pediatric piglet model. Male piglets at 4-weeks of age were randomly assigned to Sham + saline, TBI + saline, or TBI + FMT treatment groups. A moderate/severe TBI was induced by controlled cortical impact and Sham pigs underwent craniectomy surgery only. FMT or saline were administered by oral gavage daily for 7 days. MRI was performed 1 day (1D) and 7 days (7D) post TBI. Fecal and cecal samples were collected for 16S rRNA gene sequencing. Ipsilateral brain and ileum tissue samples were collected for histological assessment. Gait and behavior testing were conducted at multiple timepoints. MRI showed that FMT treated animals demonstrated decreased lesion volume and hemorrhage volume at 7D post TBI as compared to 1D post TBI. Histological analysis revealed improved neuron and oligodendrocyte survival and restored ileum tissue morphology at 7D post TBI in FMT treated animals. Microbiome analysis indicated decreased dysbiosis in FMT treated animals with an increase in multiple probiotic Lactobacilli species, associated with anti-inflammatory therapeutic effects, in the cecum of the FMT treated animals, while non-treated TBI animals showed an increase in pathogenic bacteria, associated with inflammation and disease such in feces. FMT mediated enhanced cellular and tissue recovery resulted in improved motor function including stride and step length and voluntary motor activity in FMT treated animals. Here we report for the first time in a highly translatable pediatric piglet TBI model, the potential of FMT treatment to significantly limit cellular and tissue damage leading to improved functional outcomes following a TBI.

Tanshinone IIA-loaded nanoparticles and neural stem cell combination therapy improves gut homeostasis and recovery in a pig ischemic stroke model.

Impaired gut homeostasis is associated with stroke often presenting with leaky gut syndrome and increased gut, brain, and systemic inflammation that further exacerbates brain damage. We previously reported that intracisternal administration of Tanshinone IIA-loaded nanoparticles (Tan IIA-NPs) and transplantation of induced pluripotent stem cell-derived neural stem cells (iNSCs) led to enhanced neuroprotective and regenerative activity and improved recovery in a pig stroke model. We hypothesized that Tan IIA-NP + iNSC combination therapy-mediated stroke recovery may also have an impact on gut inflammation and integrity in the stroke pigs. Ischemic stroke was induced, and male Yucatan pigs received PBS + PBS (Control, n = 6) or Tan IIA-NP + iNSC (Treatment, n = 6) treatment. The Tan IIA-NP + iNSC treatment reduced expression of jejunal TNF-α, TNF-α receptor1, and phosphorylated IkBα while increasing the expression of jejunal occludin, claudin1, and ZO-1 at 12 weeks post-treatment (PT). Treated pigs had higher fecal short-chain fatty acid (SCFAs) levels than their counterparts throughout the study period, and fecal SCFAs levels were negatively correlated with jejunal inflammation. Interestingly, fecal SCFAs levels were also negatively correlated with brain lesion volume and midline shift at 12 weeks PT. Collectively, the anti-inflammatory and neuroregenerative treatment resulted in increased SCFAs levels, tight junction protein expression, and decreased inflammation in the gut.

Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs.

The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals, a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents, has opened the doors for in-depth study of iPSC tumorigenicity, autologous transplantation, and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSC-derived chimeras possessed large numbers of tumors, rising significant concerns about the safety of iPSC therapies. Additionally, piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g., knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study, we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition, gross and histological examination of necropsied porcine chimeras at 2, 7, and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C-MYC gene expression, further implicating C-MYC as a cause of tumorigenicity. The development of germline-competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals.

Changes in Oral Microbial Diversity in a Piglet Model of Traumatic Brain Injury.

Dynamic changes in the oral microbiome have gained attention due to their potential diagnostic role in neurological diseases such as Alzheimer's disease and Parkinson's disease. Traumatic brain injury (TBI) is a leading cause of death and disability in the United States, but no studies have examined the changes in oral microbiome during the acute stage of TBI using a clinically translational pig model. Crossbred piglets (4-5 weeks old, male) underwent either a controlled cortical impact (TBI, n = 6) or sham surgery (sham, n = 6). The oral microbiome parameters were quantified from the upper and lower gingiva, both buccal mucosa, and floor of the mouth pre-surgery and 1, 3, and 7 days post-surgery (PS) using the 16S rRNA gene. Faith's phylogenetic diversity was significantly lower in the TBI piglets at 7 days PS compared to those of sham, and beta diversity at 1, 3, and 7 days PS was significantly different between TBI and sham piglets. However, no significant changes in the taxonomic composition of the oral microbiome were observed following TBI compared to sham. Further studies are needed to investigate the potential diagnostic role of the oral microbiome during the chronic stage of TBI with a larger number of subjects.

Tanshinone IIA-Loaded Nanoparticle and Neural Stem Cell Therapy Enhances Recovery in a Pig Ischemic Stroke Model.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) are a multimodal stroke therapeutic that possess neuroprotective, regenerative, and cell replacement capabilities post-ischemia. However, long-term engraftment and efficacy of iNSCs is limited by the cytotoxic microenvironment post-stroke. Tanshinone IIA (Tan IIA) is a therapeutic that demonstrates anti-inflammatory and antioxidative effects in rodent ischemic stroke models and stroke patients. Therefore, pretreatment with Tan IIA may create a microenvironment that is more conducive to the long-term survival of iNSCs. In this study, we evaluated the potential of Tan IIA drug-loaded nanoparticles (Tan IIA-NPs) to improve iNSC engraftment and efficacy, thus potentially leading to enhanced cellular, tissue, and functional recovery in a translational pig ischemic stroke model. Twenty-two pigs underwent middle cerebral artery occlusion (MCAO) and were randomly assigned to a PBS + PBS, PBS + iNSC, or Tan IIA-NP + iNSC treatment group. Magnetic resonance imaging (MRI), modified Rankin Scale neurological evaluation, and immunohistochemistry were performed over a 12-week study period. Immunohistochemistry indicated pretreatment with Tan IIA-NPs increased iNSC survivability. Furthermore, Tan IIA-NPs increased iNSC neuronal differentiation and decreased iNSC reactive astrocyte differentiation. Tan IIA-NP + iNSC treatment enhanced endogenous neuroprotective and regenerative activities by decreasing the intracerebral cellular immune response, preserving endogenous neurons, and increasing neuroblast formation. MRI assessments revealed Tan IIA-NP + iNSC treatment reduced lesion volumes and midline shift. Tissue preservation and recovery corresponded with significant improvements in neurological recovery. This study demonstrated pretreatment with Tan IIA-NPs increased iNSC engraftment, enhanced cellular and tissue recovery, and improved neurological function in a translational pig stroke model.

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives.

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.

Detecting functional connectivity disruptions in a translational pediatric traumatic brain injury porcine model using resting-state and task-based fMRI.

Functional magnetic resonance imaging (fMRI) has significant potential to evaluate changes in brain network activity after traumatic brain injury (TBI) and enable early prognosis of potential functional (e.g., motor, cognitive, behavior) deficits. In this study, resting-state and task-based fMRI (rs- and tb-fMRI) were utilized to examine network changes in a pediatric porcine TBI model that has increased predictive potential in the development of novel therapies. rs- and tb-fMRI were performed one day post-TBI in piglets. Activation maps were generated using group independent component analysis (ICA) and sparse dictionary learning (sDL). Activation maps were compared to pig reference functional connectivity atlases and evaluated using Pearson spatial correlation coefficients and mean ratios. Nonparametric permutation analyses were used to determine significantly different activation areas between the TBI and healthy control groups. Significantly lower Pearson values and mean ratios were observed in the visual, executive control, and sensorimotor networks for TBI piglets compared to controls. Significant differences were also observed within several specific individual anatomical structures within each network. In conclusion, both rs- and tb-fMRI demonstrate the ability to detect functional connectivity disruptions in a translational TBI piglet model, and these disruptions can be traced to specific affected anatomical structures.