Lessons and Applications of Omics Research in Diabetes Epidemiology.

PURPOSE OF REVIEW: Recent advances in genomic technology and molecular techniques have greatly facilitated the identification of disease biomarkers, advanced understanding of pathogenesis of different common diseases, and heralded the dawn of precision medicine. Much of these advances in the area of diabetes have been made possible through deep phenotyping of epidemiological cohorts, and analysis of the different omics data in relation to detailed clinical information. In this review, we aim to provide an overview on how omics research could be incorporated into the design of current and future epidemiological studies. RECENT FINDINGS: We provide an up-to-date review of the current understanding in the area of genetic, epigenetic, proteomic and metabolomic markers for diabetes and related outcomes, including polygenic risk scores. We have drawn on key examples from the literature, as well as our own experience of conducting omics research using the Hong Kong Diabetes Register and Hong Kong Diabetes Biobank, as well as other cohorts, to illustrate the potential of omics research in diabetes. Recent studies highlight the opportunity, as well as potential benefit, to incorporate molecular profiling in the design and set-up of diabetes epidemiology studies, which can also advance understanding on the heterogeneity of diabetes. Learnings from these examples should facilitate other researchers to consider incorporating research on omics technologies into their work to advance the field and our understanding of diabetes and its related co-morbidities. Insights from these studies would be important for future development of precision medicine in diabetes.

Interactome Profiling of DNA Damage Response (DDR) Mediators with Immunoprecipitation-Mass Spectrometry.

Immunoprecipitation-mass spectrometry (IP-MS) is a versatile tool to probe for global protein-protein interactions (PPIs) in biological samples. Such interactions coordinate complex biological processes, such as the DNA damage response (DDR). Induction of DNA damage activates signaling networks where posttranslational modifications cause PPI that facilitate DNA repair and cell cycle coordination. Protein interactome profiling of DDR sensors, transducers, and effectors has the potential to identify novel DDR mechanisms that could advance our understanding and treatment of diseases associated with DDR defects, such as cancer. The protocol described here is a routine PPI analysis procedure that can be performed on samples stimulated with DNA damage. All processes and reagents are optimized for maximum sensitivity on the interactome and minimal contamination for the mass spectrometer.

Does plant ecosystem thermoregulation occur? An extratropical assessment at different spatial and temporal scales.

To what degree plant ecosystems thermoregulate their canopy temperature (Tc ) is critical to assess ecosystems' metabolisms and resilience with climate change, but remains controversial, with opinions from no to moderate thermoregulation capability. With global datasets of Tc , air temperature (Ta ), and other environmental and biotic variables from FLUXNET and satellites, we tested the 'limited homeothermy' hypothesis (indicated by Tc & Ta regression slope < 1 or Tc < Ta around midday) across global extratropics, including temporal and spatial dimensions. Across daily to weekly and monthly timescales, over 80% of sites/ecosystems have slopes ≥1 or Tc > Ta around midday, rejecting the above hypothesis. For those sites unsupporting the hypothesis, their Tc -Ta difference (ΔT) exhibits considerable seasonality that shows negative, partial correlations with leaf area index, implying a certain degree of thermoregulation capability. Spatially, site-mean ΔT exhibits larger variations than the slope indicator, suggesting ΔT is a more sensitive indicator for detecting thermoregulatory differences across biomes. Furthermore, this large spatial-wide ΔT variation (0-6°C) is primarily explained by environmental variables (38%) and secondarily by biotic factors (15%). These results demonstrate diverse thermoregulation patterns across global extratropics, with most ecosystems negating the 'limited homeothermy' hypothesis, but their thermoregulation still occurs, implying that slope < 1 or Tc < Ta are not necessary conditions for plant thermoregulation.

Outcomes of fracture-related infections - do organism, depth of involvement, and temporality count?

Purpose: To determine mortality and outcomes of patients diagnosed with fracture-related infections (FRIs).Methods: FRI patients treated at a trauma centre between 2001 and 2020 were analysed. The primary outcome was 1-year mortality; mortality associations with FRI organism, depth of involvement, and temporality were investigated with multivariable survival analysis. Healthcare-associated and serological outcomes were reported as secondary outcomes. Results: 311 FRIs with mean age of 67.0 and median Charlson comorbidity index of 0 were analysed. Methicillin-sensitive Staphylococcus aureus (MSSA) (29.9%) was the most frequently implicated organism. The majority of FRIs were deep infections (62.7%). FRIs were diagnosed at a median of 40 (IQR 15-200) days post index surgery. The mean follow-up was 5.9 years. One-year mortality amounted to 17.7%. MSSA FRIs were associated with better survival (adj HR 0.34, 95%CI 0.15-0.76, p = 0.008). There was no difference in survivorship between deep or superficial FRI (adj HR 0.86, 95%CI 0.62-1.19, p = 0.353) or in relation to onset time (adj HR 1.0, 95%CI 0.99-1.00, p = 0.943). Implant removal or debridement alone was performed in 61.7% and 17% respectively. Antibiotics was prescribed for 53 (IQR 23-110) days, and patients were hospitalised for 39 (IQR 19-78) days. CRP and ESR normalised in 70.3% (median 46 days) and 53.8% (median 86 days) patients respectively. Conclusion: Fracture-related infections are associated with significant mortality and morbidity regardless of depth and temporality. Non-MSSA FRIs are associated with inferior survival.

Combined Alcohol Exposure and KRAS Mutation in Human Pancreatic Ductal Epithelial Cells Induces Proliferation and Alters Subtype Signatures Determined by Multi-Omics Analysis.

Pancreatic Ductal adenocarcinoma (PDAC) is an aggressive cancer commonly exhibiting KRAS-activating mutations. Alcohol contributes to the risk of developing PDAC in humans, and murine models have shown alcohol consumption in the context of KRAS mutation in the pancreas promotes the development of PDAC. The molecular signatures in pancreas cells altered by alcohol exposure in the context of mutant KRAS could identify pathways related to the etiology of PDAC. In this study, we evaluated the combined effects of alcohol exposure and KRAS mutation status on the transcriptome and proteome of pancreatic HPNE cell models. These analyses identified alterations in transcription and translational processes in mutant KRAS cells exposed to alcohol. In addition, multi-omics analysis suggests an increase in the correlation between mRNA transcript and protein abundance in cells exposed to alcohol with an underlying KRAS mutation. Through differential co-expression, SERPINE1 was found to be influential for PDAC development in the context of mutant KRAS and ethanol. In terms of PDAC subtypes, alcohol conditioning of HPNE cells expressing mutant KRAS decreases the Inflammatory subtype signature and increases the Proliferative and Metabolic signatures, as we previously observed in patient samples. The alterations in molecular subtypes were associated with an increased sensitivity to chemotherapeutic agents gemcitabine, irinotecan, and oxaliplatin. These results provide a framework for distinguishing the molecular dysregulation associated with combined alcohol and mutant KRAS in a pancreatic cell line model.

An optimized co-immunoprecipitation protocol for the analysis of endogenous protein-protein interactions in cell lines using mass spectrometry.

This protocol represents an optimized proteomics-based protocol for the endogenous protein enrichment and protein-protein interaction analysis. This 2-step protocol consists of: 1) co-immunoprecipitation of the bait protein; 2) the bait-protein interactions analysis using LC-MS/MS. Here, we used Dynabeads® for the enrichment of the target protein (the bait) and its interactors. We have tested the protocol using several different cell lines. Our conclusion is that the protocol is applicable to different cell lines and species. For complete details on the use and execution of this protocol, please refer to Lagundzin et al. (2019).

Aminopyrazole based CDK9 PROTAC sensitizes pancreatic cancer cells to venetoclax.

Cyclin-dependent kinase 9 (CDK9) is a member of the cyclin-dependent kinase (CDK) family which is involved in transcriptional regulation of several genes, including the oncogene Myc, and is a validated target for pancreatic cancer. Here we report the development of an aminopyrazole based proteolysis targeting chimera (PROTAC 2) that selectively degrades CDK9 (DC50 = 158 ± 6 nM). Mass spectrometry-based kinome profiling shows PROTAC 2 selectively degrades CDK9 in MiaPaCa2 cells and sensitizes them to Venetoclax mediated growth inhibition.

Ctdp1 deficiency leads to early embryonic lethality in mice and defects in cell cycle progression in MEFs.

RNA polymerase II subunit A Carboxy-Terminal Domain Phosphatase 1 (CTDP1), a member of the haloacid dehalogenase superfamily phosphatases, has a defined role in transcriptional regulation, but emerging evidence suggests an expanded functional repertoire in the cell cycle and DNA damage response. In humans, a splice site mutation in CTDP1 gives rise to the rare Congenital Cataracts Facial Dysmorphism and Neuropathy syndrome, and recent evidence from our lab indicates CTDP1 is required for breast cancer growth and proliferation. To explore the physiological function of CTDP1 in a mammalian system, we generated a conditional Ctdp1 knockout mouse model by insertion of loxP sites upstream of exon 3 and downstream of exon 4. Biallelic deletion of Ctdp1 results in lethality before embryonic day 7.5, with morphological features indicating embryo cell death and resorption. However, Ctdp1+/- mice are haplosufficient for phenotypic traits including body weight, hematological parameters, exploratory and locomotive functions. To investigate the potential mechanisms of the embryonic death caused by biallelic Ctdp1 knockout, mouse embryonic fibroblasts (MEFs) were established from Ctdp1+/+ and Ctdp1flox/flox mice. Lentivirus delivered Cre-mediated biallelic deletion of Ctdp1 in MEFs results in cell death preceded by impaired proliferation characterized by an increase in G1- and G2-phase populations and a reduction in the S-phase population. These cell cycle alterations caused by deletion of Ctdp1 are associated with an increase in p27 protein expression and a decrease in phosphorylated RB, phosphorylated Histone H3, and Cyclin B expression. Together, these results reveal that Ctdp1 plays an essential role in early mouse embryo development and cell growth and survival in part by regulating the cell cycle.

Low- and High-Drag Intermittencies in Turbulent Channel Flows.

Recent direct numerical simulations (DNS) and experiments in turbulent channel flow have found intermittent low- and high-drag events in Newtonian fluid flows, at Reτ=uτh/ν between 70 and 100, where uτ, h and ν are the friction velocity, channel half-height and kinematic viscosity, respectively. These intervals of low-drag and high-drag have been termed "hibernating" and "hyperactive", respectively, and in this paper, a further investigation of these intermittent events is conducted using experimental and numerical techniques. For experiments, simultaneous measurements of wall shear stress and velocity are carried out in a channel flow facility using hot-film anemometry (HFA) and laser Doppler velocimetry (LDV), respectively, for Reτ between 70 and 250. For numerical simulations, DNS of a channel flow is performed in an extended domain at Reτ = 70 and 85. These intermittent events are selected by carrying out conditional sampling of the wall shear stress data based on a combined threshold magnitude and time-duration criteria. The use of three different scalings (so-called outer, inner and mixed) for the time-duration criterion for the conditional events is explored. It is found that if the time-duration criterion is kept constant in inner units, the frequency of occurrence of these conditional events remain insensitive to Reynolds number. There exists an exponential distribution of frequency of occurrence of the conditional events with respect to their duration, implying a potentially memoryless process. An explanation for the presence of a spike (or dip) in the ensemble-averaged wall shear stress data before and after the low-drag (or high-drag) events is investigated. During the low-drag events, the conditionally-averaged streamwise velocities get closer to Virk's maximum drag reduction (MDR) asymptote, near the wall, for all Reynolds numbers studied. Reynolds shear stress (RSS) characteristics during these conditional events are investigated for Reτ = 70 and 85. Except very close to the wall, the conditionally-averaged RSS is higher than the time-averaged value during the low-drag events.

Implementation of a multiple-fraction concatenation strategy in an online two-dimensional high-/low-pH reversed-phase/reversed-phase liquid chromatography platform for qualitative and quantitative shotgun proteomic analyses.

Multidimensional liquid chromatography is the mainstay separation technique used for shotgun proteomic analyses. The application of a multiple-fraction concatenation (MFC) strategy can result in a more disperse and consistent peptide elution profile across different fractions, when compared with a conventional strategy. Herein, we present the first automated online RP-RP platform implementing an MFC strategy to facilitate robust, unattended, routine proteomic analyses. The improved duty cycle utilization of the MFC strategy led to an increase of 9% in the separation space occupancy and increases of approximately 10% in the identification of both proteins and peptides. The peptides uniquely identified by the MFC strategy were significantly biased toward those of acidic nature, with increased precursor signals leading to improved MS/MS spectral quality and enhanced acidic peptide identification. These improvements in qualitative analysis using the MFC strategy were also extended to quantitative analysis. When the acquired proteome was quantified with a normalized spectral abundance factor, the additionally acquired acidic peptides were a critical factor leading to enhanced reproducibility of quantitation using the MFC strategy. With merits of superior qualitative and quantitative characteristics over the conventional strategy, the MFC strategy appears to be a highly amenable technique for enhancing the separation capacity for routine proteomic analyses.

UBR5 HECT domain mutations identified in mantle cell lymphoma control maturation of B cells.

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next-generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including mutations in the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5, with the majority of mutations within the HECT domain of the protein that can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B-cell maturation and activation, we generated a conditional mutant disrupting UBR5's C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and upregulation of proteins associated with messenger RNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B-cell maturation by stabilization of spliceosome components during B-cell development and suggests UBR5 mutations play a role in MCL transformation.

The Proteomic Landscape of Pancreatic Ductal Adenocarcinoma Liver Metastases Identifies Molecular Subtypes and Associations with Clinical Response.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC. EXPERIMENTAL DESIGN: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status. RESULTS: Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance. CONCLUSIONS: These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.

Delineating the role of FANCA in glucose-stimulated insulin secretion in ß cells through its protein interactome.

Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas ß cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucose-stimulated insulin secretion (GSIS). This study reveals that FANCA or FANCB knockdown impairs GSIS in human pancreas ß cell line EndoC-ßH3. To identify potential pathways by which FANCA might regulate GSIS, we employed a proteomics approach to identify FANCA protein interactions in EndoC-ßH3 differentially regulated in response to elevated glucose levels. Glucose-dependent changes in the FANCA interaction network were observed, including increased association with other FA family proteins, suggesting an activation of the DNA damage response in response to elevated glucose levels. Reactive oxygen species increase in response to glucose stimulation and are necessary for GSIS in EndoC-ßH3 cells. Glucose-induced activation of the DNA damage response was also observed as an increase in the DNA damage foci marker γ-H2AX and dependent upon the presence of reactive oxygen species. These results illuminate the role of FANCA in GSIS and its protein interactions regulated by glucose stimulation that may explain the prevalence of ß cell-specific endocrinopathies in FA patients.

3D printing with 2D colloids: designing rheology protocols to predict 'printability' of soft-materials.

Additive manufacturing (AM) techniques and so-called 2D materials have undergone an explosive growth in the past decade. The former opens multiple possibilities in the manufacturing of multifunctional complex structures, and the latter on a wide range of applications from energy to water purification. Extrusion-based 3D printing, also known as Direct Ink Writing (DIW), robocasting, and often simply 3D printing, provides a unique approach to introduce advanced and high-added-value materials with limited availability into lab-scale manufacturing. On the other hand, 2D colloids of graphene oxide (GO) exhibit a fascinating rheology and can aid the processing of different materials to develop 'printable' formulations. This work provides an in-depth rheological study of GO suspensions with a wide range of behaviours from Newtonian-like to viscoelastic 'printable' soft solids. The combination of extensional and shear rheology reveals the network formation process as GO concentration increases from <0.1 vol% to 3 vol%. Our results also demonstrate that the quantification of 'printability' can be based on three rheology parameters: the stiffness of the network via the storage modulus (G'), the solid-to-liquid transition or flow stress (σf), and the flow transition index, which relates the flow and yield stresses (FTI = σf/σy).

Characterization of the Molecular Mechanisms Underlying the Chronic Phase of Stroke in a Cynomolgus Monkey Model of Induced Cerebral Ischemia.

Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.

Does the sensorimotor system minimize prediction error or select the most likely prediction during object lifting?

The human sensorimotor system is routinely capable of making accurate predictions about an object's weight, which allows for energetically efficient lifts and prevents objects from being dropped. Often, however, poor predictions arise when the weight of an object can vary and sensory cues about object weight are sparse (e.g., picking up an opaque water bottle). The question arises, what strategies does the sensorimotor system use to make weight predictions when one is dealing with an object whose weight may vary? For example, does the sensorimotor system use a strategy that minimizes prediction error (minimal squared error) or one that selects the weight that is most likely to be correct (maximum a posteriori)? In this study we dissociated the predictions of these two strategies by having participants lift an object whose weight varied according to a skewed probability distribution. We found, using a small range of weight uncertainty, that four indexes of sensorimotor prediction (grip force rate, grip force, load force rate, and load force) were consistent with a feedforward strategy that minimizes the square of prediction errors. These findings match research in the visuomotor system, suggesting parallels in underlying processes. We interpret our findings within a Bayesian framework and discuss the potential benefits of using a minimal squared error strategy. NEW & NOTEWORTHY: Using a novel experimental model of object lifting, we tested whether the sensorimotor system models the weight of objects by minimizing lifting errors or by selecting the statistically most likely weight. We found that the sensorimotor system minimizes the square of prediction errors for object lifting. This parallels the results of studies that investigated visually guided reaching, suggesting an overlap in the underlying mechanisms between tasks that involve different sensory systems.

Tetramethylpyrazine nitrone, a multifunctional neuroprotective agent for ischemic stroke therapy.

TBN, a novel tetramethylpyrazine derivative armed with a powerful free radical-scavenging nitrone moiety, has been reported to reduce cerebral infarction in rats through multi-functional mechanisms of action. Here we study the therapeutic effects of TBN on non-human primate model of stroke. Thirty male Cynomolgus macaques were subjected to stroke with 4 hours ischemia and then reperfusion. TBN were injected intravenously at 3 or 6 hours after the onset of ischemia. Cerebral infarction was examined by magnetic resonance imaging at 1 and 4 weeks post ischemia. Neurological severity scores were evaluated during 4 weeks observation. At the end of experiment, protein markers associated with the stroke injury and TBN treatment were screened by quantitative proteomics. We found that TBN readily penetrated the blood brain barrier and reached effective therapeutic concentration after intravenous administration. It significantly reduced brain infarction and modestly preserved the neurological function of stroke-affected arm. TBN suppressed over-expression of neuroinflammatory marker vimentin and decreased the numbers of GFAP-positive cells, while reversed down-regulation of myelination-associated protein 2', 3'-cyclic-nucleotide 3'-phosphodiesterase and increased the numbers of NeuN-positive cells in the ipsilateral peri-infarct area. TBN may serve as a promising new clinical candidate for the treatment of ischemic stroke.

Developmental toxicity of the common UV filter, benophenone-2, in zebrafish embryos.

Benozophenone (BP) type UV filters are extensively used in the personal care products to provide protection against the harmful effects of UV radiation. BPs are one of the primary components in the UV filter family, in which benophenone-2 (BP2) is widely used as a UV filter reagent in the sunscreen. Humans used these personal care products directly on skin and the chemicals will be washed away to the water system. BP2 has been identified as one of the endocrine disruptor chemicals, which can inference the synthesis, metabolism, and action of endogenous hormones. Environmentally, it has been found to contaminate water worldwide. In this study, we aimed to unfold the possible developmental toxicology of this chemical. Zebrafish are used as the screening model to perform in situ hybridization staining to investigate the effects of BP2 on segmentation, brain regionalization, and facial formation at four developmental stages (10-12 somite, prim-5, 2 and 5 days post-fertilization). Results showed 40 µM (9.85 mg L-1) or above BP2 exposure in zebrafish embryos for 5 days resulted in lipid accumulation in the yolk sac and facial malformation via affecting the lipid processing and the expression of cranial neural crest cells respectively. To conclude, the study alarmed its potential developmental toxicities at high dosage exposure.

Topical Therapy with Mesenchymal Stem Cells Following an Acute Experimental Head Injury Has Benefits in Motor-Behavioral Tests for Rodents.

BACKGROUND: The neuroprotective effects of mesenchymal stem cells (MSCs) have been reported in rodent and in preliminary clinical studies. MSCs are usually transplanted to patients by systemic infusion. However, only a few of the infused MSCs are delivered to the brain because of pulmonary trapping and the blood-brain barrier. In this study, MSCs were topically applied to the site of traumatic brain injury (TBI) and the neuroprotective effects were assessed. MATERIALS AND METHODS: TBI was induced in Sprague-Dawley (SD) rats with an electromagnetically controlled cortical impact device after craniotomy was performed between the bregma and lambda, 1 mm lateral to the midline. We applied 1.5 million MSCs, derived from the adipose tissue of transgenic green fluorescent protein (GFP)-SD rats, to the exposed cerebral cortex at the injured site. The MSCs were held in position by a thin layer of fibrin. Neurological function in the test (n = 10) and control (n = 10) animals was evaluated using the rotarod test, the water maze test, and gait analysis at different time points. RESULTS: Within 5 days following topical application, GFP-positive cells were found in the brain parenchyma. These cells co-expressed with markers of Glial fibrillary acidic protein (GFAP), nestin, and NeuN. There was less neuronal death in CA1 and CA3 of the hippocampus in the test animals. Neurological functional recovery was significantly improved. CONCLUSION: Topically applied MSCs can migrate to the injured brain parenchyma and offer neuroprotective effects.