Dicer1 Ablation Impairs Responsiveness of Cerebellar Granule Neuron Precursors to Sonic Hedgehog and Disrupts Expression of Distinct Cell Cycle Regulator Genes.

Granule neuron precursors (GNPs) proliferate under the influence of Sonic hedgehog (Shh) that is secreted by Purkinje neurons during early postnatal cerebellar development. To investigate microRNA (miRNA) function in this developmental process, we conditionally deleted the Dicer1 gene under the activity of human glial fibrillary acidic protein (hGFAP) promoter. We report that Dicer1-ablated GNPs display decreased proliferation and survival at early postnatal stages and that the proliferation defect of mutant GNPs cannot be rescued by treatment of an Shh agonist in vitro as assayed by 5-bromo-2'-deoxyuridine (BrdU) pulse labeling and Shh target gene expression detection. Further analysis reveals that the expression of distinct cell cycle regulator genes including cell cycle inhibitor, CDKN1a (p21), selectively increases in Dicer1-ablated GNPs. Subsequently, we demonstrate that miR-17-5p exhibits high expression level in the developing cerebellum and that transfection of a synthetic miR-17-5p mimic downregulates p21 protein expression in GNPs and promotes proliferation of GNPs in culture. Therefore, Dicer1 ablation impairs Shh-induced GNP proliferation by disrupting the expression of distinct cell cycle regulator genes that are targets of miR-17∼92 cluster members. This study establishes a molecular link between miRNAs and cell cycle progression in the proliferating GNPs during normal cerebellar development and may facilitate miRNA application in treating medulloblastoma.

MicroRNA-mediated non-cell-autonomous regulation of cortical radial glial transformation revealed by a Dicer1 knockout mouse model.

Radial glia (RG), as neurogenic progenitors and neuronal migration scaffolds, play critical roles during cortical neurogenesis. RG transformation into astrocytes, marking the transition from developmental to physiological function of these cells, is an important step during cortical development. In this study, we aim to determine the roles of microRNAs (miRNAs) during this biological process. In a conditional Dicer1-null mouse where Dicer1 is deleted in both RG and their neuronal progeny, we observe delayed RG transformation as revealed by the persistence of their radial processes, and reduced number and complexity of translocated RG cell bodies in the postnatal cerebral cortex. Downregulation of Notch1 signaling is crucial to RG transformation, and consistently we find that Notch1 signaling is enhanced in the Dicer1-null cerebral cortex. In addition, we show that, among the Notch1 ligands, Jagged2 (Jag2) is preferentially upregulated in the postnatal Dicer1-null cerebral cortex as well as primary embryonic cortical cultures with instant Dicer1 deletion. Functionally, Dicer1-deleted postnatal cerebellar cells with elevated Jag2 expression stimulate a stronger Notch1 signaling in a RG clone L2.3 when co-cultured than control cells. Therefore, we unravel a novel non-cell-autonomous mechanism that regulates RG transformation by modulating Notch1 signaling via miRNA-mediated suppression of the Nocth1 ligand Jag2. Furthermore, we validate Jag2 as a miR-124 target gene and demonstrate in vitro that Jag2 expression is highly sensitive to Dicer1 deletion. Finally, we propose a new concept of MiRNA-Sensitive target genes, identification of which may unravel a unique mode of miRNA-mediated gene expression regulation.

[Research on regulation function of γ-secretase inhibitor DAPT on the differentiation of neural precursor cell line].

This study aims to investigate the effect of γ-Secretase Inhibitor DAPT, (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), on the differentiation of neural precursor cells and the production of neurons in the neural precursor cell line GE6. GE6 was cultured in medium with 4 µmol/L DAPT added as the experimental group and the untreated medium separately as the control group. After 4 days of differentiation, we carried out the following experiments. We used immuno-fluorescent staining to observe the ratio of Tuj1, GFAP and O4 positive cells. We also used qRT-PCR to detect the effect of the DAPT on Tuj1 and GFAP mRNA transcription in the GE6. The results of immuno-fluorescent staining indicated that the Tuj1 ratio of experimental group was higher compared to that of the control group, but the GFAP and O4 ratio of experimental group was lower than that of the control group. The differences were statistically significant (P < 0.05). The result of qRT-PCR was in accordance with immunofluorescent staining results. It was well concluded that DAPT could promote the neurogenic differentiation of neural precursor cell line rather than leading to gliogenic differentiation. More neurons could be obtained for transplantation with the addition of DAPT.

[Expression of MicroRNAs of An Interneuron Precursor Cell Line GE6 in Various Differentiation Conditions].

The purpose of this study was to identify specific microRNAs (miRNAs) during differentiation and maturation of interneurons and to predict their possible functions by analyzing the expression of miRNAs during in vitro differentiation of the rat interneuron precursor cell line GE6. In the experiment, the interneuron precursor cell line GE6 was cultured under three different conditions, i. e. the first was that had not added growth factors and the normal differentiation cultured for 4 days (Ge6_4d); the second was that cultured with bone morphogenetic protein-2 (BMP2) for 4 days (Ge6_bmp2); and the third was that cultured with sonic hedgehog (SHH) for 4 days (Ge6_ shh). In addition, another group of undifferentiated GE6 (Ge6_u) was applied as a control. We found in this study that the expression levels of a large number of miRNAs changed significantly during GE6 differentiation. The expression levels of miR-710, miR-290-5p and miR-3473 increased in the GE6 cells with secreted factor BMP2. These miRNAs may play important regulatory roles during interneuron differentiation.

Functional requirement of dicer1 and miR-17-5p in reactive astrocyte proliferation after spinal cord injury in the mouse.

Reactive astrogliosis after spinal cord injury (SCI) contributes to glial scar formation that impedes axonal regeneration. The mechanisms underlying reactive astrocyte proliferation upon injury remain partially understood. MicroRNAs (miRNAs) function as a major class of post-transcriptional gene expression regulators that participate in many biological processes. However, miRNA function during reactive astrogliosis, particularly in injury-induced astrocyte proliferation, has not been carefully examined. In this study, we conditionally deleted Dicer1 gene encoding an enzyme that is required for mature miRNA generation, and examined the proliferative behavior of Dicer1-null reactive astrocytes in the transected mouse spinal cord. We found that injury-induced proliferation is blocked in Dicer1-null astrocytes. Previous reports indicate that miR-17-5p family members are upregulated during SCI. We therefore tested functional contribution of miR-17-5p to the proliferation of reactive astrocytes in vitro. Our results showed that a synthetic miR-17-5p mimic is able to rescue the proliferation defect of Dicer1-null astrocytes, while an antisense inhibitor of miR-17-5p blocked lipopolysaccharide-induced astrocytic proliferation. Similar results are also observed in leukemia inhibitory factor (LIF)-treated astroglial cultures suggesting that miR-17-5p particularly modulates reactive astrocyte proliferation initiated by LIF presumably via the JAK/STAT3 pathway. Furthermore, overexpression of miR-17-5p leads to decrease of several cell cycle regulators in cultured astroglia and astrocytoma cell line C6. Our conclusion is that miRNAs are indispensable to the injury-induced reactive astrocyte proliferation, and that miR-17-5p may be a major player regulating this pathological process by affecting cell cycle machinery.

[Study on the anti-apoptosis effect of insulin-like growth factor I on the regulation of neural precursor cells survival].

OBJECTIVE: To investigate the effect of insulin-like growth factor I (IGF-I) on the anti-apoptosis of neural precursor cells. METHODS: Rat neural precursor cells (Ge6) were used as experiment object. 10 ng/mL IGF- I were applied to cultured Ge6 differentiated 2 d, 4 d, 6 d and the untreated cells as control. Quantitative reverse transcriptase PCR (qRT-PCR) was used to detect the effect of the IGF-I on caspase-3 mRNA in the Ge6 of different groups and Immunofluorescent staining were applied to obverse the positive rate of caspase-3. RESULTS: qRT-PCR demonstrated that caspase-3 mRNA level of experimental group decreased compared to that of the control group, the differences between experimental group cultured for 4 d (2.007 +/- 0.297), 6 d (1.194 +/- 0.244) and the control group cultured for 4 d (5.530 +/- 1.027), 6 d (5.371 +/- 0.513) were statistically significant (P < 0.05). Immunofluorescent staining indicated that the caspase-3 positive rate of experimental group cultured for 2 d, 4 d and 6 d were reduced compared to that of the control group, the differences between experimental group cultured for 4 d (3.5% +/- 10.2%), 6 d (5.1% +/- 0.1%) and the control group cultured for 4 d (7.0% +/- 10.5%), 6 d (7.4% +/- 0.4%) were statistically significant (P < 0.05). CONCLUSION: IGF-I plays an anti-apoptosis role on neural precursor cells.

Controlling the Expression Level of the Neuronal Reprogramming Factors for a Successful Reprogramming Outcome.

Neuronal reprogramming is a promising approach for making major advancement in regenerative medicine. Distinct from the approach of induced pluripotent stem cells, neuronal reprogramming converts non-neuronal cells to neurons without going through a primitive stem cell stage. In vivo neuronal reprogramming brings this approach to a higher level by changing the cell fate of glial cells to neurons in neural tissue through overexpressing reprogramming factors. Despite the ongoing debate over the validation and interpretation of newly generated neurons, in vivo neuronal reprogramming is still a feasible approach and has the potential to become clinical treatment with further optimization and refinement. Here, we discuss the major neuronal reprogramming factors (mostly pro-neurogenic transcription factors during development), especially the significance of their expression levels during neurogenesis and the reprogramming process focusing on NeuroD1. In the developing central nervous system, these pro-neurogenic transcription factors usually elicit distinct spatiotemporal expression patterns that are critical to their function in generating mature neurons. We argue that these dynamic expression patterns may be similarly needed in the process of reprogramming adult cells into neurons and further into mature neurons with subtype identities. We also summarize the existing approaches and propose new ones that control gene expression levels for a successful reprogramming outcome.

MicroRNA-375 is induced during astrocyte-to-neuron reprogramming and promotes survival of reprogrammed neurons when overexpressed.

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. MicroRNAs (miRNAs), as post-transcriptional regulators of gene expression, play crucial roles during development and under various pathological conditions. To understand the function of miRNAs during AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte (HA) cultures upon NeuroD1 overexpression. Bioinformatics analyses showed that NeuroD1 not only activates essential neuronal genes to initiate reprogramming process but also induces miRNA changes in HA. Among the upregulated miRNAs, we identified miR-375 and its targets, neuronal ELAVL genes ( nELAVLs ), which encode a family of RNA-binding proteins and are also upregulated by NeuroD1. We further showed that manipulating miR-375 level regulates nELAVLs expression during NeuroD1-mediated reprogramming. Interestingly, miR-375/ nELAVLs are also induced by reprogramming factors Neurog2 and ASCL1 in HA suggesting a conserved function to neuronal reprogramming, and by NeuroD1 in the mouse astrocyte culture and spinal cord. Functionally, we showed that miR-375 overexpression improves NeuroD1-mediated reprogramming efficiency by promoting cell survival at early stages in HA even in cultures treated with the chemotherapy drug Cisplatin. Moreover, miR-375 overexpression doesn't appear to compromise maturation of the reprogrammed neurons in long term HA cultures. Lastly, overexpression of miR-375-refractory ELAVL4 induces apoptosis and reverses the cell survival-promoting effect of miR-375 during AtN reprogramming. Together, we demonstrate a neuro-protective role of miR-375 during NeuroD1-mediated AtN reprogramming and suggest a strategy of combinatory overexpression of NeuroD1 and miR-375 for improving neuronal reprogramming efficiency.

MicroRNA-375 Is Induced during Astrocyte-to-Neuron Reprogramming and Promotes Survival of Reprogrammed Neurons when Overexpressed.

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. To understand the function of miRNAs during the AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte (HA) cultures upon NeuroD1 overexpression. Bioinformatics analyses showed that NeuroD1 not only activated essential neuronal genes to initiate the reprogramming process but also induced miRNA changes in HA. Among the upregulated miRNAs, we identified miR-375 and its targets, neuronal ELAVL genes (nELAVLs), which encode a family of RNA-binding proteins and were also upregulated by NeuroD1. We further showed that manipulating the miR-375 level regulated nELAVLs' expression during NeuroD1-mediated reprogramming. Interestingly, miR-375/nELAVLs were also induced by the reprogramming factors Neurog2 and ASCL1 in HA, suggesting a conserved function to neuronal reprogramming, and by NeuroD1 in the mouse astrocyte culture and spinal cord. Functionally, we showed that miR-375 overexpression improved NeuroD1-mediated reprogramming efficiency by promoting cell survival at early stages in HA and did not appear to compromise the maturation of the reprogrammed neurons. Lastly, overexpression of miR-375-refractory ELAVL4 induced apoptosis and reversed the cell survival-promoting effect of miR-375 during AtN reprogramming. Together, we demonstrated a neuroprotective role of miR-375 during NeuroD1-mediated AtN reprogramming.

Dicer1 and MiR-9 are required for proper Notch1 signaling and the Bergmann glial phenotype in the developing mouse cerebellum.

MicroRNAs (miRNAs) have important roles in the development of the central nervous system (CNS). Several reports indicate that tissue development and cellular differentiation in the developing forebrain are disrupted in the absence of miRNAs. However, the functions of miRNAs during cerebellar development have not been systematically characterized. Here, we conditionally knocked out the Dicer1 gene under the control of the human glial fibrillary acidic protein (hGFAP) promoter to examine the effect of miRNAs in the developing cerebellum. We particularly focused on the phenotype of Bergmann glia (BG). The hGFAP-Cre activity was detected as early as embryonic day 13.5 (E13.5) at the rhombic lip (RL) in the cerebellar plate, and later in several postnatal cerebellar cell types, including BG. Dicer1 ablation induces a smaller and less developed cerebellum, accompanied by aberrant BG morphology. Notch1 signaling appears to be blocked in Dicer1-ablated BG, with reduced expression of the Notch1 target gene, brain lipid binding protein (BLBP). Using neuronal co-culture assays, we showed an intrinsic effect of Dicer1 on BG morphology and Notch1 target gene expression. We further identified miR-9 as being differentially expressed in BG and showed that miR-9 is a critical, but not the only, miRNA component of the Notch1 signaling pathway in cultured BG cells.

Neuronal reprogramming in treating spinal cord injury.

Spinal cord injury represents a devastating central nervous system injury that could impair the mobility and sensory function of afflicted patients. The hallmarks of spinal cord injury include neuroinflammation, axonal degeneration, neuronal loss, and reactive gliosis. Furthermore, the formation of a glial scar at the injury site elicits an inhibitory environment for potential neuroregeneration. Besides axonal regeneration, a significant challenge in treating spinal cord injury is to replenish the neurons lost during the pathological process. However, despite decades of research efforts, current strategies including stem cell transplantation have not resulted in a successful clinical therapy. Furthermore, stem cell transplantation faces serious hurdles such as immunorejection of the transplanted cells and ethical issues. In vivo neuronal reprogramming is a recently developed technology and leading a major breakthrough in regenerative medicine. This innovative technology converts endogenous glial cells into functional neurons for injury repair in the central nervous system. The feasibility of in vivo neuronal reprogramming has been demonstrated successfully in models of different neurological disorders including spinal cord injury by numerous laboratories. Several reprogramming factors, mainly the pro-neural transcription factors, have been utilized to reprogram endogenous glial cells into functional neurons with distinct phenotypes. So far, the literature on in vivo neuronal reprogramming in the model of spinal cord injury is still small. In this review, we summarize a limited number of such reports and discuss several questions that we think are important for applying in vivo neuronal reprogramming in the research field of spinal cord injury as well as other central nervous system disorders.

Establishing a Herpesvirus Quiescent Infection in Differentiated Human Dorsal Root Ganglion Neuronal Cell Line Mediated by Micro-RNA Overexpression.

HSV-1 is a neurotropic pathogen associated with severe encephalitis, excruciating orofacial sensation, and other chronic neuropathic complications. After the acute infection, the virus may establish a lifelong latency in the neurons of trigeminal ganglia (TG) and other sensory and autonomic ganglia, including the dorsal root ganglia (DRG), etc. The reactivation occurred periodically by a variety of physical or emotional stressors. We have been developing a human DRG neuronal cell-culture model HD10.6, which mimics the mature neurons for latency and reactivation with robust neuronal physiology. We found that miR124 overexpression without acyclovir (ACV) could maintain the virus in a quiescent infection, with the accumulation of latency-associate transcript (LAT). The immediate-early (IE) gene ICP0, on the other hand, was very low and the latent viruses could be reactivated by trichostatin A (TSA) treatment. Together, these observations suggested a putative role of microRNA in promoting HSV-1 latency in human neurons.

Regeneration of Functional Neurons After Spinal Cord Injury via in situ NeuroD1-Mediated Astrocyte-to-Neuron Conversion.

Spinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimer's disease by overexpressing a single neural transcription factor NeuroD1. Here we demonstrate regeneration of spinal cord neurons from reactive astrocytes after SCI through AAV NeuroD1-based gene therapy. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (~95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate after conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model with a long delay after injury, allowing future studies to further evaluate this in vivo reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift from classical axonal regeneration to neuronal regeneration for spinal cord repair, using in vivo astrocyte-to-neuron conversion technology to regenerate functional new neurons in the gray matter.

Durability of the Bond between CFRP and Concrete Exposed to Thermal Cycles.

The bond between carbon fiber reinforced polymer (CFRP) and concrete is significantly and adversely affected by thermal cycles in air and water. In the present study, the effects of thermal cycles in air or water on the bond performance between CFRP and concrete were examined. A single-lap shear test was adopted to evaluate the performance of the CFRP-concrete bond. A number of 270 thermal cycles in air increased the interfacial fracture energy of the CFRP plate- and CFRP sheet-concrete by 35% and 20%, respectively while 270 thermal cycles in water reduced the interfacial fracture energy of the CFRP plate⁻ and CFRP sheet-concrete by 9% and 46%, respectively. Thermal cycles in water caused the failure mode to change from concrete cohesive failure to primer-concrete interfacial debonding. The failure modes of CFRP-concrete exposed to thermal cycles in air still occurred in concrete. A reduction factor for the CFRP-concrete structure for thermal cycles in water was proposed.

Huwe1 is a novel mediator of protection of neural progenitor L2.3 cells against oxygen­glucose deprivation injury.

Hypoxic­ischemic encephalopathy is one of the most notable causes of brain injury in newborns. Cerebral ischemia and reperfusion lead to neuronal damage and neurological disability. In vitro and in vivo analyses have indicated that E3 ubiquitin protein ligase (Huwe1) is important for the process of neurogenesis during brain development; however, the exact biological function and the underlying mechanism of Huwe1 remain controversial. In the present study, neural progenitor cells, L2.3, of which we previously generated from rat E14.5 cortex, were used to investigate the role of Huwe1 and its effects on the downstream N­Myc­Delta­like 3­Notch1 signaling pathway during oxygen­glucose deprivation (OGD). To evaluate the role of Huwe1 in L2.3 cells, transduction, cell viability, lactate dehydrogenase, 5­bromo­2'deoxyurine incorporation, western blotting and immunocytochemical assays were performed. The results of the present study indicated that Huwe1 rescued L2.3 cells from OGD­induced insults by inhibiting proliferation and inducing neuronal differentiation. In addition, Huwe1 was suggested to mediate the survival of L2.3 cells by inhibiting the activation of the N­Myc­Notch1 signaling pathway. Of note, the effects of Huwe1 on Notch1 signaling were completely abolished by knockdown of N­Myc, indicating that Huwe1 may require N­Myc to suppress the activation of the Notch1 signaling in L2.3 cells. The determination of the neuroprotective function of the Huwe1­N­Myc­Notch1 axis may provide insight into novel potential therapeutic targets for the treatment of ischemic stroke.

Recycling polyethylene terephthalate wastes as short fibers in Strain-Hardening Cementitious Composites (SHCC).

As an important portion of the total plastic waste bulk but lack of reuse and recycling, the enormous amounts of polyethylene terephthalate (PET) solid wastes have led to serious environmental issues. This study explores the feasibility of recycling PET solid wastes as short fibers in Strain-Hardening Cementitious Composites (SHCCs), which exhibit strain-hardening and multiple cracking under tension, and therefore have clear advantages over conventional concrete for many construction applications. Based on micromechanical modeling, fiber dispersion and alkali resistance, the size of recycled PET fibers was first determined. Then the hydrophobic PET surface was treated with NaOH solution followed by a silane coupling agent to achieve the dual purpose of improving the fiber/matrix interfacial frictional bond (from 0.64 MPa to 0.80 MPa) and enhancing the alkali resistance for applications in alkaline cementitious environment. With surface treatment, recycling PET wastes as fibers in SHCCs is a promising approach to significantly reduce the material cost of SHCCs while disposing hazardous PET wastes in construction industry.

Detection of unexpected frauds: Screening and quantification of maleic acid in cassava starch by Fourier transform near-infrared spectroscopy.

Fourier transform near-infrared (FT-NIR) spectroscopy and chemometrics were adopted for the rapid analysis of a toxic additive, maleic acid (MA), which has emerged as a new extraneous adulterant in cassava starch (CS). After developing an untargeted screening method for MA detection in CS using one-class partial least squares (OCPLS), multivariate calibration models were subsequently developed using least squares support vector machine (LS-SVM) to quantitatively analyze MA. As a result, the OCPLS model using the second-order derivative (D2) spectra detected 0.6%(w/w) adulterated MA in CS, with a sensitivity of 0.954 and specificity of 0.956. The root mean squared error of prediction (RMSEP) was 0.192(w/w, %) by using the standard normal variate (SNV) transformation LS-SVM. In conclusion, the potential of FT-NIR spectroscopy and chemometrics was demonstrated for application in rapid screening and quantitative analysis of MA in CS, which also implies that they have other promising applications for untargeted analysis.

Automatic time-shift alignment method for chromatographic data analysis.

Time shift among samples remains a significant challenge in data analysis, such as quality control of natural plant extracts and metabolic profiling analysis, because this phenomenon may lead to invalid conclusions. In this work, we propose a new time shift alignment method, namely, automatic time-shift alignment (ATSA), for complicated chromatographic data analysis. This technique comprised the following alignment stages: (1) automatic baseline correction and peak detection stage for providing useful chromatographic information; (2) preliminary alignment stage through adaptive segment partition to correct alignment for the entire chromatogram; and (3) precise alignment stage based on test chromatographic peak information to accurately align time shift. In ATSA, the chromatographic peak information of both reference and test samples can be completely employed for time-shift alignment to determine segment boundaries and avoid loss of information. ATSA was used to analyze a complicated chromatographic dataset. The obtained correlation coefficients among samples and data analysis efficiency indicated that the influences of time shift can be considerably reduced by ATSA; thus accurate conclusion could be obtained.

Pharmacokinetic Analysis of Four Bioactive Iridoid and Secoiridoid Glycoside Components of Radix Gentianae Macrophyllae and Their Synergistic Excretion by HPLC-DAD Combined with Second-Order Calibration.

An HPLC-DAD method combined with second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm with the aid of region selection was developed to simultaneously and quantitatively characterize the synergistic relationships and cumulative excretion of the four bioactive ingredients of Radix Gentianae Macrophyllae in vivo. Although the analytes spectra substantially overlapped with that of the biological matrix, the overlapping profiles between analytes and co-eluting interferences can be successfully separated and accurately quantified by the ATLD method on the basis of the strength of region selection. The proposed approach not only determined the content change but also revealed the synergistic relationships and the cumulative excretion in vivo of the four ingredients in urine and feces samples collected at different excretion time intervals. In addition, several statistical parameters were employed to evaluate the accuracy and precision of the method. Quantitative results were confirmed by HPLC-mass spectrometry. Satisfactory results indicated that the proposed approach can be utilized to investigate the pharmacokinetics of Radix Gentianae Macrophyllae excretion in vivo.

In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells.

Neuroregeneration in the CNS has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this Perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart, and liver and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient's own internal cells for tissue repair.