Amphetamine disrupts dopamine axon growth in adolescence by a sex-specific mechanism in mice.

Initiating drug use during adolescence increases the risk of developing addiction or other psychopathologies later in life, with long-term outcomes varying according to sex and exact timing of use. The cellular and molecular underpinnings explaining this differential sensitivity to detrimental drug effects remain unexplained. The Netrin-1/DCC guidance cue system segregates cortical and limbic dopamine pathways in adolescence. Here we show that amphetamine, by dysregulating Netrin-1/DCC signaling, triggers ectopic growth of mesolimbic dopamine axons to the prefrontal cortex, only in early-adolescent male mice, underlying a male-specific vulnerability to enduring cognitive deficits. In adolescent females, compensatory changes in Netrin-1 protect against the deleterious consequences of amphetamine on dopamine connectivity and cognitive outcomes. Netrin-1/DCC signaling functions as a molecular switch which can be differentially regulated by the same drug experience as function of an individual's sex and adolescent age, and lead to divergent long-term outcomes associated with vulnerable or resilient phenotypes.

Awakening the dormant: Role of axonal guidance cues in stress-induced reorganization of the adult prefrontal cortex leading to depression-like behavior.

Major depressive disorder (MDD) is a chronic and disabling disorder affecting roughly 280 million people worldwide. While multiple brain areas have been implicated, dysfunction of prefrontal cortex (PFC) circuitry has been consistently documented in MDD, as well as in animal models for stress-induced depression-like behavioral states. During brain development, axonal guidance cues organize neuronal wiring by directing axonal pathfinding and arborization, dendritic growth, and synapse formation. Guidance cue systems continue to be expressed in the adult brain and are emerging as important mediators of synaptic plasticity and fine-tuning of mature neural networks. Dysregulation or interference of guidance cues has been linked to depression-like behavioral abnormalities in rodents and MDD in humans. In this review, we focus on the emerging role of guidance cues in stress-induced changes in adult prefrontal cortex circuitry and in precipitating depression-like behaviors. We discuss how modulating axonal guidance cue systems could be a novel approach for precision medicine and the treatment of depression.

The Mitochondrial Pyruvate Carrier Coupling Glycolysis and the Tricarboxylic Acid Cycle Is Required for the Asexual Reproduction of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite capable of infecting humans and animals. The organism has extraordinary metabolic resilience that allows it to establish parasitism in varied nutritional milieus of diverse host cells. Our earlier work has shown that, despite flexibility in the usage of glucose and glutamine as the major carbon precursors, the production of pyruvate by glycolytic enzymes is central to the parasite's growth. Pyruvate is metabolized in a number of subcellular compartments, including the mitochondrion, apicoplast, and cytosol. With the objective of examining the mechanism and importance of the mitochondrial pool of pyruvate imported from the cytosol, we identified the conserved mitochondrial pyruvate carrier (MPC) complex, consisting of two subunits, MPC1 and MPC2, in T. gondii. The two parasite proteins could complement a yeast mutant deficient in growth on leucine and valine. Genetic ablation of either one or both subunits reduced the parasite's growth, mimicking the deletion of branched-chain ketoacid dehydrogenase (BCKDH), which has been reported to convert pyruvate into acetyl-coenzyme A (CoA) in the mitochondrion. Metabolic labeling of the MPC mutants by isotopic glucose revealed impaired synthesis of acetyl-CoA, correlating with a global decrease in carbon flux through glycolysis and the tricarboxylic acid (TCA) cycle. Disruption of MPC proteins exerted only a modest effect on the parasite's virulence in mice, further highlighting its metabolic flexibility. In brief, our work reveals the modus operandi of pyruvate transport from the cytosol to the mitochondrion in the parasite, providing the missing link between glycolysis and the TCA cycle in T. gondii. IMPORTANCE T. gondii is a zoonotic parasite capable of infecting many warm-blooded organisms, including humans. Among others, a feature that allows it to parasitize multiple hosts is its exceptional metabolic plasticity. Although T. gondii can utilize different carbon sources, pyruvate homeostasis is critical for parasite growth. Pyruvate is produced primarily in the cytosol but metabolized in other organelles, such as the mitochondrion and apicoplast. The mechanism of import and physiological significance of pyruvate in these organelles remains unclear. Here, we identified the transporter of cytosol-derived pyruvate into the mitochondrion and studied its constituent subunits and their relevance. Our results show that cytosolic pyruvate is a major source of acetyl-CoA in the mitochondrion and that the mitochondrial pyruvate transporter is needed for optimal parasite growth. The mutants lacking the transporter are viable and virulent in a mouse model, underscoring the metabolic plasticity in the parasite's mitochondrion.

Antagonistic Strain Bacillus halotolerans Jk-25 Mediates the Biocontrol of Wheat Common Root Rot Caused by Bipolaris sorokiniana.

Common root rot caused by Bipolaris sorokiniana infestation in wheat is one of the main reasons for yield reduction in wheat crops worldwide. The bacterium strain JK-25 used in the current investigation was isolated from wheat rhizosphere soil and was later identified as Bacillus halotolerans based on its morphological, physiological, biochemical, and molecular properties. The strain showed significant antagonism to B. sorokiniana, Fusarium oxysporum, Fusarium graminearum, and Rhizoctonia zeae. Inhibition of B. sorokiniana mycelial dry weight and spore germination rate by JK-25 fermentation supernatant reached 60% and 88%, respectively. The crude extract of JK-25 was found, by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), to contain the surfactin that exerted an inhibitory effect on B. sorokiniana. The disruption of mycelial cell membranes was observed under laser scanning confocal microscope (LSCM) after treatment of B. sorokiniana mycelium with the crude extract. The antioxidant enzyme activity of B. sorokiniana was significantly reduced and the oxidation product malondialdehyde (MDA) content increased after treatment with the crude extract. The incidence of root rot was significantly reduced in pot experiments with the addition of JK-25 culture fermentation supernatant, which had a significant biological control effect of 72.06%. Its ability to produce siderophores may help to promote wheat growth and the production of proteases and pectinases may also be part of the strain's role in suppressing pathogens. These results demonstrate the excellent antagonistic effect of JK-25 against B. sorokiniana and suggest that this strain has great potential as a resource for biological control of wheat root rot strains.

Rapid metabolic reprogramming mediated by the AMP-activated protein kinase during the lytic cycle of Toxoplasma gondii.

The ubiquitous pathogen Toxoplasma gondii has a complex lifestyle with different metabolic activities at different stages that are intimately linked to the parasitic environments. Here we identified the eukaryotic regulator of cellular homeostasis AMP-activated protein kinase (AMPK) in Toxoplasma and discovered its role in metabolic programming during parasite's lytic cycle. The catalytic subunit AMPKα is quickly phosphorylated after the release of intracellular parasites to extracellular environments, driving energy-producing catabolism to power parasite motility and invasion into host cells. Once inside host cells, AMPKα phosphorylation is reduced to basal level to promote a balance between energy production and biomass synthesis, allowing robust parasite replication. AMPKγ depletion abolishes AMPKα phosphorylation and suppresses parasite growth, which can be partially rescued by overexpressing wildtype AMPKα but not the phosphorylation mutants. Thus, through the cyclic reprogramming by AMPK, the parasites' metabolic needs at each stage are satisfied and the lytic cycle progresses robustly.

Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth.

Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.

The beta subunit of AMP-activated protein kinase is critical for cell cycle progression and parasite development in Toxoplasma gondii.

Toxoplasma gondii is a widespread eukaryotic pathogen that causes life-threatening diseases in humans and diverse animals. It has a complex life cycle with multiple developmental stages, which are timely adjusted according to growth conditions. But the regulatory mechanisms are largely unknown. Here we show that the AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in eukaryotes, plays crucial roles in controlling the cell cycle progression and bradyzoite development in Toxoplasma. Deleting the ß regulatory subunit of AMPK in the type II strain ME49 caused massive DNA damage and increased spontaneous conversion to bradyzoites (parasites at chronic infection stage), leading to severe growth arrest and reduced virulence of the parasites. Under alkaline stress, all Δampkß mutants converted to a bradyzoite-like state but the cell division pattern was significantly impaired, resulting in compromised parasite viability. Moreover, we found that phosphorylation of the catalytic subunit AMPKα was greatly increased in alkaline stressed parasites, whereas AMPKß deletion mutants failed to do so. Phosphoproteomics found that many proteins with predicted roles in cell cycle and cell division regulation were differentially phosphorylated after AMPKß deletion, under both normal and alkaline stress conditions. Together, these results suggest that the parasite AMPK has critical roles in safeguarding cell cycle progression, and guiding the proper exist of the cell cycle to form mature bradyzoites when the parasites are stressed. Consistent with this model, growth of parasites was not significantly altered when AMPKß was deleted in a strain that was naturally reluctant to bradyzoite development.

Molecular characterization of the genome-wide BOR transporter family and their responses to boron conditions in common wheat (Triticum aestivum L.).

Boron (B) deficiency is an agricultural problem that causes significant yield losses in many countries. B transporters (BORs) are responsible for B uptake and distribution and play important roles in yield formation. A comprehensive analysis of the BOR family members in common wheat is still lacking. In the present study, to clarify the molecular characterization and response to B status, genome-wide TaBOR genes and expression patterns were investigated. Fourteen TaBOR genes were identified in common wheat by a homology search. The corresponding phylogenetic tree indicated that 14 TaBOR genes were separately classified into subfamilies of TaBOR1, TaBOR3, and TaBOR4. All TaBOR genes had 12-14 extrons and 11-13 introns. Most TaBOR proteins contained 10 conserved motifs, and motifs 1, 2, 3, 4, and 6 constituted the conserved bicarbonate (HCO3 -) domain. Fourteen TaBOR genes were mapped on 13 chromosomes mainly distributed in the first, third, fifth, and seventh homologous groups. The promoters of TaBOR genes consisted of phytohormones, light responses, and stress-related cis-elements. GO analysis indicated that TaBOR genes were enriched in terms of transmembrane transport and ion homeostasis. TaBOR genes showed diverse expression profiles in different tissues. The members of the TaBOR1 subfamily showed high expression in grains, leaves, roots, stems, and spikes, but members of the TaBOR4 subfamily were highly expressed only in spikes and grains. RT-qPCR indicated that TaBOR1-5A, TaBOR1-5B, and TaBOR1-5D were induced by low B concentrations and had much higher expression in roots than in shoots. TaBOR3-3A, TaBOR3-3B, TaBOR3-3D, TaBOR4-1A, TaBOR4-1B, TaBOR4-1D, and TaBOR3-4B were induced by low and high B concentrations and had high expression in roots and shoots. TaBOR3-4D and TaBOR3-7B were upregulated by low and high B concentrations, respectively, but had expression only in roots. Our results provide basic information on the TaBOR family, which is beneficial for elucidating the functions of TaBOR genes to overcome the problem of B deficiency.

Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii.

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.

Small RNA and degradome profiling involved in seed development and oil synthesis of Brassica napus.

MicroRNAs (miRNAs) play a prominent role in post-transcriptional gene expression regulation and have been involved in various biological and metabolic processes to regulate gene expression. For Brassica napus, improving seed-weight and oil-content is the main breeding goal. In order to better understand the regulation mechanism of miRNAs during seed-weight formation and oil-content accumulation in B. napus, in this study, a high-throughput sequencing technology was used to profile miRNAs expression of Brassica napus immature seeds from one to six weeks after flowering. A total of 1,276 miRNAs, including 1,248 novel and 28 known miRNAs, were obtained from both the high-seed-weight with low-oil-content RNA pool (S03) and the low-seed-weight with high-oil-content RNA pool (S04). Analysis of their expression profiles disclosed that 300 novel and two known miRNAs were differentially expressed between S03 and S04. For degradome analysis, 57 genes with 64 degradation sites were predicted to be targeted for degradation by these miRNAs. Further bioinformatics analysis indicated that these differentially expressed miRNAs might participate in regulation of myriad cellular and molecular processes, during seed development and oil synthesis. Finally, 6 target genes with potential roles in regulation of seed development and 9 other targets in seed oil synthesis, were further confirmed as candidate genes from small RNA and degradome sequencing.

Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy

Abstract 14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ) of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH). The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy.

14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ) of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH). The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

Delayed Liver Regeneration after Partial Hepatectomy in Aged Nos2 Knockout Mice.

OBJECTIVE: Patients over 60 years of age have higher mortality and morbidity after major liver resections. Nitric oxide (NO) derived from the catalytic activity of Nos2 plays a beneficial role in liver regeneration (LR) after partial hepatectomy (PH). In this experiment, we evaluated the effect of Nos2 knockout (KO) on LR in aged mice after PH. MATERIALS AND METHODS: In this experimental study, 52 two-year-old Nos2 KO and 46 the same age wild-type (WT) C57BL/6J mice were subjected to 2/3 PH. Liver tissues were collected at 11 time points after PH. Mice survival ratio and liver coefficient (liver-weight/ body-weight) was calculated. Transcript and protein levels were estimated by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. RESULTS: The aged Nos2 KO mice had lower survival ratio (P=0.039) and liver coefficient (P=0.002) at the termination phase. Nos2 transcript level was obviously increased after PH in WT mice and undetected in the Nos2 KO mice. During LR, the expression at the transcript level of Cyclin D1, Cyclin A2 and Cyclin B1 and protein expression level of proliferation marker Ki67 and proliferation-associated transcription factors JNK1, NF-kB and STAT3 were decreased or delayed. The expression of pro-apoptotic proteins, CASPASE3, CASPASE9 and BAX, was increased in the Nos2 KO mice. CONCLUSION: Decreased survival ratio and impaired LR in aged Nos2 KO mice is probably due to decreased liver cell proliferation and increased liver cell apoptosis.

Overexpression of transcription factor Foxa2 and Hnf1α induced rat bone mesenchymal stem cells into hepatocytes.

Hepatocytes differentiated from induced pluripotent stem cells and adult stem cells could be utilized as a tool for the study of liver diseases, screening for drug metabolism and hepatotoxicity. Thus further investigation of the method to efficiently generate hepatocytes is in great need. Bone Mesenchymal Stem Cells (BMSCs) were collected from rat femurs and tibias. FOXA2 and HNF1α genes were constructed into a lentiviral vector and introduced into BMSCs by a lentivirus-mediated overexpression system. Three weeks after the induction, the expressions of FOXA2 and HNF1α, and liver specific genes were analyzed, and hepatocyte-function related assays were performed. Overexpression of both FOXA2 and HNF1α induced the BMSCs to differentiate into hepatocyte-like cells (HLCs). Hepatocyte-specific gene and protein were detected by RT-PCR, Western Blot and Immunofluorescence. These HLCs also exerted some typical hepatocyte functions such as glycogen storage, indocyanine green absorption and lipid accumulation. The combination of FOXA2 and HNF1α can effectively induce BMSCs to differentiate into HLCs. This is a novel and efficient method to prepare HLCs within a short timeline.

Analysis of the ways and methods of signaling pathways in regulating cell cycle of NIH3T3 at transcriptional level.

BACKGROUND: To analyze the ways and methods of signaling pathways in regulating cell cycle progression of NIH3T3 at transcriptional level, we modeled cell cycle of NIH3T3 and found that G1 phase of NIH3T3 cell cycle was at 5-15 h after synchronization, S phase at 15-21 h, G2 phase at 21-22 h, M phase at 22-25 h. RESULTS: Mouse Genome 430 2.0 microarray was used to detect the gene expression profiles of the model, and results showed remarkable changes in the expressions of 64 cell cycle genes and 960 genes associated with other physiological activity during the cell cycle of NIH3T3. For the next step, IPA software was used to analyze the physiological activities, cell cycle genes-associated signal transduction activities and their regulatory roles of these genes in cell cycle progression, and our results indicated that the reported genes were involved in 17 signaling pathways in the regulation of cell cycle progression. Newfound genes such as PKC, RAS, PP2A, NGR and PI3K etc. belong to the functional category of molecular mechanism of cancer, cyclins and cell cycle regulation HER-2 signaling in breast cancer signaling pathways. These newfound genes could promote DNA damage repairment and DNA replication progress, regulate the metabolism of protein, and maintain the cell cycle progression of NIH3T3 modulating the reported genes CCND1 and C-FOS. CONCLUSION: All of the aforementioned signaling pathways interacted with the cell cycle network, indicating that NIH3T3 cell cycle was regulated by a number of signaling pathways.

Interleukin 18 augments growth ability via NF-κB and p38/ATF2 pathways by targeting cyclin B1, cyclin B2, cyclin A2, and Bcl-2 in BRL-3A rat liver cells.

Interleukin 18 (IL-18) is a pleiotropic cytokine and capable of stimulating proliferation of certain cell types. Nonetheless, its effect on normal liver cells cultured remains unclear. In the present study, we discovered that IL-18 expression level was remarkably elevated at 3.3 and 8.6h after synchronized BRL-3A rat liver cells (G0 phase) re-entering the cell cycle. In addition, recombinant rat IL-18 (rrIL-18) at dosages 5-10 ng/ml increased the cell viability compared to untreated cells (with medium only) at 24 and 48 h (P<0.05). At the same time, the percentage of BrdU-labeling cells was also significantly increased (P<0.01). On the other hand, knockdown of IL-18 expression with short interference RNA (siRNA), the cell viability began to decline at 24h and significantly decreased compared to negative control (NC) at 48 and 72 h after transfection (P<0.05). Meanwhile, the number of cells in division phase (G2/M) was reduced in parallel. Further, after treatment with rrIL-18 (5 ng/ml), IL-18 and its receptor subunit IL-18Rα increased both at mRNA and protein levels. Moreover, the expression levels of adaptor molecule MyD88, transcription factor NF-κB and its downstream targets cyclin B1 and cyclin B2 were remarkably enhanced in BRL-3A cells stimulated by rrIL-18. Furthermore, transcription factor ATF2 and its targeted genes cyclin A2, Bcl-2 were also markedly increased after treatment with rrIL-18. These results demonstrated that IL-18 can augment cell proliferation via NF-κB and p38/ATF2 pathway by targeting cyclin B1, cyclin B2, cyclin A2 and Bcl-2 in BRL-3A rat liver cells.

Differential proteome analysis of the cell differentiation regulated by BCC, CRH, CXCR4, GnRH, GPCR, IL1 signaling pathways in Chinese fire-bellied newt limb regeneration.

Following amputation, the newt has the remarkable ability to regenerate its limb, and this process involves dedifferentiation, proliferation and differentiation. To investigate the potential proteome during a dynamic network of Chinese fire-bellied newt limb regeneration (CNLR), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrum (MS) were applied to examine changes in the proteome that occurred at 11 time points after amputation. Meanwhile, several proteins were selected to validate their expression levels by Western blot. The results revealed that 1476 proteins had significantly changed as compared to the control group. Gene Ontology annotation and protein network analysis by Ingenuity Pathway Analysis 9.0 (IPA) software suggested that the differentially expressed proteins were involved in 33 kinds of physiological activities including signal transduction, cell proliferation, cell differentiation, etc. Among these proteins, 407 proteins participated in cell differentiation with 212 proteins in the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte, and 37 proteins participated in signaling pathways of BCC, CRH, CXCR4, GnRH, GPCR and IL1 which regulated cell differentiation and redifferentiation. On the other hand, the signal transduction activity and cell differentiation activity were analyzed by IPA based on the changes in the expression of these proteins. The results showed that BCC, CRH, CXCR4, GnRH, GPCR and IL1 signaling pathways played an important role in regulating the differentiation of skin cell, myocyte, neurocyte, chondrocyte and osteocyte during CNLR.

Induction of Functional Hepatocyte-Like Cells by Overexpression of FOXA3 and HNF4α in Rat Bone Marrow Mesenchymal Stem Cells.

Liver regeneration has always been of great therapeutic interest, particularly to generate hepatocytes from other types of cells including adult stem cells and induced pluripotent stem cells. In this study, it was found that overexpression of both FOXA3 and HNF4α can convert rat bone marrow mesenchymal stem cells (BMSCs) to functional hepatocyte-like cells (HLCs). The results show that these HLCs had the typical hepatocyte morphology with unique hepatocyte markers such as ALB, G6P, KRT18, TAT, TTR, KRT19 and AFP. Simultaneously, they also exhibited hepatic functions such as glycogen storage, indocyanine green absorption, and cytoplasmic accumulation of neutral triglycerides and lipids. In addition to that, these functions could be maintained in the process of cell culture, thus suggesting that FOXA3 and HNF4α are powerful transcription factors for inducing rat BMSCs to differentiate into functional HLCs, and this study will provide a novel method to generate HLCs.