Elucidation of triacylglycerol catabolism in Yarrowia lipolytica: How cells balance acetyl-CoA and excess reducing equivalents.

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from ß-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.

Galactoglycerolipid Lipase PGD1 Is Involved in Thylakoid Membrane Remodeling in Response to Adverse Environmental Conditions in Chlamydomonas.

Photosynthesis occurs in the thylakoid membrane, where the predominant lipid is monogalactosyldiacylglycerol (MGDG). As environmental conditions change, photosynthetic membranes have to adjust. In this study, we used a loss-of-function Chlamydomonas reinhardtii mutant deficient in the MGDG-specific lipase PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION1) to investigate the link between MGDG turnover, chloroplast ultrastructure, and the production of reactive oxygen species (ROS) in response to different adverse environmental conditions. The pgd1 mutant showed altered MGDG abundance and acyl composition and altered abundance of photosynthesis complexes, with an increased PSII/PSI ratio. Transmission electron microscopy showed hyperstacking of the thylakoid grana in the pgd1 mutant. The mutant also exhibited increased ROS production during N deprivation and high light exposure. Supplementation with bicarbonate or treatment with the photosynthetic electron transport blocker DCMU protected the cells against oxidative stress in the light and reverted chlorosis of pgd1 cells during N deprivation. Furthermore, exposure to stress conditions such as cold and high osmolarity induced the expression of PGD1, and loss of PGD1 in the mutant led to increased ROS production and inhibited cell growth. These findings suggest that PGD1 plays essential roles in maintaining appropriate thylakoid membrane composition and structure, thereby affecting growth and stress tolerance when cells are challenged under adverse conditions.

Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5.

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein-protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.

Rapid labeling of intracellular His-tagged proteins in living cells.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Stress-induced neutral lipid biosynthesis in microalgae - Molecular, cellular and physiological insights.

Photosynthetic microalgae have promise as biofuel feedstock. Under certain conditions, they produce substantial amounts of neutral lipids, mainly in the form of triacylglycerols (TAGs), which can be converted to fuels. Much of our current knowledge on the genetic and molecular basis of algal neutral lipid metabolism derives mainly from studies of plants, i.e. seed tissues, and to a lesser extent from direct studies of algal lipid metabolism. Thus, the knowledge of TAG synthesis and the cellular trafficking of TAG precursors in algal cells is to a large extent based on genome predictions, and most aspects of TAG metabolism have yet to be experimentally verified. The biofuel prospects of microalgae have raised the interest in mechanistic studies of algal TAG biosynthesis in recent years and resulted in an increasing number of publications on lipid metabolism in microalgae. In this review we summarize the current findings on genetic, molecular and physiological studies of TAG accumulation in microalgae. Special emphasis is on the functional analysis of key genes involved in TAG synthesis, molecular mechanisms of regulation of TAG biosynthesis, as well as on possible mechanisms of lipid droplet formation in microalgal cells. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae.

Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis.

Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction.

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre-treatment with melatonin, an endogenous hormone with newly found anti-oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 µM melatonin pre-treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD-1), as well as pro-angiogenic and mitogenic factors like insulin-like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre-treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti-apoptosis kinases like p-Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre-treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.

Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and ß-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, ß-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.

Expression of Arabidopsis acyl-CoA-binding proteins AtACBP1 and AtACBP4 confers Pb(II) accumulation in Brassica juncea roots.

In Arabidopsis thaliana, the expression of two genes encoding acyl-CoA-binding proteins (ACBPs) AtACBP1 and AtACBP4, were observed to be induced by lead [Pb(II)] in shoots and roots in qRT-PCR analyses. Quantitative GUS (ß-glucuronidase) activity assays confirmed induction of AtACBP1pro::GUS by Pb(II). Electrophoretic mobility shift assays (EMSAs) revealed that Pas elements in the 5'-flanking region of AtACBP1 were responsive to Pb(II) treatment. AtACBP1 and AtACBP4 were further compared in Pb(II) uptake using Brassica juncea, a potential candidate for phytoremediation given its rapid growth, large roots, high biomass and good capacity to accumulate heavy metals. Results from atomic absorption analyses on transgenic B. juncea expressing AtACBP1 or AtACBP4 indicated Pb(II) accumulation in roots. Subsequent Pb(II)-tracing assays demonstrated Pb(II) accumulation in the cytosol of root tips and vascular tissues of transgenic B. juncea AtACBP1-overexpressors (OXs) and AtACBP4-OXs and transgenic Arabidopsis AtACBP1-OXs. Transgenic Arabidopsis AtACBP1-OXs sequestered Pb(II) in the trichomes and displayed tolerance to hydrogen peroxide (H2 O2 ) treatment. In addition, AtACBP1 and AtACBP4 were H2 O2 -induced in the roots of wild-type Arabidopsis, while lipid hydroperoxide (LOOH) measurements of B. juncea AtACBP1-OX and AtACBP4-OX roots suggested that AtACBP1 and AtACBP4 can protect lipids against Pb(II)-induced lipid peroxidation.

Intraarticular gene transfer of SPRY2 suppresses adjuvant-induced arthritis in rats.

AKT and ERK pathways have been implicated as therapeutic targets for human rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) inhibition, and thus RA treatment. Sprouty2 (SPRY2) has been known as a tumor suppressor by blocking both ERK and AKT signaling cascades. Whether SPRY2 can function as a suppressor of tumor-like inflammatory FLS and RA through negatively regulating AKT and ERK activation has not been reported. The purpose of this study was to determine whether SPRY2 might have antiarthritic effects in experimental animal model of RA. We first determined that expression of SPRY2 mRNA was decreased in FLS from patients with RA compared with patients with osteoarthritis (OA). Further studies demonstrated that intraarticular gene transfer with AdSPRY2, the recombinant adenovirus containing SPRY2 complementary DNA, resulted in a significant suppression of rat adjuvant-induced arthritis (AIA) compared with the control AdGFP, the adenoviral vector encoding green fluorescent protein, as reflected in both clinical and histological observations. AdSPRY2 suppressed the production of proinflammatory cytokines and matrix metalloproteinases (MMPs), and the activation of ERK and AKT signals in AIA ankle joints. These results suggest that using SPRY2 to block the AKT and ERK pathways effectively reduces the inflammatory responses and arthritic progression in AIA. Thus, the development of an immunoregulatory strategy based on SPRY2 may therefore have therapeutic potential in the treatment of RA.

The stem cell adjuvant with Exendin-4 repairs the heart after myocardial infarction via STAT3 activation.

The poor survival of cells in ischaemic myocardium is a major obstacle for stem cell therapy. Exendin-4 holds the potential of cardioprotective effect based on its pleiotropic activity. This study investigated whether Exendin-4 in conjunction with adipose-derived stem cells (ADSCs) could improve the stem cell survival and contribute to myocardial repairs after infarction. Myocardial infarction (MI) was induced by the left anterior descending artery ligation in adult male Sprague-Dawley rats. ADSCs carrying double-fusion reporter gene [firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)] were quickly injected into border zone of MI in rats treated with or without Exendin-4. Exendin-4 enhanced the survival of transplanted ADSCs, as demonstrated by the longitudinal in vivo bioluminescence imaging. Moreover, ADSCs adjuvant with Exendin-4 decreased oxidative stress, apoptosis and fibrosis. They also improved myocardial viability and cardiac function and increased the differentiation rates of ADSCs into cardiomyocytes and vascular smooth muscle cells in vivo. Then, ADSCs were exposed to hydrogen peroxide/serum deprivation (H(2)O(2)/SD) to mimic the ischaemic environment in vitro. Results showed that Exendin-4 decreased the apoptosis and enhanced the paracrine effect of ADSCs. In addition, Exendin-4 activated signal transducers and activators of transcription 3 (STAT3) through the phosphorylation of Akt and ERK1/2. Furthermore, Exendin-4 increased the anti-apoptotic protein Bcl-2, but decreased the pro-apoptotic protein Bax of ADSCs. In conclusion, Exendin-4 could improve the survival and therapeutic efficacy of transplanted ADSCs through STAT3 activation via the phosphorylation of Akt and ERK1/2. This study suggests the potential application of Exendin-4 for stem cell-based heart regeneration.

Arabidopsis acyl-CoA-binding protein ACBP1 participates in the regulation of seed germination and seedling development.

A family of six genes encoding acyl-CoA-binding proteins (ACBPs), ACBP1-ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1-over-expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over-production in 12-day-old seedlings up-regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress-responsive genes: ABA-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH-TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12-day-old seedlings of ACBP1-over-expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two-hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA-mediated seed germination and seedling development.

Metabolic Engineering of Escherichia coli for Production of 2,5-Dimethylpyrazine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD+ and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

Effect of Astragalus membranaceus on left ventricular remodeling in HFrEF: a systematic review and meta-analysis.

Background: Left ventricular remodeling (LVR) is a key factor leading to the onset and progression of heart failure with reduced ejection fraction (HFrEF). Improving LVR can delay the progression of HFrEF and improve quality of life. Objective: To evaluate the improvement effect of Astragalus membranaceus (A. membranaceus) on LVR in patients with HFrEF. Method: We retrieved randomized controlled trials (RCTs) of A. membranaceus in treating HFrEF from eight Chinese and English databases, up until 31 October 2023. To assess the quality of the literature, we utilized the bias risk tool from the Cochrane Handbook. For meta-analysis, we employed Review Manager 5.4.1 software. Additionally, we performed sensitivity analysis and publication bias assessment using Stata 17.0 software. Result: Totally 1,565 patients were included in 19 RCTs. Compared to conventional treatment (CT), the combination therapy of A. membranaceus with CT demonstrated significant improvements in LVR, specifically increasing left ventricular ejection fraction (LVEF, MD = 5.82, 95% CI: 4.61 to 7.03, p < 0.00001), decreasing left ventricular end-diastolic diameter (LVEDD, MD = -4.05, 95% CI: -6.09 to -2.01, p = 0.0001), and left ventricular end-systolic diameter (LVESD, MD = -12.24, 95% CI: -15.24 to -9.24, p < 0.00001). The combination therapy of A. membranaceus with CT also improved clinical efficacy (RR = 4.81, 95% CI: 3.31 to 7.00, p < 0.00001), reduced brain natriuretic peptide (BNP, MD = -113.57, 95% CI: -146.91 to -81.22, p < 0.00001) level, and increased 6-min walking distance (6-MWD, MD = 67.62, 95% CI: 41.63 to 93.60, p < 0.00001). In addition, the combination therapy of A. membranaceus with CT mitigated inflammatory responses by reducing tumor necrosis factor-alpha (TNF-α, MD = -16.83, 95% CI: -22.96 to -10.71, p < 0.00001), interleukin-6 (IL-6, MD = -29.19, 95% CI: -36.08 to -22.30, p < 0.00001), and high-sensitivity C-reactive protein (hs-CRP, MD = -0.98, 95% CI: -1.43 to -0.52, p < 0.0001). Notably, the combination therapy of A. membranaceus with CT did not increase the incidence of adverse reactions (RR = 0.86, 95% CI: 0.25 to 2.96, p = 0.81). Conclusion: This systematic review and meta-analysis revealed that the combination therapy of A. membranaceus with CT has more advantages than CT alone in improving LVR and clinical efficacy in HFrEF patients, without increasing the incidence of adverse reactions. However, due to the limited quality of included studies, more high-quality investigations are required to provide reliable evidence for clinical use. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=397571, Identifier: CRD42023397571.

Light irradiation changes the regulation pattern of BtCrgA on carotenogenesis in Blakeslea trispora.

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

Discovery of a brain-permeable bromodomain and extra terminal domain (BET) inhibitor with selectivity for BD1 for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease and lacks effective therapeutic agents. Dysregulation of transcription mediated by bromodomain and extra-terminal domain (BET) proteins containing two different bromodomains (BD1 and BD2) is an important factor in multiple diseases, including MS. Herein, we identified a series of BD1-biased inhibitors, in which compound 16 showed nanomolar potency for BD1 (Kd = 230 nM) and a 60-fold selectivity for BRD4 BD1 over BD2. The co-crystal structure of BRD4 BD1 with 16 indicated that the hydrogen bond interaction of 16 with BD1-specific Asp145 is important for BD1 selectivity. 16 showed favorable brain distribution in mice and PK properties in rats. 16 was able to inhibit microglia activation and had significant therapeutic effects on EAE mice including improvement of spinal cord inflammatory conditions and demyelination protection. Overall, these results suggest that brain-permeable BD1 inhibitors have the potential to be further investigated as therapeutic agents for MS.

The tumourigenicity of iPS cells and their differentiated derivates.

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC-derivates), iPSC-derivated cardiomyocyte (iPSC-CMs) were subcutaneously injected into the back of nude mice. Non-invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC-derivates and iPSC-CMs survived in receipts. Both iPSCs and iPSC-derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC-CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC-derivates transplanted mice, but not in iPSC-CM transplanted ones. In vitro study showed the long-term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC-like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage-specific differentiation is necessary prior to therapeutic use of iPSCs.

Immunohistochemical characterization and functional identification of mammary gland telocytes in the self-assembly of reconstituted breast cancer tissue in vitro.

Telocyte (TC) as a special stromal cell exists in mammary gland and might play an important role in the balance of epithelium-stroma of mammary gland. Considering that different types of breast interstitial cells influence the development and progression of breast cancer, TCs may have its distinct role in this process. We here studied the roles of TCs in the self-assembly of reconstituted breast cancer tissue. We co-cultured primary isolated TCs and other breast stromal cells with breast cancer EMT-6 cells in collagen/Matrigel scaffolds to reconstitute breast cancer tissue in vitro. Using histology methods, we investigated the immunohistochemical characteristics and potential functions of TCs in reconstituted breast cancer tissue. TCs in primary mammary gland stromal cells with long and thin overlapping cytoplasmic processes, expressed c-kit/CD117, CD34 and vimentin in reconstitute breast cancer tissue. The transmission electron microscopy showed that the telocyte-like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells through membrane-to-membrane contact. Compared with cancer tissue sheets of EMT-6 alone, PCNA proliferation index analysis and TUNEL assay showed that TCs and other breast stromal cells facilitated the formation of typical nest structure, promoted the proliferation of breast cancer cells, and inhibited their apoptosis. In conclusion, we successfully reconstituted breast cancer tissue in vitro, and it seems to be attractive that TCs had potential functions in self-assembly of EMT-6/stromal cells reconstituted breast cancer tissue.

Interactions between Arabidopsis acyl-CoA-binding proteins and their protein partners.

Protein-protein interactions are at the core of cellular interactomics and are essential for various biological functions. Since proteins commonly function as macromolecular complexes, it is important to identify their interacting partners to better understand their function and the significance in these interactions. The acyl-CoA-binding proteins (ACBPs) of eukaryotes show conservation in the presence of a lipid-binding acyl-CoA-binding domain. In Arabidopsis thaliana, four of six members from the AtACBP family possess ankyrin repeats (AtACBP1 and AtACBP2) or kelch motifs (AtACBP4 and AtACBP5), which can potentially mediate protein-protein interactions. Through yeast two-hybrid screens, a dozen putative protein partners interacting with AtACBPs have been isolated from an Arabidopsis cDNA library. Investigations in the past decade on the interaction between AtACBPs and their protein partners have revealed novel roles for AtACBPs, including functions in mediating oxidative stress responses, heavy metal tolerance and oxygen sensing. Recent progress and current questions on AtACBPs and their interactors are discussed in this review.