Low-intensity pulsed ultrasound modulates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord, and there are no effective drug treatments. Low-intensity pulsed ultrasound (LIPUS) has garnered attention as a promising noninvasive neuromodulation method. In this study, we investigate its effects on the motor cortex and underlying mechanisms using the SOD1G93A mouse model of ALS. Our results show that LIPUS treatment delays disease onset and prolongs lifespan in ALS mice. LIPUS significantly increases cerebral blood flow in the motor cortex by preserving vascular endothelial cell integrity and increasing microvascular density, which may be mediated via the ion channel TRPV4. RNA sequencing analysis reveals that LIPUS substantially reduces the expression of genes associated with neuroinflammation. These findings suggest that LIPUS applied to the motor cortex may represent a potentially effective therapeutic tool for the treatment of ALS.

Characterizing Cellular Physiological States with Three-Dimensional Shape Descriptors for Cell Membranes.

The shape of a cell as defined by its membrane can be closely associated with its physiological state. For example, the irregular shapes of cancerous cells and elongated shapes of neuron cells often reflect specific functions, such as cell motility and cell communication. However, it remains unclear whether and which cell shape descriptors can characterize different cellular physiological states. In this study, 12 geometric shape descriptors for a three-dimensional (3D) object were collected from the previous literature and tested with a public dataset of ~400,000 independent 3D cell regions segmented based on fluorescent labeling of the cell membranes in Caenorhabditis elegans embryos. It is revealed that those shape descriptors can faithfully characterize cellular physiological states, including (1) cell division (cytokinesis), along with an abrupt increase in the elongation ratio; (2) a negative correlation of cell migration speed with cell sphericity; (3) cell lineage specification with symmetrically patterned cell shape changes; and (4) cell fate specification with differential gene expression and differential cell shapes. The descriptors established may be used to identify and predict the diverse physiological states in numerous cells, which could be used for not only studying developmental morphogenesis but also diagnosing human disease (e.g., the rapid detection of abnormal cells).

Tumor budding is an optimal indictor of occult cervical metastasis in clinical early-stage buccal mucosa squamous cell carcinoma.

BACKGROUND: Buccal mucosa squamous cell carcinoma (BMSCC) is an aggressive disease. This study investigated the clinicopathological significance of tumor budding (TB), depth of invasion (DOI), and mode of invasion (MOI) on occult cervical metastasis (CM) of BMSCC. METHODS: Seventy-one cT1-2N0 BMSCC patients were included in this retrospective study. TB, DOI, MOI, and other clinicopathological features were reviewed. Risk factors for occult CM, locoregional recurrence-free survival (LRRFS), and overall survival (OS) were analyzed using logistic regression and Cox's proportional hazard models, respectively. RESULTS: Multivariate analysis with the logistic regression model revealed that MOI, DOI, and TB were significantly associated with occult CM in early-stage BMSCC after adjusting for variates. However, multivariate analysis with the Cox's proportional hazard model found only TB to be a prognostic factor for LRRFS (hazard ratio 15.03, 95% confidence interval [CI] 1.94-116.66; p = 0.01; trend test p = 0.03). No significant association was found between MOI, DOI, or TB and OS. CONCLUSIONS: The optimal predictor of occult CM and prognosis of early-stage BMSCC is TB, which may assist clinicians in identifying patients at high risk of cervical metastasis.

Molecular mechanism for activation of the 26S proteasome by ZFAND5.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Thallium adsorption on three iron (hydr)oxides and Tl isotopic fractionation induced by adsorption on ferrihydrite.

Thallium (Tl) is an extraordinarily toxic metal, which is usually present with Tl(I) and highly mobile in aquatic environment. Limited knowledge is available on the adsorption and isotopic variations of Tl(I) to Fe-(hydr)oxides. Herein, the adsorption behavior and mechanism of Tl(I) on representative Fe-(hydr)oxides, i.e. goethite, hematite, and ferrihydrite, were comparatively investigated kineticly and isothermally, additional to crystal structure modelling and Tl isotope composition (205Tl/203Tl). The results showed that ferrihydrite exhibited overall higher Tl(I) adsorption capacity (1.11-10.86 mg/kg) than goethite (0.21-1.83 mg/kg) and hematite (0.14-2.35 mg/kg), and adsorption by the three prevalent Fe-minerals presented strong pH and ionic strength dependence. The magnitude of Tl isotopic fractionation during Tl(I) adsorption to ferrihydrite (αsolid-solution ≈ 1.00022-1.00037) was smaller than previously observed fractionation between Mn oxides and aqueous Tl(I) (αsolid-solution ≈ 1.0002-1.0015). The notable difference is likely that whether oxidation of Tl(I) occurred during Tl adsorption to the mineral surfaces. This study found a small but detectable Tl isotopic fractionation during Tl(I) adsorption to ferrihydrite and heavier Tl isotope was slightly preferentially adsorbed on surface of ferrihydrite, which was attributed to the formation of inner-sphere complex between Tl and ≡Fe-OH. The findings offer a new understanding of the migration and fate of 205Tl/203Tl during Tl(I) adsorption to Fe (hydr)oxides.

Human umbilical cord mesenchymal stem cells for psoriasis: a phase 1/2a, single-arm study.

Psoriasis is a common, chronic immune-mediated systemic disease that had no effective and durable treatment. Mesenchymal stem cells (MSCs) have immunomodulatory properties. Therefore, we performed a phase 1/2a, single-arm clinical trial to evaluate the safety and efficacy of human umbilical cord-derived MSCs (UMSCs) in the treatment of psoriasis and to preliminarily explore the possible mechanisms. Seventeen patients with psoriasis were enrolled and received UMSC infusions. Adverse events, laboratory parameters, PASI, and PGA were analyzed. We did not observe obvious side effects during the treatment and 6-month follow-up. A total of 47.1% (8/17) of the psoriasis patients had at least 40% improvement in the PASI score, and 17.6% (3/17) had no sign of disease or minimal disease based on the PGA score. And the efficiency was 25% (2/8) for males and 66.7% (6/9) for females. After UMSC transplantation (UMSCT), the frequencies of Tregs and CD4+ memory T cells were significantly increased, and the frequencies of T helper (Th) 17 and CD4+ naive T cells were significantly decreased in peripheral blood (PB) of psoriasis patients. And all responders showed significant increases in Tregs and CD4+ memory T cells, and significant decreases in Th17 cells and serum IL-17 level after UMSCT. And baseline level of Tregs in responders were significantly lower than those in nonresponders. In conclusion, allogeneic UMSCT is safe and partially effective in psoriasis patients, and level of Tregs may be used as a potent biomarker to predict the clinical efficacy of UMSCT. Trial registration Clinical Trials NCT03765957.

Biophysical studies of cancer cells' traverse-vessel behaviors under different pressures revealed cells' motion state transition.

Circulating tumor cells (CTCs) survive in the bloodstream and then seed and invade to foster tumor metastasis. The arrest of cancer cells is favored by permissive flow forces and geometrical constraints. Through the use of high-throughput microfluidic devices designed to mimic capillary-sized vessels, we applied pressure differences to cancer cells (MCF-7 cell line) and recorded the cell traverse-vessel behaviors. Our results showed that cancer cells transform from a Newtonian droplet state to an adhesion/migration state when cancer cells traverse artificial vessels. To explain these phenomena, a modified Newtonian droplet model was also proposed. These phenomena and the modified model may reveal how CTCs in the blood seed and invade vessels under suitable conditions.

Unexpected enrichment of thallium and its geochemical behaviors in soils impacted by historically industrial activities using lead­zinc carbonate minerals.

Thallium is a trace metal with severe toxicity. Contamination of thallium (Tl) generated by steel and non-ferrous metals industry is gaining growing concern worldwide. However, little is known on Tl contamination owing to industrial activities using carbonate minerals. This study revealed abundant geochemical mobile/bioavailable Tl (> 65.7%, in average; mostly in oxidizable fraction) in soils from a carbonate-hosted PbZn ore utilizing area in China for the first time. Unexpected Tl enrichment was observed in soil accompanying with 3655, 7820, 100.1, 27.3 and 29.9 mg/kg (in average) of Pb, Zn, As, Cd and Sb, respectively. Characterization using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis further confirmed that historical industrial activities impose anthropogenic catastrophic effects on the local agricultural soil system. The ecological and health risk assessment of heavy metal(loid)s in soils proclaimed serious potential non-carcinogenic risks of Pb and V to adults, and Pb, Tl and As to children. Sequential extraction analysis showed that Tl, as well as Pb, Zn, Mn, Co, and Cd, mainly existed in the mobile fractions (exchangeable/acid-extractable, reducible and oxidizable), indicating an ecological risk of biological accumulation of multiple metal(loid)s in this area. These findings provide a theoretical basis for taking appropriate remediation measures in order to ensure safety of soils in such industrial areas likewise.

Ultrahigh Flux and Strong Affinity Poly(N-vinylformamide)-Grafted Polypropylene Membranes for Continuous Removal of Organic Micropollutants from Water.

The rapid and effective removal of organic micropollutants (OMPs) from water remains a huge challenge for traditional water treatment techniques. Compared with powder adsorbents such as polymers and nanomaterials, the free-standing adsorptive membrane is possible for large-scale applications and shows promise in removing OMPs. Herein, inspired by aquatic plants, a novel free-standing adsorptive membrane (NPPM) with high water flux, strong adsorption affinity, and excellent reproducibility was prepared by one-step UV surface grafting. N-Vinylformamide (NVF) was employed to introduce multiple hydrophilic and hydrogen bonding sites on the surface of commercial polypropylene fiber membranes (PPM). The NPPM exhibits excellent water permeability and ultrahigh water flux (up to 40 000 L/(m2 h)) and could continuously remove a broad spectrum of OMPs from water. Its adsorption performance is 5-100 times higher than that of PPM and commercial membranes. Even in natural water sources such as tap water and river water, the NPPM shows unchanged adsorption performance and high OMPs removal efficiency (>95%). Notably, the NPPM has excellent regeneration performance and can be regenerated by hot water elution, which provides an environmentally friendly regeneration method without involving any organic solvent. Moreover, the synergy between hydrogen bonding and hydrophobic interaction is revealed, and the hydrophobic interaction provided by the hydrophobic substrate is proved to play a fundamental role in OMPs adsorption. The strong hydrogen bonds between the grafts and the OMPs are demonstrated by variable-temperature FTIR spectroscopy (vt-FTIR), 13C nuclear magnetic resonance spectroscopy (13C NMR), and simulation calculations. The strong hydrogen bonds could increase the enthalpy change and enhance the adsorption affinity, so the NPPM has a strong adsorption affinity, which is 100 times that of similar adsorption membranes. This study not only presents an adsorptive membrane with great commercial potential in the rapid remediation of a water source but also opens a pathway to develop an adsorptive membrane with high water flux and strong adsorption affinity.

Facile and Safe Synthesis of Novel Self-Pored Amine-Functionalized Polystyrene with Nanoscale Bicontinuous Morphology.

The chloromethyl-functionalized polystyrene is the most commonly used ammonium cation precursor for making anion exchange resins (AER) and membranes (AEM). However, the chloromethylation of polystyrene or styrene involves highly toxic and carcinogenic raw materials (e.g., chloromethyl ether) and the resultant ammonium cation structural motif is not stable enough in alkaline media. Herein, we present a novel self-pored amine-functionalized polystyrene, which may provide a safe, convenient, and green process to make polystyrene-based AER and AEM. It is realized by hydrolysis of the copolymer obtained via random copolymerization of N-vinylformamide (NVF) with styrene (St). The composition and structure of the NVF-St copolymer could be controlled by monomeric ratio, and the copolymers with high NVF content could form bicontinuous morphology at sub-100 nm levels. Such bicontinuous morphology allows the copolymers to be swollen in water and self-pored by freeze-drying, yielding a large specific surface area. Thus, the copolymer exhibits high adsorption capacity (226 mg/g for bisphenol A). Further, the amine-functionalized polystyrene has all-carbon backbone and hydrophilic/hydrophobic microphase separation morphology. It can be quaternized to produce ammonium cations and would be an excellent precursor for making AEM and AER with good alkaline stability and smooth ion transport channels. Therefore, the present strategy may open a new pathway to develop porous alkaline stable AER and AEM without using metal catalysts, organic pore-forming agents, and carcinogenic raw materials.

A novel microfluidic device integrating focus-separation speed reduction design and trap arrays for high-throughput capture of circulating tumor cells.

Isolation and analysis of circulating tumor cells (CTCs) from peripheral blood provides a potential way to detect and characterize cancer. Existing technologies to separate or capture CTCs from whole blood still have issues with sample throughput, separation efficiency or stable efficiency at different flow rates. Here, we proposed a new concept to capture rare CTCs from blood by integrating a triangular prism array-based capture apparatus with streamline-based focus-separation speed reduction design. The focus-separation design could focus and maintain CTCs, while removing a considerable proportion of liquid (98.9%) containing other blood cells to the outlet, therefore, a high CTC capture efficiency could be achieved in the trap arrays with a high initial flow rate. It is worth mentioning that the new design works well over a wide range of flow rates, so it does not require the stability of the flow rate. The results showed that this novel integrated chip can achieve a sample throughput from 5 to 40 mL h-1 with a stable and high CTC capture efficiency (up to 94.8%) and high purity (up to 4 log white blood cells/WBC depletion). The clinical experiment showed that CTCs including CTC clusters were detected in 11/11 (100.0%) patients (mean = 31 CTCs mL-1, median = 25 CTCs mL-1). In summary, our chip enriches and captures CTCs based on physical properties, and it is simple, cheap, fast, and efficient and has low requirements on flow rate, which is very suitable for large-scale application of CTC testing in clinics.

Essentiality of CTNNB1 in Malignant Transformation of Human Embryonic Stem Cells under Long-Term Suboptimal Conditions.

Human embryonic stem cells (hESCs) gradually accumulate abnormal karyotypes during long-term suboptimal culture, which hinder their application in regenerative medicine. Previous studies demonstrated that the activation of CTNNB1 might be implicated in this process. Hence, the hESC line with stably silenced CTNNB1 was established to further explore the role of CTNNB1 in the malignant transformation of hESCs. It was shown to play a vital role in the maintenance of the physiological properties of stem cells, such as proliferation, migration, differentiation, and telomere regulation. Furthermore, the malignant transformation of hESCs was induced by continuous exposure to 0.001 µg/ml mitomycin C (MMC). The results showed that CTNNB1 and its target genes, including proto-oncogenes CCND1 and C-MYC, were aberrantly upregulated in hESCs after MMC treatment. Moreover, the high expression of CTNNB1 accelerated cell transition from G0/G1 phase to the S phase and stimulated the growth of cells containing breakage-fusion-bridge (BFB) cycles. Conversely, CTNNB1 silencing inhibited these effects and triggered a survival crisis. The current data indicated that CTNNB1 is intimately associated with the physiological properties of stem cells; however, the aberrant expression of CTNNB1 is involved in the malignant transformation of hESCs, which might advance the process by facilitating telomere-related unstable cell proliferation. Thus, the aberrant CTNNB1 level might serve as a potential biomarker for detecting the malignant transformation of hESCs.

Bazedoxifene Suppresses Intracellular Mycobacterium tuberculosis Growth by Enhancing Autophagy.

Tuberculosis (TB) is still the leading killer caused by Mycobacterium tuberculosis infection. There is a clear need for new treatment strategy against TB. It has been reported that tamoxifen, known as a selective estrogen receptor modulator (SERM), exhibits antimycobacterial activity and inhibits M. tuberculosis growth in macrophages. However, it remains unknown whether such antimicrobial activity is a general property of all SERMs and how it works. In this study, we identified that bazedoxifene (BZA), a newer SERM, inhibits intracellular M. tuberculosis growth in macrophages. BZA treatment increases autophagosome formation and LC3B-II protein expression in M. tuberculosis-infected macrophages. We further demonstrated that the enhancement of autophagy by BZA is dependent on increased reactive oxygen species (ROS) production and associated with phosphorylation of Akt/mTOR signaling. In summary, our data reveal a previously unappreciated antimicrobial function of BZA and suggest that future investigation focusing on the mechanism of action of SERMs in macrophages may lead to new host-directed therapies against TB.IMPORTANCE Since current strategies for the treatment of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have low efficacy and highly negative side effects, research on new treatments including novel drugs is essential for curing drug-resistant tuberculosis. Host-directed therapy (HDT) has become a promising idea to modulate host cell responses to enhance protective immunity against pathogens. Bazedoxifene (BZA), which belongs to a new generation of SERMs, shows the ability to inhibit the growth of M. tuberculosis in macrophages and is associated with autophagy. Our findings reveal a previously unrecognized antibacterial function of BZA. We propose that the mechanism of SERMs action in macrophages may provide a new potential measure for host-directed therapies against TB.

A hepatocyte differentiation model reveals two subtypes of liver cancer with different oncofetal properties and therapeutic targets.

Clinical observation of the association between cancer aggressiveness and embryonic development stage implies the importance of developmental signals in cancer initiation and therapeutic resistance. However, the dynamic gene expression during organogenesis and the master oncofetal drivers are still unclear, which impeded the efficient elimination of poor prognostic tumors, including human hepatocellular carcinoma (HCC). In this study, human embryonic stem cells were induced to differentiate into adult hepatocytes along hepatic lineages to mimic liver development in vitro. Combining transcriptomic data from liver cancer patients with the hepatocyte differentiation model, the active genes derived from different hepatic developmental stages and the tumor tissues were selected. Bioinformatic analysis followed by experimental assays was used to validate the tumor subtype-specific oncofetal signatures and potential therapeutic values. Hierarchical clustering analysis revealed the existence of two subtypes of liver cancer with different oncofetal properties. The gene signatures and their clinical significance were further validated in an independent clinical cohort and The Cancer Genome Atlas database. Upstream activator analysis and functional screening further identified E2F1 and SMAD3 as master transcriptional regulators. Small-molecule inhibitors specifically targeting the oncofetal drivers extensively down-regulated subtype-specific developmental signaling and inhibited tumorigenicity. Liver cancer cells and primary HCC tumors with different oncofetal properties also showed selective vulnerability to their specific inhibitors. Further precise targeting of the tumor initiating steps and driving events according to subtype-specific biomarkers might eliminate tumor progression and provide novel therapeutic strategy.

Expression and Significance of Periostin in Tissues and Serum in Oral Leukoplakia and Squamous Cell Carcinoma.

Objective: This study aims to investigate the expression changes of periostin (PN or OSF-2) in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), and analyze its significance in the development of OSCC. Study Design: The expression of periostin was detected from tissue specimens and serum obtained from normal mucosa, OLK and OSCC by immunohistochemistry, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction. Results: Periostin was significantly overexpressed in OLK and OSCC, when compared with normal controls (p < 0.05). Furthermore, the overexpression of periostin was positively correlated with TNM stage, depth of invasion, and lymph node metastasis (p < 0.05). Conclusion: The overexpression of periostin may be involved in the carcinogenesis process of OLK, which may be used as a marker for detecting OLK. In addition, periostin serum levels can be used as a potential indicator of invasion and a prognosis target for OSCC.

Generation of NERCe003-A-3, a p53 compound heterozygous mutation human embryonic stem cell line, by CRISPR/Cas9 editing.

p53 is a tumor suppressor gene involved mainly in the regulation of the G1/S cell cycle phase, DNA repair, and senescence. Although p53 is frequently altered in human cancer, the consequences of its depletion in human embryonic stem cells (hESCs) are unknown. We generated NERCe003-A-3, a p53 knockout hESC line, from the normal NERCe003-A hESC line by using CRISPR/Cas9 editing. This cell line maintained a normal 46, XY karyotype. Further analysis suggested that the cells expressed pluripotency-related markers and had the capacity to differentiate in vitro into derivatives of all three germ layers.

Prevalence and authenticity of de-novo segmental aneuploidy (>16 Mb) in human blastocysts as detected by next-generation sequencing.

RESEARCH QUESTION: What is the prevalence and authenticity of de-novo segmental aneuploidies (>16 Mb) detected by next-generation sequencing (NGS) in human preimplantation blastocysts? DESIGN: Between April 2013 and June 2016, 5735 blastocysts from 1854 couples (average age 33.11 ± 5.65 years) underwent preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) or for aneuploidy (PGT-A) using NGS on trophectoderm (TE) biopsy samples. The prevalence of de-novo segmental aneuploidy was calculated from these results. Forty blastocysts with de-novo segmental aneuploidy detected by NGS, which had been donated for research, were warmed for further fluorescence in-situ hybridization (FISH) analysis to confirm their authenticity. RESULTS: The frequency of de-novo segmental aneuploidies in blastocysts was 10.13% (581/5735); the phenomenon was not related to maternal age and occurred on all chromosomes. Of the 40 donated blastocysts, 39 were successfully warmed and fixed for FISH analysis at the single-cell level. The de-novo segmental aneuploidies identified by NGS were confirmed by FISH in all 39 blastocysts. However, the de-novo segmental aneuploidies in these blastocysts were not all pure patterns, with 66.67% (26/39) of blastocysts exhibiting mosaic patterns varying from 8.30% to 92.86% of cells with de-novo segmental aneuploidy. The concordance rate between NGS and FISH in TE and inner cell mass (ICM) samples was 47.69% (31/65). CONCLUSIONS: De-novo segmental aneuploidy above 16 Mb occurred in blastocysts and could be detected by NGS, while some aneuploidies existed as mosaics in both TE and ICM.

Parameter sensitivity analysis for a stochastic model of mitochondrial apoptosis pathway.

Understanding how gene alterations induce oncogenesis plays an important role in cancer research and may be instructive for cancer prevention and treatment. We conducted a parameter sensitivity analysis to the mitochondrial apoptosis model. Both a nonlinear bifurcation analysis of the deterministic dynamics and energy barrier analysis of the corresponding stochastic models were performed. We found that the parameter sensitivity ranking according to the change of the bifurcation-point locations in deterministic models and the change of the barrier heights from a living to death state of the cell in stochastic models are highly correlated. For the model we considered, in combination with previous knowledge that the parameters significantly affecting the system's bifurcation point are strongly associated with frequently mutated oncogenic genes, we conclude that the energy barrier height can be used as indicator of oncogenesis as well as bifurcation point. We provide a possible mechanism that may help elucidate the logic of cancer initiation from the view of stochastic dynamics and energy landscape. And we show the equivalence of energy barrier height and bifurcation-point location in determining the parameter sensitivity spectrum for the first time.

Structural mechanism for nucleotide-driven remodeling of the AAA-ATPase unfoldase in the activated human 26S proteasome.

The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all known eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of the substrate-translocation channel leading to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γ-S-bound human proteasome, in which the CP gates are asymmetrically open, visualized by cryo-EM at near-atomic resolutions. At least four nucleotides are bound to the AAA-ATPase ring in these open-gate states. Variation in nucleotide binding gives rise to an axial movement of the pore loops narrowing the substrate-translation channel, which exhibit remarkable structural transitions between the spiral-staircase and saddle-shaped-circle topologies. Gate opening in the CP is thus regulated by nucleotide-driven conformational changes of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the human proteasome holoenzyme.

FLI1 and PKC co-activation promote highly efficient differentiation of human embryonic stem cells into endothelial-like cells.

Rationale-endothelial cells (ECs) play important roles in various regeneration processes and can be used in a variety of therapeutic applications, such as cardiac regeneration, gene therapy, tissue-engineered vascular grafts and prevascularized tissue transplants. ECs can be acquired from pluripotent and adult stem cells. To acquire ECs from human embryonic stem cells (hESCs) in a fast, efficient and economic manner. We established a conditional overexpression system in hESCs based on 15 transcription factors reported to be responsible for hematopoiesis lineage. Among them, only overexpression of FLI1 could induce hESCs to a hematopoietic lineage. Moreover, simultaneous overexpression of FLI1 and activation of PKC rapidly and efficiently induced differentiation of hESCs into induced endothelial cells (iECs) within 3 days, while neither FLI1 overexpression nor PKC activation alone could derive iECs from hESCs. During induction, hESCs differentiated into spindle-like cells that were consistent in appearance with ECs. Flow cytometric analysis revealed that 92.2-98.9% and 87.2-92.6% of these cells were CD31+ and CD144+, respectively. Expression of vascular-specific genes dramatically increased, while the expression of pluripotency genes gradually decreased during induction. iECs incorporated acetylated low-density lipoproteins, strongly expressed vWF and bound UEA-1. iECs also formed capillary-like structures both in vitro and in vivo. RNA-seq analysis verified that these cells closely resembled their in vivo counterparts. Our results showed that co-activation of FLI1 and PKC could induce differentiation of hESCs into iECs in a fast, efficient and economic manner.