CDC25B induces cellular senescence and correlates with tumor suppression in a p53-dependent manner.

The phosphatase cell division cycle 25B (Cdc25B) regulates cell cycle progression. Increased Cdc25B levels are often detected in cancer cell lines and human cancers and have been implicated in contributing to tumor growth, potentially by providing cancer cells with the ability to bypass checkpoint controls. However, the specific mechanism by which increased Cdc25B impacts tumor progression is not clear. Here we analyzed The Cancer Genome Atlas (TCGA) database and found that patients with high CDC25B expression had the expected poor survival. However, we also found that high CDC25B expression had a p53-dependent tumor suppressive effect in lung cancer and possibly several other cancer types. Looking in more detail at the tumor suppressive function of Cdc25B, we found that increased Cdc25B expression caused inhibition of cell growth in human normal fibroblasts. This effect was not due to alteration of specific cell cycle stage or inhibition of apoptosis, nor by induction of the DNA damage response. Instead, increased CDC25B expression led cells into senescence. We also found that p53 was required to induce senescence, which might explain the p53-dependent tumor suppressive function of Cdc25B. Mechanistically, we found that the Cdc25B phosphatase activity was required to induce senescence. Further analysis also found that Cdc25B stabilized p53 through binding and dephosphorylating p53. Together, this study identified a tumor-suppressive function of Cdc25B that is mediated through a p53-dependent senescence pathway.

A Crucial Role of Mitochondrial Dynamics in Dehydration Resistance in Saccharomyces cerevisiae.

Mitochondria are dynamic organelles as they continuously undergo fission and fusion. These dynamic processes conduct not only mitochondrial network morphology but also activity regulation and quality control. Saccharomyces cerevisiae has a remarkable capacity to resist stress from dehydration/rehydration. Although mitochondria are noted for their role in desiccation tolerance, the mechanisms underlying these processes remains obscure. Here, we report that yeast cells that went through stationary growth phase have a better survival rate after dehydration/rehydration. Dynamic defective yeast cells with reduced mitochondrial genome cannot maintain the mitochondrial activity and survival rate of wild type cells. Our results demonstrate that yeast cells balance mitochondrial fusion and fission according to growth conditions, and the ability to adjust dynamic behavior aids the dehydration resistance by preserving mitochondria.

The serine protease inhibitor serpinB2 binds and stabilizes p21 in senescent cells.

SerpinB2 is a serine protease inhibitor also known as plasminogen activator inhibitor type 2 (PAI-2). It has been well documented that serpinB2 is an inhibitor of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). Interestingly, serpinB2 levels are increased in senescent cells and serpinB2 is thus considered a senescence biomarker. In this study, by mimicking the elevated levels of serpinB2 in senescent cells, proliferating human fibroblasts were induced into senescence. Senescence induced by serpinB2 did not relate to its extracellular function, as inhibition of serpinB2 secretion, exogenous introduced serpinB2, or a serpinB2 mutant that failed to bind to its extracellular target uPA did not affect senescence. We also showed that serpinB2 is a direct downstream target of p53 that is activated by the DNA damage response pathway. Significantly, serpinB2 bound to and stabilized p21 to mediate senescence in a proteasome-independent manner, indicating that serpinB2 has a direct role in senescence. Thus, this study reveals a unique mechanism by which serpinB2 maintains senescence through stabilization of p21 protein levels.

Knockdown of POLDIP2 suppresses tumor growth and invasion capacity and is linked to unfavorable transformation ability and metastatic feature in non-small cell lung cancer.

The main problem in the treatment of non-small cell lung cancer (NSCLC) is metastasis. Epithelial-mesenchymal transition (EMT) is known as the critical signaling in tumor progression, metastasis, and also the drug resistance. In this study, we reported a novel gene Polymerase delta-interacting protein 2 (POLDIP2) was downregulated in NSCLC tissues and first demonstrated that overexpression of POLDIP2 increased the anchorage-independent growth (AIG) and invasiveness of H1299 cells. In addition, we examined that knockdown of POLDIP2 in H1299 and A549 cells reduced tumorigenicity and metastatic capacity in vitro and also in vivo. Moreover, downregulation of the cell proliferation marker cyclin D1 and EMT markers CDH2, Slug, and Twist was showed in H1299 cells by POLDIP2 knockdown, suggesting that the inhibition of malignancy was affected by modulating key genes for tumor growth and invasiveness. Taken together, our study is the first study that demonstrated that POLDIP2 gene was function as an oncogene in NSCLC and implied the oncogenic ability might be through promoting cell proliferation or EMT.

Timely eradication of HCV viremia by PegIFN/RBV is crucial in prevention of post RFA recurrence in CHC-HCC patients.

BACKGROUND: Secondary prevention of hepatocellular carcinoma (HCC) among patients with chronic hepatitis C (CHC) who achieve sustained virologic response (SVR) with interferon-based therapy has been proved effective. However, tertiary prevention with PegIFN/RBV therapy of HCC recurrence seems limited effect in CHC-HCC patients post curative therapies. This study aims to investigate the timing and impact of PegIFN/RBV treatment on prevention of HCC recurrence in patients after RFA treatment. METHODS: From 2013 to 2016, a total of 137 CHC-HCC patients from a 508 patient based cohort receiving complete RFA treatment in Chang Gung Memorial Hospital, Linkou Medical Center were retrospectively recruited. Pre-RFA patient demographics were analyzed by cox regression analysis for prediction on tumor recurrence. Statistics analysis was performed with SPSS V.20 (IBM, USA). RESULTS: The mean age of the 137 patients were 69.6 year-old and 71.5% of patients were cirrhotic. After propensity score matching, one hundred and two patients were enrolled into the analysis. Fifty-one patients (50%) received PegIFN/RBV therapy and twenty-seven patients (52.9%) achieved SVR. Patients who could achieve SVR had lower tumor recurrence rate than non-SVR and untreated groups (29.6% vs. 66.7% vs. 49.0%, P = 0.030). The effect is more prominent in those achieve SVR prior to compared with after RFA despite not reach statistically significant (26.1% vs. 50.0%, P = 0.334). CONCLUSION: Timely treatment with SVR achievement has the lowest tumor recurrence rate in CHC-HCC patients. Secondary prevention might be even more important than tertiary prevention in CHC patients, especially regarding prevention of post RFA HCC recurrence.

Sensitive detection of cyanide using bovine serum albumin-stabilized cerium/gold nanoclusters.

A simple, sensitive, and selective fluorescence assay for the detection of CN(-) has been demonstrated using bovine serum albumin-stabilized cerium/gold nanoclusters (BSA-Ce/Au NCs). When excited at 325 nm, BSA-Ce/Au NCs have two fluorescence bands centered at 410 and 658 nm, which are assigned to BSA-Ce/Au complexes and Au NCs, respectively. Each BSA-Ce/Au NC contains 22 Au atoms and 8 Ce ions. Through etching of the Au core in BSA-Ce/Au NCs by CN(-), the fluorescence at 658 nm is quenched, while that at 410 nm enhances during the formation of complexes among BSA, Ce(4+), and [Au(CN)2](-). The circular dichroism spectra reveal that relative to BSA-Au NCs, BSA-Ce/Au NCs have looser structures of the BSA templates. As a result, it is easier for CN(-) to access the Au cores in BSA-Ce/Au NCs, allowing faster (within 15 min) etching of the Au cores by CN(-). At pH 12.0, this assay allows the detection of CN(-) down to 50 nM, with linearity over 0.1-15 µM. This assay has been applied to the determination of the concentrations of CN(-) in spiked drinking water and pond water samples.

The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors.

Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity.

Accumulation and toxicity of intravenously-injected functionalized graphene oxide in mice.

Graphene and its functionalized derivatives have recently emerged as interesting nanomaterials with promising applications in biomedicine. In this study, the long-term in vivo biodistribution of intravenously injected nanographene oxide (NGO) functionalized with poly sodium 4-styrenesulfonate (PSS) was systematically examined and the potential toxicity over 6 months of NGO-PSS nanoparticles was investigated. Our results showed that the nanoparticles mainly accumulate in the lung, liver and spleen, where they persist for at least 6 months. These nanoparticles result in acute liver injury and chronic inflammation of the lung, liver and spleen, as evidenced by blood biochemistry results and histological examinations.

Enhancing the Repair of Substantial Volumetric Muscle Loss by Creating Different Levels of Blood Vessel Networks Using Pre-Vascularized Nerve Hydrogel Implants.

Volumetric muscle loss (VML), a severe muscle tissue loss from trauma or surgery, results in scarring, limited regeneration, and significant fibrosis, leading to lasting reductions in muscle mass and function. A promising approach for VML recovery involves restoring vascular and neural networks at the injury site, a process not extensively studied yet. Collagen hydrogels have been investigated as scaffolds for blood vessel formation due to their biocompatibility, but reconstructing blood vessels and guiding innervation at the injury site is still difficult. In this study, collagen hydrogels with varied densities of vessel-forming cells are implanted subcutaneously in mice, generating pre-vascularized hydrogels with diverse vessel densities (0-145 numbers/mm2) within a week. These hydrogels, after being transplanted into muscle injury sites, are assessed for muscle repair capabilities. Results showed that hydrogels with high microvessel densities, filling the wound area, effectively reconnected with host vasculature and neural networks, promoting neovascularization and muscle integration, and addressing about 63% of the VML.

Reconstructing vascular networks promotes the repair of skeletal muscle following volumetric muscle loss by pre-vascularized tissue constructs.

Current treatment for complex and large-scale volumetric muscle loss (VML) injuries remains a limited success and have substantial disadvantages, due to the irreversible loss of muscle mass, slow muscle regeneration, and rapid formation of non-functional fibrosis scars. These VML injuries are accompanied by denervation and the destruction of native vasculature which increases difficulties in the functional restoration of muscle. Here, reconstruction of the vascular network at the injury site was offered as a possible solution for improving the repair of muscle defects through the timely supply of nutrients and oxygen to surrounding cells. A hydrogel-based tissue construct containing various densities of the vascular network was successfully created in the subcutaneous space of mice by manipulating hydrogel properties, and then implanted into the VML injury site. One month after implantation, the mouse treated with the highly vascularized tissue had extensive muscle repair at the injury site and only spent a shorter time completing the inclined plane tests. These findings suggest that the reconstruction of the functional vascular network at the VML injury site accelerated muscle fiber repair through a timely supply of sufficient blood and avoided invasion by host fibroblasts.

SOX1 Functions as a Tumor Suppressor by Repressing HES1 in Lung Cancer.

The development of lung cancer is a complex process that involves many genetic and epigenetic changes. Sex-determining region Y (SRY)-box (SOX) genes encode a family of proteins that are involved in the regulation of embryonic development and cell fate determination. SOX1 is hypermethylated in human cancers. However, the role of SOX1 in the development of lung cancer is unclear. We used quantitative methylation-specific polymerase chain reaction (MSP), quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, and web tools to confirm the frequent epigenetic silencing of SOX1 in lung cancer. Stable overexpression of SOX1 repressed cell proliferation, anchorage-independent growth, and invasion in vitro as well as cancer growth and metastasis in a xenograft mouse model. Knockdown of SOX1 by the withdrawal of doxycycline partly restored the malignant phenotype of inducible SOX1-expressing NSCLC cells. Next, we discovered the potential downstream pathways of SOX1 using RNA-seq analysis and identified HES1 as a direct target of SOX1 using chromatin immunoprecipitation (ChIP)-PCR. Furthermore, we performed phenotypic rescue experiments to prove that overexpression of HES1-FLAG in SOX1-expressing H1299 cells partly reversed the tumor-suppressive effect. Taken together, these data demonstrated that SOX1 acts as a tumor suppressor by directly inhibiting HES1 during the development of NSCLC.

Engineering large and geometrically controlled vascularized nerve tissue in collagen hydrogels to restore large-sized volumetric muscle loss.

Developing scalable vascularized and innervated tissue is a critical challenge for the successful clinical application of tissue-engineered constructs. Collagen hydrogels are extensively utilized in cell-mediated vascular network formation because of their naturally excellent biological properties. However, the substantial increase in hydrogel contraction induced by populated cells limits their long-term use. Previous studies attempted to mitigate this issue by concentrating collagen pre-polymer solutions or synthesizing covalently crosslinked collagen hydrogels. However, these methods only partially reduce hydrogel contraction while hindering blood vessel formation within the hydrogels. To address this challenge, we introduced additional support in the form of a supportive spacer to counteract the contraction forces of populated cells and prevent hydrogel contraction. This approach was found to promote cell spreading, resist hydrogel contraction, control hydrogel/tissue geometry, and even facilitate the engineering of functional blood vessels and host nerve growth in just one week. Subsequently, implanting these engineered tissues into muscle defect sites resulted in timely anastomosis with the host vasculature, leading to enhanced myogenesis, increased muscle innervation, and the restoration of injured muscle functionality. Overall, this innovative strategy expands the applicability of collagen hydrogels in fabricating large vascularized nerve tissue constructs for repairing volumetric muscle loss (∼63 %) and restoring muscle function.

Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels.

The generation of functional, 3D vascular networks is a fundamental prerequisite for the development of many future tissue engineering-based therapies. Current approaches in vascular network bioengineering are largely carried out using natural hydrogels as embedding scaffolds. However, most natural hydrogels present a poor mechanical stability and a suboptimal durability, which are critical limitations that hamper their widespread applicability. The search for improved hydrogels has become a priority in tissue engineering research. Here, the suitability of a photopolymerizable gelatin methacrylate (GelMA) hydrogel to support human progenitor cell-based formation of vascular networks is demonstrated. Using GelMA as the embedding scaffold, it is shown that 3D constructs containing human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs) generate extensive capillary-like networks in vitro. These vascular structures contain distinct lumens that are formed by the fusion of ECFC intracellular vacuoles in a process of vascular morphogenesis. The process of vascular network formation is dependent on the presence of MSCs, which differentiate into perivascular cells occupying abluminal positions within the network. Importantly, it is shown that implantation of cell-laden GelMA hydrogels into immunodeficient mice results in a rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, it is shown that the degree of methacrylation of the GelMA can be used to modulate the cellular behavior and the extent of vascular network formation both in vitro and in vivo. These data suggest that GelMA hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues.

Viscous Fingering as a Rapid 3D Pattering Technique for Engineering Cell-Laden Vascular-Like Constructs.

Tissues are much larger than the diffusion limit distance, so rapidly providing blood vessels to supply embedded cells inside tissues with sufficient nutrients and oxygen is regarded as a major strategy for the success of bioengineered large and thick tissue constructs. Here, a patterning technique, viscous fingering, is developed to bioengineer vascularized-like tissues within a few minutes. By controlling viscosity, flow rate, and the volume of photo-cross-linkable prepolymer, macro- and microscale vascular network structures can be precisely engineered using the Hele-Shaw cell that is designed in this study. After cross-linking, a vascular-like gel with fingering structures is formed between the bottom and top base gels, creating a sandwich-like structure. Cells can be incorporated into the fingers, bases, or both gels. The spreading and growth direction of the embedded cells are successfully controlled and guided by manipulating the physical properties of the fingering and base gels individually. Moreover, fingering is generated, connected, and surrounded prepared cell-laden microgels in base prepolymers to form prevascularized tissue-like constructs. Taken together, the 3D cell patterning technique extends the potential for modeling and fabricating large and stackable vascularized tissue-like constructs for both ex vivo and in vivo applications.

Use artificial neural network to recommend the lumbar spinal endoscopic surgical corridor.

Objectives: The transforaminal and interlaminar approaches are the two main surgical corridors of full endoscopic lumbar surgery. However, there are no quantifying methods for assessing the best surgical approach for each patient. This study aimed to establish an artificial intelligence (AI) model using an artificial neural network (ANN). Materials and Methods: Patients who underwent full endoscopic lumbar spinal surgery were enrolled in this research. Fourteen pre-operative factors were fed into the ANN. A three-layer deep neural network was constructed. Patient data were divided into the training, validation, and testing datasets. Results: There were 899 patients enrolled. The accuracy of the training, validation, and test datasets were 87.3%, 85.5%, and 85.0%, respectively. The positive predictive values for the transforaminal and interlaminar approaches were 85.1% and 89.1%, respectively. The area under the curve of the receiver operating characteristic was 0.91. The SHapley Additive exPlanations algorithm was utilized to explain the relative importance of each factor. The surgical lumbar level was the most important factor, followed by herniated disc localization and migrating disc zone level. Conclusion: ANN can effectively learn from the choice of an experienced spinal endoscopic surgeon and can accurately predict the appropriate surgical approach.

Strategy for improving cell-mediated vascularized soft tissue formation in a hydrogen peroxide-triggered chemically-crosslinked hydrogel.

The physically-crosslinked collagen hydrogels can provide suitable microenvironments for cell-based functional vascular network formation due to their biodegradability, biocompatibility, and good diffusion properties. However, encapsulation of cells into collagen hydrogels results in extensive contraction and rapid degradation of hydrogels, an effect known from their utilization as a pre-vascularized graft in vivo. Various types of chemically-crosslinked collagen-based hydrogels have been successfully synthesized to decrease volume contraction, retard the degradation rate, and increase mechanical tunability. However, these hydrogels failed to form vascularized tissues with uniformly distributed microvessels in vivo. Here, the enzymatically chemically-crosslinked collagen-Phenolic hydrogel was used as a model to determine and overcome the difficulties in engineering vascular networks. Results showed that a longer duration of inflammation and excessive levels of hydrogen peroxide limited the capability for blood vessel forming cells-mediated vasculature formation in vivo. Lowering the unreacted amount of crosslinkers reduced the densities of infiltrating host myeloid cells by half on days 2-4 after implantation, but blood vessels remained at low density and were mainly located on the edge of the implanted constructs. Co-implantation of a designed spacer with cell-laden hydrogel maintained the structural integrity of the hydrogel and increased the degree of hypoxia in embedded cells. These effects resulted in a two-fold increase in the density of perfused blood vessels in the hydrogel. Results agreed with computer-based simulations. Collectively, our findings suggest that simultaneous reduction of the crosslinker-induced host immune response and increase in hypoxia in hydrogen peroxide-triggered chemically-crosslinked hydrogels can effectively improve the formation of cell-mediated functional vascular networks.

A strategy to engineer vascularized tissue constructs by optimizing and maintaining the geometry.

The success of engineered tissues is limited by the need for rapid perfusion of a functional vascular network that can control tissue engraftment and promote survival after implantation. Diabetic conditions pose an additional challenge, because high glucose and lipid concentrations cause an aggressive oxidative environment that impairs vessel remodeling and stabilization and impedes integration of engineered constructs into surrounding tissues. Thus, to achieve rapid vasculogenesis, angiogenesis, and anastomosis, hydrogels incorporating cells in their structure have been developed to facilitate formation of functional vascular networks within implants. However, their transport diffusivity decreases with increasing thickness, preventing the formation of a thick vascularized tissue. To address this, we used diffusion-based computational simulations to optimize the geometry of hydrogel structures. The results show that the micro-patterned constructs improved diffusion, thus supporting cell viability, and spreading while retaining their mechanical properties. Thick cell-laden bulk, linear, or hexagonal infill patterned hydrogels were implanted; and structural stability due to skin mobility was improved by the protective spacer. Larger and thicker perfused vascular networks formed in the hexagonal structures (∼17 mm diameter, ∼1.5 mm thickness) in both normal and diabetic mice on day 3, and they became functional and uniformly distributed on day 7. Moreover, transplanted islets were rapidly integrated subcutaneously in this engineered functional vascular bed, which significantly enhanced islet viability and insulin secretion. In summary, we developed a promising strategy for generating large, thick vascularized tissue constructs, which may support transplanted islet cells. These constructs showed potential for engineering other vascularized tissues in regenerative therapy. STATEMENT OF SIGNIFICANCE: Diffusion-based computational simulations were used to optimize the geometry of hydrogel structures, i.e., hexagonal cell-laden hydrogels. To maintain the hydrogel's structural integrity, a spacer was designed and co-implanted subcutaneously to increase the permeability of explants. The spacer provides the structural integrity to improve the permeability of the implanted hydrogel. Otherwise, the implanted hydrogel may be easily squeezed and deformed by compression from the skin mobility of mice. Here, we successfully developed a cell-based strategy for rapidly generating large, functional vasculature (diameter ∼17 mm and thickness ∼1.5 mm) in both normal and diabetic mice and demonstrated its advantages over currently available methods in a clinically-relevant animal model. This concept could serve as a basis for engineering and repairing other tissues in animals.

Dynamic patterns of microRNA expression during acute myeloid leukemia state-transition.

MicroRNAs (miRNAs) have been shown to hold prognostic value in acute myeloid leukemia (AML); however, the temporal dynamics of miRNA expression in AML are poorly understood. Using serial samples from a mouse model of AML to generate time-series miRNA sequencing data, we are the first to show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. We modeled AML state-transition as a particle undergoing Brownian motion in a quasi-potential and validated the AML state-space and state-transition model to accurately predict time to AML in an independent cohort of mice. The critical points of the model provided a framework to align samples from mice that developed AML at different rates. Our mathematical approach allowed discovery of dynamic processes involved during AML development and, if translated to humans, has the potential to predict an individual's disease trajectory.

Kazinol Q from Broussonetia kazinoki enhances cell death induced by Cu(II) through increased reactive oxygen species.

The ability of the flavan kazinol Q (KQ) to induce DNA breakage in the presence of Cu(II) was examined by agarose gel electrophoresis using supercoiled plasmid DNA. In KQ-mediated DNA breakage reaction, the involvement of reactive oxygen species (ROS), H(2)O(2) and O(2)- was established by the inhibition of DNA breakage by catalase and revealed DNA breakage by superoxide dismutase (SOD). The cell viability of gastric carcinoma SCM-1 cells treated with various concentrations of KQ was significantly decreased by cotreatment with Cu(II). Treatment of SCM-1 cells with 300 µM Cu(II) enhanced the necrosis induced by 100 µM KQ. Treatment of SCM-1 cells with 100 mM KQ in the presence of 300 mM Cu(II) increased the generation of H(2)O(2). Taken together, the above finding suggested that KQ cotreatment with Cu(II) produced increased amounts of H(2)O(2), thus enhancing subsequent cell death due to necrosis.

Dynamically and Spatially Controllable Albumin-Based Hydrogels for the Prevention of Postoperative Adhesion.

Since the degree of severity and the geometry of wounds vary, it is necessary to prepare an antiadhesive hydrogel that possesses dynamically controllable material properties, exhibits biodegradability, and possesses drug-releasing properties. Injectable, oxygen peroxide-sensitive, and photo-cross-linkable hydrogels that permit in situ dynamic and spatial control of their physicochemical properties were synthesized for the prevention of postoperative adhesion. Albumin is the most abundant protein in blood serum and serves as a carrier for several molecules that exhibit poor water solubility. It is therefore a suitable biomaterial for the fabrication of hydrogels since it presents a low risk of life-threatening complications and does not require immunosuppressive therapy for preventing graft rejection. The physicochemical properties of this hydrogel can then be spatially postadjusted via transdermal exposure to light to release drugs, depending on what is required for the injury. A significant reduction in postoperative peritoneal adhesion was observed in an animal model involving severe sidewall and bowel abrasions. This study demonstrated that the fabricated dually cross-linked, albumin-based hydrogels have great potential in such applications because they showed a low immune response, easy handling, full wound coverage, and tunable biodegradability. Precise spatial and controllable drug-release profiles may also be achieved via in situ transdermal post-tuning of the biomaterials, depending on the injury.