Injectable Self-Oxygenating Cardio-Protective and Tissue Adhesive Silk-Based Hydrogel for Alleviating Ischemia After Mi Injury.

Myocardial infarction (MI) is a significant cardiovascular disease that restricts blood flow, resulting in massive cell death and leading to stiff and noncontractile fibrotic scar tissue formation. Recently, sustained oxygen release in the MI area has shown regeneration ability; however, improving its therapeutic efficiency for regenerative medicine remains challenging. Here, a combinatorial strategy for cardiac repair by developing cardioprotective and oxygenating hybrid hydrogels that locally sustain the release of stromal cell-derived factor-1 alpha (SDF) and oxygen for simultaneous activation of neovascularization at the infarct area is presented. A sustained release of oxygen and SDF from injectable, mechanically robust, and tissue-adhesive silk-based hybrid hydrogels is achieved. Enhanced endothelialization under normoxia and anoxia is observed. Furthermore, there is a marked improvement in vascularization that leads to an increment in cardiomyocyte survival by ≈30% and a reduction of the fibrotic scar formation in an MI animal rodent model. Improved left ventricular systolic and diastolic functions by ≈10% and 20%, respectively, with a ≈25% higher ejection fraction on day 7 are also observed. Therefore, local delivery of therapeutic oxygenating and cardioprotective hydrogels demonstrates beneficial effects on cardiac functional recovery for reparative therapy.

Spontaneous differentiation of human induced pluripotent stem cells to odorant-responsive olfactory sensory neurons.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPSCs), can differentiate into almost all cell types and are anticipated to have significant applications in the field of regenerative medicine. However, there are no reports of successfully directing iPSCs to become functional olfactory sensory neurons (OSNs) capable of selectively receiving odorant compounds. In this study, we employed dual SMAD inhibition and fibroblast growth factor 8 (FGF-8, reported to dictate olfactory fates) along with N-2 and B-27 supplements in the culture medium to efficiently induce the differentiation of iPSCs into neuronal cells with olfactory function through olfactory placode. Temporal gene expression and expression of OSN-specific markers during differentiation indicated that the expression of olfactory marker proteins and various olfactory receptors (ORs), which are markers of mature OSNs, was observed after approximately one month of differentiation culture, irrespective of the differentiation cues, suggesting differentiation into OSNs. Cells that exhibited specific responses to odorant compounds were identified after administering odorant compounds to differentiated iPSC-derived OSNs. This suggests the spontaneous generation of functional OSNs expressing diverse ORs that respond to odorant compounds from iPSCs.

A review on 3D printing functional brain model.

Modern neuroscience increasingly relies on 3D models to study neural circuitry, nerve regeneration, and neural disease. Several different biofabrication approaches have been explored to create 3D neural tissue model structures. Among them, 3D bioprinting has shown to have great potential to emerge as a high-throughput/high precision biofabrication strategy that can address the growing need for 3D neural models. Here, we have reviewed the design principles for neural tissue engineering. The main challenge to adapt printing technologies for biofabrication of neural tissue models is the development of neural bioink, i.e., a biomaterial with printability and gelation properties and also suitable for neural tissue culture. This review shines light on a vast range of biomaterials as well as the fundamentals of 3D neural tissue printing. Also, advances in 3D bioprinting technologies are reviewed especially for bioprinted neural models. Finally, the techniques used to evaluate the fabricated 2D and 3D neural models are discussed and compared in terms of feasibility and functionality.

Impact of Diabetes on Patient Outcomes in Breast Cancer Patients.

Background: Diabetes and the etiology of breast cancer are clearly associated. However, the impact of diabetes on prognosis is not yet understood. Therefore, we conducted a retrospective cohort study to examine the relationship between diabetes and patient outcomes in breast cancer patients. Methods: We investigated 332 Japanese women with breast cancer who underwent curative surgery at our hospital. Patients without sufficient clinical information including hemoglobin A1c (HbA1c) and those with an observation period of less than 1 year were excluded. Results: Among the patients examined, 106 had diabetes at the time of their breast cancer diagnosis. Among the 296 patients with invasive breast carcinoma, 36 patients developed distant metastases during the mean observation period of 45 months. Sixteen patients died due to breast cancer, while 13 died of other causes. Multivariate analysis revealed that diabetes, tumor size, and estrogen receptor (ER) status were independent factors related to distant metastasis-free survival (DMFS) (p = 0.038, p < 0.001, and p = 0.006, respectively). Kaplan-Meier curve analysis revealed that diabetes negatively affected the outcomes of ER-negative breast cancer patients both in DMFS and overall survival (p = 0.045 and p = 0.029, respectively). Meanwhile, patient outcomes did not differ according to the level of HbA1c in diabetes patients. Conclusion: Patients with diabetes had a significantly shorter DMFS, and the negative effect of diabetes on patient outcomes was more evident in ER-negative breast cancer. Our data indicates the importance of primary prevention of diabetes for breast cancer patients.

3D bioprinted human iPSC-derived somatosensory constructs with functional and highly purified sensory neuron networks.

Engineering three-dimensional (3D) sensible tissue constructs, along with the complex microarchitecture wiring of the sensory nervous system, has been an ongoing challenge in the tissue engineering field. By combining 3D bioprinting and human pluripotent stem cell (hPSC) technologies, sensible tissue constructs could be engineered in a rapid, precise, and controllable manner to replicate 3D microarchitectures and mechanosensory functionalities of the native sensory tissue (e.g. response to external stimuli). Here, we introduce a biofabrication approach to create complex 3D microarchitecture wirings. We develop an hPSC-sensory neuron (SN) laden bioink using highly purified and functional SN populations to 3D bioprint microarchitecture wirings that demonstrate responsiveness to warm/cold sense-inducing chemicals and mechanical stress. Specifically, we tailor a conventional differentiation strategy to our purification method by utilizing p75 cell surface marker and DAPT treatment along with neuronal growth factors in order to selectively differentiate neural crest cells into SNs. To create spatial resolution in 3D architectures and grow SNs in custom patterns and directions, an induced pluripotent stem cell (iPSC)-SN-laden gelatin bioink was printed on laminin-coated substrates using extrusion-based bioprinting technique. Then the printed constructs were covered with a collagen matrix that guided SNs growing in the printed micropattern. Using a sacrificial bioprinting technique, the iPSC-SNs were seeded into the hollow microchannels created by sacrificial gelatin ink printed in the gelatin methacryloyl supporting bath, thereby demonstrating controllability over axon guidance in curved lines up to several tens of centimeters in length on 2D substrates and in straight microchannels in 3D matrices. Therefore, this biofabrication approach could be amenable to incorporate sensible SN networks into the engineered skin equivalents, regenerative skin implants, and augmented somatosensory neuro-prosthetics that have the potential to regenerate sensible functions by connecting host neuron systems in injured areas.

Toward a neurospheroid niche model: optimizing embedded 3D bioprinting for fabrication of neurospheroid brain-like co-culture constructs.

A crucial step in creating reliablein vitroplatforms for neural development and disorder studies is the reproduction of the multicellular three-dimensional (3D) brain microenvironment and the capturing of cell-cell interactions within the model. The power of self-organization of diverse cell types into brain spheroids could be harnessed to study mechanisms underlying brain development trajectory and diseases. A challenge of current 3D organoid and spheroid models grown in petri-dishes is the lack of control over cellular localization and diversity. To overcome this limitation, neural spheroids can be patterned into customizable 3D structures using microfabrication. We developed a 3D brain-like co-culture construct using embedded 3D bioprinting as a flexible solution for composing heterogenous neural populations with neurospheroids and glia. Specifically, neurospheroid-laden free-standing 3D structures were fabricated in an engineered astrocyte-laden support bath resembling a neural stem cell niche environment. A photo-crosslinkable bioink and a thermal-healing supporting bath were engineered to mimic the mechanical modulus of soft tissue while supporting the formation of self-organizing neurospheroids within elaborate 3D networks. Moreover, bioprinted neurospheroid-laden structures exhibited the capability to differentiate into neuronal cells. These brain-like co-cultures could provide a reproducible platform for modeling neurological diseases, neural regeneration, and drug development and repurposing.

Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium-on-a-Chip Application.

Bioprinting holds great promise towards engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, we have developed a novel and mechanically robust biomaterial-ink based on non-mulberry silk fibroin protein. The silk-based ink demonstrated suitable mechanical properties required in terms of elasticity and stiffness (~40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allowed the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which were able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin-43, and maintain synchronously beating of cardiomyocytes. The printed constructs were found to be non-immunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue-on-a-chip, the silk-based ink was used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC-CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. We confirmed the ability of silk-based supporting hydrogel towards maturation and viability of hiPSC-CSs and endothelial cells, and for applications in evaluating drug toxicity.

Plastid-targeted forms of restriction endonucleases enhance the plastid genome rearrangement rate and trigger the reorganization of its genomic architecture.

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid-targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double-stranded breaks (DSB) at their recognition sites in ptDNAs. Genome-wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology-mediated recombination and microhomology-mediated end-joining, and less by non-homologous end-joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small-scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.

Nontoxic organic solvents identified using an a priori approach with Hansen solubility parameters.

Nontoxicities of organic solvents were predicted using Hansen solubility parameters and various truly nontoxic solvents were identified according to the prediction. The nontoxicity of solvents originates from their exhibition of poor solubility or low diffusivity toward cell constituents and good stability against hydrolytic degradation.

Comprehensive study on cellular morphologies, proliferation, motility, and epithelial-mesenchymal transition of breast cancer cells incubated on electrospun polymeric fiber substrates.

The progress of microenvironment-mediated tumor progression in an artificial extracellular matrix explores the design criteria to understand the cancer progression mechanism and metastatic potential. This study was aimed at examining the combination of both surface topographies (fiber alignments) and different stiffness of polymeric substrates (PLLA and PCL) to evaluate the effects on the cellular morphologies, proliferation, motility, and gene expression regarding epithelial to mesenchymal transition (EMT) of two different types of breast cancer cells (MDA-MB-231 and MCF-7). The cellular morphologies (roundness and nuclear elongation factor), E-cadherin and vimentin expression, and cellular motility in terms of cellular migration speed, persistent time, and diffusivity have been comprehensively discussed. We demonstrated that the microenvironment of cell culture substrates influences cancer progression and metastatic potential.

[A Case of Axillary Nodal Metastasis of Adenocarcinoma of an Unknown Primary Site Effectively Treated with Anti-HER2plus AC plus Paclitaxel Therapy].

A 68-year-old woman who presented with a left axillary mass was admitted. A computed tomography scan showed swelling ofthe left axillary and supraclavicular lymph nodes, but magnetic resonance imaging(MRI)and fluorodeoxyglucose positron emission tomography(FDG-PET)did not reveal these primary sites. Histological findings of the axillary mass revealed a HER2-positive adenocarcinoma. We diagnosed the patient with axillary nodal metastasis ofadenocarcinoma ofan unknown primary site and treated her with neoadjuvant chemotherapy including paclitaxel and trastuzumab followed by doxorubicin and cyclophosphamide. The lesions almost disappeared after 3 courses of chemotherapy and she showed a pathologically complete response(CR)after surgery. The patient has been recurrence-free since the operation owing to treatment with adjuvant trastuzumab.

Impact of metallothionein gene polymorphisms on the risk of lung cancer in a Japanese population.

Metallothioneins (MTs) are cysteine-rich proteins that act as antioxidants. A case-control study was conducted to assess the effects of gene polymorphisms in the MT region on the risk of lung cancer in Japanese subjects: 769 lung cancer cases and 939 non-cancer controls. Associations were evaluated using logistic regression models with adjustment for potential confounders (age, sex, and lifestyle factors including smoking, drinking, and green-yellow vegetable intake). We found five polymorphisms in the MT-1 gene region that showed statistically significant associations with lung cancer. Of these polymorphisms, rs7196890 showed the strongest association (odds ratio: 1.30, P = 0.004, 95% confidence interval: 1.09-1.55). The impact of the polymorphism decreased with the increase of smoking, and virtually no association with lung cancer was observed among heavy smokers whose pack-year values were 30 or more (odds ratio: 1.02, P = 0.93, 95% confidence interval: 0.67-1.55). These results suggest that polymorphisms in the MT gene are moderately associated with the risk of lung cancer and that the associations are modified by lifestyle factors.

[Usefulness of bevacizumab with paclitaxel for advanced breast cancer - a case report].

A 55-year-old woman with an exudative, necrotizing left breast tumor consulted Ibaraki Prefectural Central Hospital and Cancer Center. We diagnosed her as advanced ER+, PgR-, HER2- invasive ductal carcinoma of the left breast by tumor needle biopsy. FDG-PET/CT revealed multiple lymph node, pulmonary, bone, and hepatic metastases. Systemic chemotherapy with biweekly bevacizumab and weekly paclitaxel(PTX)was administered. The chemotherapy induced a widespread tumor lysis in her left chest wall. We continued chemotherapy, and the ulcer has been healing gradually. We recognized that bevacizumab with PTX successfully brought about a rapid, good local response, and improved the patient's quality of life.

Classification of polycyclic aromatic hydrocarbons based on mutagenicity in lung tissue through DNA microarray.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. Exposure to PAHs raises the risk of lung cancer and inflammatory and allergic disorders such as asthma. DNA microarray technologies have been applied to research on toxicogenomics in the recent years. To evaluate the mutagenicity of PAHs and constituents of environmental pollutants in lung tissue, including metabolic activation, human alveolar epithelial type II cells (A549) were treated with nonmutagenic PAH pyrene and with the mutagenic PAHs benzo-[a]-pyrene, 1-nitropyrene, or 1,8-dinitropyrene. Comparison of genome-wide microarray expression profiles between a nonmutagenic and a mutagenic PAH-treated group revealed that xenobiotic response genes such as CYP1B1 were commonly upregulated in two groups and that DNA damage induced genes, especially p53-downstream genes such as p21 (CDKN1A) were upregulated only in the mutagenic PAH-treated group. Pretreatment with cytochrome P450 inhibitor α-naphthoflavone or p53 inhibitor pifithrin-α inhibited the benzo-[a]-pyrene-induced p21 expression. These data suggest that when PAHs enter the cells, lung epithelium induces PAH metabolic activating enzymes, and then the DNA damages-recognition signal is converged with p53 downstream genes. This metabolic activation and DNA damage is induced in lung epithelium, and the mutagenicity of PAHs can be classified by DNA microarray expression profiles.

A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish). This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf), knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1), and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant) revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

Rapid decrease in active tension generated by C2C12 myotubes after termination of artificial exercise.

We found that the active tension of C2C12 myotubes that had been subjected to artificial exercise for ~10 days decreased rapidly after termination of the artificial exercise. When differentiated C2C12 myotubes were subjected to continuous 1 Hz artificial exercise for ~10 days, the active tension increased to ~4× compared to that before application of the artificial exercise, as reported previously. On termination of artificial exercise, the active tension decreased rapidly, the level reaching that before application of the artificial exercise within 8 h. Concomitant with the decrease in the active tension, an increase in the amount of ubiquitinated proteins was observed. Real time RT-PCR revealed that the expression of several genes associated with atrophy, namely Smc6, Vegfa, Jarid2, Kitl, Cds2, Inmt, Fasn, Neurl, Topors, and Cul2, were also changed after termination of artificial exercise. These results indicate that termination of artificial exercise induced atrophy-like responses of C2C12 myotubes. Here we found that during the decrease in active tension, the sarcomere structure, especially the thin filament structure, decayed rapidly after termination of artificial exercise. On reapplication of the artificial exercise, the active tension was restored rapidly, within 8 h, concomitant with reformation of the sarcomere structure. These results indicate that disassembly of the sarcomere structure may be one of the reasons for the active tension decrease during disuse muscle atrophy.

Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.

BACKGROUND: Obesity is a multifactorial disorder influenced by genetic and environmental factors. Animal models of obesity are required to help us understand the signaling pathways underlying this condition. Zebrafish possess many structural and functional similarities with humans and have been used to model various human diseases, including a genetic model of obesity. The purpose of this study was to establish a zebrafish model of diet-induced obesity (DIO). RESULTS: Zebrafish were assigned into two dietary groups. One group of zebrafish was overfed with Artemia (60 mg dry weight/day/fish), a living prey consisting of a relatively high amount of fat. The other group of zebrafish was fed with Artemia sufficient to meet their energy requirements (5 mg dry weight/day/fish). Zebrafish were fed under these dietary protocols for 8 weeks. The zebrafish overfed with Artemia exhibited increased body mass index, which was calculated by dividing the body weight by the square of the body length, hypertriglyceridemia and hepatosteatosis, unlike the control zebrafish. Calorie restriction for 2 weeks was applied to zebrafish after the 8-week overfeeding period. The increased body weight and plasma triglyceride level were improved by calorie restriction. We also performed comparative transcriptome analysis of visceral adipose tissue from DIO zebrafish, DIO rats, DIO mice and obese humans. This analysis revealed that obese zebrafish and mammals share common pathophysiological pathways related to the coagulation cascade and lipid metabolism. Furthermore, several regulators were identified in zebrafish and mammals, including APOH, IL-6 and IL-1ß in the coagulation cascade, and SREBF1, PPARα/γ, NR1H3 and LEP in lipid metabolism. CONCLUSION: We established a zebrafish model of DIO that shared common pathophysiological pathways with mammalian obesity. The DIO zebrafish can be used to identify putative pharmacological targets and to test novel drugs for the treatment of human obesity.

In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives.

BACKGROUND: The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae. RESULTS: We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1µg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells. CONCLUSIONS: The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.

Synergistic induction of heme oxygenase-1 by nicaraven after subarachnoid hemorrhage to prevent delayed cerebral vasospasm.

Cerebral vasospasm remains a major cause of morbidity and mortality in patients with subarachnoid hemorrhage. Heme oxygenase-1 (HO-1) is an oxidative stress-inducible enzyme with multiple protective functions against vascular and neurological diseases, including delayed cerebral vasospasm. In the present study, intravenous administration (i.v.) of nicaraven (1 mg/kg/min, for 2 days after subarachnoid hemorrhage) ameliorated delayed cerebral vasospasm in rat subarachnoid hemorrhage models, marked synergistic induction of HO-1 protein (> 2.5-fold than 'subarachnoid hemorrhage with saline i.v.'), and elicited a rapid increase of cGMP accumulation in the basilar arteries. In the sham-operated rats, nicaraven could not induce HO-1 expression. Antisense HO-1 oligodeoxynucleotides abrogated this HO-1 induction and the antivasospastic effect of nicaraven. In vitro study using Hela cells, nicaraven enhanced the human HO-1 promoter (-4.5 kbp) activity, which was pre-activated with the blood component oxyhemoglobin to mimic the ability of subarachnoid hemorrhage. These results suggest that this enhanced HO-1 expression through a combination of pathological state and pharmacological agent could be an effective strategy to improve the prognosis of heme- and oxidative stress-induced diseases, such as delayed cerebral vasospasm.

Zebrafish beta-adrenergic receptor mRNA expression and control of pigmentation.

Beta adrenergic receptors (beta-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of beta-ARs in zebrafish are, however, largely unknown. We have identified zebrafish beta-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these beta-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.