Human papillomavirus (HPV) is the most important causative agent of cervical cancers worldwide. However, our understanding of how the vaginal microbiota might be associated with HPV infection is limited. In addition, the influence of human genetic and physiological factors on the vaginal microbiota is unclear. Studies on twins and their families provide the ideal settings to investigate the complicated nature of human microbiota. This study investigated the vaginal microbiota of 68 HPV-infected or uninfected female twins and their families using 454-pyrosequencing analysis targeting the variable region (V2–V3) of the bacterial 16S rRNA gene. Analysis of the vaginal microbiota from both premenopausal women and HPV-discordant twins indicated that HPV-positive women had significantly higher microbial diversity with a lower proportion of Lactobacillus spp. than HPV-negative women. Fusobacteria, including Sneathia spp., were identified as a possible microbiological marker associated with HPV infection. The vaginal microbiotas of twin pairs were significantly more similar to each other than to those from unrelated individuals. In addition, there were marked significant differences from those of their mother, possibly due to differences in menopausal status. Postmenopausal women had a lower proportion of Lactobacillus spp. and a significantly higher microbiota diversity. This study indicated that HPV infection was associated with the composition of the vaginal microbiota, which is influenced by multiple host factors such as genetics and menopause. The potential biological markers identified in this study could provide insight into HPV pathogenesis and may represent biological targets for diagnostics.

Spinal cord injury (SCI) is one of the most devastating injuries. Treatment strategies for SCI are required to overcome comprehensive issues. Implantation of biomaterial scaffolds and stem cells has been demonstrated to be a promising strategy. However, a comprehensive recovery effect is difficult to achieve. In the comprehensive treatment process, the specific roles of the implanted scaffolds and of stem cells in combined strategy are usually neglected. In this study, a peptide-modified scaffold is developed based on hyaluronic acid and an adhesive peptide PPFLMLLKGSTR. Synchrotron radiation micro computed tomography measurement provides insights to the three-dimensional inner topographical property and perspective porous structure of the scaffold. The modified scaffold significantly improves cellular survival and adhesive growth of mesenchymal stem cells during 3D culture in vitro. After implantation in transected spinal cord, the modified scaffold and mesenchymal stems are found to function in synergy to restore injured spinal cord tissue, with respective strengths. Hindlimb motor function scores exhibit the most significant impact of the composite implant at 2 weeks post injury, which is the time secondary injury factors begin to take hold. Investigation on the secondary injury factors including inflammatory response and astrocyte overactivity at 10 days post injury reveals the possible underlying reason. Implants of the scaffold, cells, and especially the combination of both elicit inhibitory effects on these adverse factors. The study develops a promising implant for spinal cord tissue engineering and reveals the roles of the scaffold and stem cells. More importantly, the results provide the first understanding of the bioactive peptide PPFLMLLKGSTR concerning its functions on mesenchymal stem cells and spinal cord tissue restoration.

In this study, we present a method for assembling biofunctionalized paper into a multiform structured scaffold system for reliable tissue regeneration using an origami-based approach. The surface of a paper was conformally modified with a poly(styrene-comaleic anhydride) layer via initiated chemical vapor deposition followed by the immobilization of poly-L-lysine (PLL) and deposition of Ca 2+ . This procedure ensures the formation of alginate hydrogel on the paper due to Ca 2+ diffusion. Furthermore, strong adhesion of the alginate hydrogel on the paper onto the paper substrate was achieved due to an electrostatic interaction between the alginate and PLL. The developed scaffold system was versatile and allowed area-selective cell seeding. Also, the hydrogel-laden paper could be folded freely into 3D tissue-like structures using a simple origamibased method. The cylindrically constructed paper scaffold system with chondrocytes was applied into a three-ring defect trachea in rabbits. The transplanted engineered tissues replaced the native trachea without stenosis after 4 wks. As for the custom-built scaffold system, the hydrogel-laden paper system will provide a robust and facile method for the formation of tissues mimicking native tissue constructs.paper scaffolds | origami | tissue engineering | initiated chemical vapor deposition | hydrogel T he living organ changes its shape from a sheet-like arrangement with primitive cells to mature three-dimensional (3D) structures through morphogenetic processes (1-3). To date, a wide range of biomaterials have been used for the total or partial replacement of damaged organs and/or tissue structures (4-7). As the functions of the living organ are realized by periodic changes in the spatial arrangement of tissue elements, multiform scaffold systems mimicking the native tissue are desired. Moldcasting and electrospinning, among various other methods, have been introduced to fabricate diverse scaffolds (8, 9). These fabrication processes, however, possess limitations for organlike structure productions. Although recent progress in tissue engineering has focused on using 3D printer schemes, there are still limitations such as the shortage of appropriate printing materials and technical challenges related to the sensitivity of living cells (10-12).Paper-based scaffolds have been used previously for cell culture platforms (13), high-throughput biochemical assay platforms (14), and a point-of-care diagnostic system (15). As a nature-originated substrate, paper has attracted enormous research interest for applications in tissue engineering (16). Cellulose-based paper may serve as a promising material for tissue engineering as it contains macroporous structures that allow nutrient transport and oxygenation (13). In this regard, paper origami is a simple alternative approach for fabricating a multiform scaffold. Based on computeraided design (CAD) planar figures, a variety of shaped scaffolds could be designed using biofunctionalized paper.In this report, a vapor-phase method, init...

Acquired resistance (AQR) to drug treatment occurs frequently in cancer patients and remains an impediment to successful therapy. The aim of this study was to gain insight into how AQR arises following the application of PI3K/mTOR inhibitors. H1975 lung cancer cells with EGFR T790M mutations that confer resistance to EGFR inhibitors underwent prolonged treatment with the PI3K/mTOR inhibitor, BEZ235. Monoclonal cells with stable and increased resistance to BEZ235 were obtained after 8 months treatment. These AQR clones showed class-specific resistance to PI3K/mTOR inhibitors, reduced G1 cell cycle arrest and impedance of migration following PI3K/mTOR inhibition, reduced PTEN expression and increased Akt and S6RP phosphorylation. Transcriptome analysis revealed the AQR clones had increased expression of the metabolite transporters SLC16A9 and SLC16A7, suggestive of altered cell metabolism. Subsequent experiments revealed that AQR clones possess features consistent with elevated glycolysis, including increased levels of glucose, lactate, glutamine, glucose dependence, GLUT1 expression, and rates of post-glucose extracellular acidification, and decreased levels of reactive oxygen species and rates of oxygen consumption. Combination treatment of BEZ235 with the glycolysis inhibitor 3-bromopyruvate was synergistic in AQR clones, but only additive in parental cells. DNA sequencing revealed the presence of a mitochondrial DNA (mtDNA) MT-C01 variant in AQR but not parental cells. Depletion of mitochondrial DNA in parental cells induced resistance to BEZ235 and other PI3K/mTOR inhibitors, and was accompanied by increased glycolysis. The results of this study provide the first evidence that a metabolic switch associated with mtDNA mutation can be an underlying mechanism for AQR.

Adult stem cells age during long-term in vitro culture, and neural stem cells (NSCs), which can self-renew and differentiate into neurons and glial cells, also display reduced differentiation potential after repeated passaging. However, the mechanistic details underlying this process remain unclear. In this study, we found that long-term in vitro culture of NSCs resulted in aging-related upregulation of inflammatory-and endoplasmic reticulum (ER) stress-related genes, including the proinflammatory cytokines interleukin (IL)1β and IL6, the senescence-associated enzyme matrix metallopeptidase 13 (MMP13), and the ER stress-responsive transcription factor activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, the cyclic and transient induction of four reprogramming factors (POU domain, class 5, transcription factor 1, also known as octamer-binding transcription factor 4; SRY [sex determining region Y]-box 2; Kruppel-like factor 4; and myelocytomatosis oncogene; collectively referred to as OSKM) can inhibit NSC aging, as indicated by the decreased expression of the inflammatory and ER stress-related genes. We used ROSA-4F NSCs, which express OSKM from only one allele, to minimize the potential for full reprogramming or tumor formation during NSC rejuvenation. We expect that this novel rejuvenation method will enhance the potential of NSCs as a clinical approach to the treatment of neurological diseases.

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During embryonic development, cells undergo changes in gene expression, signaling pathway activation/inactivation, metabolism, and intracellular organelle structures, which are mediated by mitochondria. Mitochondria continuously switch their morphology between elongated tubular and fragmented globular via mitochondrial fusion and fission. Mitochondrial fusion is mediated by proteins encoded by Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is mediated by proteins encoded by Fis1 and Dnm1L. Here, we investigated the expression patterns of mitochondria-related genes during the differentiation of mouse embryonic stem cells (ESCs). Pluripotent ESCs maintain stemness in the presence of leukemia inhibitory factor (LIF) via the JAK-STAT3 pathway but lose pluripotency and differentiate in response to the withdrawal of LIF. We analyzed the expression levels of mitochondrial fusion-and fission-related genes during the differentiation of ESCs. We hypothesized that mitochondrial fusion genes would be overexpressed while the fission genes would be downregulated during the differentiation of ESCs. Though the mitochondria exhibited an elongated morphology in ESCs differentiating in response to LIF withdrawal, only the expression of Mfn2 was increased and that of Dnm1L was decreased as expected, the other exceptions being Mfn1, Opa1, and Fis1. Next, by comparing gene expression and mitochondrial morphology, we proposed an index that could precisely represent mitochondrial changes during the differentiation of pluripotent stem cells by analyzing the expression ratios of three fusion-and two fissionrelated genes. Surprisingly, increased Mfn2/Dnm1L ratio was correlated with elongation of mitochondria during the differentiation of ESCs. Moreover, application of this index to other specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic fibroblasts (MEFs) showed increased Mfn2/Dnm1L ratio compared to ESCs. Thus, we suggest that the Mfn2/Dnm1L ratio could reflect changes in mitochondrial morphology according to the extent of differentiation.

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