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Background: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized.

Methods: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model.

Results: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model.

Conclusions: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

This study was to examine the formation of advanced glycation end-products (AGEs) in black tea during drying process at 90, 120, and 150 degrees C for 1 h. Nine AGEs including N epsilon-(carboxyethyl)-L-lysine (CEL), N epsilon-(carboxymethyl)-L-lysine (CML), three isomers of methylglyoxal-hydroimidazolones (MG-Hs), three isomers of glyoxal-hydroimidazolones (GO-Hs), and argpyrimidine were quantified by using HPLC-MS/MS with isotope-labelled internal standard. Results showed that each AGE during the drying process of 150 degrees C was significantly higher (p < 0.05) than at 90 and 120 degrees C, and argpyrimidine was only found in the treatment of 150 degrees C. MG-H1/3 was first quantified as the major AGE during drying at 120- 150 degrees C, the content respectively reached to (39.66 +/- 2.61) mu g/g and (58.88 +/- 1.76) mu g/g after 1 h drying, where CML content only had (19.86 +/- 1.02) mu g/g and (23.71 +/- 1.40) mu g/g. This study indicated that arginine derived-AGEs are the key components of black tea AGEs.

Corneal tissue engineering is an alternative way to solve the problem of lack of corneal donor tissue in corneal transplantation. Keratocytes with a normal phenotype and function in tissue-engineered cornea would be critical for corneal regeneration. Although the role of extracellular/substrate material stiffness is well-known for the regulation of the cell phenotype and cell behavior in many different cell types, its effects in keratocyte culture have not yet been thoroughly studied. This project studied the effect of substrate stiffness on the keratocyte phenotype marker expression and typical cell behavior (cell adhesion, proliferation, and migration), and the possible mechanisms involved. Human primary keratocytes were cultured on tissue culture plastic (TCP, similar to 10(6) kPa) or on plates with the stiffness equivalent of physiological human corneal stroma (25 kPa) or vitreous body (1 kPa). The expression of keratocyte phenotype markers, cell adhesion, proliferation, and migration were compared. The results showed that the stiffness of the substrate material regulates the phenotype marker expression and cell behavior of cultured keratocytes. Physiological corneal stiffness (25 kPa) superiorly preserved the cell phenotype when compared to the TCP and 1 kPa group. Keratocytes had a larger cell area when cultured on 25 kPa plates as compared to on TCP. Treatment of cells with NSC 23766 (Rac1 inhibitor) mimicked the response in the cell phenotype and behavior seen in the transition from soft materials to stiff materials, including the cytoskeletal structure, expression of keratocyte phenotype markers, and cell behavior. In conclusion, this study shows that substrate stiffness regulates the cell phenotype marker expression and cell behavior of keratocytes by Rac1-mediated cytoskeletal reorganization. This knowledge contributes to the development of corneal tissue engineering.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1 beta(IL-1 beta) on corneal wound healing. However, the effect of IL-1 beta on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1 beta induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-kappa B pathway is involved in the IL-1 beta-induced changes of keratocyte phenotype, and that the selective IKK beta inhibitor TPCA-1, which inhibits NF-kappa B, can preserve keratocyte phenotype under IL-1 beta simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

Osteoarthritis (OA) is a degenerative joint disorder and one of the most prevalent diseases among the elderly population. Due to the limited spontaneous healing capacity of articular cartilage, it still remains challenging to find satisfactory treatment for OA. This review covers the emerging trends of pharmacologic therapies for OA such as traditional OA drugs (acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, serotonin-norepinephrine reuptake inhibitors (SNRIs), intra-articular injections of corticosteroids, and dietary supplements), which are effective in pain relief but not in reversing damage, and are frequently associated with adverse events. Alternatively, disease-modifying drugs provide promising alternatives for the management of OA. The development of these emerging OA therapeutic agents requires a comprehensive understanding of the pathophysiology of OA progression. The process of cartilage anabolism/catabolism, subchondral bone remodeling and synovial inflammation are identified as potential targets. These emerging OA drugs such as bone morphogenetic protein-7 (BMP-7), fibroblast growth factor-18 (FGF-18), human serum albumin (HSA), interleukin-1 (IL-1) inhibitor, h-Nerve growth factor (beta-NGF) antibody, matrix extracellular phosphoglycoprotein (MERE) and inverse agonist of retinoic acid-related orphan receptor alpha (ROR alpha) etc. have shown potential to modify progression of OA with minimal adverse effects. However, large-scale randomized controlled trials (RCTs) are needed to investigate the safety and efficacy before translation from bench to bedside.

Aims To explore the role of corneal-shaped static mechanical strain on the differentiation of human periodontal ligament stem cells (PDLSCs) into keratocytes and the possible synergistic effects of mechanics and inducing medium. Methods PDLSCs were exposed to 3% static dome-shaped mechanical strain in a Flexcell Tension System for 3 days and 7 days. Keratocyte phenotype was determined by gene expression of keratocyte markers. Keratocyte differentiation (inducing) medium was introduced in the Flexcell system, either continuously or intermittently combined with mechanical stimulation. The synergistic effects of mechanics and inducing medium on keratocyte differentiation was evaluated by gene and protein expression of keratocyte markers. Finally, a multilamellar cell sheet was assembled by seeding PDLSCs on a collagen membrane and inducing keratocyte differentiation. The transparency of the cell sheet was assessed, and typical markers of native human corneal stroma were evaluated by immunofluorescence staining. Results Dome-shaped mechanical stimulation promoted PDLSCs to differentiate into keratocytes, as shown by the upregulation of ALDH3A1, CD34, LUM, COL I and COL V. The expression of integrins were also upregulated after mechanical stimulation, including integrin alpha 1, alpha 2, beta 1 and non-muscle myosin II B. A synergistic effect of mechanics and inducing medium was found on keratocyte differentiation. The cell sheets were assembled under the treatment of mechanics and inducing medium simultaneously. The cell sheets were transparent, multilamellar and expressed typical markers of corneal stroma. Conclusion Dome-shaped mechanical stimulation promotes differentiation of PDLSCs into keratocytes and has synergistic effects with inducing medium. Multilamellar cell sheets that resemble native human corneal stroma show potential for future clinical applications.

High concentration of ascorbic acid (vitamin C) has been found in corneal epithelium of various species. However, the specific functions and mechanisms of ascorbic acid in the repair of corneal epithelium are not clear. In this study, it was found that ascorbic acid accelerates corneal epithelial wound healing in vivo in mouse. In addition, ascorbic acid enhanced the stemness of cultured mouse corneal epithelial stem/progenitor cells (TKE2) in vitro, as shown by elevated clone formation ability and increased expression of stemness markers (especially p63 and SOX2). The contribution of ascorbic acid on the stemness enhancement was not dependent on the promotion of Akt phosphorylation, as concluded by using Akt inhibitor, nor was the stemness found to be dependent on the regulation of oxidative stress, as seen by the use of two other antioxidants (GMEE and NAC). However, ascorbic acid was found to promote extracellular matrix (ECM) production, and by using two collagen synthesis inhibitors (AzC and CIS), the increased expression of p63 and SOX2 by ascorbic acid was decreased by around 50%, showing that the increased stemness by ascorbic acid can be attributed to its regulation of ECM components. Moreover, the expression of p63 and SOX2 was elevated when TKE2 cells were cultured on collagen I coated plates, a situation that mimics the in vivo situation as collagen I is the main component in the corneal stroma. This study shows direct therapeutic benefits of ascorbic acid on corneal epithelial wound healing and provides new insights into the mechanisms involved.

Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor Fos was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7-days-old rats. It was further confirmed that expression of Fos decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of Fos also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell-sheet and a three-dimensional cell culture model, the expression of Fos was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in Fos-overexpressed tendon stem/progenitor cells (TSPCs), and Fos knock-down gave opposite results. In situ rat tendon repair experiments found more normal tendon-like tissue formed and higher tendon markers expression at 4 weeks postimplantation of Fos-overexpressed TSPCs derived nonscaffold engineering tendon (cell-sheet), as compared with the control group. This study identifies Fos as a new marker and functional driver in the early stage teno-lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration.

Background: We aimed to generate a bioengineered multi-lamellar human corneal stroma tissue in vitro by differentiating periodontal ligament stem cells (PDLSCs) towards keratocytes on an aligned silk membrane.

Methods: Human PDLSCs were isolated and identified. The neuropeptide substance P (SP) was added in keratocyte differentiation medium (KDM) to evaluate its effect on keratocyte differentiation of PDLSCs. PDLSCs were then seeded on patterned silk membrane and cultured with KDM and SP. Cell alignment was evaluated and the expression of extracellular matrix (ECM) components of corneal stroma was detected. Finally, multi-lamellar tissue was constructed in vitro by PDLSCs seeded on patterned silk membranes, which were stacked orthogonally and stimulated by KDM supplemented with SP for 18 days. Sections were prepared and subsequently stained with hematoxylin and eosin or antibodies for immunofluorescence observation of human corneal stroma-related proteins.

Results: SP promoted the expression of corneal stroma-related collagens (collagen types I, III, V, and VI) during the differentiation induced by KDM. Patterned silk membrane guided cell alignment of PDLSCs, and important ECM components of the corneal stroma were shown to be deposited by the cells. The constructed multi-lamellar tissue was found to support cells growing between every two layers and expressing the main type of collagens (collagen types I and V) and proteoglycans (lumican and keratocan) of normal human corneal stroma.

Conclusions: Multi-lamellar human corneal stroma-like tissue can be constructed successfully in vitro by PDLSCs seeded on orthogonally aligned, multi-layered silk membranes with SP supplementation, which shows potential for future corneal tissue engineering.

The optimal functionality of the native corneal stroma is mainly dependent on the well-ordered arrangement of extracellular matrix (ECM) and the pressurized structure. In order to develop an in vitro corneal model, it is crucial to mimic the in vivo microenvironment of the cornea. In this study, the influence of surface topography and mechanical strain on keratocyte phenotype and ECM formation within a biomimetic 3D corneal model is studied. By modifying the surface topography of materials, it is found that patterned silk fibroin film with 600 grooves mm(-1) optimally supports cell alignment and ECM arrangement. Furthermore, treatment with 3% dome-shaped mechanical strain, which resembles the shape and mechanics of native cornea, significantly enhances the expression of keratocyte markers as compared to flat-shaped strain. Accordingly, a biomimetic 3D corneal model, in the form of a collagen-modified, silk fibroin-patterned construct subjected to 3% dome-shaped strain, is created. Compared to traditional 2D cultures, it supports a significantly higher expression of keratocyte and ECM markers, and in conclusion better maintains keratocyte phenotype, alignment, and fusiform cell shape. Therefore, the novel biomimetic 3D corneal model developed in this study serves as a useful in vitro 3D culture model to improve current 2D cultures for corneal studies.