Oxidative stress modulating nanomaterials and their biochemical roles in nanomedicine.

Many pathological conditions are predominantly associated with oxidative stress, arising from reactive oxygen species (ROS); therefore, the modulation of redox activities has been a key strategy to restore normal tissue functions. Current approaches involve establishing a favorable cellular redox environment through the administration of therapeutic drugs and redox-active nanomaterials (RANs). In particular, RANs not only provide a stable and reliable means of therapeutic delivery but also possess the capacity to finely tune various interconnected components, including radicals, enzymes, proteins, transcription factors, and metabolites. Here, we discuss the roles that engineered RANs play in a spectrum of pathological conditions, such as cancer, neurodegenerative diseases, infections, and inflammation. We visualize the dual functions of RANs as both generator and scavenger of ROS, emphasizing their profound impact on diverse cellular functions. The focus of this review is solely on inorganic redox-active nanomaterials (inorganic RANs). Additionally, we deliberate on the challenges associated with current RANs-based approaches and propose potential research directions for their future clinical translation.

Influence of Gelatin Source and Bloom Number on Gelatin Methacryloyl Hydrogels Mechanical and Biological Properties for Muscle Regeneration.

Approximately half of an adult human's body weight is made up of muscles. Thus, restoring the functionality and aesthetics of lost muscle tissue is critical. The body is usually able to repair minor muscle injuries. However, when volumetric muscle loss occurs due to tumour extraction, for instance, the body will form fibrous tissue instead. Gelatin methacryloyl (GelMA) hydrogels have been applied for drug delivery, tissue adhesive, and various tissue engineering applications due to their tuneable mechanical properties. Here, we have synthesised GelMA from different gelatin sources (i.e., porcine, bovine, and fish) with varying bloom numbers, which refers to the gel strength, and investigated for the influence of the source of gelatin and the bloom number on biological activities and mechanical properties. The results indicated that the source of the gelatin and variable bloom numbers have an impact on GelMA hydrogel properties. Furthermore, our findings established that the bovine-derived gelatin methacryloyl (B-GelMA) has better mechanical properties than the other varieties composed of porcine and fish with 60 kPa, 40 kPa, and 10 kPa in bovine, porcine, and fish, respectively. Additionally, it showed a noticeably greater swelling ratio (SR) ~1100% and a reduced rate of degradation, improving the stability of hydrogels and giving cells adequate time to divide and proliferate to compensate for muscle loss. Furthermore, the bloom number of gelatin was also proven to influence the mechanical properties of GelMA. Interestingly, although GelMA made of fish had the lowest mechanical strength and gel stability, it demonstrated excellent biological properties. Overall, the results emphasise the importance of gelatin source and bloom number, allowing GelMA hydrogels to have a wide range of mechanical and excellent biological properties and making them suitable for various muscle tissue regeneration applications.

Discovery of novel small molecule inhibitors of S100P with in vitro anti-metastatic effects on pancreatic cancer cells.

S100P, a calcium-binding protein, is known to advance tumor progression and metastasis in pancreatic and several other cancers. Herein is described the in silico identification of a putative binding pocket of S100P to identify, synthesize and evaluate novel small molecules with the potential to selectively bind S100P and inhibit its activation of cell survival and metastatic pathways. The virtual screening of a drug-like database against the S100P model led to the identification of over 100 clusters of diverse scaffolds. A representative test set identified a number of structurally unrelated hits that inhibit S100P-RAGE interaction, measured by ELISA, and reduce in vitro cell invasion selectively in S100P-expressing pancreatic cancer cells at 10 µM. This study establishes a proof of concept in the potential for rational design of small molecule S100P inhibitors for drug candidate development.

SOX10 Single Transcription Factor-Based Fast and Efficient Generation of Oligodendrocytes from Human Pluripotent Stem Cells.

Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109-1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

Prostate Cancer Patients' Preferences for Intermittent vs. Continuous Androgen Deprivation-A Pilot Institutional Study.

INTRODUCTION: With locally advanced, recurrent, and metastatic prostate cancer patients, patient preference between intermittent (IAD) and continuous (CAD) androgen deprivation therapy has not been investigated. The goal of the study was to determine patients' preference for IAD vs. CAD therapy. The secondary aim was to elucidate demographic or treatment variables that may affect a patient's preference for one type of hormonal treatment. MATERIALS AND METHODS: Using a tradeoff model that demonstrates the difference in outcome between IAD and CAD, a survey questionnaire was developed and administered to prostate cancer patients at the Odette Cancer Centre at Sunnybrook Health Sciences Centre in Toronto, Ontario, Canada. Only patients who had (1) locally advanced prostate cancer, (2) been previously treated for prostate cancer with relapsing prostate-specific antigen, or (3) slow metastatic disease were asked to participate. Data related to patients' demographic information and their decisional preference factors were collected. RESULTS AND CONCLUSIONS: Overall, 36 of 53 (68%) patients completed the survey. Most patients favoured IAD (n = 32) over CAD (n = 4). Patients currently on radical treatment (adjuvant hormone therapy and radiation therapy) preferred CAD compared with patients who were not on radical treatment (P = .044). Patients with high (>20 ng/L) pretreatment prostate-specific antigen showed preference for CAD; however, this was not statistically significant (P = 0.07). Patients from both groups viewed quality of life as the strongest influence on their treatment preference, but had diverging opinions on side effects and general well being. The results of this pilot study could serve as a guide for future studies; a larger study combined with qualitative methodology may better address patients' needs and minimize any regret over their hormonal treatment.

Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9.

BACKGROUND: The therapeutic efficacy of arsenic trioxide (As2O3) in acute myeloid leukemia (AML) is modest, which is partly related to its limited intracellular uptake into the leukemic cells. As2O3 enters cells via the transmembrane protein aquaglyceroporin 9 (AQP9). Azacytidine, a demethylating agent that is approved for the treatment of AML, has been shown to have synergistic effect with As2O3. We tested the hypothesis that azacytidine might up-regulate AQP9 and enhances As2O3-mediated cytotoxicity in AML. METHODS: Arsenic-induced cytotoxicity, the expression of AQP9, and the intracellular uptake of As2O3 were determined in AML cell lines and primary AML cells with or without azacytidine pre-treatment. The mechanism of AQP9 up-regulation was then investigated by examining the expression of transcription factors for AQP9 gene and the methylation status of their gene promoters. RESULTS: As2O3-induced cytotoxicity in AML cell lines was significantly enhanced after azacytidine pre-treatment as a result of AQP9 up-regulation, leading to increased arsenic uptake and hence intracellular concentration. Blocking AQP9-mediated As2O3 uptake with mercury chloride abrogated the sensitization effect of azacytidine. AQP9 promoter does not contain CpG islands. Instead, azacytidine pre-treatment led to increased expression of HNF1A, a transcription activator of AQP9, through demethylation of HNF1A promoter. HNF1 knockdown abrogated azacytidine-induced AQP9 up-regulation and almost completely blocked intracellular As2O3 entry, confirming that azacytidine enhanced As2O3-mediated cell death via up-regulation of HNF1A and hence increased AQP9 and As2O3 intracellular concentration. Azacytidine sensitization to As2O3 treatment was re-capitulated also in primary AML samples. Finally, azacytidine did not enhance arsenic toxicity in a liver cell line, where HNF1A was largely unmethylated. CONCLUSIONS: Azacytidine sensitizes AML cells to As2O3 treatment, and our results provide proof-of-principle evidence that pharmacological up-regulation of AQP9 potentially expands the therapeutic spectrum of As2O3. Further clinical trial should evaluate the efficacy of azacytidine in combination with As2O3 in the treatment of AML.

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing.

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and ß1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of ß1 and ß3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1.

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.

The road to regenerative liver therapies: the triumphs, trials and tribulations.

The liver is one of the few organs that possess a high capacity to regenerate after liver failure or liver damage. The parenchymal cells of the liver, hepatocytes, contribute to the majority of the regeneration process. Thus, hepatocyte transplantation presents an alternative method to treating liver damage. However, shortage of hepatocytes and difficulties in maintaining primary hepatocytes still remain key obstacles that researchers must overcome before hepatocyte transplantation can be used in clinical practice. The unique properties of pluripotent stem cells (PSCs) and induced pluripotent stem cells (iPSCs) have provided an alternative approach to generating enough functional hepatocytes for cellular therapy. In this review, we will present a brief overview on the current state of hepatocyte differentiation from PSCs and iPSCs. Studies of liver regenerative processes using different cell sources (adult liver stem cells, hepatoblasts, hepatic progenitor cells, etc.) will be described in detail as well as how this knowledge can be applied towards optimizing culture conditions for the maintenance and differentiation of these cells towards hepatocytes. As the outlook of stem cell-derived therapy begins to look more plausible, researchers will need to address the challenges we must overcome in order to translate stem cell research to clinical applications.

Neo-vascularization of the stroke cavity by implantation of human neural stem cells on VEGF-releasing PLGA microparticles.

Replacing the tissue lost after a stroke potentially provides a new neural substrate to promote recovery. However, significant neurobiological and biotechnological challenges need to be overcome to make this possibility into a reality. Human neural stem cells (hNSCs) can differentiate into mature brain cells, but require a structural support that retains them within the cavity and affords the formation of a de novo tissue. Nevertheless, in our previous work, even after a week, this primitive tissue is void of a vasculature that could sustain its long-term viability. Therefore, tissue engineering strategies are required to develop a vasculature. Vascular endothelial growth factor (VEGF) is known to promote the proliferation and migration of endothelial cells during angio- and arteriogenesis. VEGF by itself here did not affect viability or differentiation of hNSCs, whereas growing cells on poly(D,L-lactic acid-co-glycolic acid) (PLGA) microparticles, with or without VEGF, doubled astrocytic and neuronal differentiation. Secretion of a burst and a sustained delivery of VEGF from the microparticles in vivo attracted endothelial cells from the host into this primitive tissue and in parts established a neovasculature, whereas in other parts endothelial cells were merely interspersed with hNSCs. There was also evidence of a hypervascularization indicating that further work will be required to establish an adequate level of vascularization. It is therefore possible to develop a putative neovasculature within de novo tissue that is forming inside a tissue cavity caused by a stroke.

Absence of NPM1 promoter hypermethylation in human myelodysplastic syndrome.

Npm1(+/-) heterozygous mice develop a haematological disorder with features resembling human myelodysplastic syndrome (MDS). Promoter hypermethylation of the NPM1 gene may lead to suppressed gene transcription and hence functional haploinsufficiency, which contributes to the development of MDS. Thirty-one patients with MDS and eight normal individuals were studied for promoter methylation and mRNA expression of NPM1. Methylation-specific PCR (MSP), COBRA and bisulfite sequencing were used to examine the NPM1 methylation status. Quantitative PCR was used to assess the expression of NPM1. NPM1 DNA methylation was rare, occurring in one of 31 cases as determined by MSP. There was no significant difference in NPM1 mRNA expression between MDS and normal blood samples. In conclusion, the finding suggests that NPM1 methylation is rare in MDS and does not play a major role in its pathogenesis.

Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization.

OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Attachment of stem cells to scaffold particles for intra-cerebral transplantation.

Cell-replacement therapy and tissue regeneration using stem cells are of great interest to recover histological damage caused by neuro-degenerative disease or traumatic insults to the brain. To date, the main intra-cerebral delivery for these cells has been as a suspension in media through a thin needle. However, this does not provide cells with a support system that would allow tissue regeneration. Scaffold particles are needed to provide structural support to cells to form de novo tissue. In this 16-d protocol, we describe the generation and functionalization of poly (D,L-lactic-co-glycolic) acid (PLGA) particles to enhance cell attachment, the attachment procedure to avoid clumping and aggregation of cells and particles, and their preparation for intra-cerebral injection through a thin needle. Although the stem cell-scaffold transplantation is more complicated and labor-intensive than cell suspensions, it affords de novo tissue generation inside the brain and hence provides a significant step forward in traumatic brain repair.

The effect of delivery via narrow-bore needles on mesenchymal cells.

AIMS: Recently, there have been numerous preclinical and human studies investigating the regenerative capacity of cell suspensions following their direct injection into a target organ: the fundamental parameters for successful (clinical) cell therapy. At present, limited data exist in the identification of factors important for the survival of these cells (i.e., morphology, viability and proliferation rates) during and following their ejection via narrow-bore needles. MATERIALS & METHODS: Primary murine mesenchymal stem cells (mMSCs) were isolated, expanded and processed into a concentrated cell suspension consisting of either HBSS or HBSS supplemented with the antioxidant n-acetyl-cysteine. This suspension was then ejected from a 10 microl Hamilton syringe, via a variety of bore-sized needles, at different ejection rates. Cell characteristics including viability, spreading and attachment, apoptosis and proliferative ability were then assessed. RESULTS: Following manipulation within a syringe, a decrease in the viability and cell spreading of mMSCs and a concurrent increase in the production of the caspase-3 protein, an early regulatory event in apoptosis, occurs. These detrimental effects were found to be increased when the cells were left in the syringe chamber for increased periods of time, and were similar at 5 microl/min and 1 microl/min ejection rates. However, on increasing the needle bore diameter, a significant reduction in these characteristics was observed. By comparison, mMSCs that were left to stand at room temperature (18-20 degrees C), but were not manipulated within a syringe, showed a significantly greater viability compared with manipulated cells. However, cells kept at 4 degrees C demonstrated a decreased viability compared with manipulated cells. When the mMSC were incubated with n-acetyl-cysteine, a known antioxidant, no significant change in caspase-3 production or cell spreading was observed. CONCLUSIONS: This study highlights potential parameters, such as minimizing the time period the cells are within the syringe and the use of wider-bore needles, involved in maintaining the high viable cell density required for the delivery of cell suspensions for cell therapy applications.

Prevalence of neural tube defects in economically and socially deprived area of China.

OBJECTIVE: This paper aims to understand the situation and epidemiology trend of neural tube defects in Guizhou province, China from 1996 to 2004. STUDY DESIGN: Pregnant women from 17 hospitals in Guizhou province were chosen for investigation of perinatal infants from January 1996 to December 2004. RESULTS: Of 1,208 birth defect cases studied in this 9-year period, a total of 122 cases were identified as neural tube defects (NTD), making up a 10.10% of the total birth defects. The average prevalence rate of NTD was 12.21 per 10,000 births; however, there is no significant difference between the genders. In this study, the age of mothers during pregnancy was shown to be one of the major factors affecting the prevalence rate of NTD and the differences between infants with or without NTD and the usage of folic acid as a supplement during pregnancy were found to be statistical significant. CONCLUSION: NTD was found to be the most common and serious form of human birth defect in Guizhou province. For prevention, folic acid supplement is thought to be efficacious in inhibiting NTD, and thus, it is essential for people in socially deprived areas to effectively reduce the prevalence of NTD in China.

Coxsackievirus B3 proteases 2A and 3C induce apoptotic cell death through mitochondrial injury and cleavage of eIF4GI but not DAP5/p97/NAT1.

By transfection of Coxsackievirus B3 (CVB3) individual protease gene into HeLa cells, we demonstrated that 2A(pro) and 3C(pro) induced apoptosis through multiple converging pathways. Firstly, both 2A(pro) and 3C(pro) induced caspase-8-mediated activation of caspase-3 and dramatically reduced cell viability. Secondly, they both activated the intrinsic mitochondria-mediated apoptosis pathway leading to cytochrome c release from mitochondria and activation of caspase-9. However, 3C(pro) induced these events via both up-regulation of Bax and cleavage of Bid, and 2A(pro) induced these events via cleavage of Bid only. Nevertheless, neither altered Bcl-2 expression. Thirdly, both proteases induced cell death through cleavage or down regulation of cellular factors for translation and transcription: both 2A(pro) and 3C(pro) cleaved eukaryotic translation initiation factor 4GI but their cleavage products are different, indicating different cleavage sites; further, both 2A(pro) and 3C(pro) down-regulated cyclic AMP responsive element binding protein, a transcription factor, with 2A(pro) exhibiting a stronger effect than 3C(pro). Surprisingly, neither could cleave DAP5/p97/NAT1, a translation regulator, although this cleavage was observed during CVB3 infection and could not be blocked by caspase inhibitor z-VAD-fmk. Taken together, these data suggest that 2A(pro) and 3C(pro) induce apoptosis through both activation of proapoptotic mediators and suppression of translation and transcription.