Neurosphere culture derived from aged hippocampal dentate gyrus.

The neurosphere assay is the gold standard for determining proliferative and differentiation potential of neural progenitor cells (NPCs) in neurogenesis studies 1-3 . While several in vitro assays have been developed to model the process of neurogenesis, they have predominantly used embryonic and early postnatal NPCs derived from the dentate gyrus (DG). A limitation of these approaches is that they do not provide insight into adult-born NPCs, which are modeled to affect hippocampal function and diseases later in life. Here, we show a novel free-floating neurosphere culture system using NPCs isolated from the DG of mature adult and aged mice. The protocol outlines detailed steps on the isolation, propagation, and maintenance of neurospheres from adult and aged (>12 months old) mouse brain and how to differentiate cultured neurospheres into neurons and astrocytes. Culturing adult and aged NPCs provides an important in vitro model to (1) investigate cellular and molecular properties of this unique cell population and (2) expand the understanding of plasticity in the adult and aging brain. This protocol requires ∼2 hours to complete dissection, dissociation and culture plating, while differentiation to neuronal and astrocytic lineages takes 9 days. By focusing on neurospheres obtained from animals at later ages this model facilitates investigation of important biological questions related to development and differentiation of hippocampal neurons generated throughout adult life.

Attenuation Aß1-42-induced neurotoxicity in neuronal cell by 660nm and 810nm LED light irradiation.

Oligomeric amyloid-ß 1-42 (Aß1-42) has a close correlation with neurodegenerative disorder especially Alzheimer's disease (AD). It induces oxidative stress and mitochondrial damage in neurons. Therefore, it is used to generate AD-like in vitro model for studying neurotoxicity and neuroprotection against amyloid-ß. A low-level light therapy (LLLT) is a non-invasive method that has been used to treat several neurodegenerative disorders. In this study, the red wavelength (660nm) and near infrared wavelength (810nm) at energy densities of 1, 3, and 5 J/cm2 were used to modulate biochemical processes in the neural cells. The exposure of Aß1-42 resulted in cell death, increased intracellular reactive oxygen species (ROS), and retracted neurite outgrowth. We showed that both of LLLT wavelengths could protect neurons form Aß1-42-induced neurotoxicity in a biphasic manner. The treatment of LLLT at 3 J/cm2 potentially alleviated cell death and recovered neurite outgrowth. In addition, the treatment of LLLT following Aß1-42 exposure could attenuate the intracellular ROS generation and Ca2+ influx. Interestingly, both wavelengths could induce minimal level of ROS generation. However, they did not affect cell viability. In addition, LLLT also stimulated Ca2+ influx, but not altered mitochondrial membrane potential. This finding indicated LLLT may protect neurons through the stimulation of secondary signaling messengers such as ROS and Ca2+. The increase of these secondary messengers was in a functional level and did not harmful to the cells. These results suggested the use of LLLT as a tool to modulate the neuronal toxicity following Aß1-42 accumulation in AD's brain.

HPLC analysis and in vitro antioxidant mediated through cell migration effect of C.hystrix water extract on human keratinocytes and fibroblasts.

Citrus hystrix or kaffir lime is a native tropical plant containing a high level of phenolic and flavonoid compounds. Its fruits are used as a food ingredient to enhance the sour-sweet scent and flavor in many dishes. Due to its polyphenol-containing, it has also been used as traditional medicine for health benefits including oral and gum health, stress relief, hair care, and skincare. In this study, we demonstrated the antioxidant activity of C. hystrix water extract and its effect on human keratinocyte and fibroblast migration. The extract showed a high amount of phenolic and flavonoid contents. The HPLC analysis indicated the presence of gallic acid, catechin, caffeic acid, rutin, and quercetin. We showed that C. hystrix water extract exhibited free radical scavenging capacity, determined by DPPH assay, with IC50 of 14.91 mg/mL, and nitrite radical scavenging capacity, determined by NO assay, with IC50 of 4.46 mg/mL. The C. hystrix water extract displayed unnoticeable toxicity at all tested doses. We showed that the treatment of water extracts as low as 50 µg/mL decreased the reactive oxygen species (ROS) from H2O2-induced ROS formation in both cell lines. Besides, C. hystrix water extract promoted cell migration in a dose-dependent manner. Together, these results demonstrated the positive benefit of C. hystrix water extract as a wound-healing accelerator. Its health benefits may be due to the antioxidant capability of its phytochemical compounds contained in C. hystrix water extract that enhances the migration of two major cell types: fibroblast and keratinocytes, responsible for the proliferation and remodeling phase of wound healing.

Photo-Protective and Anti-Inflammatory Effects of Antidesma thwaitesianum Müll. Arg. Fruit Extract against UVB-Induced Keratinocyte Cell Damage.

The main cause of most skin cancers is damage from UVB from sunlight, which penetrate the skin surface and induce inflammation. For this reason, this study aims to identify natural products with photo-protection properties and their mode of action by using the UVB-irradiated HaCaT keratinocyte model. Antidesma thwaitesianum fruit extracts at 25, 50, and 100 µg/mL recovered cell viability following UVB exposure in a dose-dependent manner. Cell survival was associated with the reduction in intracellular ROS and NO. In addition, we showed that the pre-treatment with the fruit extract lowered the phosphorylation level of two MAPK-signaling pathways: p38 MAPKs and JNKs. The resulting lower MAPK activation decreased their downstream pro-inflammatory cascade through COX-2 expression and subsequently reduced the PGE2 proinflammatory mediator level. The photoprotective effects of the fruit extract were correlated with the presence of polyphenolic compounds, including cyanidin, ferulic acid, caffeic acid, vanillic acid, and protocatechuic acid, which have been previously described as antioxidant and anti-inflammation. Together, we demonstrated that the pre-treatment with the fruit extract had photo-protection by inhibiting oxidative stress and subsequently lowered stress-induced MAPK responses. Therefore, this fresh fruit is worthy of investigation to be utilized as a skincare ingredient for preventing UVB-induced skin damage.

An in vitro workflow of neuron-laden agarose-laminin hydrogel for studying small molecule-induced amyloidogenic condition.

In vitro studies have been popularly used to determine the cellular and molecular mechanisms for many decades. However, the traditional two-dimension (2D) cell culture which grows cells on a flat surface does not fully recapitulate the pathological phenotypes. Alternatively, the three-dimension (3D) cell culture provides cell-cell and cell-ECM interaction that better mimics tissue-like structure. Thus, it has gained increasing attention recently. Yet, the expenses, time-consuming, and complications of cellular and biomolecular analysis are still major limitations of 3D culture. Herein, we describe a cost-effective and simplified workflow of the 3D neuronal cell-laden agarose-laminin preparation and the isolation of cells, RNAs, and proteins from the scaffold. To study the effects of the amyloidogenic condition in neurons, we utilized a neuron-like cell line, SH-SY5Y, and induced the amyloidogenic condition by using an amyloid forty-two inducer (Aftin-4). The effectiveness of RNAs, proteins and cells isolation from 3D scaffold enables us to investigate the cellular and molecular mechanisms underlying amyloidogenic cascade in neuronal cells. The results show that SH-SY5Y cultured in agarose-laminin scaffold differentiated to a mature TUJ1-expressing neuron cell on day 7. Furthermore, the gene expression profile from the Aftin-4-induced amyloidogenic condition revealed the expression of relevant gene-encoding proteins in the amyloidogenic pathway, including APP, BACE1, PS1, and PS2. This platform could induce the amyloid-beta 42 secretion and entrap secreted proteins in the scaffold. The induction of amyloidogenic conditions in a 3D culture facilitates the interaction between secreted amyloid-beta and neurons, which makes it resembles the pathological environment in Alzheimer's brain. Together, this workflow is applicable for studying the cellular and molecular analysis of amyloid-induced neuronal toxicity, such as those occurred in Alzheimer's disease progression. Importantly, our method is cost-effective, reproducible, and easy to manipulate.

The Leaf Extract of Coccinia grandis (L.) Voigt Accelerated In Vitro Wound Healing by Reducing Oxidative Stress Injury.

The impairment in the regulation of the physiological process in the inflammatory phase of wound healing results in oxidative stress damage, which increases the severity and extends the healing time. In this study, we aimed to evaluate the radical scavenging properties of Coccinia leaf extract and its ability to ameliorate a migration process in vitro. Coccinia is a medicinal plant that was used in ancient times for relieving insect bite itching and swelling. However, the role of Coccinia leaf extract as an antioxidant related to the process of wound healing has never been studied. In this study, we demonstrated that the leaf extract possessed antioxidant properties that acted as a proton donor to neutralize reactive oxygen species with the IC50 value of 4.85 mg/mL of the extract. It could chelate iron with the IC50 value of 21.39 mg/mL of the extract. The leaf extract protected the human fibroblasts and keratinocytes from hydrogen peroxide-induced oxidative stress by increasing cell survival rate by more than 20% in all test doses. The protective property was dose-dependently correlated with the decrease in reactive oxygen species formation. In addition, the leaf extract enhanced the cell migration rate of fibroblasts and keratinocytes up to 23% compared with vehicle control. The results suggested that Coccinia leaf extract may be a potential herb for increasing the wound healing process with its antioxidant capacity and can be used as an herbal ingredient for the utilization of skincare products.

Daily White kwao krua dietary supplement alleviates LDL oxidative susceptibility, plasma LDL level and improves vasculature in a hypercholesterolemia rabbit model.

BACKGROUND AND AIM: White kwao krua is an edible plant that grows in Southeast Asia. It is very rich in natural phytoestrogens. Previous clinical studies revealed that the use of White kwao krua as a hormone replacement therapy has beneficial effects on the lipid profile of menopause women. In this present study, we utilized the hypercholesterolemia rabbit model to demonstrate the effect of White kwao krua on the daily intake of high-fat diet. EXPERIMENTAL PROCEDURE: We induced hypercholesterolemia in rabbits by feeding with high-fat diet (1% cholesterol-containing diet). The animals were maintained 12 weeks for the experimentation. The White kwao krua supplement was administered 100 mg/kg/day, and the effects were monitored comparing with Statins and turmeric. Blood was collected periodically to monitor the plasma cholesterol level and the oxidative susceptibility of isolated LDL-cholesterol. At the end of the experiment, the aorta was collected from the animal and performed endothelial-dependent relaxation and endothelial-independent relaxation assays. The relative ratio of intima to media layer was microscopically evaluated from hematoxylin/eosin-stained tissues. RESULTS AND CONCLUSION: We showed that the White kwao krua supplement reduced LDL-cholesterol about 40% compared with high-fat diet consumption alone. Administration of White kwao krua had significantly prolonged the susceptibility of LDL-cholesterol to oxidation. Besides, it led to the improvement of vascular function by recovering endothelium-dependent relaxation and alleviating vascular structure impairment induced by high-fat dietary intake. Together, we suggest that White kwao krua should be used as a dietary supplement to reduce the atherogenesis in high-fat dietary consumption. SECTION: Dietary therapy/nutrients supplements. TAXONOMY: Inflammation, Disease.

Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro.

BACKGROUND: Oligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals. METHODS: Our protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq). RESULTS: We demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice. CONCLUSIONS: This is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs.

Regulation by SoxR of mfsA, Which Encodes a Major Facilitator Protein Involved in Paraquat Resistance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia MfsA (Smlt1083) is an efflux pump in the major facilitator superfamily (MFS). Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin) treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084) is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression.

Copper chloride induces antioxidant gene expression but reduces ability to mediate H2O2 toxicity in Xanthomonas campestris.

Copper (Cu)-based biocides are currently used as control measures for both fungal and bacterial diseases in agricultural fields. In this communication, we show that exposure of the bacterial plant pathogen Xanthomonas campestris to nonlethal concentrations of Cu(2+) ions (75 µM) enhanced expression of genes in OxyR, OhrR and IscR regulons. High levels of catalase, Ohr peroxidase and superoxide dismutase diminished Cu(2+)-induced gene expression, suggesting that the production of hydrogen peroxide (H2O2) and organic hydroperoxides is responsible for Cu(2+)-induced gene expression. Despite high expression of antioxidant genes, the CuCl2-treated cells were more susceptible to H2O2 killing treatment than the uninduced cells. This phenotype arose from lowered catalase activity in the CuCl2-pretreated cells. Thus, exposure to a nonlethal dose of Cu(2+) renders X. campestris vulnerable to H2O2, even when various genes for peroxide-metabolizing enzymes are highly expressed. Moreover, CuCl2-pretreated cells are sensitive to treatment with the redox cycling drug, menadione. No physiological cross-protection response was observed in CuCl2-treated cells in a subsequent challenge with killing concentrations of an organic hydroperoxide. As H2O2 production is an important initial plant immune response, defects in H2O2 protection are likely to reduce bacterial survival in plant hosts and enhance the usefulness of copper biocides in controlling bacterial pathogens.

Novel roles of SoxR, a transcriptional regulator from Xanthomonas campestris, in sensing redox-cycling drugs and regulating a protective gene that have overall implications for bacterial stress physiology and virulence on a host plant.

In Xanthomonas campestris pv. campestris, SoxR likely functions as a sensor of redox-cycling drugs and as a transcriptional regulator. Oxidized SoxR binds directly to its target site and activates the expression of xcc0300, a gene that has protective roles against the toxicity of redox-cycling compounds. In addition, SoxR acts as a noninducible repressor of its own expression. X. campestris pv. campestris requires SoxR both for protection against redox-cycling drugs and for full virulence on a host plant. The X. campestris model of the gene regulation and physiological roles of SoxR represents a novel variant of existing bacterial SoxR models.

Evaluation of the virulence of Xanthomonas campestris pv. campestris mutant strains lacking functional genes in the OxyR regulon.

Xanthomonas campestris pv. campestris causes black rot in cruciferous crops. Hydrogen peroxide (H(2)O(2)) production and accumulation is an important initial response in plant defense against invading microbes. The role of genes involved in the bacterial H(2)O(2) protection system in pathogenicity was evaluated. Mutants of katA (encoding a monofunctional catalase) and, to a lesser extent, katG (encoding a catalase-peroxidase) and oxyR (encoding a H(2)O(2) sensor and a transcription regulator), are hypersensitive to H(2)O(2) treatments that mimic the plant H(2)O(2) burst. These data correlate with the results of pathogenicity testing that show katA, katG, and oxyR mutants are avirulent on a compatible plant. Moreover, exposure to H(2)O(2) (1, 2, and 4 mM) highly induces the expression of genes in the OxyR regulon, including katA, katG, and ahpC. The avirulent phenotype of the oxyR mutant is partly because of its inability to mount an adaptive response upon exposure to an H(2)O(2) burst. Our data provide insights into important roles of a transcription regulator and other genes involved in peroxide stress protection in the virulence of X. campestris pv. campestris.