Human Tissue Culture Research Articles

Discovery of a Highly Selective MC1R Agonists Pentapeptide to Be Used as a Skin Pigmentation Enhancer and with Potential Anti-Aging Properties.

One of the first lines of cutaneous defense against photoaging is (a) the synthesis of melanin and (b) the initiation of an oxidative stress response to protect skin against the harmful effects of solar radiation. Safe and selective means to stimulate epidermal pigmentation associated with oxidative stress defense are; however, scarce. Activation of the melanocortin-1 receptor (MC1R) on epidermal melanocytes represents a key step in cutaneous pigmentation initiation and, additionally, it regulates cellular defense mechanisms like oxidative stress and DNA-repair. Thus, making the activation of MC1R an attractive strategy for modulating skin pigmentation and oxidative stress. In this context, we designed and synthesized pentapeptides that act as MC1R agonists. These peptides bound, with high potency, to MC1R and activated cAMP synthesis in CHO cells expressing human MC1R. Using one lead pentapeptide, we could show that this activation of MC1R was specific as testing the activation of other G-protein coupled receptors, including the MC-receptor family, was negative. In vitro efficacy on mouse melanoma cells showed similar potency as for the synthetic MC1R agonist alpha-melanocyte stimulating hormone (NDP-alpha-MSH). Moreover, we could reproduce this activity in human skin tissue culture. The lead pentapeptide was able to induce ex-vivo protein expression of key melanogenesis markers melanocyte inducing transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP-1). Concerning oxidative stress response, we found that the pentapeptide enhanced the activation of Nrf2 after UVA-irradiation. Our results make this pentapeptide an ideal candidate as a skin pigmentation enhancer that mimics alpha-MSH and may also have anti-photoaging effects on the skin.

Deep sequencing and automated histochemistry of human tissue slice cultures improve their usability as preclinical model for cancer research

Cancer research requires models closely resembling the tumor in the patient. Human tissue cultures can overcome interspecies limitations of animal models or the loss of tissue architecture in in vitro models. However, analysis of tissue slices is often limited to histology. Here, we demonstrate that slices are also suitable for whole transcriptome sequencing and present a method for automated histochemistry of whole slices. Tumor and peritumoral tissue from a patient with glioblastoma was processed to slice cultures, which were treated with standard therapy including temozolomide and X-irradiation. Then, RNA sequencing and automated histochemistry were performed. RNA sequencing was successfully accomplished with a sequencing depth of 243 to 368 x 106 reads per sample. Comparing tumor and peritumoral tissue, we identified 1888 genes significantly downregulated and 2382 genes upregulated in tumor. Treatment significantly downregulated 2017 genes, whereas 1399 genes were upregulated. Pathway analysis revealed changes in the expression profile of treated glioblastoma tissue pointing towards downregulated proliferation. This was confirmed by automated analysis of whole tissue slices stained for Ki67. In conclusion, we demonstrate that RNA sequencing of tissue slices is possible and that histochemical analysis of whole tissue slices can be automated which increases the usability of this preclinical model.

SDF-1 preconditioned HPC scaffolds mobilize cartilage-derived progenitors and stimulate meniscal fibrocartilage repair in human explant tissue culture

ABSTRACTPurpose: The purpose of this study was to characterize the influence of SDF-1 on cell migration/adhesion and temporal gene expression of human cartilage mesenchymal progenitor cells (C-PCs); and to utilize SDF-1 conditioned mesenchymal progenitors to stimulate reintegration of human meniscus fibrocartilage breaks.Materials and Methods: Characterization of SDF-1-induced cell migration was achieved using hydroxypropyl cellulose (HPC) scaffolds pretreated with SDF-1. Fluorescence microscopy and cell counting were used to visualize and quantify the extent of cell migration into scaffolds, respectively. Relative mRNA expression analysis was used to characterize the temporal effects of SDF-1 on C-PCs. Tissue reintegration experiments were conducted using cylindrical human meniscal tissue punches, which were then placed back together with an HPC scaffold embedded with C-PCs. Tensile testing was used to evaluate the extent of tissue reintegration stimulated by human mesenchymal progenitors.Results: C-PCs migrate into scaffolds in response to SDF-1 with the same efficiency as mesenchymal progenitors from human marrow (BM-MSCs). SDF-1 treatment of C-PCs did not significantly alter the expression of early and late stage chondrogenic differentiation genes. Scaffolds containing SDF-1 pre-conditioned C-PCs successfully adhered to fibrocartilage breaks and migrated from the scaffold into the tissue. Tensile testing demonstrated that SDF-1 preconditioned C-PCs stimulate reintegration of fibrocartilage tears.Conclusion: C-PCs migrate in response to SDF-1. Exposure to SDF-1 does not significantly alter the unique mRNA profile of C-PCs that make them desirable for cartilaginous tissue repair applications. SDF-1 pretreated mesenchymal progenitors successfully disperse into injured tissues to help facilitate tissue reintegration.

Assessing Triplet Repeat Expansions in Human SVG-A Cell Culture.

Determining the molecular mechanisms that contribute to trinucleotide repeat (TNR) expansions is essential to understanding the origin of genetically inherited diseases, such as Huntington's disease, and to inform efforts in developing therapeutic treatments. As one resource to probe the mechanisms of TNR expansions, we describe an expansion assay in human tissue culture cells. The cell line SVG-A, derived from human astrocytes, has the important property of supporting expansions in culture, unlike many cell lines derived from patients. SVG-A cells are also amenable to standard genetic and biochemical techniques such as siRNA, CRISPR-Cas9 and enzymatic inhibitors. This combination of features allows for mechanistic studies of TNR expansions, using the quantitative genetic assay described here as a readout. The SVG-A assay has correctly identified key proteins that drive expansions and it has facilitated testing of enzymatic inhibitors that suppress expansions as potential therapeutics. This chapter describes how repeat expansions are detected, visualized, and quantified.

Alternative Experimental Models for Studying Influenza Proteins, Host-Virus Interactions and Anti-Influenza Drugs.

Ninety years after the discovery of the virus causing the influenza disease, this malady remains one of the biggest public health threats to mankind. Currently available drugs and vaccines only partially reduce deaths and hospitalizations. Some of the reasons for this disturbing situation stem from the sophistication of the viral machinery, but another reason is the lack of a complete understanding of the molecular and physiological basis of viral infections and host–pathogen interactions. Even the functions of the influenza proteins, their mechanisms of action and interaction with host proteins have not been fully revealed. These questions have traditionally been studied in mammalian animal models, mainly ferrets and mice (as well as pigs and non-human primates) and in cell lines. Although obviously relevant as models to humans, these experimental systems are very complex and are not conveniently accessible to various genetic, molecular and biochemical approaches. The fact that influenza remains an unsolved problem, in combination with the limitations of the conventional experimental models, motivated increasing attempts to use the power of other models, such as low eukaryotes, including invertebrate, and primary cell cultures. In this review, we summarized the efforts to study influenza in yeast, Drosophila, zebrafish and primary human tissue cultures and the major contributions these studies have made toward a better understanding of the disease. We feel that these models are still under-utilized and we highlight the unique potential each model has for better comprehending virus–host interactions and viral protein function.

629 The ATP-dependent chromatin remodeler BRG1 controls epidermal keratinocytes migration during human cutaneous wound healing

SWI/SNF ATP-dependent chromatin remodelling complexes alter nucleosome structure, positioning and chromatin compaction state resulting in target gene activation or repression. The SWI/SNF complexes contain either BRG1 or BRM as the core ATPase together with other common and variable subunits. BRG1 is required for epidermal terminal differentiation in both mice and human; and for hair follicle stem cell activation during mouse hair regeneration and cutaneous wound healing. However, the role of SWI/SNF complexes in human skin wound healing remains unknown. Here, we demonstrate that genes encoding SWI/SNF complex subunits are expressed in human epidermis, and BRG1 is substantially upregulated in hyper-proliferating and migrating keratinocytes in healing cutaneous wounds. siRNA mediated BRG1 gene suppression leads to the substantial retardation of the wound closure in the full thickness human excision wounded skin tissue culture model and in scratched human primary keratinocyte monolayers. Furthermore, we showed that the retardation in the keratinocyte migration with no changes in cellular proliferation or apoptosis. Micro-array based transcriptome analysis revealed that BRG1 is required for full induction of genes encoding several signaling molecules, transcription factors and structural proteins involved in changes in cellular adhesion and cytoskeletal organization, driving keratinocyte migration during skin wound healing, including the stress cytokeratins 16 and 17. In summary, our data uncovered an essential role of the BRG1 chromatin remodeler in induction of the transcription programme driving keratinocyte migration during skin wound closure and re-epithelisation.

Characterization of Plasma Membrane Localization and Phosphorylation Status of Organic Anion Transporting Polypeptide (OATP) 1B1 c.521 T>C Nonsynonymous Single-Nucleotide Polymorphism.

Membrane transport protein organic anion transporting polypeptide (OATP) 1B1 mediates hepatic uptake of many drugs (e.g. statins). The OATP1B1 c.521T > C (p. V174A) polymorphism has reduced transport activity. Conflicting in vitro results exist regarding whether V174A-OATP1B1 has reduced plasma membrane localization; no such data has been reported in physiologically relevant human liver tissue. Other potential changes, such as phosphorylation, of the V174A-OATP1B1 protein have not been explored. Current studies characterized the plasma membrane localization of V174A-OATP1B1 in genotyped human liver tissue and cell culture and compared the phosphorylation status of V174A- and wild-type (WT)-OATP1B1. Localization of V174A- and WT-OATP1B1 were determined in OATP1B1 c.521T > C genotyped human liver tissue (n = 79) by immunohistochemistry and in transporter-overexpressing human embryonic kidney (HEK) 293 and HeLa cells by surface biotinylation and confocal microscopy. Phosphorylation and transport of OATP1B1 was determined using 32P-orthophosphate labeling and [3H]estradiol-17β-glucuronide accumulation, respectively. All three methods demonstrated predominant plasma membrane localization of both V174A- and WT-OATP1B1 in human liver tissue and in cell culture. Compared to WT-OATP1B1, the V174A-OATP1B1 has significantly increased phosphorylation and reduced transport. We report novel findings of increased phosphorylation, but not impaired membrane localization, in association with the reduced transport function of the V174A-OATP1B1.

Transcriptional networks in the human epididymis.

The epithelial lining of the human epididymis is critical for sperm maturation. This process requires distinct specialized functions in the head, body, and tail of the duct. These region-specific properties are maintained by distinct gene expression profiles which are governed by transcription factor networks, non-coding RNAs, and other factors. We used genome-wide protocols including DNase-seq, RNA-seq and ChIP-seq to characterize open (active) chromatin, the transcriptome and occupancy of specific transcription factors (TFs) respectively, in caput, corpus, and cauda segments of adult human epididymis tissue and primary human epididymis epithelial (HEE) cell cultures derived from them. RNA-seq following TF depletion or activation, combined with gene ontology analysis also determined TF targets. Among regional differentially expressed transcripts were epithelial-selective transcription factors (TFs), microRNAs, and antiviral response genes. Caput-enriched TFs included hepatocyte nuclear factor 1 (HNF1) and the androgen receptor (AR), both of which were also predicted to occupy cis-regulatory elements identified as open chromatin in HEE cells. HNF1 targets were identified genome-wide using ChIP-seq, in HEE cells. Next, siRNA-mediated depletion of HNF1 revealed a pivotal role for this TF in coordinating epithelial water and solute transport in caput epithelium. The importance of AR in HEE cells was shown by AR ChIP-seq, and by RNA-seq after synthetic androgen (R1881) treatment. AR has a distinct transcriptional program in the HEE cells and likely recruits different co-factors (RUNX1 and CEBPβ) in comparison to those used in prostate epithelium. Our data identify many transcription factors that regulate the development and differentiation of HEE cells. Moreover, a comparison between immature and adult HEE cells showed key TFs in the transition to fully differentiated function of this epithelium. These data may help identify new targets to treat male infertility and have the potential to open new avenues for male contraception.

Olive Leaf Extract (OleaVita) Suppresses Inflammatory Cytokine Production and NLRP3 Inflammasomes in Human Placenta.

The placenta is essential for pregnancy and produces both pro-inflammatory and anti-inflammatory cytokines. Excessive production of inflammatory cytokines, involving interleukin-1β (IL-1β), IL-6, and IL-8, from placental tissues is associated with pregnancy complications. Olive leaf extract has several health benefits, including anti-inflammatory functions. OleaVita is a new commercial olive leaf extract; it is hypothesized to suppress placental inflammation. In human placental tissue culture, OleaVita treatment inhibited the secretion of inflammatory cytokines and NF-κB p65 protein expression. OleaVita also suppressed toll-like receptor ligands-induced IL-1β secretion in human placental tissues. IL-1β is regulated by the NLRP3 inflammasomes, a pivotal regulator of various diseases. OleaVita significantly decreased NLRP3 and pro-IL-1β protein expression, suggesting that it has an inhibitory effect on NLRP3 inflammasome activation. Thus, OleaVita is beneficial as an inhibitor of inflammation and NLRP3 inflammasome activation, and may be used as a supplement for the treatment and prevention of inflammatory diseases.

SUN-LB045 Microfluidic and Static Culture of Human Testicular Tissue

Testicular tissue culture has been used as a method to study testicular physiology for over 100 years. However, most of these investigations rely upon rodent tissues, and the translation of these techniques to the human model has proven comparably challenging. This is a critical gap in our fundamental knowledge of human testis biology and limits the value of testicular tissues currently being cryopreserved for men and boys requiring fertility preservation. To meet these twin objectives, maintaining or maturing human testicular tissues ex vivo must be accomplished using new technologies. Microfluidics is an emerging technology that has been developed to support cells ex vivo and has resulted in longer lived tissue function, including the murine ovary and human female reproductive tract. Recent work in the microfluidic culture of murine testes has also demonstrated superior germ cell maintenance comparatively with static culture. For these reasons, we hypothesized that the microfluidic culture will similarly improve and extend the viability of cultured human testicular tissue, while also enabling investigations into endocrine function through a non-invasive manner. To test our hypothesis, we cultured human testicular tissue in both microfluidic and static conditions. Our microfluidic culture system is composed of inert materials (optimal for the recovery of steroid and peptide hormones), and was run at a 40 μL/hour flow rate. Adult human tissues were donated through an IRB-approved autopsy protocol. Tissues were cultured at an air-liquid interface in both culture conditions for upwards of 2 weeks and in the presence of gonadotropins (hCG and FSH). At the completion of culture, tissues were fixed and processed for histological analysis, and major cell types and structures were identified. Secreted testosterone and Inhibin B within culture media was quantified via enzyme-linked immunosorbent assay. Cultured tissues demonstrated a dramatic reduction in germ cell number in both static and microfluidic conditions. However, tissues cultured in microfluidics demonstrated reduced tubule basement membrane breakdown and structural degeneration. Tissues cultured in both conditions were productive of secreted testosterone and inhibin B. In conclusion, the present study has explored the utility of microfluidics for human testicular culture. Similar to other tissues, microfluidic culture supported higher viability and better tissue integrity compared to static. Furthermore, microfluidic culture was a convenient way to study secreted hormones over time. Dissimilar to murine microfluidic reports, germ cell populations were not maintained in our studies, yet work is continuing to improve this outcome. Funding: This work was funded by a UH3 grant under the NIEHS and a F31 grant under the NICHD at the National Institutes of Health. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

HoxA10 Facilitates SHP-1-Catalyzed Dephosphorylation of p38 MAPK/STAT3 To Repress Hepatitis B Virus Replication by a Feedback Regulatory Mechanism

Hepatitis B virus (HBV) infection is the leading cause of chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). This study reveals a distinct mechanism underlying the regulation of HBV replication. HBV activates homeobox A10 (HoxA10) in human hepatocytes, leukocytes, peripheral blood mononuclear cells (PBMCs), HepG2-NTCP cells, leukocytes isolated from CHB patients, and HBV-associated HCC tissues. HoxA10 in turn represses HBV replication in human hepatocytes, HepG2-NTCP cells, and BALB/c mice. Interestingly, we show that during early HBV infection, p38 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) were activated to facilitate HBV replication; however, during late HBV infection, HoxA10 was induced to attenuate HBV replication. Detailed studies reveal that HoxA10 binds to p38 MAPK, recruits SH2-containing protein tyrosine phosphatase 1 (SHP-1) to facilitate SHP-1 in catalyzing dephosphorylation of p38 MAPK/STAT3, and thereby attenuates p38 MAPK/STAT3 activation and HBV replication. Furthermore, HoxA10 binds to the HBV enhancer element I (EnhI)/X promoter, competes with STAT3 for binding of the promoter, and thereby represses HBV transcription. Taken together, these results show that HoxA10 attenuates HBV replication through repressing the p38 MAPK/STAT3 pathway by two approaches: HoxA10 interacts with p38 MAPK and recruits SHP-1 to repress HBV replication, and HoxA10 binds to the EnhI/X promoter and competes with STAT3 to attenuate HBV transcription. Thus, the function of HoxA10 is similar to the action of interferon (IFN) in terms of inhibition of HBV infection; however, the mechanism of HoxA10-mediated repression of HBV replication is different from the mechanism underlying IFN-induced inhibition of HBV infection.IMPORTANCE Two billion people have been infected with HBV worldwide; about 240 million infected patients developed chronic hepatitis B (CHB), and 650,000 die each year from liver cirrhosis (LC) or hepatocellular carcinoma (HCC). This work elucidates a mechanism underlying the control of HBV replication. HBV infection activates HoxA10, a regulator of cell differentiation and cancer progression, in human cells and patients with CHB and HCC. HoxA10 subsequently inhibits HBV replication in human tissue culture cells and mice. Additionally, HoxA10 interacts with p38 MAPK to repress the activation of p38 MAPK and STAT3 and recruits and facilitates SHP-1 to catalyze the dephosphorylation of p38 MAPK and STAT3. Moreover, HoxA10 competes with STAT3 for binding of the HBV X promoter to repress HBV transcription. Thus, this work reveals a negative regulatory mechanism underlying the control of HBV replication and provides new insights into the development of potential agents to control HBV infection.

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue's stiffness, which is crucial for subsequent slicing. 500 µm thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

A Multimaterial Microphysiological Platform Enabled by Rapid Casting of Elastic Microwires.

Due to escalating drug developmental costs and limitations of cardiotoxicity screening, there is an urgent need to develop robust in vitro 3D tissue culture platforms that can both facilitate the culture of human cardiac tissues and provide noninvasive functional readouts predictive of cardiotoxicity in clinical settings. However, such platforms commonly require complex fabrication procedures that are difficult to scale up to high-throughput testing platforms. Here, innovative multimaterial processing into a scalable and functional platform is proposed in the format of a 96-well plate. Three classes of materials are integrated into the platform. An array of soft elastic microwires is used both as anchors for tissue formation as well as sensors for recording tissue contraction. Conductive carbon electrodes are embedded into the plate to drive electrical stimulation for tissue maturation and pace tissue contraction during drug testing. The bulk of the device is made of rigid polystyrene plastic to eliminate drug-absorbing polydimethylsiloxane (PDMS). The platform has higher throughput than the current state-of-the-art devices, at a significantly reduced cost of manufacturing and tissue production.

Combined inhibition of sonic Hedgehog signaling and histone deacetylase is an effective treatment for liver cancer.

Clinical trials have revealed that inhibition of sonic Hedgehog (SHH) signaling or histone deacetylase (HDAC) holds promise as a treatment for liver cancer. Based on our previous results, it was hypothesized that dual inhibition of SHH and HDAC may contribute to more efficient targeting of this disease. The effect of SHH inhibitor vismodegib as a single‑agent or in combination with HDAC inhibitor entinostat was evaluated by Cell Counting Kit‑8 (CCK‑8) and flow cytometric assays, as well as immunoblotting. The synergistic effect on cell viability was assessed by combination indexes. Ex vivo cultured liver cancer tissues from a patient were treated with vismodegib as a single‑agent or in combination with entinostat, and analyzed by histological and immunohistochemical methods. The results revealed that the dual use of the SHH inhibitor and the HDAC inhibitor effectively synergized to inhibit proliferation, and promote apoptosis in liver cancer cells. Furthermore, the effect of the combination of these drugs was confirmed in an ex vivo culture of human liver cancer tissue. Mechanistically, combined use of SHH and HDAC inhibitors resulted in significantly greater downregulation of SHH and PI3K/mTOR signaling. In conclusion, the combined use of SHH signaling and HDAC inhibitors may be an effective therapeutic strategy for liver cancer.

Interactions between cancer-associated fibroblasts and tumor cells promote MCL-1 dependency in estrogen receptor-positive breast cancers

Selective inhibition of BCL-2 is expected to enhance therapeutic vulnerability in luminal estrogen receptor-positive breast cancers. We show here that the BCL-2 dependency of luminal tumor cells is nevertheless mitigated by breast cancer-associated fibroblasts (bCAFs) in a manner that defines MCL-1 as another critical therapeutic target. bCAFs favor MCL-1 expression and apoptotic resistance in luminal cancer cells in a IL-6 dependent manner while their own, robust, survival also relies on MCL-1. Studies based on ex vivo cultures of human luminal breast cancer tissues further argue that the contribution of stroma-derived signals to MCL-1 expression shapes BCL-2 dependency. Thus, MCL-1 inhibitors are beneficial for targeted apoptosis of breast tumor ecosystems, even in a subtype where MCL-1 dependency is not intrinsically driven by oncogenic pathways.

Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry.

Histone acetylation plays important roles in the regulation of DNA transcription, repair, and replication. Here we detail a method for quantitative detection of specific histone modifications in the nascent chromatin at or behind replication forks in vivo in cultured cells. The method involves labeling DNA with EdU, using Click chemistry to biotinylate EdU moieties in DNA, and then using in situ proximity ligation assay (PLA) to selectively visualize co-localization of EdU with a modified histone of choice recognized by a modification-specific antibody. We focus on detection of acetylated histones H3 and H4 in the nascent chromatin of cultured human cells as a specific example of the method's application. Notably, the method is fully applicable to studies of histones or nonhistone proteins expected to be present on nascent DNA or at replication forks, and has been successfully used in model organisms and human tissue culture.

Investigating ROS, RNS, and H2S-Sensitive Signaling Proteins.

The modification of proteins is a key way to alter their activity and function. Often thiols, cysteine residues, on proteins are attractive targets for such modification. Assuming that the thiol group is accessible then reactions may take place with a range of chemicals found in cells. These may include reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reactive nitrogen species such as nitric oxide (NO), hydrogen sulfide (H2S), or glutathione. Such modifications often are instrumental to important cellular signaling processes, which ultimately result in modification of physiology of the organism. Therefore, there is a need to be able to identify such modifications. There are a variety of techniques to find proteins which may be altered in this way but here the focus is on two approaches: firstly, the use of fluorescent thiol derivatives and the subsequent use of mass spectrometry to identify the thiols involved; secondly the confirmation of such changes using biochemical assays and genetic mutants. The discussion will be based on the use of two model organisms: firstly the plant Arabidopsis thaliana (both as cell cultures and whole plants) and secondly the nematode worm Caenorhabditis elegans. However, these tools, as described, may be used in a much wider range of biological systems, including human and human tissue cultures.

The effects of the nanosphere carrier for the combined carmustine-busulfan trastuzumab in human breast cancer tissue culture

The effects of the nanosphere carrier for the combined carmustine-busulfan trastuzumab in human breast cancer tissue culture

Indigenous irrigants as potent antimicrobials in endodontic treatment: An in vitro study.

The success of an endodontic treatment depends on effective disinfection and complete sealing of root canal. The irrigants that are currently used in the field of endodontics have certain limitations, so the quest for an ideal root canal irrigant continues. Nowadays, the use of herbal extracts such as Triphala and liquorice are used for their potent antimicrobial activity and less side effects. This study aims (1) to evaluate the antimicrobial efficacy of Triphala and liquorice against Enterococcus faecalis.(2) To determine any cytotoxic effect on isolated human periodontal ligament (PDL) fibroblasts. The antimicrobial efficacy of Triphala and liquorice extracts was analyzed at different concentrations (12.5, 25, 50, 75, and 100 mg/ml) using the well-diffusion method. Three percent sodium hypochlorite (NaOCl) and distilled water were taken as positive and negative controls. Minimum inhibitory concentration of the active extract was determined by the broth dilution assay. Human PDL fibroblast tissue culture was used to assess the cytotoxicity of the preparations. The data thus obtained were subjected to statistical analysis. The results showed that the mean antimicrobial efficacy of Triphala and liquorice at 50 mg/ml is 20.33 and 9.33, respectively, which are statistically significant (P < 0.0001) as compared with a concentration 12.5 and 25 mg/ml. 50 mg/ml showed significant results (P < 0.001) on comparing with hypochlorite. Triphala and liquorice showed no cytotoxic effect as compared to NaOCl on human PDL fibroblasts. Among the three tested materials Triphala showed the highest antimicrobial efficacy followed by NaOCl and liquorice.

In-vitro culture of freeze-thawed human ovarian cortical tissue on agar scaffold

In-vitro culture of freeze-thawed human ovarian cortical tissue on agar scaffold