Culture Time Points Research Articles

Response Mechanism of Microbial Communities in River Sediments under Different Hydrodynamic Conditions

This paper studies the response of sedimentary microbial communities to different hydrodynamic conditions through laboratory experiments. The hydrodynamic conditions and flow field distribution in the experimental tank were measured and simulated by acoustic Doppler flowmeter and FLUENT software. The sediment DNA of different culture time and different flow velocity points was extracted and amplified by PCR amplification and high-throughput sequencing. The OTU sequence of the microbial organism.

The effect of different surface treatment methods on the physical, chemical and biological performances of a PGA scaffold.

In order to improve the adhesion between a PGA scaffold and islet cells, it is necessary to find a suitable method to modify the scaffold. In this study, the PGA scaffold surface was modified by plasma, polylysine coating and plasma combined with polylysine coating (P–P-PGA). The surface adhesion of the modified PGA scaffold was examined, and the stretchability and infiltration of the PGA scaffold were also tested. Then, the PGA scaffold treated under the optimal treatment conditions was selected to co-culture with rat islet cells, and the survival activity of the rat islet cells on the untreated PGA scaffold and the P–P-PGA scaffold was examined via the MTT method. Rhodamine staining and DAPI staining were used to detect the number of islet cells adhered to four groups of scaffolds at different culture time points. The PGA-islet graft in the leg muscle of rats was stained with HE to perform the PGA-islet graft pathological examination. The experimental results showed that when the plasma treatment power was 240 W, the processing time was 4 min; the concentration of the polylysine coating solution was 2 mg ml−1, the tensile strength of the PGA scaffold was 320.45 MPa and the amount of infiltration of the PGA scaffold by the serum medium presented the maximum value: 3.17 g g−1. The MTT survival activity test results showed that after 3 d of culture, the survival activity of the islet cells of the treated PGA scaffold culture group (2.02 ± 0.13) was significantly different from the survival activity of the islet cells of the untreated PGA scaffold culture group (1.93 ± 0.10). The survival activities of the islet cells in the experimental groups (1.60 ± 0.13, 1.40 ± 0.12) were still higher than those of the control groups (0.96 ± 013, 0.69 ± 0.09) at 15 and 21 d. The results of the rhodamine and DAPI staining showed that with the increase in culture time, the number of the adherent cells in each group increased, and the number of the adherent islet cells in the experimental group was higher than that in the untreated group. The HE staining results showed that the islet cells on the P–P-PGA scaffold were more than those on the untreated PGA scaffold. After modification of the PGA scaffold, the adhesion of the islet cells improved, which was conducive to the growth of islet cells. These results confirmed that the plasma combined with polylysine coating treatment could enhance the adhesion of the PGA scaffold surface, so that the scaffold and the islet cells exhibited better adhesion and biocompatibility, and the modified PGA scaffold (P–P-PGA) could be used as a promising islet cell scaffold.

Cryopreservation Impacts Cell Functionality of Long Term Expanded Adipose-Derived Stem Cells

Objective: Adipose-Derived Stem Cells (ADSCs) are less immunogenic cells and have a heterogenic cytokine secretion profile making them a better candidate for cell-based immunotherapy. Even innately or stimulated, Interleukin-6 (IL-6) and Toll-like Receptor 2 (TLR2) secreted by ADSC were modulated and led to different inflammatory mechanism pathways through different inflammatory factors secretion. These properties can be very useful in the treatment of inflammation-associated chronic diseases. To be used, ex-vivo ADSC expansion is a critical issue prior to transplantation or cryopreservation. Functional cell changes have been reported during culture expansion being susceptible to interact with freezing/thawing effects leading to doubt on cell therapeutic outcomes. The aim of this study is to identify the effect of freezing/thawing at the different time point of expansion culture on IL-6 and TLR2 secretion.Methods: ADSC were collected from young female donors, expanded in culture and cryopreserved in Foetal Bovine Serum (FBS) and Dimethylsulfoxide (DMSO) after each passage for 6 months to a year. ADSC was then tested for proliferation, clonogenicity, cytokine gene expression and assessment before cryopreservation (fresh) and after thawing and cultured until confluence (frozen/thawed). ADSC preserved at Passage 0 (P0) were thawed and tested after confluence at P1.Results: Cryopreserved ADSC as P1 resulted in increased clonogenicity, total RNA and protein secretion compared to the fresh ones. Relative Quantification (RQ) and cytokine assessment of IL-6, IL-10, Tumor Necrosis Factor (TNF)-α and TLR2 revealed a moderate up-regulation of TLR2 while significantly higher IL-6 secretion levels were observed in long term expanded and cryopreserved ADSC.Conclusion: Our results suggested that cryopreserved ADSC long term expanded in culture were functionally different and might have impaired immunosuppressive properties through modulation of the inflammatory responses by IL-6 and TLR2 activation.

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed the gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that enable fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species, aspen, pine, and spruce, under various culture conditions. We examined both gene expression (transcription levels) and RNA editing (posttranscriptional modification of RNA, which can potentially yield different proteins from the same gene). We found that F. pinicola is able to modify both gene expression and RNA editing profiles across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This work provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

Ischemic heart disease, also known as coronary artery disease (CAD), poses a challenge for regenerative medicine. iPSC technology might lead to a breakthrough due to the possibility of directed cell differentiation delivering a new powerful source of human autologous cardiomyocytes. One of the factors supporting proper cell maturation is in vitro culture duration. In this study, primary human skeletal muscle myoblasts were selected as a myogenic cell type reservoir for genetic iPSC reprogramming. Skeletal muscle myoblasts have similar ontogeny embryogenetic pathways (myoblasts vs. cardiomyocytes), and thus, a greater chance of myocardial development might be expected, with maintenance of acquired myogenic cardiac cell characteristics, from the differentiation process when iPSCs of myoblastoid origin are obtained. Analyses of cell morphological and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) performed at two in vitro culture time points spanning the early stages of cardiac development (day 20 versus 40 of cell in vitro culture) confirmed the ability of the obtained myogenic cells to acquire adult features of differentiated cardiomyocytes. Prolonged 40-day iPSC-derived cardiomyocytes (iPSC-CMs) revealed progressive cellular hypertrophy; a better-developed contractile apparatus; expression of marker genes similar to human myocardial ventricular cells, including a statistically significant CX43 increase, an MHC isoform switch, and a troponin I isoform transition; more efficient intercellular calcium handling; and a stronger response to β-adrenergic stimulation.

An RNA-seq based transcriptomic investigation into the productivity and growth variants with Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used in producing monoclonal antibodies (mAbs), however, these cell lines can show variants associated with productivity and growth. Fundamental understanding of cellular mechanisms can benefit high mAbs production while maintaining robust cell growth. In this study, RNA sequencing (RNA-seq) based approach was used to study the transcriptomic space among three mAb-producing CHO cell lines. A phenotypic contrast of higher productivity but lower growth was found with one cell line versus the other two cell lines. The messenger RNAs (mRNAs) from those cells at two different time points in culture were sequenced to seek the gene functions possibly associated with the phenotypic differences. It was found that the mAb transcripts from each cell line were correlated with the mAb production level, indicating a strong determination of production by the expression level of gene of interest (GOI). Further exploration in the global transcriptome showed that the high producers had more expression with genes related with secretion and protein transportation. In the meantime, the slower growth of the high producers was linked to higher regulation to cell growth as well as lower gene expression on biosynthesis, central metabolism and nutrient transport.

Insights into the generation of monoclonal antibody acidic charge variants during Chinese hamster ovary cell cultures.

Charge variation is one of the most important heterogeneities during monoclonal antibody (mAb) manufacturing and this study presents insights into the generation of acidic charge variants during cell culture processes. Since acidic variants generate both intracellularly and extracellularly, main charge fraction collected by weak cation exchange chromatography (WCX) was incubated in harvested cell supernatant (HCS) to simulate and investigate the extracellular process firstly. It is found that the main fraction was degraded rapidly into acidic variants rather than basic variants extracellularly, and the degradation sites were located in both Fab and Fc fragments indicated by papain digestion. Besides, certain process parameters were investigated as their potential roles in the extracellular process. As a result, media composition showed significant influence on degradation while culture time point did not, suggesting that the extracellular process was a spontaneous process without enzyme catalysis. Additionally, kinetics study reveals that the extracellular process was a pseudo first-order reaction. The E app value (21.59kcal/mol) estimated from the Arrhenius equation suggests that the extracellular degradation might be mainly attributed to asparagine deamidation. Furthermore, we established an acidic variants generation model, indicating that the extracellular process plays a dominant role in modulating the final acidic variant level. This study provides better understanding for controlling product heterogeneity in mAb manufacturing.

Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical Aspergillus fumigatus in different culture conditions based on ribosomal proteins.

Fast and accurate discrimination of Aspergillus fumigatus is significant, since misidentification may lead to inappropriate clinical therapy. This study assessed VITEK mass spectrometry (MS) V3.0 for A. fumigatus identification using extracted fungal ribosomal proteins. A total of 52 isolates preliminarily identified as A. fumigatus by traditional morphological methods were inoculated in three different culture media and cultured at two different temperatures. The specific spectral fingerprints of different culture time points (48, 72, 96, and 120 h) were obtained. Of all strains, 88.5% (46/52) were discriminated as A. fumigatus, while the remaining 11.5% (6/52) produced results inconsistent with morphological analysis. Molecular sequencing, as a reference method for species identification, was used to validate the morphological analysis and matrix-assisted laser desorption/ionization time of flight MS. Chi-square tests (χ2 test, P=0.05) demonstrated that the culture medium and incubation temperature had no effects on identification accuracy; however, identification accuracy of the strains in the 48-h group was lower than that in other groups. In addition, we found that ribosomal proteins extracted from A. fumigatus can be stored in different environments for at least 1 week, with their profiles remaining stable and strain identification results showing no change. This is beneficial for medical institutions with no mass spectrometer at hand. Overall, this study showed the powerful ability of VITEK MS V 3.0 in identifying A. fumigatus.

Determination of the Culture Time Point to Induce Corneal Epithelial Differentiation in Induced Pluripotent Stem Cells

BackgroundLimbal stem cells (LSC) are progenitor cells in the ocular surface that renew the corneal epithelium. Limbal stem cell deficiency usually induces blindness through the loss of corneal transparency, and bilateral cases do not an accurate treatment because of the lack of an autologous source of stem cells. MethodsInduced pluripotent stem cells (iPSC) are promising for use in cell therapy because of their autologous origin and the capability to differentiate into corneal epithelial cells. However, there are not standardized protocols to achieve a complete corneal epithelial differentiation. We examined the expression of several markers in a human episomal iPSC line after an induction period from embryoid bodies. ResultsProgenitor LSC and corneal epithelial differentiation markers, some extracellular matrix protein adhesion molecules, and wingless signaling pathway were studied. Overall, LSC progenitor and corneal epithelium differentiation markers increased after maintaining cell culture in specific conditions for 14 days, whereas pluripotency markers decreased. ConclusionsOur approach indicated that the optimal time point to initiate iPSC differentiation into LSC and corneal phenotypes, with the use of specific medium, is from 14 days after initial embryoid bodies treatment induction.

Pure Glaucoma Drugs Are Toxic to Immortalized Human Corneal Epithelial Cells, but They Do Not Destabilize Lipid Membranes.

Most pure glaucoma drugs (pGDs) are hydrophobic substances intended to reduce elevated intraocular pressure. The aims of our study were to determine the toxicity of pGDs (brimonidine tartrate, brinzolamide, latanoprost, timolol maleate, and pilocarpine hydrochloride) on ocular surface cells and to establish whether their toxicity is subsequent to cellular membrane destabilization. The toxicity of clinically efficient doses of pGDs was measured at different time points in a cell culture of human corneal epithelial cells using a redox indicator. pGD interaction with the plasma membrane was analyzed using a hemolysis assay and liposome electrokinetic chromatography. The capacity of pGDs to induce endoplasmic reticulum stress was investigated by immunoblotting. The toxicity assay showed that all pGDs decrease the viability of the epithelial cells to variable degrees. Early toxicity was measured for 4% pilocarpine and 0.15% brimonidine with 60% cell death at 4 hours, whereas 2% pilocarpine and 0.005% latanoprost showed almost 100% toxicity but only after 16 hours. The hemolysis assay and liposome electrokinetic chromatography experiments suggested that interaction between pGDs and lipid membranes is weak and cannot explain cell death through lysis. Immunoblotting revealed that the drugs activate endoplasmic reticulum stress and, with the exception of pilocarpine, have the capacity to induce apoptosis through upregulation of C/EBP homologous protein. Our study indicates that all studied pGDs decrease the viability of the corneal epithelial cells, but none of the tested compounds were able to destabilize cellular membranes. The pGDs seem to be internalized and can induce apoptosis through C/EBP homologous protein recruitment.

Switch From Fetal to Adult SCN5A Isoform in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Unmasks the Cellular Phenotype of a Conduction Disease-Causing Mutation.

BackgroundHuman induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) can recapitulate features of ion channel mutations causing inherited rhythm disease. However, the lack of maturity of these cells is considered a significant limitation of the model. Prolonged culture of hiPSC‐CMs promotes maturation of these cells. We studied the electrophysiological effects of the I230T mutation in the sodium channel gene SCN5A in hiPSC‐CMs generated from a homozygous (I230Thomo) and a heterozygous (I230Thet) individual from a family with recessive cardiac conduction disease. Since the I230T mutation occurs in the developmentally regulated “adult” isoform of SCN5A, we investigated the relationship between the expression fraction of the adult SCN5A isoform and the electrophysiological phenotype at different time points in culture.Methods and ResultsAfter a culture period of 20 days, sodium current (IN a) was mildly reduced in I230Thomo hiPSC‐CMs compared with control hiPSC‐CMs, while I230Thet hiPSC‐CMs displayed no reduction in IN a. This coincided with a relatively high expression fraction of the “fetal” SCN5A isoform compared with the adult isoform as measured by quantitative polymerase chain reaction. Following prolonged culture to 66 days, the fraction of adult SCN5A isoform increased; this was paralleled by a marked decrease in IN a in I230Thomo hiPSC‐CMs, in line with the severe clinical phenotype in homozygous patients. At this time in culture, I230Thet hiPSC‐CMs displayed an intermediate loss of IN a, compatible with a gene dosage effect.ConclusionsProlonged culture of hiPSC‐CMs leads to an increased expression fraction of the adult sodium channel isoform. This new aspect of electrophysiological immaturity should be taken into account in studies that focus on the effects of SCN5A mutations in hiPSC‐CMs.

Abstract LB-093: Simultaneous measurement of global methylation and copy number alterations in human colorectal cancer samples

Abstract Epigenomic changes are commonly observed in cancer. We developed a next-generation sequencing (NGS) assay which can identify subtle changes in global methylation as well as copy number alterations (CNAs). The assay measures the methylation level of some repeat elements, covering over 25% of all CpG in the genome. Genomic DNA extracted from 18 pairs of matched colorectal tumor to normal tissue was tested (four adenocarcinomas, including one associated with inflammatory bowel disease, and fourteen adenomas, including one adenoma from a Lynch syndrome patient, one familial adenomatous polyposis (FAP) and two sessile serrated adenomas (SSA)). We also assayed organoids established from these tissues at early (2 month) and late (more than 6 months) timepoints in culture. Briefly, bisulfite conversion and enrichment of the repeats was performed. The Illumina compatible products were sequenced on a HiSEQ 2500. Analysis was performed using Bismark (Krueger F., Babraham institute) and Nexus copy number (BioDiscovery) to determine the global methylation and the CNAs, respectively. Finally, somatic and germline variants were determined by targeted sequencing of 71 genes using the QIAseq colorectal cancer panel (QIAGEN). Our data showed that all adenocarcinoma presented hypomethylation and CNAs as well as multiple somatic variants in APC, KRAS, TP53, SMAD4 and PIK3CA. Six out of the fourteen adenomas presented CNAs and nine showed hypomethylation. The most frequently observed copy number gain affected the chromosomes 8q and 13. Eight adenomas presenting somatic mutations in APC also exhibited distinct global hypomethylation. Previous publications have reported that mutations in APC precede global hypomethylation. Interestingly, one adenoma showed significant hypomethylation but no detectable CNAs or APC mutations. The two SSA samples showed the characteristic BRAF V600E mutations and the FAP sample presented germline mutation in APC. The FAP and the SSA samples did not show hypomethylation or CNAs. Most organoids presented CNAs and somatic mutations similar to their matched tissue even after 2 years in culture. However, few organoids developed new CNAs and somatic variants. Monitoring the changes in organoid may provide important information on tumor progression. This assay measured CNAs and methylation in colorectal cancer samples, findings which may assist in determining the status of the disease and provide guidance to the appropriate action. Our NGS assay interrogates a significant portion of the genome leading to a more accurate and sensitive evaluation of the state of these cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Julie C. Laliberte, Michael K. Dame, Durga Attili, Bodrul Islam, Kevin Kim, Jessica Zhang, Erica L. Katz, Gina M. Newsome, Priya H. Dedhia, Adele Kruger, Tobias Mann, Tom Goodman, Jeffrey Buis, Dean E. Brenner, James Varani, Jason R. Spence, Justin A. Colacino, Jay Stoerker. Simultaneous measurement of global methylation and copy number alterations in human colorectal cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-093. doi:10.1158/1538-7445.AM2017-LB-093

Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating side effect of radiation treatment. A mouse model of radiation-induced mucositis leads to weight loss and tissue damage, reflecting the human ailment as it responds to keratinocyte growth factor (KGF), the standard-of-care treatment. Cultured intestinal crypt organoids allowed the development of an assay monitoring the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay resembles the mouse model as KGF and roof plate-specific spondin-1 (RSPO1) enhanced crypt organoid recovery following radiation. Screening identified compounds that increased the survival of organoids postradiation. Testing of these compounds revealed that the organoids changed their responses over time. Unbiased transcriptome analysis was performed on crypt organoid cultures at various time points in culture to investigate this adaptive behavior. A number of genes and pathways were found to be modulated over time, providing a rationale for the altered sensitivity of the organoid cultures. This report describes an in vitro assay that reflects aspects of human disease. The assay was used to identify bioactive compounds, which served as probes to interrogate the biology of crypt organoids over prolonged culture. The pathways that are changing over time may offer potential targets for treatment of mucositis.

Influences of abaR gene on biofilm formation of Acinetobacter baumannii

Influences of abaR gene on biofilm formation of Acinetobacter baumannii

Assessment of real-time PCR cycle threshold values in Microsporum canis culture-positive and culture-negative cats in an animal shelter: a field study.

Objectives Real-time PCR provides quantitative information, recorded as the cycle threshold (Ct) value, about the number of organisms detected in a diagnostic sample. The Ct value correlates with the number of copies of the target organism in an inversely proportional and exponential relationship. The aim of the study was to determine whether Ct values could be used to distinguish between culture-positive and culture-negative samples. Methods This was a retrospective analysis of Ct values from dermatophyte PCR results in cats with suspicious skin lesions or suspected exposure to dermatophytosis. Results One hundred and thirty-two samples were included. Using culture as the gold standard, 28 were true positives, 12 were false positives and 92 were true negatives. The area under the curve for the pretreatment time point was 96.8% (95% confidence interval [CI] 94.2-99.5) compared with 74.3% (95% CI 52.6-96.0) for pooled data during treatment. Before treatment, a Ct cut-off of <35.7 (approximate DNA count 300) provided a sensitivity of 92.3% and specificity of 95.2%. There was no reliable cut-off Ct value between culture-positive and culture-negative samples during treatment. Ct values prior to treatment differed significantly between the true-positive and false-positive groups ( P = 0.0056). There was a significant difference between the pretreatment and first and second negative culture time points ( P = 0.0002 and P <0.0001, respectively). However, there was substantial overlap between Ct values for true positives and true negatives, and for pre- and intra-treatment time points. Conclusions and relevance Ct values had limited usefulness for distinguishing between culture-positive and culture-negative cases when field study samples were analyzed. In addition, Ct values were less reliable than fungal culture for determining mycological cure.

Adhesion and migration of CHO cells on micropatterned single layer graphene

Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

Antimicrobial activity and regulation of CXCL9 and CXCL10 in oral keratinocytes.

Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 are dysregulated in oral inflammatory conditions, and it is not known if these chemokines target microorganisms that form oral biofilm. The aim of this study was to investigate the antimicrobial activity of CXCL9 and CXCL10 on oral microflora and their expression profiles in oral keratinocytes following exposure to inflammatory and infectious stimuli. Streptococcus sanguinis was used as a model and Escherichia coli as a positive control. The antimicrobial effect of CXCL9/CXCL10 was tested using a radial diffusion assay. mRNA transcripts were isolated from lipopolysaccharide (LPS)-treated and untreated (control) oral keratinocyte cell lines at 2-, 4-, 6-, and 8-h time-points of culture. The CXCL9/10 expression profile in the presence or absence of interferon-γ (IFN-γ) was assessed using semiquantitative PCR. Although both chemokines demonstrated antimicrobial activity, CXCL9 was the most effective chemokine against both S. sanguinis and E coli. mRNA for CXCL10 was expressed in control cells and its production was enhanced at all time-points following stimulation with LPS. Conversely, CXCL9 mRNA was not expressed in control or LPS-stimulated cells. Finally, stimulation with IFN-γ enhanced basal expression of both CXCL9 and CXCL10 in oral keratinocytes. Chemokines derived from oral epithelium, particularly CXCL9, demonstrate antimicrobial properties. Bacterial and inflammatory-stimulated up-regulation of CXCL9/10 could represent a key element in oral bacterial colonization homeostasis and host-defense mechanisms.

A comparative study on monomer elution and cytotoxicity of different adhesive restoration materials

This study evaluated monomer release and cytotoxicity of different adhesive restoration materials used for dental restorations. The extracts (1, 2, and 7 days) of three types of adhesive dental restoration materials, [Quixfill (QF), Silorane Restorative (SR), and Ketac N 100 Restorative (KR)], and the adhesive resins, [XP Bond (XP), Silorane Primer (SP), Ketac N 100 Primer (KP), and Silorane Bond (SB)] were analyzed using high performance liquid chromatography/mass spectrometry (HPLC-MS). The cytotoxicity levels were determined at different time points (24, 48, and 72 h) of cell culture using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. All adhesive resin materials showed monomer release at varying amounts with the highest release after 7 days. The lowest amount of release was observed in QF and the highest with KP. Bis-Phenol A (BPA) was not detected in SP and KR that contain bisphenol-A diglycidyl ether dimethacrylate (bis-GMA). Decamethylpenthasiloxane (D5) was not eluted from SR. Except for SR and QF, all other adhesive restoration materials showed different degrees of toxicity along with different monomer release kinetics. The correlation between the monomer release and cytotoxicity of the materials indicated that the cytotoxicity of the materials increased with the monomer release (Spearman’s rho correlation coefficient – r). The correlation after 48 h was statistically significant (r = −0.342, p = 0.017).