Control Primer Research Articles

A Novel Graphite Fluoride/Bioactive Glass-containing Orthodontic Primer with Antibacterial and Remineralization Properties: An In-vitro Study.

This study aimed to develop a novel orthodontic primer that incorporated graphite fluoride (GF) and Bioactive glass (BAG) and to investigate its cell viability, bonding strength, and enamel damage, as well as its antibacterial and remineralization properties. Nine groups were prepared by adding different concentrations of GF (1, 2, and 4 wt.%) and BAG (1, 3, and 5 wt.%) to Transbond™ XT orthodontic primer. The prepared primers were compared to the control primer in terms of cell viability, shear bond strength (SBS), adhesive remnant index (ARI), enamel damage index (EDI), and antibacterial test. Then, the groups with better antibacterial properties (GFBAG 1-1, GFBAG 4-1, GFBAG 4-3, GFBAG 4-5) were evaluated for the remineralization properties. All the prepared orthodontic primers with different concentrations of GF/BAG revealed acceptable cell viability levels, with comparable SBS and ARI values to the control primer (p>0.05). Simultaneously, the EDI was reduced, while the antibacterial properties were significantly enhanced when compared to the control group (p0.05). The result of remineralization properties revealed that the selected groups had significantly higher remineralization ability than the control group; this was most pronounced in the GFBAG 4-3 group. All the prepared GF/BAG orthodontic primers are biologically safe with adequate SBS, ARI, and EDI values for clinical application with enhanced antibacterial properties. The GFBAG 4-3 experimental primer reveals the best antibacterial and remineralization properties which require further in-vitro and in-vivo investigations as a preventive measure of white spot lesions.

Development of a graphene oxide/hydroxyapatite-containing orthodontic primer: An in-vitro study

Fixed orthodontic appliances increase the risk of enamel demineralization and the development of white spot lesions (WSLs). This study aimed to develop a novel orthodontic primer that incorporates graphene oxide (GO) and hydroxyapatite (HA), investigate their cell viability, bonding strength, and enamel damage, and evaluate their antibacterial and remineralization properties. Nine groups were prepared by adding different concentrations of GO (0.1, 0.25, and 0.5 wt%) and HA (2, 5, and 7 wt%) to Transbond™ XT orthodontic primer. After observing the surface morphology of the material by a Field Emission Scanning Electron Microscope (FESEM), the phase of the materials by X-ray diffraction (XRD), and their chemical compositions by Energy Dispersive X-ray spectroscopy (EDX). The prepared primers were compared to the control primer group in terms of cell viability, shear bond strength (SBS), adhesive remnant index (ARI), enamel damage index (EDI), and antibacterial test. Then the groups with higher antibacterial properties (GOHA 0.1–2, GOHA 0.1–5, GOHA 0.25–2) were selected to evaluate their remineralization properties. Primers with all the prepared concentrations of GO/HA revealed acceptable cell viability levels within the limit of the ISO standards with comparable SBS and ARI values with the control primer (p > 0.05). Simultaneously, the EDI significantly reduced, and the antibacterial properties enhanced when compared to the control group (p < 0.05). The result of remineralization properties revealed that the selected groups had significantly higher remineralization ability than the control group which was more pronounced in GOHA 0.25–2 group. All the prepared orthodontic primers with different concentrations of GO/HA are safe biologically with adequate SBS, ARI, and EDI values for clinical application with enhanced antibacterial properties. The GOHA 0.25–2 experimental primer reveals the best antibacterial and remineralization properties which require further in-vitro and in-vivo investigation as a preventive measure of WSL.

A next generation sequencing assay combining Ixodes species identification with pathogen detection to support tick surveillance efforts in the United States

The burden of tick-borne diseases continues to increase in the United States. Tick surveillance has been implemented to monitor changes in the distribution and prevalence of human disease-causing pathogens in ticks that frequently bite humans. Such efforts require accurate identification of ticks to species and highly sensitive and specific assays that can detect and differentiate pathogens from genetically similar microbes in ticks that have not been demonstrated to be pathogenic in humans. We describe a modification to a next generation sequencing pathogen detection assay that includes a target that accurately identifies Ixodes ticks to species. We show that the replacement of internal control primers used to ensure assay performance with primers that also act as an internal control and can additionally differentiate tick species, retains high sensitivity and specificity, improves efficiency, and reduces costs by eliminating the need to run separate assays to screen for pathogens and for tick identification.

Single and combined neurotoxic, cytotoxic and genotoxic effects of 5GHz MIMO waves and computed tomography irradiation in male Wistar rats

ABSTRACT A significant public concern is how technologies that emit electromagnetic waves interact and affect the biota because they are linked to the dysregulation of genes involved in neurotransmission, oxidative stress, and normal cellular function. Standard methods have been used to study how the combination of electromagnetic waves from 5 GHz radio and computed tomography (CT) irradiation affects whole blood parameters, neurobehavioural profiles, genomic DNA, and p53 gene expression in Wistar rats grouped into five, namely, I-Negative control, II-sham, III-5 GHz only, IV-5 GHz + CT, V-CT. The 5 GHz router was connected to the internet using an ethernet cable and the specific absorbance rate (SAR) was measured as 0.54W/kg and 24 V/0.5A power density, while CT parameters were set at 140 K.v, 300 mA, 5.3 cv at a 1.0-s speed for 60 s. Genomic DNA was isolated from rats’ cerebral cortex, while target gene and internal control primers (GAPDH) were synthesized for tumor suppressor (p53) gene expression and electrophoresed on a 1.2% agarose gel. We found that CT irradiation had gross effects on platelets, white blood cell counts, memory, hepatic and testicular histoarchitectures compared to the 5 GHz-only group. However, there was a loss of p53 (exons 5–7) gene bands in electrophoresed data with increased micronucleated polychromatic erythrocyte count in the 5 GHz group. Regardless of the interferential interaction in the combination group, the deleterious effects of non-ionizing and ionizing irradiation in the single and combined exposure groups predict functional abnormalities and dysregulated cellular processes from high electromagnetic fields exposure in biological systems.

One-step amplification refractory mutation system-PCR/high-resolution melting curve assay for carrier detection of red blood cell membranopathy caused by common SPTB mutations.

Hereditary pyropoikilocytosis (HPP) is the most common cause of non-thalassemic severe inherited hemolytic anemia in Thai population. Up to 90% of affected patients harbor biallelic mutations of SPTB Providence (SPTB c.6055T>C), SPTB Buffalo (SPTB c.6074T>G), and SPTB Chiang Mai (SPTB c.6224A>G). This study aimed to develop a simple assay for mass screening of the three common SPTB mutations and to study their carrier frequencies in a healthy Thai population. We combined multiplex amplification refractory mutation system-PCR (ARMS-PCR) and high-resolution melting (HRM) curve analysis to create a one-step single-tube assay. The primers were designed to generate products with different melting temperatures in the presence of 6055C, 6074G, and 6224G. Internal control primers were added for quality control. Residual samples from blood donors and healthy adolescents were collected and tested for the three common SPTB mutations using the newly developed assay. Optimized multiplex ARMS-PCR/HRM curve assay yielded well-separated melt curves to detect the three SPTB mutations with 4-h turnaround time. The assay was validated in screening of 2261 non-repetitive blood donors and 89 adolescents, in which 10 (0.43%), 2 (0.09%), and 3 (0.13%) individuals were identified as carriers of SPTB Providence, SPTB Buffalo, and SPTB Chiang Mai, respectively. All mutated SPTB and 20 random wild-type samples were confirmed using Sanger sequencing with 100% accuracy. The novel ARMS-PCR/HRM curve assay is simple, accurate, and time-effective for mass screening of the common SPTB mutations. This can be employed to prevent HPP birth in a Thai population.

Evaluation of Antimicrobial and Mechanical Properties of a Novel Propolis-Modified Orthodontic Primer: An In-Vitro Study.

Introduction Accumulation of cariogenic biofilm around the bracket surface and the enamel adhesive interface leads to the formation of white spot lesions which poses an arduous challenge in orthodontics today. The aim of this study was to do a comparative evaluation of the antimicrobial, cytotoxic and mechanical properties of a novel propolis-modified orthodontic primer with a control primer. Materials and methods This in-vitro study involved two groups (group A: propolis-modified primer and group B: control primer). Antibacterial properties against Streptococcus mutans were evaluated using the agar well diffusion technique to measure the zone of inhibition and mic was evaluated using the two-fold diffusion technique. 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) fibroblast assay was done to evaluate the cytotoxicity. After bonding brackets on extracted natural teeth (premolars) the shear bond strength (SBS), contact angle (CA) and adhesive remnant index (ARI) were evaluated for both groups. Statistical analysis was done using Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, IBM Corp., Version 23.0, Armonk, NY), and an independent t-test was performed. Results The propolis-modified primer when compared to the control primer had higher zone of inhibition values and lower minimum inhibitory concentration (MIC) values. The MTT fibroblast assay showed that the cell viability % shown by the propolis primer was more than the control primer. There was no statistically significant difference between the two primers for SBS (p>0.05), CA (p>0.05) and ARI (p>0.05) (p=0.05). Conclusion Thepropolis-modified primer showed higher antibacterial activity against S. mutans at a lower inhibitory concentration, with less cytotoxicity and no effect on the SBS, CAand ARI scores.

EFFECTIVENESS OF DIETARY INTERVENTIONS IN MANAGING GESTATIONAL DIABETES: A META-ANALYSIS

Notwithstanding its high pervasiveness and rise, gestational diabetes mellitus (GDM) is a worldwide wellbeing worry because of its potential consequences for the two moms' and their kids' wellbeing. The reason for this study was to lead a deliberate survey and organization meta-analysis of the collaborations among five GDM counteraction techniques. The productivity of dietary administration methodologies for gestational diabetes was concentrated on in this survey. An exhaustive writing search was finished to order information from the very outset to June 30,2021.Research on medicines for preventing GDM, as genuine work, dietary intervention, ideal for biotic mediation, joined intervention, and inositol supplementation, was restricted to randomized control primers and semi randomized control starters circulated in English. Contrasted with normal treatment, dietary directing emphatically diminished the frequency of GDM. No mediation was effective in diminishing the mother's fasting glucose. 8,545 patients from 20 randomized control studies were remembered for the organization meta-analysis. Actual activity in addition to probiotic mediation fundamentally diminished the rate of GDM when contrasted with fake treatment. The gamble of GDM was not fundamentally different by dietary intercession, actual work and diet mediation, or inositol supplementation. This study exhibits the variety of dietary mediation conveyance techniques utilized in preliminaries to forestall GDM in overweight and corpulent ladies. It is trying to sum up the components of effective healthful interventions because of irregularity in both the supplier and the substance of the interventions.

SINGLE AND COMBINED EFFECTS OF 5GHZ FREQUENCY WAVES AND COMPUTED TOMOGRAPHY(CT) SCAN DOSES ON WHOLE BLOOD PARAMETRES, MEMORY AND P53 GENE EXPRESSION IN RATS

AbstractBackgroundExponential increase has emerged globally in the use of non‐ionizing and ionizing radio‐frequency electromagnetic fields(Rf‐emfs) for a wide range of applications, daily activities, communication, diagnostics , therapeutics to mention but a few. However, a significant public concern is how these technologies interacts and affects the biota as electromagnetic field (EMF) emissions is linked to dysregulation of genes involved in neurotransmission,oxidative stress and normal cellular function.MethodsStandard methods (y‐maze, novel object recognition tests, automated hematology analyzer, PCR etc), were used to study how the combination of EM waves from 5Ghz radio and CT irradiation affects whole blood parameters, neurobehavioural profiles (memory and cognition), DNA fragmentation and p53 gene expression in Wistar rats which were grouped into five namely: I‐Negative control, II‐sham, III‐5Ghz only , IV‐ 5Ghz+CT, V‐CT only for fourteen days. Genomic DNA was isolated from rats’ cerebral cortex while target gene and internal control primers (GAPDH) were synthesized for p53 gene exons expression and electrophoresed on a 2.5% agarose gel.ResultsWe found that CT irradiation had higher modulatory effects on platelets, white blood cell counts and memory impairments compared to 5GHz only group. However, there was gross DNA damage and complete loss of p53 (exons 5‐7) gene expression profiles in the 5GHz while the CT irradiated rats expressed mutant p53 gene. The effect of CT irradiation in pre‐exposed 5GHz rats revealed mutant p53(exons 5‐7), elevated memory and cognition impairments amongst others .ConclusionDeleterious single effects of 5GHz was observed in p53 gene expression profiles and cognition with an interferential interaction in its combination with CT irradiation leading to mutant p53 gene expression . The impaired neurobehavioural and p53 gene profiles as a result of the single and combined 5GHz and CT exposure predicts functional radiation waves emission effects, changes in resultant protein and dysregulated cellular functions.

An accurate genetic assay to identify human neutrophil antigen 2 deficiency.

We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans. HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now. A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T. In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null. The novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency.

Antibacterial Effect of Various Fermentation Products and Identification of Differentially Expressed Genes of E. coli

Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) are typical opportunistic pathogens. Moreover, these bacteria are known to possess multidrug-resistant (MDR) properties. This study investigates the antimicrobial activity of six fermented products, which have varying efficacies against P. aeruginosa, E. coli, and S. aureus. To identify novel candidate genes, differential expression analysis was performed using an annealing control primer. In the disk diffusion method, Fig vinegar (FV) and Diospyros kaki Thunb. vinegar (DTV) showed the greatest increase in inhibition compared to other fermented products, whereas fermented Korean traditional nature herb (FKTNH) had no antibacterial effect. This study identified down-regulation of Escherichia coli O157:H7 OmpW gene for outer membrane protein W, whereas gene for synthetic construct Lao1 gene for L-amino acid oxidase were up-regulated in E. coli treated with 5% FV. Consuming fermented vinegar helps to prevent bacterial infections. In particular, FV and DTV are potentially useful alternative natural products for multidrug resistance. Furthermore, both are expected to be used as effective natural antimicrobial agents, such as disinfectants.

Ionizing Radiation from Radiopharmaceuticals and the Human Gut Microbiota: An Ex Vivo Approach

This study aimed to determine the effect of three widely used radiopharmaceuticals with intestinal excretion on selected relevant bacteria that are part of the human gut microbiota, using an ex vivo approach. Fecal samples obtained from healthy volunteers were analyzed. Each sample was divided into four smaller aliquots. One served as the non-irradiated control. The other three were homogenized with three radiopharmaceutical solutions ([131I]NaI, [99mTc]NaTcO4, and [223Ra]RaCl2). Relative quantification of each taxa was determined by the 2−ΔΔC method, using the ribosomal gene 16S as an internal control (primers 534/385). Twelve fecal samples were analysed: three controls and nine irradiated. Our experiment showed fold changes in all analyzed taxa with all radiopharmaceuticals, but results were more significant with I-131, ranging from 1.87–83.58; whereas no relevant differences were found with Tc-99m and Ra-223, ranging from 0.98–1.58 and 0.83–1.97, respectively. This study corroborates limited existing research on how ionizing radiation changes the gut microbiota composition, providing novel data regarding the ex vivo effect of radiopharmaceuticals. Our findings justify the need for future larger scale projects.

Effect of calcium fluoride nanoparticles in prevention of demineralization during orthodontic fixed appliance treatment: a randomized clinical trial.

White spot lesions (WSLs) are the most common complications of fixed appliance orthodontic treatment. To evaluate the effectiveness of calcium fluoride nanoparticles-containing orthodontic primer (nCaF2-primer) in preventing the incidence of WSLs during orthodontic treatment. Single-centre, double-blinded, split-mouth, randomized clinical trial. The sample involved 31 orthodontic patients (≥12 years). Participants were recruited using a simple nonstratified randomization. Data collection, measurements, and analysis were performed blindly. Outcome measures included comparing the effect of nCaF2-primer with control primer (Transbond) regarding the degree of demineralization (DIAGNOdent pen), Streptococcus mutans (S. mutans) bacterial counting [real-time polymerase chain reaction device (PCR)], and WSLs incidence (pre- and post-operative photographs). The measurements were performed before bonding, 1, 3, and 6 months after bonding and after appliance removal. A two-way repeated measure analysis of variance test (for DIAGNOdent pen scores), and Wilcoxon signed-rank test (for the difference between bacterial counting and WSLs incidence) were used (P < 0.05). Thirty-one patients were recruited and randomized (mean age 17.9 ± 2.45 years). For the primary outcome (DIAGNOdent pen scores) and secondary outcome of S. mutans counting: 31 patients (310 teeth for each group) were included in scoring at T1 and T3, and 30 patients (300 teeth) were included at T6. While for the photographic scores, 26 patients were included after bracket bonding. The demineralization scores showed significant differences at all-time intervals within the 6 months after bracket bonding which was more noticeable after the first month. There was a significant difference in bacterial count between the two primer groups at the T1 only. Regarding photographic scores, there were no significant differences in the WSLs incidence between the two primers groups after brackets removal. No harm was detected during treatment, except the usual pain/gingival irritation. nCaF2-primer effectively decreased demineralization scores within the 6 months after bracket bonding. Moreover, it significantly reduced S. mutans colonization after the first month. However, the tested primer did not have an extra advantage in preventing WSLs development at the clinical level after appliance removal. The trial was registered with ClinicalTrials.gov on 8 May 2021 (registration number: NCT04994314).

Abstract 2363: Transcription factors of keratin 18 gene in MDA-MB-231Br (brain-colonizing) breast cancer cells

Abstract Among all cancer types, breast cancer has the highest incidence rate and second highest mortality rate in females in the United States. Breast cancer patients with brain metastasis have a poor prognosis, with a median survival time of less than 1 year despite treatment. Decreasing the prevalence of brain metastasis would improve the overall prognosis of patients. We previously built a novel mouse model of breast cancer brain metastasis by intracardiac injection of the MDA-MB-231 (triple-negative breast cancer cell line) cells in mice. The brain colonizing counterpart of the parental cells primarily metastasizes to the brain, which is called the MDA-MB-231Br cell line. We have also shown that keratin 18 (an epithelial marker) plays a crucial role in the brain metastasis process as the MDA-MB-231Br cells have lower expression of the keratin 18 gene compared to their parental MDA-MBA-231 cells. Therefore, it is important to understand the transcriptional control of the keratin 18 gene, in order to fully understand the role of keratin 18 in regulating brain metastasis of breast cancer. We hypothesize that keratin 18 gene expression differs between these two cell lines due to the differential occupation of the gene by transcription factors such as Fli-1 or ETS-1. In this study, we analyzed the pattern of chromatin occupation of transcription factors ETS-1 and Fli-1 to determine whether they are responsible for the altered expression of the keratin 18 gene in MDA-MB-231Br cells.The Chromatin Immunoprecipitation (ChIP) was performed using the Pierce Magnetic ChIP Kit to analyze the presence of transcription factors Fli-1 and ETS-1 with enzymatic and mechanical shearing combined. The ChIP Kit included the positive control anti-RNA Polymerase II Antibody and GAPDH control primers. We optimized the experimental conditions including ChIP analysis for MDA-MB-231Br cells, detection of expected positive control results, and sonication settings to break apart the chromatin. We measured the ChIP mediated enrichment of the intron 1 region of the keratin 18 gene, as the DNA is hypermethylated in this region in MDA-MB-231Br cells. The primers were designed and used as follows: forward: 5′-GATCATCGAGGACCTGAGGG-3′ and reverse: 5′-GGGGAGCAGATCCTTCTTAGC-3′. Our results indicated that an ETS transcription factor occupies intron 1 of the keratin 18 gene in the MDA-MB-231Br cells, also showing that ChIP is an effective method to examine transcription factor binding. For future studies, we plan to perform similar experiments with the parental MDA-MB-231 cells. Then a quantitative PCR will be used to measure DNA binding sites of the transcription factors and determine if there is a significant difference in the level of chromatin occupation between the regular and brain-colonizing breast cancer cells. Our studies are necessary to understand the role of the keratin 18 gene in regulating tumor metastasis at the molecular level. Citation Format: Anissa Elice Johnson, Sagardeep Singh, Paul Lockman, Tuoen Liu, Gabor Szalai. Transcription factors of keratin 18 gene in MDA-MB-231Br (brain-colonizing) breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2363.

PCR optimization for the detection of bunchy top virus of abaca (Musa textilis Nee) in Eastern Visayas Philippines

The bunchy top virus in Eastern Visayas has serverly reduced abaca production. Early and accurate detection of plant viral pathogens is an essential and crucial component for disease management. At present, there are no standard PCR conditions in the Eastern Visayas region for detecting the bunchy top virus at an early stage using PCR. Thus, optimization for the detection was carried out to assist in disease management. Different annealing temperatures (57, 60 and 65oC), gel concentrations (1, 1.5 and 2%), and running conditions (80, 90 and 100 volts) were tested using My TaqTM DNA Polymerase (Bioline, USA). The annealing temperatures of 57oC and 60oC resulted in DNA amplification as indicated by the presence of bands but absence of bands at 65oC. The higher voltages of 90 and 100 volts resulted in smears and distorted DNA bands with 1% and 1.5% agarose; thus 2% agarose gel was used to resolve small DNA fragments (100bp to 3kb). Electrophoresis using 80 volts for 45min successfully separated the DNA bands. The amplification of the product with internal control primers indicated the absence of PCR inhibitors in the abaca-extracted DNA samples. This confirmed the negative PCR reaction as indicative of the absence of the virus. The optimized PCR conditions could be applied by students and researchers for the early detection of bunchy top virus in the National Abaca Research Center Germplasm collection and the region.

Identification of differentially expressed transcripts for trunk formation in sago palm using annealing control primer GeneFishing technique

Identification of differentially expressed transcripts for trunk formation in sago palm using annealing control primer GeneFishing technique

Captopril inhibits matrix metalloproteinase activity and improves dentin bonding durability.

We investigated the inhibitory effects of captopril on matrix metalloproteinases (MMPs) and its effect as a primer on dentin bonding durability. One hundred fifty human third molars were selected. Flat surfaces of the middle dentin were exposed, etched 15s, and followed by pretreatment with a primer for 60s, including distilled water (control, the negative control primer), 2% chlorhexidine digluconate (CHD,the positive control primer), and captopril solution. Inhibitory effects of primers on MMPs were evaluated by hydroxyproline and gelatinase activity tests. All primers were applied on dentin followed by bonding. Some of the samples were sliced into slabs, placed in a fluorescent solution containing gelatin, and incubated for in situ zymography. Some were cut into sticks, and after aging for 1day, 12months, or 24months, microtensile bonding strength was tested. Some were cut into slabs, aged for 1day, 12months, or 24months, and taken out for nanoleakage tests to reveal interface defects. Hydroxyproline and gelatinase activity analyses showed that captopril exerted better inhibitory effects on MMPs, relative to 2% CHD (p < 0.05). A 0.2% captopril aqueous solution (0.2% CapW) was chosen to apply to the dentin. In situ zymography showed that inhibitory effects of captopril on gelatinase were significantly higher compared to 2% CHD (p < 0.01). Microtensile strength revealed that the bonding effects of the 0.2% CapW group lasted longer, compared to the control and 2% CHD groups (p < 0.05). Interface defects, detected by nanoleakage, were significantly reduced in the 0.2% CapW group, compared to the control and 2% CHD groups (p < 0.05). Captopril inhibits dentin MMP activities and effectively improves dentin bonding durability. Captopril is a promising dentin bonding primer for improving bonding durability.

Detection of Vector Copy Number in Bicistronic CD19xCD22 CAR T Cell Products with Digital PCR

Chimeric antigen receptor (CAR) T cell therapy is a rapidly evolving immunotherapeutic treatment modality for adult and pediatric patients with a variety of cancers, which has been most extensively investigated in B-cell malignancies. Given that CAR T cell immunotherapy involves changing the genetic composition of a patient's T cells, this living drug presents unique safety and quality control challenges. Vector copy number (VCN), a measurement of transgene copies within a CAR T cell product, is a product-specific characteristic that must be quantified prior to patient administration as high VCN increases the risk of insertional mutagenesis.Historically, VCN assessment in CAR T cell products has been performed via qPCR. qPCR is reliable along a broad range of concentrations but has inherent limitations in its lower limit of detection and limit of quantification. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared to qPCR include simplicity, reproducibility, lower limit of detection, and definitive quantification.In this present study, we developed an assay for analysis of the novel bicistronic UCD19x22 CAR T cell construct, which was developed in the laboratory of Dr. Terry Fry at the University of Colorado and will be moving in to clinical trials later this year. Custom primer-probe assays were designed using Primer Express v3.0.1 and the ThermoFisher Custom TaqMan Assay Design Tool. As an internal control, forward and reverse primers as well as a VIC-labeled probe specific to human albumin (NCBI gene 213, HGNC:399) were designed. Primers and a FAM-labeled probe assay, specific for the bicistronic CD19x22 CAR T cell product, were designed at the junction site between the two distinct CARs. This study compares two different digital PCR modalities: (1) droplet digital PCR (ddPCR) via the BioRad QX200 system which utilizes water-in-oil droplet partitions and (2) the QIAcuity digital PCR system utilizing a nanoplate-based partitioning platform. While dPCR is a newer methodology compared to ddPCR, the two apply parallel procedures, data generation, and analyses. The primer/probe assay was validated with qPCR, dPCR and ddPCR using patient samples from preclinical CAR T cell manufacturing production runs, as well as Jurkat cell subclones which stably express this bicistronic CAR T product.We successfully developed an assay to specifically detect and quantify our bicistronic CD19xCD22 CAR transgene. ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product without any signal detected in samples containing untransduced T cells or T cells transduced with CD19 only CARs. Additionally, our assay gives accurate, precise, and reproducible CAR T cell VCN measurements across qPCR, dPCR, and ddPCR modalities. We demonstrate that digital PCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, and that specific assays can be developed for detection of unique constructs. Future studies will evaluate the utility of this assay with digital PCR modalities in measuring CAR T cell persistence in clinical trial patient samples after receiving this novel CAR T cell product. [Display omitted] DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

Evaluation of a newly developed calcium fluoride nanoparticles-containing orthodontic primer: An in-vitro study

ObjectivesFluoride and calcium ions have the ability to reduce the formation of white spot lesions (WSL) in enamel. This study aimed to develop a novel orthodontic primer that incorporates calcium fluoride nanoparticles (nCaF2) and investigate its cytotoxic and physical properties as this primer could enhance the remineralization process when used with the conventional acid etching technique. Materials and methodsThree groups of the newly introduced primers were prepared by adding and mixing nCaF2 (Nanoshell Company, USA at 5%, 10%, 20% in weight) to Transbond™ XT orthodontic primers (3M-Unitek, Monrovia, USA). X-Ray Diffraction (XRD) was performed to verify and evaluate the phase of the nanopowders. Field Emission Scanning Electron Microscope (FESEM) observation was used to assess the homogeneity of tested primers, and Energy Dispersive X-Ray Spectroscopy (EDS) was performed to analyze elements of the prepared samples. The three primer groups were compared to the control primer (without the addition of nCaF2) in terms of cytotoxic behavior, homogeneity, agglomeration, shear bond strength (SBS), and adhesive remnant index (ARI). ResultsnCaF2 primers with all the prepared concentrations revealed good homogeneity with no apparent agglomeration after four months of mixing. Cytotoxicity of the new primers was higher than that of the control primer, but it was within the accepted limits of the ISO standards (70% cell viability). While the SBS and ARI values were comparable with the control primer (p > 0.05). ConclusionsThe newly developed orthodontic primers with different concentrations of nCaF2 (5%, 10%, and 20%) showed a homogenous distribution of nCaF2 within the primers with no apparent agglomeration, acceptable cytotoxic level, adequate SBS, and ARI. Future clinical testing of nCaF2-containing orthodontic primer as a preventive measure for WSLs during fixed orthodontic treatment is suggested.

Effects of ‘in-service’ conditions – mimicked hull roughness ranges and biofilms – on the surface and the hydrodynamic characteristics of foul-release type coatings

To develop a better understanding of ‘in-service’ performance of modern marine coatings, this study explored the combined effects of different roughness ranges of foul-release coating (FRC) and light biofouling (slime) on the surface, boundary layer and drag characteristics under a range of ‘in-service’ conditions. Natural and laboratory biofilms were grown dynamically on FRC panels by exposing panels in facilities dedicated to realistic fouling culture. The boundary layer experiments were conducted in a circulating water tunnel. Boundary layer similarity-law scaling was used to predict the combined effects of coating roughness and biofilms on the added frictional resistance (% and added required effective power ) for a benchmark KRISO container ship (KCS) and a bulk carrier. The increase in due to the presence of biofilms on commercial FRC is estimated to be between 7% and 16% depending on the biofilm type, biofilm thickness and percentage coverage. Significant increases in effective power are estimated for non-fouling control primers with heavy fouling. Moreover, the paper suggests updated roughness allowances () for two vessel types assuming FRCs on their hulls with more representative hull roughness ranges and fluffy biofilms.

Development of TaqMan real-time RT-PCR for sensitive detection of diverse Raspberry ringspot virus isolates

Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61–98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.