A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Mass Treatment of Flooded Archival Materials by Gamma Radiation

This work describes mass recovery processes of flooded archival materials at industrial scale. The presence of fungi on paper represents a threat to the integrity of the document because they degrade cellulose, one of the main components of paper. Gamma radiation treatments are investigated as mass disinfection agents for their high penetrating power, speed of treatment, and absence of risk due to chemical residuals. We compared two different recovery processes: thermal drying followed by gamma irradiation and gamma irradiation followed by thermal drying. Both these processes were conducted simultaneously on naturally contaminated archival items and on paper specimens artificially contaminated with species test. Efficacy was assessed by culture method and ATP assay, right after the treatments and after four years of storage at room temperature. Coupling gamma irradiation with a drying step with dry heat at 55–60 °C reduces the fungal loads on natural items up to levels close to the detection limits, and the reduction is maintained after four years. On artificial specimens, spore germination is completely inhibited, mycelia growth is also highly affected, but the melanised test species appear to be more resistant. A synergistic effect between gamma irradiation, water content, and thermal drying is highlighted in this paper.

The Anti-Proliferative Lichen-Compound Protolichesterinic Acid Inhibits Oxidative Phosphorylation and Is Processed via the Mercapturic Pathway in Cancer Cells

The lichen compound protolichesterinic acid (PA) has an anti-proliferative effect against several cancer cell lines of different origin. This effect cannot be explained by the known inhibitory activity of PA against 5- and 12-lipoxygenases. The aim was therefore to search for mechanisms for the anti-proliferative activity of PA. Two cancer cell lines of different origin, both sensitive to anti-proliferative effects of PA, were selected for this study, T-47D from breast cancer and AsPC-1 from pancreatic cancer. Morphological changes were assessed by transmission electron microscopy, HPLC coupled with TOF spectrometry was used for metabolomics, mitochondrial function was measured using the Agilent Seahorse XFp Real-time ATP assay and glucose/lactate levels by radiometry. Levels of glutathione, NADP/NADPH and reactive oxygen species [ROS] were measured by luminescence. Following exposure to PA both cell lines showed structural changes in mitochondria that were in line with a measured reduction in oxidative phosphorylation and increased glycolysis. These changes were more marked in T-47D, which had poorer mitochondrial function at baseline. PA was processed and expelled from the cells via the mercapturic pathway, which consumes glutathione. Nevertheless, glutathione levels were increased after 24 hours of exposure to PA, implying enhanced synthesis. Redox balance was not much affected and ROS levels were not increased. We conclude that PA is metabolically processed and expelled from cells, leading indirectly to increased glutathione levels with minimal effects on redox balance. The most marked effect was on mitochondrial structure and metabolic function implying that effects of PA may depend on mitochondrial fitness.

Abstract 402: Defining Diverse Disease Pathomechanisms Across Thick And Thin Filament Hypertrophic Cardiomyopathy Variants.

Hypertrophic cardiomyopathy (HCM) affects as many as ~1 in 500 individuals, and is often typified by hyperdynamic contraction and poor cellular relaxation. HCM can be caused by mutations in a variety of key contractile proteins of the sarcomere. A large proportion of these variants are found in MYBPC3, MYH7, TNNT2, and TNNI3. These genes encode proteins that control cardiac muscle contraction at the thick (MYBPC3 and MYH7) and thin filaments (TNNT2 and TNNI3) of the sarcomere. In this study we use human induced pluripotent stem cell derived cardiomyocytes to model HCM across all of these genes. We do this to define key mechanistic differences between thick and thin filament HCM. We define sarcomeric contractility (SarcTrack) calcium transients (CalTrack) and myosin states using the mant-ATP assay. We use the parametric data from these experimental studies in iPSC-CMs to model possible disease mechanisms in silico. Our experimental analysis highlights that both thick and thin filament HCM variants cause cellular hypercontractility, with slowed cellular relaxation. We find that thick filament HCM variants drive cellular HCM phenotypes by destabilising the myosin interacting heads motif (IHM), showing a marked reduction in the super relaxed state of myosin. Counterintuitively thin filament based HCM variants show a reduction in DRX myosin. When applying Mavacamten the allosteric myosin ATPase inhibitor to our thin and thick filament HCM variant iPSC-CMs we find a dichotomy of cellular responses. The thick filament variants studied all show a clear resolution of cellular HCM. However, not all cellular phenotypes of thin filament HCM are corrected by Mavacamten treatment, although there is benefit. We conclude that causal mechanisms of thick filament HCM are well corrected at the molecular and cellular level by Mavacamten, but these causal mechanisms in thin filament based HCM are not suitably corrected. We highlight key mechanistic pharmacological targets for thin filament variants that could add cellular benefit to HCM phenotype resolution.

Early (5-Day) Onset of Diabetes Mellitus Causes Degeneration of Photoreceptor Cells, Overexpression of Incretins, and Increased Cellular Bioenergetics in Rat Retina

The effects of early (5-day) onset of diabetes mellitus (DM) on retina ultrastructure and cellular bioenergetics were examined. The retinas of streptozotocin-induced diabetic rats were compared to those of non-diabetic rats using light and transmission electron microscopy. Tissue localization of glucagon-like-peptide-1 (GLP-1), exendin-4 (EXE-4), and catalase (CAT) in non-diabetic and diabetic rat retinas was conducted using immunohistochemistry, while the retinal and plasma concentration of GLP-1, EXE-4, and CAT were measured with ELISA. Lipid profiles and kidney and liver function markers were measured from the blood of non-diabetic and diabetic rats with an automated biochemical analyzer. Oxygen consumption was monitored using a phosphorescence analyzer, and the adenosine triphosphate (ATP) level was determined using the Enliten ATP assay kit. Blood glucose and cholesterol levels were significantly higher in diabetic rats compared to control. The number of degenerated photoreceptor cells was significantly higher in the diabetic rat retina. Tissue levels of EXE-4, GLP-1 and CAT were significantly (p = 0.002) higher in diabetic rat retina compared to non-diabetic controls. Retinal cellular respiration was 50% higher (p = 0.004) in diabetic (0.53 ± 0.16 µM O2 min−1 mg−1, n = 10) than in non-diabetic rats (0.35 ± 0.07 µM O2 min−1 mg−1, n = 11). Retinal cellular ATP was 76% higher (p = 0.077) in diabetic (205 ± 113 pmol mg−1, n = 10) than in non-diabetic rats (116 ± 99 pmol mg−1, n = 12). Thus, acute (5-day) or early onslaught of diabetes-induced hyperglycemia increased incretins and antioxidant levels and oxidative phosphorylation. All of these events could transiently preserve retinal function during the early phase of the progression of diabetes.

Rapid Hygiene Assay Sensitive to Cumulative Adenylate Homologues Exhibits Equal or Higher Frequencies of Soil Contamination Detection than Assay Limited to ATP Detection

Based upon regulatory and food industry-driven food safety standards, there is a need for rapid, accurate methods for assessing sanitary conditions. A commonly utilized assay is based on the assessment of the biochemical molecule, adenosine triphosphate (ATP). A more recent assay (AXP) targets the cumulative presence of ATP and its dephosphorylated homologues, adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Yet, there is little information that compares the practical performance of these two assays. This work examined these two assay types with a comparative study in a Grade A dairy foods processing plant and a licensed and inspected meat processing facility. A total of 1,920 concomitant analyses were conducted with main variables of assay type, processing facility type and hygiene zone category. Statistical process control methodology was used to calculate 95% confidence control limits (CL); data beyond those limits were considered contamination events. Results demonstrated that, overall, the AXP assay detected contamination events approximately two times more often than the ATP-only based assay. This increase in the rate of contamination event detection was especially more prevalent in the meat processing facility where, across all hygienic zones, there were 38 vs. 85 contaminations events detected for the ATP and AXP assays, respectively. Across hygiene zones, the AXP data displayed either an equal or increased incidence of soil detection compared to data from the ATP assay. This study provides applied evidence that assays solely dependent on ATP concentrations are less able to detect soil contaminants under conditions that favor ATP dephosphorylation reactions.

House Dust Mite Induces IL-33 Release From Human Nasal Epithelial Cells via P2Y Purinergic Signals and ERK/P38 MAPK Pathways.

BackgroundAllergic rhinitis (AR) is an inflammatory disease of the nasal mucosa, which is triggered by stimulations of environmental allergens such as house dust mite (HDM). Th2-type proinflammatory factor interleukin 33 (IL-33) plays an important role in the pathogenesis of AR, but it remains unknown how IL-33 products in human nasal epithelial cells (HNECs) mediated by HDM. MethodsWe investigated the effect of HDM allergens by analyzing the accumulation of Ca2+ levels and IL-33 release in HNECs. Involvements of Adenosine triphosphate (ATP)-dependent activation of P2Y-PLC-IP3 pathways, downstream of Ca2+ signaling and P38/ERK pathways were studied, using the P2Y-PLC-IP3 pathways agonists, the calcium chelators, and P38/ERK pathways inhibitors. ResultsDer p induced expression of IL-33 mRNA and protein in HNECs via ERK/P38 pathways. Average 69.4% of co-localization of quinacrine and Lyso-tracker fluorescent puncta revealed that ATP was mainly stored in the lysosomes of nasal epithelial cells. After stimulation with Der p, ATP released from lysosomes by P2Y-PLC-IP3-Ca2+pathways. The ATP assay of HNECs culture supernatants implied an acute accumulation of extracellular ATP immediately after the Der p stimulation. Using P2Y-PLC-IP3 signaling inhibitors, we found that the Der p-induced IL-33 release was dependent on ATP-P2Y-PLC-IP3 signaling, followed by abolishing the ERK/P38 pathways. ConclusionDer p induced an acute accumulation of extracellular ATP which activated PY2-PLC-IP3 pathways to induce Ca2+ releasing from endoplasmic reticulum (ER), and intracellular Ca2+ induced ATP release from lysosomes from HNECs. ATP activated PY2-PLC-IP3 pathways followed by transactivation of ERK/P38 pathways which induced the expression of IL-33 mRNA and protein.

Hypoxia-induced PTTG3P contributes to colorectal cancer glycolysis and M2 phenotype of macrophage

Long noncoding RNAs (lncRNAs) play critical factors in tumor progression and are ectopically expressed in malignant tumors. Until now, lncRNA PTTG3P biological function in colorectal cancer (CRC) needs further to be clarified. qRT-PCR was used to measure the PTTG3P level and CCK-8, glucose uptake, lactate assay, ATP assay, ECAR assay, and xenograft mice model were adopted to evaluate the glycolysis and proliferation, and macrophage polarization were determined in CRC cells. Xenograft experiments were utilized to analyze tumor growth. Ectopic expression of PTTG3P was involved in CRC and related to dismal prognosis. Through gain-of-function and loss-of-function approaches, PTTG3P enhanced cell proliferation and glycolysis through YAP1. Further, LDHA knockdown or glycolysis inhibitor (2-DG,3-BG) recovered PTTG3P-induced proliferation. And PTTG3P overexpression could facilitate M2 polarization of macrophages. Silenced PTTG3P decreased the level of inflammatory cytokines TNF-α, IL-1β, and IL-6, and low PTTG3P expression related with CD8+ T, NK, and TFH cell infiltration. Besides, HIF1A could increase PTTG3P expression by binding to the PTTG3P promoter region. Hypoxia-induced PTTG3P contributes to glycolysis and M2 phenotype of macrophage, which proposes a novel approach for clinical treatment.

Effectiveness of photon-initiated photoacoustic streaming in root canal models with different diameters or tapers

Background This study aimed to compare the use of photon-initiated photoacoustic streaming (PIPS) and conventional needle irrigation (CNI) in conjunction with different concentrations of sodium hypochlorite (NaOCl) to remove Enterococcus faecalis (E. faecalis) suspended bacteria and biofilms from root canal systems with different diameters or tapers. Methods Artificial root canal samples (n = 480) were randomly divided into three groups (n = 160/group). The canals were prepared to fit file sizes #10/.02, #25/.02, or #25/.06. The size #10/.02 group was incubated for seven days. The size #25/.02 or #25/.06 group was incubated for 2 days. A stable biological model of E. faecalis infection was established. The root canals were washed with distilled water or with 1%, 2%, or 5.25% NaOCl combined with CNI or PIPS. Bacterial suspensions and biofilms were assessed using an ATP assay kit and fluorescence microscopy. Image-Pro Plus was used to analyse the average fluorescence intensity to determine the most suitable root canal irrigation solution. Results In the CNI and PIPS groups, the ATP value of the 5.25% NaOCl subgroup was the lowest, followed by that of the 2% and 1% NaOCl subgroups. The ATP value of the distilled water subgroup was the highest (P < 0.05). When the root canal taper was 0.02, the ATP value of the #10/.02 + PIPS group was significantly lower than that of the #25/.02 + CNI group (P < 0.05). The average fluorescence intensity of the #10/.02 + PIPS group was lower than that of the #25/.02 + CNI group (P < 0.05). When the apical diameter was #25, the ATP value of the 0.02 taper in the PIPS group was lower than that of the 0.06 taper in the CNI group (P < 0.05), and the average fluorescence intensity of the 0.02 taper + PIPS group was lower than that of the 0.06 taper + CNI group (P < 0.05). PIPS combined with 2% and 5.25% NaOCl effectively improved the long-term antibacterial effect after irrigation and re-culture for 6 h. Conclusions Compared with CNI, PIPS has greater ability to remove bacteria in root canals with a small preparation diameter and a small taper. PIPS with 2% and 5.25% NaOCl exhibited superior antibacterial and bacteriostatic effects.