Low Dilutions Research Articles

Diluted versus potentitized probes of silver nitrate (10e-2 to 10e-10) and wheat germination

Corinne Kraus, Ute Knobloch, Scherer Waltraud, Peter Christian Endler
 Interuniversity College for Health and Development Graz / Castle of Seggau, Austria
 
 
 Background
 In 1926, an influence of a homeopathically prepared high dilution of silver nitrate on the growth of coleoptiles of wheat seedlings was described (Kolisko 1926). Later, in an extensive series of experiments, wheat was observed under the influence of extremely diluted agitated silver nitrate (10e-23, “24x”). Stalk lengths clearly indicate that development is enhanced by the probe silver nitrate 24x as compared to control (Scherer et al. 2015). A preliminary experiment was performed in early autumn 2015 on stalk growth of wheat seedlings treated with (not potentized) dilutions of silver nitrate 10e-6 to 10e-10 (“6e to 10e”), compared to potentized silver nitrate 6x to 10x (N = 100 per group). A clear, albeit not statistically significant trend was observed of 6x-stalks being longer (23.4 + 16.2 mm) than 6e-stalks (13.0 + 10.9 mm).
 
 Objective
 The aim of this study was to investigate the influence of diluted versus potentized low dilutions of silver nitrate (10e-2 to 10e-10) on wheat germination.
 
 Method
 The experiments were performed in late autumn 2015 on wheat grain (Triticum aestivum L., Capo variety). The grains were observed under the influence of aqueous solutions 10-2 to 10-10 part per weight of silver nitrate, either diluted in steps of 1 : 10 in distilled water by mere pipetting (probes “2e – 10e”), or diluted and agitated in steps of 1:10 (to create potentized probes “2x – 10x”). Untreated distilled water (“w”) served as an additional control. All probes were applied blindly. 100 grains were observed per treatment group in each of the groups resulting a total of 2,000 grains. Grains were placed in glass dishes, probes were added and dishes were covered with lids and placed in drawers . The following endpoint criteria were defined: K1 = visible emergence of sprout material, K2 = lifting of the operculum and emergence of the sprout and W1 = development of three roots. 
  
 
 Result
 Germination rates K1 of seedlings treated with “w”-probes (blue), with “e”-probes ranging from 2e to 10e (black) and with “x”-probes ranging from 2x to 10x (red) at the measuring points 20h, 24h and 28h (from left to right for each of the probes). 
 
 In K1, K2 and W, there is an obvious increase of germination rates from the high to the lower concentrations of silver nitrate, both in the “e” and in the “x”-groups and observable at 20h, 24h and 28h (p < 0.01). In contrast, germination rates of the two „w“-probes are practically alike (p > 0,05) 
 When “e” and “x”-data are compared, germination rates are higher under the influence of “x” than under the influence of “e” (p < 0.01 for the pooled “x”-values compared to the pooled “e”-values with regard to K1 as well as K2 as well as W). 
 
 Conclusion: A significant difference was found between wheat grains treated with mere dilutions compared to grains treated with potentised dilutions. 
 
 References
 1. Endler PC, Belavite P, Bonamin L,Jäger T, Mazon S. Replication of fundamental research models in ultra high dilutions 1994 and 2015 – update on a bibliometric study. Special issue Homeopathy London. 2015 a Oktober;104(4):234-45.
 2. Endler PC, Schulte J, Stock-Schroeer B, Stephen S. Ultra high Dilution 1994 revisited 2015 – the state of follow-up research. Special issue Homeopathy London. 2015 b Oktober;104(4):223-6.
 3. Kolisko L. Physiologischer Nachweis der Wirksamkeit kleinster Entitäten bei 7 Metallen – Wirkung von Licht und Pflanzen auf das Pflanzenwachstum. Dornach Schweiz: Philosophisch-Anthroposophischer Verlag am Goetheanum; 1926.
 4. Kraus C, Knobloch U. Diluted versus diluted and agitated probes of silver nitrate (10-2 to 10-10) and wheat germination, Thesis (MSc); branch campus UCN at Interuniversity College Graz / Schloss Seggau; 2016.
 5. Scherer-Pongratz W., Endler P.C., Lothaller H., Stephen S. Wheat and ultra high diluted silver nitrate – further experiments and re-analysis of data. Special issue Homeopathy London. 2015;104(4):246-9.

The production of hyperimmune pseudotuberculosis sera

Background . Pseudotuberculosis remains a serious public health problem, which dictates the need to develop simple and rapid diagnostic methods for this disease. The effectiveness of the latter largely depends on the activity and specificity of the diagnostic sera. Currently, in our country, a diagnostic serum for Yersinia pseudotuberculosis O-monovalent (serotype I, III) is produced with a regulated area of application: for an approximate agglutination reaction on glass (Pasteur NIIEM, St. Petersburg). Preparation of pseudotuberculosis sera for a wider scope of their use, in particular, as a source of specific antibodies in the design of diagnostic immunobiological preparations and test systems for pseudotuberculosis, is relevant and in demand in healthcare practice. Aims . To obtain hyperimmune pseudotuberculosis sera, promising for use in the practice of laboratory studies for pseudotuberculosis. Materials and methods. Chinchilla rabbits aged 3–6 months, weighing 2.5–3.0 kg served as animal producers of pseudotuberculosis sera. As immunogens, we used a corpuscular antigen (CAg) - a suspension of cells of the epidemically significant Yersinia pseudotuberculosis 3704 O: 1b strain isolated by an outbreak of pseudotuberculosis in the town of Zima, Irkutsk region, inactivated by boiling, and a preparation obtained from the outer membranes, containing the main surface immunogen of the strain. For the adsorption of experimental sera, in order to minimize the nonspecific response, we used heterologous microorganisms Escherichiacoli, Salmonella typhimurium, Shigella flexneri and Y. enterocolitica O:3, which have a similarity of surface antigenic structures with the causative agent of pseudotuberculosis. The presence of specific antibodies in experimental pseudotuberculosis sera was determined in a volumetric agglutination reaction. Results . Optimal schemes of rabbit immunization were selected, which made it possible to obtain hyperimmune sera against Y. pseudotuberculosis 3704 O:1b antigens with an agglutination activity of 1:3200-1:6400. In low dilutions of experimental sera (1:100–1:400), obtained against corpuscular antigen and outer membrane antigens (Escherichia coli, Salmonella typhimurium, Shigella flexneri , Y. enterocolitica О:3, S . enteritidis Gartnery) was observed in the agglutination reaction (AR). After the adsorption of experimental pseudotuberculosis sera by cells of heterologous strains, there was no cross-reaction with the indicated microorganisms in the agglutination reaction. Conclusions . The obtained hyperimmune adsorbed sera against the boiling-inactivated Y. pseudotuberculosi s 3704 O: 1b corpuscular antigen and outer membrane antigens can be used as a source of specific antibodies in the design of diagnostic immunobiological preparations for the detection of pseudotuberculosis microbe, as well as in the monitoring of the epidemic situation.

Aperture: alignment-free detection of structural variations and viral integrations in circulating tumor DNA.

The identification of structural variations (SVs) and viral integrations in circulating tumor DNA (ctDNA) is a key step in precision oncology that may assist clinicians in treatment selection and monitoring. However, due to the short fragment size of ctDNA, it is challenging to accurately detect low-frequency SVs or SVs involving complex junctions in ctDNA sequencing data. Here, we describe Aperture, a new fast SV caller that applies a unique strategy of $k$-mer-based searching, binary label-based breakpoint detection and candidate clustering to detect SVs and viral integrations with high sensitivity, especially when junctions span repetitive regions. Aperture also employs a barcode-based filter to ensure specificity. Compared with existing methods, Aperture exhibits superior sensitivity and specificity in simulated, reference and real data tests, especially at low dilutions. Additionally, Aperture is able to predict sites of viral integration and identify complex SVs involving novel insertions and repetitive sequences in real patient data. Aperture is freely available at https://github.com/liuhc8/Aperture.

The Relative Toxicity of Road Salt Alternatives to Freshwater Mussels; Examining the Potential Risk of Eco-Friendly De-icing Products to Sensitive Aquatic Species

The negative impact of road salt on freshwater ecosystems has led to an interest in “eco-friendly” de-icing products. Biota, including freshwater mussels that have heightened salt sensitivity, would be expected to benefit from a transition to alternative de-icing products. However, it was unknown whether the alternatives themselves pose a risk. The toxicity of three road salt alternatives including a salt brine, a beet juice, and a brine–beet juice product were examined. Lampsilis fasciola glochidia (larvae) were exposed to dilutions (0–2%) of de-icing products. On a per volume basis, beet juice products were significantly more toxic than brine with 48 h EC50s (95% confidence intervals) as follows: brine, 0.42% (0.35–0.50%); beet juice, 0.020% (0.018–0.022%); and brine–beet juice, 0.034% (0.028–0.039%). Unlike brine, beet juice toxicity did not correspond with the concentration of chloride in the exposure. While elevated trace metals (Cu, Fe, Zn) and reduced water quality occurred in the 1% and 2% beet juice exposures, toxicity occurred at much lower dilutions (≤0.05%). The toxicity of beet juice products aligned with glochidia potassium EC50s. Based on toxicity and application rates, beet juice de-icing products pose more of a hazard to early life stage mussels than traditional products and could contribute substantial potassium to receiving environments.

Nigella sativa oil inhibits proliferation and stimulates apoptosis of human lymphocytes in vitro

The study aimed to examine the in vitro influence of Nigella sativa oil on human lymphocytes. Cells were stimulated with a monoclonal anti-CD3 antibody in the presence of serial oil ethanol dilutions. Then their proliferation and apoptosis rates were assessed using flow cytometry. Our results demonstrate that the lowest dilutions (1:1 and 1:10) of Nigella sativa oil inhibited lymphocytes’ proliferation. The number of cell divisions was 8, 1.25, 1.88 after stimulation with anti-CD3, or its combination with 1:1 and 1:10 oil dilution. The percentage of proliferating cells was 92.48%, 8.75%, 24.3% after stimulation with anti-CD3 antibody, or its combination with 1:1 and 1:10 oil dilution. The mean percentage of living cells was 81% after stimulation with anti-CD3, 13.6%, 19.9% in the presence of 1:1 and 1:10 oil dilution. The preliminary studies show that black seed oil has a potent antiproliferative and proapoptotic effect on human lymphocytes in vitro.

Intravenous Fluid Administration and the Coagulation System.

Intravenous fluid administration in veterinary patients can alter coagulation function by several mechanisms. Both crystalloid and colloid fluids cause hemodilution, reducing platelet count and plasma coagulation protein concentrations. Hemodilution is associated with a hypercoagulable effect at low dilutions and a hypocoagulable effect at higher dilutions. Composition of crystalloid fluids likely has a minor effect, primarily dependent on fluid ion composition. Hypertonic crystalloids may also cause hypocoagulability. Colloids, both synthetic and natural, can cause hypocoagulability by several mechanisms beyond the effects of hemodilution. These include impaired platelet function, decreased plasma coagulation factor activity, impaired fibrin formation and crosslinking, and accelerated fibrinolysis. The vast majority of the veterinary literature investigates the hypocoagulable effects of hydroxyethyl starch–containing fluids using in vitro, experimental, and clinical studies. However, results are inconsistent, likely due to the varying doses and physicochemical properties of the specific fluid products across studies. In addition, some evidence exists for hypocoagulable effects of gelatin and albumin solutions. There is also evidence that these colloids increase the risk of clinical bleeding in people. Limitations of the veterinary evidence for the hypocoagulable effects of colloid fluids include a predominance of in vitro studies and in vivo studies using healthy subjects, which exclude the interaction of the effects of illness. Therefore, clinical relevance of these effects, especially for low-molecular-weight hydroxyethyl starch, is unknown. Firm recommendations about the most appropriate fluid to use in clinical scenarios cannot be made, although it is prudent to limit the dose of synthetic colloid in at-risk patients. Clinicians should closely monitor relevant coagulation assays and for evidence of hemorrhage in at-risk patients receiving any type of fluid therapy, especially in large volumes.

Evaluation of influenza A and B cold-adapted reassortant virus reproduction in trivalent live influenza vaccines

BackgroundThe objective of the present study was to compare reproduction of trivalent LAIV vaccine strains in MDCK cells and to perform quantitative RT-PCR analysis of trivalent LAIV replication after inoculation in mice. MethodsWe applied a reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). We confirmed the PCR data in ELISA using staining of MDCK monolayer with mouse monoclonal antibodies to hemagglutinin. ResultsThe viral load during the reproduction of mono-vaccines and trivalent LAIV in MDCK cells was similar at low dilutions. The content of vaccine viruses was evaluated using quantitative RT-PCR analysis in the nasal turbinate and lungs of CBA mice on day 3 after intranasal immunization. It was shown that despite the almost complete absence of reproduction of the A/H3N2 virus in mice, the immune response of A/H3N2-specific antibodies was formed at the same level as to other viruses. In MDCK cells, a decreased infectious titers of vaccine viruses in trivalent LAIV compared to mono-vaccines was demonstrated except for B/Yamagata virus. ConclusionRT-PCR analysis is applicable to assess the growth characteristics of cold-adapted reassortant influenza viruses in vitro and in mice. The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. In mice, decrease in infectious titers did not lead to a decline of the immunogenicity.

Assessment of trivalent live influenza vaccines in MDCK cell line

We applied a one-step reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs).The cold-adapted reassortant influenza viruses A/H1N1 pdm09, A/H3N2, B/Yamagata and B/Victoria, included in the composition of the LAIV in 2015-2016, 2017-2018 and 2018-2019 flu season were studied for reproductive activity in MDCK cells as part of a mono-vaccine and tri-vaccine. For this we have developed a set of specific primers and probes. Method validation was performed using ELISA-test after mouse monoclonal antibodies to hemagglutinin (HA) staining of MDCK monolayer.Influenza B viruses B/Yamagata and B/Victoria were studied in MDCK cells in mono-infection and coinfection with different multiplicity of infection (MOI) using quantitative rRT-PCR.•RT-PCR analysis was adjusted to assess the growth characteristics of cold-adapted reassortant influenza viruses in MDCK cell line. The greatest suppression in the composition of the tri-vaccine was exposed to the H1N1 pdm09 LAIV component.•Influenza B viruses are least suppressed in trivalent LAIV. Influenza viruses B/Yamagata and B/Victoria reproduced as part of a mixed preparation not lower, if not better than as a mono-preparation at an MOI of 0.1. At an MOI of 0.01, the reproduction of both B/Yamagata and B/Victoria in the mixture was reduced compared to mono-vaccine.•The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. This indicates that it is undesirable to reduce the titers of vaccine viruses, including at the stages of transportation and storage of LAIV

Protective properties in Hymenaea martiana Hayne against multi-drug-resistant Staphylococcus aureus

Antibiotic resistance represents a widespread problem in milk production. The identification of compounds for a topically applied ointment used in mastitis therapy remains elusive. Compounds from the genus Hymenaea can be administered in cases of multi-drug-resistant Staphylococcus aureus infection for ruminant species, but the protective properties are not well known. Wi this research the aim is verify the protective effects of H. martiana against S. aureus infection in bovine mammary epithelial cell line (MAC-T) and to obtain an antioxidant profile evaluation in vitro. The MAC-T cells were challenged with S. aureus after being exposed to the extract of the H. martiana in the protective assay. For the verification of the viability of the MAC-T cells, the MTT assay was performed, and was used dilutions of the plant extract, starting at 2.5%. The extract of H. martiana was evaluated for antioxidant aspect in different diluition by FRAP, ORAC and DPPH. A variety of flavonoids (quercetin, luteolin, etc.) have been identified as the main components by using mass spectrometry, reinforcing our in vitro findings that flavonoids, especially quercetin, have a medicinal profile capable of killing mastitis-causing bacteria. An excellent antioxidant pattern was observed in the 2.5% solution; however, membrane integrity in MAC-T cells was compromised. Those findings suggest low dilutions of H. martiana extract has a desirable protective effect from S. aureus pathogenesis. Our in vitro studies can be gleaned upon for further in vivo studies.

Optimization of mixing in a Chinese dome digester for tropical regions

Optimization of mixing in a Chinese dome digester for tropical regions

Abstract 6116: Role of secretory factors from obese-derived omentum in supporting ovarian cancer progression

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty peritoneal tissue that encloses the abdomen. This finding coincides with some evidence that ovarian cancer is more aggressive in the obese setting with worse overall survival. While it has been shown that ovarian cancer cells invade towards adipocytes in vitro, the mechanism by which they support formation of secondary tumors is not well understood. We hypothesize that soluble factors enriched in the obese setting are activating signaling pathways in tumor cells to drive migration and proliferation. To evaluate this hypothesis we co-cultured primary human omental adipocytes derived from obese female donors with ovarian cancer cell lines and profiled secretory factors (cytokines and adipokines) responsible for increasing tumor initiation in vivo and clonogenicity, proliferation, and drug resistance in vitro. Secretion of cytokines IL-6, IL-8, and CCL2 by adipocytes is enhanced when co-cultured with ovarian cancer cells. A subcutaneous tumor model showed that low dilutions of ovarian cancer cells require adipocytes for engraftment and tumor formation. A transplantation assay showed that ovarian cancer cells require the presence of adipocytes for sustained tumor-initiation capacity. Studies are underway to investigate adipocyte-secreted factors in this process. Our findings highlight the role of adipocytes in the ovarian tumor microenvironment and uncover signaling factors that may enhance ovarian cancer progression. Understanding these processes will enable the design of more effective therapies for treating ovarian cancer. Citation Format: Jennifer Waters, Mikella Robinson, Samuel F. Gilbert, Logan J. Alexander, Carrie D. House. Role of secretory factors from obese-derived omentum in supporting ovarian cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6116.

Abstract 4941: Identification and isolation of ovarian cancer stem cell populations using a novel functional reporter may provide new insights into ovarian cancer cell dynamics and drug resistance

Abstract Background: Ovarian cancer is the most lethal gynecologic malignancy mainly due to a high prevalence of chemotherapy-resistant recurrent disease. This mechanism is unknown but recent studies have pointed to a small population of chemoresistant cancer stem cells (CSC) as the driver of these recurrences. Traditional surface markers of cancer stem cells (CD44, ALDH, CD117, etc.) have shown mixed success in identifying this subpopulation, with different cell lines and patient samples exhibiting dissimilar profiles. These markers are often related to the cell of origin and are not necessarily specific to CSCs. As such, a reporter that responds to functional features of stem cells would be beneficial in identifying the CSC subpopulation regardless of cell line and patient sample. Here we demonstrate that using a reporter to detect SOX2 and OCT4 overexpression (called SORE6) can identify the CSC subpopulation within ovarian cancer cell lines. Methods: The SORE6 reporter was stably transduced into OV90 and ACI23 ovarian cancer cell lines. The subpopulation of cells having high SOX2/OCT4 expression (SORE6+) were isolated using fluorescence assisted cell sorting (FACs) and evaluated for CSC characteristics relative to SORE6- cells. Gene expression, viability, spheroid formation capacity, and tumorigenicity was assessed for both SORE6+ and SORE6- cells. In vivo limiting dilution analyses were performed on athymic nude female mice using subcutaneous injection. Results: Compared to the bulk population which has low levels of SOX2/OCT4 expression (SORE6-), SORE6+ cells showed a 1.5-fold increase in SOX2/OCT4/NANOG transcript levels in ACI23 cells and 2 to 15-fold increase in these transcript levels in the OV90 cells. SORE6+ cells showed enhanced chemoresistance when treated with conventional chemotherapies for ovarian cancer, carboplatin and paclitaxel, although this difference was more dramatic in the OV90 cells (four-fold) relative to ACI23 (two-fold). These findings indicate that SOX2/OCT4 expression could contribute to drug resistance and clinical recurrence. SORE6+ cells also showed an increased capacity to form spheroids in low-serum conditions and again this difference was more dramatic in the OV90 cells. In vivo limiting dilution studies are underway but appear to show significant differences in tumorigenicity at low dilutions of ≤5,000 cells/mouse. Conclusion: The SORE6 reporter construct identifies CSC populations within ovarian cancer cell lines and may be a reliable tool for isolating CSCs that depend on SOX2 and OCT4 expression. Use of this reporter will enhance our understanding of mechanisms that support chemoresistance and enable the study of CSC dynamics within the tumor microenvironment, both of which have significant implications for ovarian cancer progression and treatment. Citation Format: Samuel F. Gilbert, Mikella Robinson, Logan A. Alexander, Omar Lujano-Olazaba, Jacqueline M. Lara, Jennifer A. Waters, Omid Patrus, Alex Horkowitz, Carrie D. House. Identification and isolation of ovarian cancer stem cell populations using a novel functional reporter may provide new insights into ovarian cancer cell dynamics and drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4941.

The Antibacterial Effect of QMix, a Novel Root Canal Irrigant (Ex vivo Study)

Aims: To evaluate the antibacterial effect of a novel root canal irrigant, QMix, by evaluating its effect against E. faecalis and comparing it to 17% EDTA and 2% Chlorhexidine digluconate. Materials and Methods: Minimum Inhibitory Concentration (MIC), Minimum bactericidal concentration (MBC) and direct exposure test were the techniques used. In the MIC and MBC technique, the irrigants (QMix, 17% EDTA, 2% CHX) were serially diluted in BHI broth and 0.2 mL of the tested bacterial suspensions was added. Results were obtained on the basis of turbidity and growth on agar plates. In the direct exposure test, Enterococcus faecalis were exposed to QMix, 2% Chlorhexidinedigluconate and 17% EDTA for 5 seconds, 30 seconds and 3 minutes. Following exposure, samples were taken and serially diluted and incubated anaerobically on E. faecalis selective media for 24 hours to count the resistance of the bacteria. Results: In the MIC and MBC technique, CHX showed to be more effective against E. faecalis than both QMix and EDTA, as lower dilutions were required to inhibit growth of both bacteria. Ethylene diamine tetra acid (EDTA) was the least effective. In the direct exposure test, QMix was more effective than CHX and EDTA as it was the only solution to be able to kill all bacteria. Few E. faecalis cells remained even after exposure of bacteria to 3 minutes of EDTA and CHX. There was significant difference between QMix and both EDTA and CHX in killing of E. feacalis at 5 seconds exposure (p<0.05). QMix killed more than 95% of bacteria, whereas CHX and EDTA killed fewer than 20% (p<0.05). There was no statistical significance between the irrigant solutions at 3 minutes of exposure. Conclusions: In the MIC and MBC techniques, Chlorhexidinedigluconate was found superior to both QMix and EDTA. In the Direct Exposure Test, QMix showed the best performance as it was the only irrigant solution to kill all E. faecalis cells, as well as killing more than 95% of all bacteria at 5 seconds exposure.

OR18-03 Functional TSH Receptor Antibodies Are a Biomarker for Graves’ Disease - a Prospective Trial

ObjectiveWe aimed to evaluate the clinical utility and predictive value of stimulatory (TSAb) and blocking (TBAb) TSH receptor antibodies in the management of Graves’ disease (GD).MethodsHundred well-defined, consecutive, unselected, untreated hyperthyroid patients with GD were enrolled in a prospective two-year trial. Methimazole (MMI) was administered for 24 weeks according to baseline serum concentrations of free T3/free T4. Starting dose was 5–30 mg/day. Through a titration regimen, this dose was respectively tapered or increased at each subsequent study visit as the patient became euthyroid or remained hyperthyroid. Goals of therapy were to maintain normal fT4 and TSH levels. MMI therapy was stopped at week 24. The main outcome measure was clinical response versus non-response to a 24-week MMI treatment defined as biochemical euthyroidism versus persistent hyperthyroidism at week 24 and/or relapse at weeks 36, 48, and 96. TSAb was reported as percentage of specimen-to-reference ratio (cut-off SRR% <140). Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (cut-off >40% inhibition).ResultsForty-four patients responded to MMI of whom 43% had Graves’ orbitopathy (GO) while 56 were non-responders (66% with GO, p<0.01). At baseline, undiluted serum TSAb but not thyroid binding inhibiting immunoglobulins (TBII) differentiated between thyroidal GD only versus GD+GO (p<0.001). Further, at baseline responders demonstrated marked differences in diluted TSAb titers compared with non-responders (p<0.001). All patients with a TSAb dilution titer above three did not respond to MMI treatment. In contrast, TBII dilution titers did not differentiate between responders and non-responders to MMI and serum samples became TBII negative already at low dilutions. During treatment, serum TSAb levels decreased markedly in responders (p<0.001) but increased in non-responders (p<0.01). In contrast, TBII strongly decreased in non-responders (p=0.002). All non-responders at week 24 and/or those who relapsed during the 72-week follow-up were TSAb positive at week 24. A shift from TSAb to TBAb was noted in eight patients during treatment and/or follow-up and led to remission.ConclusionsSerum TSAb levels are a biomarker for and mirror severity of GD. Their increase during MMI treatment is a marker for on-going disease activity. TSAb dilution analysis had additional predictive value.

Late development of pustular, erosive lesions in the muzzle of calves inoculated with Pseudocowpox virus

We studied the pathogenesis of Pseudocowpox virus (PCPV), a zoonotic parapoxvirus associated with mucocutaneous lesions in cattle. Inoculation of calves with PCPV isolate SD 76–65 intranasally (n = 6) or transdermally in the muzzle (n = 2) resulted in virus replication and shedding up to day 13 post-infection (pi). No local or systemic signs were observed in inoculated calves up to day 20pi, when the clinical monitoring was discontinued. However, from days 28–34 pi, seven (7/8) inoculated calves underwent an asynchronous clinical course characterized by development of a few (one or two) to countless papulo-pustular, erosive-fibrinous and scabby lesions in the muzzle, in some cases extending to the lips and gingiva. In some animals, the lesions coalesced, forming extensive fibrinotic/necrotic and scabby plaques covering almost entirely the muzzle. The clinical course lasted 8–15 days and spontaneously subsided after day 42pi. Infectious virus and/or viral DNA were detected in swabs collected from lesions of 5/8 animals between days 34 and 42pi. Histological examination of fragments collected from the muzzle lesions of two affected calves (day 36pi) revealed marked epidermal hyperplasia and severe orthokeratotic and parakeratotic hyperkeratosis, covered by thick scabs. The epidermis showed multifocal areas of keratinocyte coalescing necrosis and mild multifocal vacuolar degeneration. Sera of inoculated calves at 50pi showed partial virus neutralization at low dilutions, demonstrating seroconversion. The delayed and severe clinical course associated with virus persistence in lesions are novel findings and contribute for the understanding of PCPV pathogenesis.

Top-down evaluation of the matrix effects in microbial enumeration test uncertainty

Microbial contamination may compromise the efficacy and safety of pharmaceutical products. Microbial enumeration tests are required by most of the pharmacopeial compendia and consist in conventional pour-plate inoculation of a sample aliquot followed by incubation under appropriate conditions. Despite of this, the measurement uncertainty evaluation for microbial enumeration tests is rarely considered. Thus, the aim of this paper was to assess the matrix effects in microbial enumeration tests and their top-down uncertainty evaluation. Microbial counting methods for eighteen pharmaceutical products were validated concerning the trueness (mean recovery) and precision (repeatability and intermediate precision) using seven test microorganisms. Uncertainty factors values were found to be between 1.1 and 3.3, based on trueness and precision results. Trueness uncertainty component was the most relevant in 59% of the cases. This issue can be explained due to the matrix interference cause by preservatives or antimicrobial agents, particularly for lower dilutions when compared to higher dilutions. Thus, we concluded that the higher the matrix interference the higher the uncertainty factor, and consequently, the higher the asymmetry for the interval around the measurement.

Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.

Establishment and Qualification of A Competitive Enzyme-Linked Immunosorbent Assay for the Measurement of Heparin in Plasma Products

Abstract Heparin is used in plasma fractionation processes to prevent activation of coagulation factors. Consequently, intermediates and final products have to be measured for their heparin content. Established chromogenic heparin assays rely on measurement of the accelerated inhibitory action of antithrombin against thrombin or activated factor X. These assays, however, show limitations, especially at the low sample dilutions required to provide adequate sensitivity. These limitations, potentially biasing results, originate from the specific sample matrix including proteases or protease inhibitors or from the test sample’s color. To overcome these shortcomings, a commercially available competitive enzyme-linked immunosorbent assay (ELISA) was established, described so far to measure heparin in plasma only. Briefly, heparin contained in the sample competed with the binding of a peroxidase-labelled heparin binding protein to the wells of a heparin-coated plate. The six-point calibration curve ranging from 0.03 to 7.14 IU heparin/mL showed acceptable accuracy and reproducibility. Test samples were diluted at least 1/6 in normal human citrated plasma. Spike-recovery was carried out by adding heparin to obtain a concentration of 0.26 IU/mL in the test dilution of the respective sample. Acceptable recovery was found in all sample types investigated (final immunoglobulin G product and process intermediates, albumin and α1-proteinase inhibitor), while intra-and inter-run precision data showed relative standard deviations lower than 10%. The ELISA’s limit of quantification was 0.18 IU/mL, thus demonstrating required sensitivity. In summary, the competitive heparin ELISA allowed accurate, precise and sensitive heparin measurement in specific plasma protein matrices, which interfere with chromogenic heparin assays.

Aqueous Film-Forming Foams (AFFFs) Are Very Toxic to Aquatic Microcrustaceans

The use of aqueous film-forming foams (AFFFs) have been related to environmental contamination due to handling, storage, and use in the firefighting against class B fire. Studies have associated the use of AFFFs with toxic effects from its perfluorinated compounds (PFCs) to the aquatic ecosystem, which led the Stockholm Convention to restrict their use. In Brazil, despite the large-scale use, there is no data on employment or annual commercialization of these products. This study evaluated the toxicity of seven brands of AFFFs used in the firefighting of the petrochemical’s terminal of fuel storage in Port of Santos (Santos, Sao Paulo, Brazil) which occurred in 2015, in which more than 61,000 L of AFFFs drained into the adjacent aquatic ecosystems. The toxicity evaluation was performed by means of the acute bioassay using the freshwater microcrustacean Daphnia similis. The AFFF brands tested were considered toxic to D. similis, including at much lower dilutions than those recommended by the manufacturers. The brand that showed the lowest toxicity was Kidde Sintex® 3% × 6%, followed by Kidde Sintex® 1% × 3%, Argus Prime®, Cold Fire®, Ageofoam®, and Liovac®, and the one with the highest toxicity was F-500 fire®. These results provide valuable information for the development of public policies aimed at managing the AFFF discharge in freshwater ecosystems.

Bifurcation of pulsation instability in one-dimensional H2−O2 detonation with detailed reaction mechanism

Classical modes of one-dimensional (1D) detonation characterized by a simplified reaction model are reproduced by using a real chemical kinetics for the H2−O2 system with argon dilution. As Ar dilution is varied, the bifurcation points of pulsating instability are identified and a formed bifurcation diagram is compared with that obtained by the one-step reaction model. Eventually, the numerical results demonstrate that, for real detonations with detailed chemistry, the criterion of Ng et al. works well on prediction of the 1D detonation instability. Furthermore, the detonability limits are found respectively at low and high Ar dilutions. Above the high Ar dilution limit, detonations decays to the minimum level where long autoignition time and small heat release rate make reestablishment impossible for both 1D and 2D simulations. However, below the low Ar dilution limit, a 1D detonation cannot be sustained due to high instability, while the corresponding cellular detonation can propagate sustainably due to the role of transverse instability.