Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera (n = 91) and pre-COVID-19 control sera (n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.

Comparison of Static and Dynamic Assays When Quantifying Thermal Plasticity of Drosophilids

Numerous assays are used to quantify thermal tolerance of arthropods including dynamic ramping and static knockdown assays. The dynamic assay measures a critical temperature while the animal is gradually heated, whereas the static assay measures the time to knockdown at a constant temperature. Previous studies indicate that heat tolerance measured by both assays can be reconciled using the time × temperature interaction from “thermal tolerance landscapes” (TTLs) in unhardened animals. To investigate if this relationship remains true within hardened animals, we use a static assay to assess the effect of heat hardening treatments on heat tolerance in 10 Drosophila species. Using this TTL approach and data from the static heat knockdown experiments, we model the expected change in dynamic heat knockdown temperature (CTmax: temperature at which flies enter coma) and compare these predictions to empirical measurements of CTmax. We find that heat tolerance and hardening capacity are highly species specific and that the two assays report similar and consistent responses to heat hardening. Tested assays are therefore likely to measure the same underlying physiological trait and provide directly comparable estimates of heat tolerance. Regardless of this compliance, we discuss why and when static or dynamic assays may be more appropriate to investigate ectotherm heat tolerance.

The Cell Cycle G2/M Block Is an Indicator of Cellular Radiosensitivity

Background: Determination of the radiosensitivity of a specific tumor is essential to its precision tumor radiotherapy, but the measurement of cellular radiosensitivity with a routine colony forming assay is both labor- and time-consuming. An alternative option allowing rapid and precise prediction of radiosensitivity is necessary. Methods: In this study, we exposed 4 in vitro cultured cell lines to various doses of X-rays or carbon ions and then measured their radiosensitivities with a routine colony-forming assay, and monitored the kinetics of cell cycle distribution with routine propidium iodine staining and flow cytometry. Results: Based on the results, we correlated cellular radiosensitivity with a dynamic assay of cell cycle distribution, specifically, the negative correlation of cellular radiosensitivity with the accumulated G2/M arrested cells at 48 hours after exposure. The higher the proportion of accumulated G2/M arrested cells at 48 hours after exposure, the lower the radiosensitivity of the cell line, that is, the higher radioresistance of the cell line. Conclusion: These findings provide an optional application of regular cell cycle analysis for the prediction of tumor radiosensitivity.

THE ROLE OF INFLAMMATORY MARKERS IN THE EVALUATION OF MYOCARDIUM ALTERATIONS, DIAGNOSTICS AND PROGNOSIS IN PATIENTS WITH INFECTIVE ENDOCARDITIS

The results of the examination of 121 patients with infective endocarditis (IE), who received inpatient treatment at the Botkin State Clinical Hospital from 2008 to 2016, were presented in the article. The obtained data on primary and secondary IE with complicated and uncomplicated disease development was analyzed. Diagnostic significance of the dynamic assays of the highly sensitive C-reactive protein (hsСRP), tumor necrosis factor α (TNFα), and highly sensitive troponin I (hsTnI) in the evaluation of the infective toxic process activity, disease severity, and complications were identified. The obtained results indicate the high informative value of the dynamic assay of the levels of TNFα, hsСRP, and hsTnI in patients with IE. The authors established the correlation between the levels of the markers, and the expression of toxic and infectious syndrome manifestations, severity of the disease, and development of complications. A direct correlation was established between the increase in the pro-inflammatory cytokines levels and hsTnI level. It was shown that the increase in troponin concentration corresponded to the expressed morphofunctional alterations in patients with severe HF. Morphological study of the cardiomyocyte destruction revealed the formation of focal dystrophy, development of focal and diffuse fibrosis, indicated on the non-coronarogenic myocardium damage in patients with IE. The myocardium alterations were caused by many factors (hypoxia, microcirculation failure, permeability disorders, etc.), and the cytotoxic effect of pro-inflammatory cytokines, in particular, TNFα, played an essential role in their development. Myocardium alterations, along with the valve destruction, become an important part of the pathogenesis of hemodynamic disorders in patients with IE.

Application of a Multi-Marker Approach to Assess Vitamin B12 Status at the End of the First Trimester of Pregnancy

Background : During pregnancy, vitamin B12 (B12) utilisation increases, with deficiency being associated with adverse outcomes (Murphy 2007, VanderJagt 2011). It remains unclear however at what level vitamin B12 status becomes detrimental, and the correlation with fetal harm. Establishing B12 status during pregnancy is complicated by physiological changes such as haemodilution due to expanded blood volume, altered renal function, alterations in the vitamin B12 -binding proteins and transfer of materno-fetal B12 yet laboratories typically rely on the total abundance of B12 in serum (serum B12) asthe sole laboratory status indicator, interpreting results against reference range cut offs derived from non-pregnant populations, including men. Serum B12 assays measure the sum of holohaptocorrin and holotranscobalamin (holoTC). Other markers of B12 status are also available and are increasingly being adopted: holoTC (active B12) accounts 6-20% of bound B12, which is the only form of B12 presented for cellular uptake and used to satisfy metabolic demand; methylmalonic acid (MMA) is a by-product of methylmalonyl-CoA metabolism, the concentration of which in serum correlates inversely with tissue B12 utilisation. Methods: Over a 2 monthperiod in 2015 we used an extended panel of laboratory tests consisting of serum B12, holoTC, and MMA to determine B12 status, and assess appropriateness of our assay reference ranges, in 100 pregnant women with normal renal function at the end of the first trimester. Laboratory Analysis Serum B12 and holoTC were determined by an Abbott Architect analyzer. The serum B12 assay has a reference range of 187 - 883 ng/L. The holoTC assay has ²10% cross-reactivity from B12-binding proteins haptocorrin and apotranscobalamin. Values >128 pmol/L are above the dynamic assay range. In our laboratory holoTC results <25 pmol/L are defined as deficient and those >70 pmol/L as replete. Results 25-70 pmol/L are classified as indeterminate and a serum MMA assay subsequently performed (Sobczyńska-Malefora A et al 2014). We determined serum MMA using a Gerstel Multipurpose Sampler coupled to a liquid chromatography tandem mass spectrometer with electrospray ionization with results >270 nmol/L considered indicative of suboptimal B12 status (Harrington DJ 2016). Results: All three assays, figure 3, were performed for 92 women (insufficient sample available n=8).HoloTCand MMA results are shown in Table 1. The distribution ofholoTCand MMA concentrations are shown in Figure 1. One woman had a serum B12 below the reference range with a corresponding indeterminate holoTC and normal MMA. This set of results suggests questionable abundance of B12 that has not translated to functional deficiency. One woman had a holoTCconcentration <25pmol/L, however the corresponding serum B12 and MMA were not indicative of sub optimal B12 status. Fourteen women had aholoTC result above the dynamic assay range. Two women had an elevated MMA concentration with corresponding holoTCresults indicating questionable abundance of B12 but serum B12 within our reference limit. This triad of values indicates possible emerging suboptimal B12 status that would have been overlooked if serum B12 had been used in isolation. Discussion: B12 requirement increases in pregnancy and diet alone is unlikely to meet requirements (Murphy et al 2007). Deficiency has a detrimental impact on maternal and infant development and therefore it is useful to explore the relationship between different laboratory markers of B12 status in pregnancy (Ramirez-Velez et al 2016, Siddiqua et al 2014). The comparison of serum B12,holoTC and MMA levels at the end of the first trimester with reference ranges derived from non-pregnant adults suggests that they are broadly applicable. Serum B12 is the least sensitive marker and its use in isolation to assess B12 status is limiting. The application of holoTC and MMA in tandem can reveal previously missed deficient states. Using these markers two women in this small study were identified as possibly deficient although the true significance of a mildly elevated MMA in pregnancy requires further investigation. These data highlight the benefit of adopting a panel of markers ahead of a single test to assess B12 status. Identifying women at the end of the first trimester that may benefit from B12 replacement potentially protects from deficient states. Disclosures Robinson: Pharmacosmos: Honoraria; Amgen: Honoraria; Novartis: Honoraria.

EVALUATION OF DIFFERENT PRE-SETTING CONDITIONS IN AIRLIFT BIOREACTOR TO DETERMINE THE RESPIRATORY KINETICS OF FUNGI

in culture media and, therefore, used to obtain large volumes of fungal tissue to be used as inoculum. The implementation of controlled mycorrhization programs is dependent upon the production of commercially large volumes of inoculum. As of now, there is no such place around the globe where we can find people or companies that are able to achieve such goal. Information on the fungal growing kinetics is scant, and there are no studies that deal with the topic of oxygen transfer for the cultivation of these fungi (major impediment), making it hard to produce inoculum in large scales. Therefore, the current study used an airlift bioreactor to provide information on aspects, such as time of mixture, the effect of depressurization on the oxygen concentration readings and the delay of probe response, among others, that are fundamental for the commercial production of these fungal inocula. The study showed that the results obtained from the dynamic assay need adjustments prior to analytical interpretation. The data was obtained with operating specific air flow rates of 0.2, 0.36 and 0.52 vvm. In conclusion, the study provided essential information that can be used by others to continue the studies on the dynamic aspects of an airlift bioreactor operation intended for fungal biomass production.