Development of an anti-SARS-CoV-2 monoclonal antibody panel and its applicability as a reagent in high-throughput fluorescence reduction neutralization and immunohistochemistry assays.

Since its emergence in late 2019, coronavirus disease 2019 (COVID-19) has caused millions of deaths and socioeconomic losses. Although vaccination significantly reduced disease mortality, it has been shown that protection wanes over time, and that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) may escape vaccine-derived immunity. Therefore, serological studies are necessary to assess protection in the population and guide vaccine regimens. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. We developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we developed and characterized monoclonal antibodies (mAbs) to lower costs and reduce the assay's vulnerability to reagent shortages. Using samples of individuals vaccinated with COVID-19 and unvaccinated/pre-pandemic samples, we showed that FRNA results using commercial and in-house mAbs strongly correlated with those of the PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high-throughput alternative for measuring nAbs, the FRNA can be easily customized to assess SARS-CoV-2 VOCs. Additionally, the mAb we produced was able to detect SARS-CoV-2 in pulmonary tissues by immunohistochemistry assays.

Fatigue resistance, electrochemical corrosion and biological response of Ti-15Mo with surface modified by amorphous TiO2 nanotubes layer.

The objective of this work was a systemic evaluation of the anodizing treatment in a ß-type Ti-15Mo alloy to grow a TiO2 nanostructured layer for osseointegration improvement. The technical viability of the surface modification was assessed based on the resistance to mechanical fatigue, electrochemical corrosion, and biological response. By using an organic solution of NH4 F in ethylene glycol, a well-organized array of 90 nm diameter nanotubes was obtained with a potential of 40 V for 6 h, while undefined nanotubes of 25 nm diameter were formed with a potential of 20 V for 1 h. Nevertheless, the production of the 90 nm diameter nanotubes was followed by micrometer pits that significantly reduced the fatigue performance. The undefined nanotubes of 25 nm diameter, besides the greater cell viability and improved osteoblastic cell differentiation in comparison to the as-polished surface, were not deleterious to the fatigue and corrosion properties. This result strengthens the necessity of an overall evaluation of the anodizing treatment, particularly the fatigue resistance, before suggesting it for the design of implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 86-96, 2019.

Physicochemical, morphological, and biological analyses of Ti-15Mo alloy surface modified by laser beam irradiation.

Perform a physicochemical and morphological characterization of a Ti-15Mo alloy surface modified by laser beam irradiation and to evaluate in vitro the morphological response and proliferation of osteoblastic cells seeded onto this alloy. Disks were made of two different metals, Ti-15Mo alloy and cpTi, used as control. A total of four groups were evaluated: polished cpTi (cpTi-pol), laser-irradiated cpTi (cpTi-L), polished Ti-15Mo alloy (Ti-15Mo-pol), and laser-irradiated Ti-15Mo alloy (Ti-15Mo-L). Before and after laser irradiation of the surfaces, physicochemical and morphological analyses were performed: scanning electron microscopy (FEG-SEM), energy-dispersive spectroscopy (EDX), and X-ray diffraction (XRD). The wettability of the samples was evaluated by contact angle measurement. Murine preosteoblastic cells MC3T3-E1 were cultured onto the experimental disks for cell proliferation, morphology, and spreading analyses. Laser groups presented irregular-shaped cavities on its surface and a typical microstructured surface with large depressions (FEG-SEM). The contact angle for both laser groups was 0°, whereas for the polished groups was ≈ 77 and ≈ 78 for cpTi-pol and Ti-15Mo-pol, respectively. Cell proliferation analysis demonstrated a higher metabolic activity in the laser groups (p < 0.05). From the fluorescence microscopy, Ti-15Mo-L surface seems to induce greater cellular differentiation compared to the cpTi-L surface. The preliminary biological in vitro analyses suggested possible advantages of laser surface treatment in the Ti-15Mo alloy regarding cell proliferation and maturation.

Post-translational Modifications of Trypanosoma cruzi Canonical and Variant Histones.

Chagas disease, caused by Trypanosoma cruzi, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of T. cruzi canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modification types were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to T. cruzi or to trypanosomatids as a group and might represent good candidates for the development of antiparasitic drugs.

A prospective study of patients with refractory angina: outcomes and the role of high-sensitivity troponin T.

BACKGROUND: The predictors of cardiovascular events in patients with chronic refractory angina are limited. High-sensitivity cardiac troponin T (hs-cTnT) assays are biomarkers that may be used to determine the prognosis of patients with stable coronary artery disease. HYPOTHESIS: Hs-cTnT is a predictor of death and nonfatal myocardial infarction (MI) in patients with refractory angina. METHODS: We prospectively enrolled 117 consecutive patients in this study. A heart team ruled out myocardial revascularization feasibility after assessing recent coronary angiograms; evidence of myocardial ischemia served as an inclusion criterion. Optimal medical therapy was encouraged via outpatient visits every 6 months; plasma hs-cTnT levels were determined at baseline. The primary endpoint was the composite incidence of death and nonfatal MI. RESULTS: During a median follow-up period of 28.0 months (interquartile range, 18.0-47.5 months), an estimated 28.0-month cumulative event rate of 13.4% was determined via the Kaplan-Meier method. Univariate predictors of the composite endpoint were hs-cTnT levels and LV dysfunction. Following a multivariate analysis, only hs-cTnT was independently associated with the events in question, either as a continuous variable (hazard ratio per unit increase in the natural logarithm: 2.83, 95% confidence interval: 1.62-4.92, P < 0.001) or as a categorical variable (hazard ratio for concentrations above the 99th percentile: 5.14, 95% confidence interval: 2.05-12.91, P < 0.001). CONCLUSIONS: In patients with chronic refractory angina, plasma concentration of hs-cTnT is the strongest predictor of death and nonfatal MI. Notably, none of the outcomes in question occurred in patients with baseline plasma levels <5.0 ng/L.

Obstructive sleep apnoea is associated with myocardial injury in patients with refractory angina.

OBJECTIVE: To investigate the association between obstructive sleep apnoea (OSA) severity with markers of overnight myocardial injury in patients with refractory angina. METHODS: Patients with refractory angina were characterised clinically and they underwent ischaemia imaging stress tests by single-photon emission computed tomography (SPECT) and/or cardiac MRI. The patients were admitted to the hospital, remained under resting conditions for blood determination of high-sensitivity cardiac troponin T (hs-cTnT) at 14:00, 22:00 and after overnight polysomnography at 7:00. RESULTS: We studied 80 consecutive patients (age: 62±10 years; male: 66%; body mass index (BMI): 29.5±4 kg/m(2)) with well-established diagnosis of refractory angina. The mean apnoea-hypopnoea index (AHI) was 37±29 events/h and OSA (AHI >15 events/h) was present in 75% of the population. Morning detectable hs-cTnT and above 99th percentile was present in 88% and 36% of the population, respectively. Patients in the first to third quartiles of OSA severity did not have circadian variation of hs-cTnT. In contrast, patients in the fourth quartile (AHI ≥51 events/h) had a circadian variation of hs-cTnT with a morning peak of hs-cTnT that was two times higher than that in the remaining population (p=0.02). The highest quartile of OSA severity remained associated with the highest quartile of hs-cTnT (p=0.028) in multivariate analysis. CONCLUSION: Very severe OSA is common and independently associated with overnight myocardial injury in patients with refractory angina.

OSA and depression are common and independently associated with refractory angina in patients with coronary artery disease.

OBJECTIVE: Refractory angina is a severe form of coronary artery disease (CAD) characterized by persistent angina despite optimal medical therapy. OSA and depression are common in patients with stable CAD and may contribute to a poor prognosis. We hypothesized that OSA and depression are more common and more severe in patients with refractory angina than in patients with stable CAD. METHODS: We used standardized questionnaires and full polysomnography to compare consecutive patients with well-established refractory angina vs consecutive patients with stable CAD evaluated for coronary artery bypass graft surgery. RESULTS: Patients with refractory angina (n = 70) compared with patients with stable CAD (n = 70) were similar in sex distribution (male, 61.5% vs 75.5%; P = .07) and BMI (29.5 ± 4 kg/m2 vs 28.5 ± 4 kg/m2, P = .06), and were older (61 ± 10 y vs 57 ± 7 y, P = .013), respectively. Patients with refractory angina had significantly more symptoms of daytime sleepiness (Epworth Sleepiness Scale score, 12 ± 6 vs 8 ± 5; P < .001), had higher depression symptom scores (Beck Depression Inventory score, 19 ± 8 vs 10 ± 8; P < .001) despite greater use of antidepressants, had a higher apnea-hypopnea index (AHI) (AHI, 37 ± 30 events/h vs 23 ± 20 events/h; P = .001), higher proportion of oxygen saturation < 90% during sleep (8% ± 13 vs 4% ± 9, P = .04), and a higher proportion of severe OSA (AHI ≥ 30 events/h, 48% vs 27%; P = .009) than patients with stable CAD. OSA (P = .017), depression (P < .001), higher Epworth Sleepiness Scale score (P = .007), and lower sleep efficiency (P = .016) were independently associated with refractory angina in multivariate analysis. CONCLUSIONS: OSA and depression are independently associated with refractory angina and may contribute to poor cardiovascular outcome.

Biomedical Ti-Mo alloys with surface machined and modified by laser beam: biomechanical, histological, and histometric analysis in rabbits.

PURPOSE: In vivo bone response was assessed by removal torque, hystological and histometrical analysis on a recently developed biomedical Ti-15Mo alloy, after surface modification by laser beam irradiation, installed in the tibia of rabbits. MATERIALS AND METHODS: A total of 32 wide cylindrical Ti-15Mo dental implants were obtained (10mm × 3.75mm). The implants were divided into two groups: 1) control samples (Machined surface - MS) and 2) implants with their surface modified by Laser beam-irradiation (Test samples - LS). Six implants of each surface were used for removal torque test and 10 of each surface for histological and histometrical analysis. The implants were placed in the tibial metaphyses of rabbits. RESULTS: Average removal torque was 51.5Ncm to MS and >90Ncm to LS. Bone-to-implant-contact percentage was significantly higher for LS implants both in the cortical and marrow regions. CONCLUSIONS: The present study demonstrated that laser treated Ti-15Mo alloys are promising materials for biomedical application.

Impact of smoking on human bone apposition at different dental implant surfaces: a histologic study in type IV bone.

Smoking has adverse effects on peri-implant bone healing and can cause bone loss around successfully integrated implants placed on type IV bone. This study evaluated the influence of implant surface topography of microimplants retrieved from posterior maxilla of smokers after 2 months of unloaded healing. Seven partially edentulous patients received 2 microimplants (machined and sandblasted acid-etched surface) each during conventional implant surgery. Histometric evaluation showed that the mean bone to implant contact was 10.40 +/- 14.16% and 22.19 +/- 14.68% to machined and sandblasted acid-etched surfaces, respectively (P < .001). These data suggest that the sandblasted acid-etched surface presented better results than the machined surface after a short healing time in smokers.

Electrochemical studies on zirconium and its biocompatible alloys Ti-50Zr at.% and Zr-2.5Nb wt.% in simulated physiologic media.

Different electrochemical studies were carried out for Zr and its biocompatible alloys Ti-50Zr at.% and Zr-2.5Nb wt.% in solutions simulating physiologic media, Ringer and PBS (phosphate buffered saline) solutions. The results from rest-potential measurements showed that the three materials are spontaneously passivated in both solutions and that the Ti-50Zr alloy has the greatest tendency for spontaneous oxide formation. Some corrosion parameters (such as the pitting and repassivation potentials) were obtained via cyclic voltammetry in both solutions, revealing that the Ti-50Zr has the best corrosion protection while Zr has the worst. On the other hand, the pre-anodization (up to 8 V vs. SCE) of the alloys in a 0.15 mol/L Na2SO4 solution led to a significant improvement in their protection against pitting corrosion when exposed to the Ringer solution. Elemental analyses by EDX showed that during pitting corrosion, there is no preferential corrosion of any of the alloying elements (Zr, Ti, Nb).

Stability and Persistence of Two Formulations Containing Anticarsia gemmatalis Nuclear Polyhedrovirus (AgMNPV) / Estabilidade e Persistência de Formulações à Base de Nucleopoliedrovírus de Anticarsia gemmatalis (VPNMAg)

A estabilidade e persistência de duas formulações de VPNMAg desenvolvidas pelo Instituto Biológico e Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo foram estudadas. As formulações foram armazenadas em condições ambiente e expostas à radiação ultravioleta em condições de laboratório e à radiação solar em condições de campo. A formulação pó molhável (PM) foi preparada através da impregnação da suspensão de vírus e adjuvantes em inerte mineral (Caolin). A formulação óleo emulsionável (OE) foi preparada pela mistura da suspensão do patógeno com óleo + adjuvantes. Durante 20 meses, com intervalos de 120 dias, amostras das formulações e do vírus original eram adicionadas à dieta artificial de lagartas de quarto ínstar de Anticarsia gemmatalis (Hübner). Após 20 meses de armazenamento, a formulação OE perdeu apenas 18,3 por cento de sua atividade original, enquanto a formulação PM teve reduzida sua eficiência para 11,7 por cento após 12 meses. Nenhuma diferença significativa foi observada para as duas formulações quando expostas à luz ultravioleta. Ambas protegeram o vírus quando comparado ao tratamento controle. Em condições de campo a formulação OE teve melhor persistência com cerca de 60 por cento da atividade original presente após 14 dias da aplicação. Grande parte da formulação PM foi removida das folhas pela chuva.

The stability and the persistence of two AgMNPV formulations developed by the Instituto Biológico and the Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Brazil were studied. Formulations were stored in environmental conditions and exposed to ultraviolet radiation in laboratory and to solar radiation in the field. The wettable powder formulation (WP) was prepared by impregnating viral suspensions + adjuvants on inert mineral, chiefly kaolin. The emulsifiable oil formulation (EO) was prepared by mixing the pathogen suspension with corn oil + adjuvants. At 120-day intervals for 20 months, samples of formulations and the standard treatment (crude virus) kept in a laboratory cabinet were added to artificial diet fed to 4th-instar Anticarsia gemmatalis (Hübner) larvae. After 20 months in storage, the formulation EO lost only 18.3 percent of its original activity, whereas the formulation WP had its efficiency reduced to 11.7 percent after 12 months. No significant differences between the two formulations were observed when exposed to the ultraviolet germicidal light, and both formulations protected the virus when compared to the standard treatment. Under field conditions, the EO formulation enhanced the viral persistence, with >60 percent of the original activity present 14 days after the application. Most of the WP formulation was removed from leaves by the rain.

Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells.

Patients with solid tumors are increasingly being treated by autologous bone marrow transplantation (BMT). Although response rates appear to be increased, disease recurrence is the commonest cause of treatment failure. Whether relapse is entirely due to residual disease in the patient or arises also from infiltrating malignant cells contained in the autologous marrow transplant has not been resolved. If the latter explanation is correct, then purging would be required as part of the transplantation procedure. We used retrovirally mediated transfer of the neomycin-resistance gene to mark BM harvested from eight patients with neuroblastoma in clinical remission. The marked marrow cells were subsequently reinfused as part of an autologous BMT. At relapse, we sought the marker gene in malignant cell populations. Three patients have relapsed, and in each the marker gene was detected by phenotypic and genetic analyses of resurgent malignant cells at medullary and extramedullary sites. Analysis of neuroblast DNA for discrete marker gene integration sites suggested that at least 200 malignant cells, each capable of tumor formation, were introduced with the autologous marrow transplant and contributed to relapse. Thus, autologous BMTs administered to patients with this solid tumor may contain a multiplicity of malignant cells that subsequently contribute to relapse. The marker-gene technique we describe should permit evaluation of the mechanisms of relapse and the efficacy of purging in patients receiving autologous marrow transplantation for other solid tumors that infiltrate the marrow.

Pyrimidine-enhanced purine uptake in hepatocytes and improved survival from hypovolemic shock.

To assess the effect of pyrimidines on the incorporation of purines into rat hepatocytes, monolayer preparations of hepatocytes were incubated with radiolabeled precursors and their uptake determined. The uptake of purine precursors (inosine monophosphate, inosine, and hypoxanthine) was 27.8 +/- 1.31 nmoles per well, per 30 min. In the presence of added pyrimidines (uridine monophosphate, uridine, and orotate), this increased by 22%. Further enhancement (45%) was observed when L-dihydroxyphenylalanine (L-DOPA) plus pyridoxal-5'-phosphate was added to the purine/pyrimidine incubation medium. The effect of increased uptake of purines on survival after hypovolemic shock was also studied. Rats were bled to a blood pressure of 40 mmHg for 105 min and then treated with return of shed blood plus saline with or without purines and pyrimidines. Survival at 24 hr was 43% (15/35) in control animals, 66% (8/12) (P = 0.276) when treated with inosine monophosphate (IMP) and aspartate (60 mumoles/kg each), and 83% (10/12) (P = 0.037) for IMP with aspartate and orotate treatment (60 mumoles/kg each). Thus, the addition of orotate enhanced survival possibly by promoting salvage or by uptake of purines. Nucleotide concentrations in the livers of animals after shock which had received IMP and orotate demonstrated a return of both ATP and energy charge to normal, further indicating the value of this treatment.

Evaluation of renal functional changes after extracorporeal piezoelectric lithotripsy (EPL) by radionuclide studies.

Extracorporeal piezoelectric lithotripsy (EPL) has been recently introduced as a non-invasive method for treatment of renal and gall bladder stones. Thirty seven patients treated with EPL for renal calculi were the subjects of radionuclide renal studies. The aim was to evaluate the effects of EPL as a new technology by radionuclide renography. 99Tcm-DTPA and 99Tcm-DMSA studies were performed pre-EPL and one day and one week post-EPL. Various parameters of the sequential radionuclide studies were evaluated. One day post-EPL, changes in parenchymal transit time index, relative uptake function, glomerular filtration rate, time-activity curves and kidney dimensions were observed. At one week post-EPL, different parameters returned to baseline status in the majority of cases. The study indicates that EPL has a variety of effects on the kidney that could be demonstrated and followed by sequential radionuclide renal studies. Furthermore the study suggests spacing of EPL sessions by approximately one week.

Serum prostate-specific antigen: hourly change/24 hours compared with prostatic acid phosphatase.

Simultaneous hourly changes in serum PSA and PAP levels per twenty-four-hour period was studied in 19 urologic patients with no prostatic disease. Serum PSA was determined using Hybritech PSA-R Kits, and PAP was determined using EIA method. PSA and PAP serum levels varied independently from each other. PSA level showed no diurnal variations. The level remained stable with minor variations around a low mean of 0.907 ng/mL, SD 0.09, and CV 9.9% in 16 of 19 (84%) patients, while PAP showed changes consistent with diurnal variations in 5 of those patients. Random variations with no discernible pattern in 7 and remained more or less constant in 4 other patients. Marked random variations in PSA serum level occurred in only 3 patients in the older age group and were accompanied by diurnal variations in PAP level in 2 patients and a constant high level of PAP in 1 patient. The highest PSA level obtained was 6.9 ng/mL which is important when selecting the cut-off level. PSA did not increase appreciably after prostatic massage, and on follow-up PSA returned to premassage level after twenty-four hours (except in 1 patient).

Viability of Mycobacterium leprae: a comparison of morphological index and fluorescent staining techniques in slit-skin smears and M. leprae suspensions.

In a comparison of the estimation of Mycobacterium leprae viability by morphology and the fluorescent vital dyes FDA/EB and R123/EB, the latter techniques were more satisfactory using suspensions and slit-skin smears of M. leprae bacilli. Both FDA/EB and R123/EB seem to more accurately reflect viability after freeze/thaw cycles and heating, and are able to detect lower percentages of viable bacilli. In addition, the fluorescent vital dye techniques are both simple and less open to subjective interpretation than the conventional estimation of the morphological index.

Diurnal variation of prostatic acid phosphatase (PAP) in urologic patients with nonprostatic disease.

Variation in serum prostatic acid phosphatase (PAP) level over a twenty-four-hour period was studied in 18 urologic patients with no prostatic disease. Hourly, twenty-four-hour serum PAP level was determined using Elisa method. Seven of 18 patients (38.8%) showed appreciable change with definite diurnal pattern. PAP level was highest in early AM hours and lowest in the PM period. The difference between the mean AM and PM periods was statistically significant (P less than 0.01). Four of 18 patients showed no change over the twenty-four hours while 7 patients showed some variation without definite diurnal pattern. Such changes may lead to variation in serum PAP level and may lead to false positive results.