Rapid and sensitive diagnostic procedure for multiple detection of pandemic Coronaviridae family members SARS-CoV-2, SARS-CoV, MERS-CoV and HCoV: a translational research and cooperation between the Phan Chau Trinh University in Vietnam and University of Bari "Aldo Moro" in Italy.

OBJECTIVE: A new pandemic coronavirus causing coronavirus disease-2019 (COVID-19), initially called 2019-nCoV and successively named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The COVID-19 refers to the disease while the SARS-CoV-2 refers to the virus and is characterized by a rapid contagious capacity able to spread worldwide in a very short time. The rise in the number of infected patients and deaths is of great concern especially because symptoms are vague and similar to other forms of flu infection and corona syndrome infections characterized by fever, fatigue, dry cough, and dyspnea. According to the latest guidelines published by the World Health Organization (WHO), the diagnosis of COVID-19 must be confirmed by quantitative reverse transcription polymerase chain reaction (rRT-PCR) or gene sequencing of specimen obtained from throat, sputum and blood samples. However, the limitations due to logistics, as well as low sensitivity and specificity diagnostic tools currently available have been reported as the main cause of high incidence of either false-negative or positive results. PATIENTS AND METHODS: The purpose of the present translational research protocol is to discuss and present the original findings from our research team on new diagnostic technique to detect four Coronaviridae family members (SARS-CoV-2, SARS-CoV, HCoV and MERS-CoV), highlighting the methodology, the procedure and the possible advantages. Moreover, the authors review the current epidemiology, precautions and safety measures for health personnel to manage patients with known or suspected COVID-19 infection. RESULTS: Implementation of an effective and rapid plan of diagnosing, screening and checking is a key factor to reduce and prevent further transmission. This procedure based on rRT-PCR could be of great help to decisively validate the results obtained from more conventional diagnostic procedures such as chest computed tomography (CT) imaging and chest ultrasound. CONCLUSIONS: This translational diagnostic tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing people with suspected or confirmed SARS-CoV-2.

Nutrient composition and retention in whole turkeys with and without added solution.

Whole turkeys sold in retail outlets are typically processed with added solutions to improve their taste and tenderness. The purpose of this study was to evaluate the nutrient composition of whole turkeys with and without added solution, and to update the nutrient profile of turkey for the USDA National Nutrient Database for Standard Reference. Eleven pairs of turkeys with added solution were obtained from statistically representative retail outlets using a nationwide sampling plan developed for USDA's National Food and Nutrient Analysis Program; 4 pairs of turkeys without added solution were purchased from local food outlets. Turkeys were roasted to an internal temperature of 165°F (74°C). Values of selected nutrients in light and dark meat, including skin, were determined by USDA approved laboratories using quality assurance protocols. Both raw and cooked turkeys, with and without added solution, were compared by one-way and 2-way factorial ANOVA. The results showed a significant interaction for fat (P < 0.0001) and zinc (P = 0.0070) between turkeys that were raw and cooked and those prepared with or without added solution. Fat was higher in raw turkeys with added solution compared to without added solution. Similarly, sodium, phosphorus, and calcium values were significantly higher in turkeys with added solution (P < 0.05) than in turkeys without added solution. Data from this study will be useful for developing strategies to address sodium-related health issues, nutrition monitoring, consumption surveys, and policy development.

Enzyme-less electrochemical displacement heterogeneous immunosensor for diclofenac detection.

We describe an electrochemical immunosensor based on functionalization of a working electrode by electrografting two functional diazonium salts. The first one is a molecular probe, diclofenac, coupled with an arylamine onto which a specific antibody is immobilized by affinity interactions; the second is a redox probe (a quinone) also coupled with an arylamine, able to transduce the hapten-antibody association into a change in electroactivity. The steric hindrance induced by the antibody leads to a current decrease upon binding of the antibody on the grafted molecular probe; conversely, when diclofenac is present in solution, a displacement equilibrium occurs between the target diffusing into the solution and the grafted probe. This leads to dissociation of the antibody from the electrode surface, event which is transduced into a current increase ("signal-on" detection). The detection limit is ca. 20 fM, corresponding to 6pgL-1 diclofenac, which is competitive compared to other label-free immunosensors. We demonstrate that the sensor is selective and is able to quantify diclofenac in tap water.

A modified blade element theory for estimation of forces generated by a beetle-mimicking flapping wing system.

We present an unsteady blade element theory (BET) model to estimate the aerodynamic forces produced by a freely flying beetle and a beetle-mimicking flapping wing system. Added mass and rotational forces are included to accommodate the unsteady force. In addition to the aerodynamic forces needed to accurately estimate the time history of the forces, the inertial forces of the wings are also calculated. All of the force components are considered based on the full three-dimensional (3D) motion of the wing. The result obtained by the present BET model is validated with the data which were presented in a reference paper. The difference between the averages of the estimated forces (lift and drag) and the measured forces in the reference is about 5.7%. The BET model is also used to estimate the force produced by a freely flying beetle and a beetle-mimicking flapping wing system. The wing kinematics used in the BET calculation of a real beetle and the flapping wing system are captured using high-speed cameras. The results show that the average estimated vertical force of the beetle is reasonably close to the weight of the beetle, and the average estimated thrust of the beetle-mimicking flapping wing system is in good agreement with the measured value. Our results show that the unsteady lift and drag coefficients measured by Dickinson et al are still useful for relatively higher Reynolds number cases, and the proposed BET can be a good way to estimate the force produced by a flapping wing system.

Insights into the mechanism of magnetic particle assisted gene delivery.

In magnetic particle assisted gene delivery DNA is complexed with polymer-coated aggregated magnetic nanoparticles (AMNPs) to effect transfection. In vitro studies based on COS-7 cells were carried out using pEGFP-N1 and pMIR-REPORT-complexed, polyethylenimine (PEI)-coated iron oxide magnetic nanoparticles (MNPs). PEI-coated AMNPs (PEI-AMNPs) with average individual particle diameters of 8, 16 and 30 nm were synthesized. Normal, reverse and retention magnetic transfection experiments and cell wounding assays were performed. Our results show that the optimum magnetic field yields maximum transfection efficiency with good viability. The results of the normal, reverse and retention magnetic transfection experiments show that the highest transfection efficiency was achieved in normal magnetic transfection mode due to clustering of the PEI-AMNPs on the cells. Cell wounding assay results suggest that the mechanism of magnetic transfection is endocytosis rather than cell wounding.

[Orthostatic proteinuria and compression of the left renal vein (nutcracker syndrome)]. / Protéinurie orthostatique et phénomène de compression de la veine rénale gauche (nutcracker syndrome).

INTRODUCTION: The pathogenic mechanism of orthostatic proteinuria has not yet been clearly established. OBSERVATION: In a tall, thin, 21 year-old man, isolated proteinuria was discovered during an urological control conducted one year after a bilateral orchidopexy following left testicular torsion. Proteinuria was orthostatic. Doppler examination of the kidney revealed an entrapment of the left renal vein (nutcracker phenomenon-NCP). COMMENTS: An NCP was diagnosed in a young patient presenting with orthostatic proteinuria. By provoking modifications in intraglomerular haemodynamics, the NCP may, in nearly half of the cases, be at the origin of orthostatic proteinuria. Doppler examination is the diagnostic method of choice in the screening for NCP.

Diagnostic approach to hypercalcemia: relevance of parathyroid hormone and parathyroid hormone-related protein measurements.

Background: Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTH-rP) are two potent hypercalcemic hormones that act on the same targets. Autonomous secretion of the former is involved in primary hyperparathyroidism (PHPT), whereas the latter is responsible for humoral hypercalcemia of malignancy (HHM). Methods: From 250 consecutive, hypercalcemic serum samples sent to our laboratory for assessment of intact PTH, we were able to obtain clinical information, as well as an additional plasma sample for PTH-rP measurement, in 134 patients. At the time of sampling, patients could be classified into seven groups: cancer without known bone metastases (CaNoMeta, n=36), cancer with bone metastases (CaMeta, n=9), no evidence of cancer (noEvCa, n=71), sarcoidosis (Sarc, n=3), end-stage renal disease (ESRD, n=12), vitamin D overdose (VIT-D, n=2), and hyperthyroidism (Thyr, n=1). Results: In the CaNoMeta group, 29/36 patients had elevated PTH-rP levels, 9/36 patients had inappropriately elevated PTH levels, and 5/36 had elevated levels of both hormones. In the CaMeta group, three of the nine patients had inappropriately elevated PTH levels, two of them with concomitantly elevated PTH-rP levels. In the NoEvCa group, 63/71 patients had an inappropriate elevation of PTH levels and were diagnosed as having PHPT. Four of the 71 patients had elevated levels of both PTH and PTH-rP; three of them were in poor health and died within a short period of time. All of the ESRD patients had very high PTH and normal PTH-rP levels, except for one woman with high PTH-rP and undetectable PTH levels; she died from what later turned out to be a recurrent bladder carcinoma. In the Sarc, Vit-D, and Thyr groups, both PTH and PTH-rP levels were normal. Conclusions: (1) Elevated PTH-rP levels are a common finding in cancer patients without bone metastases. Intact PTH, however, should always be measured in hypercalcemic patients with malignancy because concurrent primary hyperparathyroidism is not rare. (2) Primary hyperparathyroidism accounts for hypercalcemia in 90% of patients without evidence of cancer whose PTH-rP levels may also be found to be elevated in a few cases, even some with surgically demonstrated parathyroid adenoma.

Sensitivity to meat protein intake and hyperoxaluria in idiopathic calcium stone formers.

BACKGROUND: High protein intake is an accepted risk factor for renal stone disease. Whether meat protein intake affects oxaluria, however, remains controversial in healthy subjects and in stone formers. This study was designed (1) to test the oxaluric response to a meat protein load in male recurrent idiopathic calcium stone formers (ICSFs) with and without mild metabolic hyperoxaluria (MMH and non-MMH, respectively), as well as in healthy controls, and (2) to seek for possible disturbed vitamin B(6) metabolism in MMH, in analogy with primary hyperoxaluria. METHODS: Twelve MMH, 8 non-MMH, and 13 healthy males were studied after five days on a high meat protein diet (HPD; 700 g meat/fish daily) following a run-in phase of five days on a moderate protein diet (MPD; 160 g meat/fish daily). In both diets, oxalate-rich nutrients were avoided, as well as sweeteners and vitamin C-containing medicines. Twenty-four-hour urinary excretion of oxalate was measured on the last day of each period, along with 4-pyridoxic acid (U(4PA)) and markers of protein intake, that is, urea, phosphate, uric acid, and sulfate. Serum pyridoxal 5' phosphate (S(P5P)) was measured after protein loading. RESULTS: Switching from MPD (0.97 +/- 0.18 g protein/kg/day) to HPD (2.26 +/- 0.38 g protein/kg/day) led to the expected rise in the urinary excretion rates of all markers of protein intake in all subjects. Concurrently, the mean urinary excretion of oxalate increased in ICSFs taken as a whole (+73 +/- 134 micromol/24 h, P = 0.024) as well as in the MMH subgroup (+100 +/- 144 micromol/24 h, P = 0.034) but not in controls (-17 +/- 63 micromol/24 h). In seven ICSFs (4 MMH and 3 non-MMH) but in none of the healthy controls (P = 0.016, chi square), an increment in oxaluria was observed and considered as significant based on the intra-assay coefficient of variation at our laboratory (8.5%). There was no difference in S(P5P)nd U(4PA)etween the groups after protein loading. CONCLUSION: Approximately one third of ICSFs with or without so-called MMH are sensitive to meat protein in terms of oxalate excretion, as opposed to healthy subjects. Mechanisms underlying this sensitivity to meat protein remain to be elucidated and do not seem to involve vitamin B(6) deficiency.

A systematic review of the relationship between overjet size and traumatic dental injuries.

The aim of this study was to aggregate the risk of traumatic dental injury due to overjet using several published papers and performing a meta-analysis on the results. The 11 articles involved in this investigation were identified by a literature search of Medline (1966-1996) and Exerpta Medica (1985-1996) databases using predetermined keywords, and inclusion and exclusion criteria. In order to assess the quality of each paper, a methodological checklist for observational studies was developed resulting in a score between 0 and 100. The relative risk of overjet, compared with a reference, was expressed as an Odds Ratio (OR). For each study, the OR was computed using the data presented and, subsequently, these ORs were pooled across studies. The effect of confounders (i.e. age, gender), which could bias the relationship between overjet and dental injury was taken into account. Furthermore, the influence of quality of the study on the pooled OR was addressed. The average methodological score was 41. From the results, it can be concluded that children with an overjet larger than 3 mm are approximately twice as much at risk of injury to anterior teeth than children with an overjet smaller than 3 mm. The effect of overjet on the risk of dental injury is less for boys than for girls in the same overjet group. In addition, risk of injury of anterior teeth tends to increase with increasing overjet size. Furthermore, the pooled OR does not seem to be affected by the quality of the studies.

Early human T cell activation events with engagement of surface MHC class II.

Major histocompatibility complex (MHC) class II are expressed on most activated human lymphocytes. They direct antigen presentation events in dendritic cells and B cells (collectively called antigen presenting cells), but the role for MHC class II in human T cells is not well understood. To understand the role of surface MHC class II and to identify the molecules involved in signaling, we have defined the early activation sequence in T cells when MHC class II are engaged by a specific antibody. Specifically, we have characterized the involvement of phosphotyrosine kinases, phospholipase C (PLC), and Ca2+ mobilization. With the engagement by either whole anti-class II antibody or its Fab fragments, the enzymatic activity of p56lck and ZAP-70 increased, but there was no increase in p59fyn activity. In addition, the intracellular free Ca2+ increased, which was due to enhanced influx and not to the mobilization of intracytoplasmic Ca2+. These events did not require cross-linking because they were not significantly augmented by the addition of antispecies antibody. The coimmunoprecipitation of tyrosine phosphorylated PLC-gamma1 with surface MHC class II suggested that PLC-gamma1 could be recruited to MHC class II after engagement. These results show the complexities of the early signals transduced by the engagement of surface MHC class II on T cells.

KrF laser-induced photobleaching effects in O(2) planar laser-induced fluorescence signals: experiment and model.

KrF excimer lasers are often employed as high-power excitation sources in planar laser-induced fluorescence (PLIF) imaging experiments to measure the distributions of O(2), OH, and H(2)O-all important species in combustion phenomena. However, due to the predissociative nature of these molecules, the high laser pumping rates typically required in such PLIF experiments may significantly deplete the ground-state population. The proper interpretation of the ground-state number density and/or the temperature from the fluorescence signals then requires the inclusion of photobleaching effects. We compare the results of a five-level rate-equation model incorporating photobleaching effects to the time-resolved PLIF signals from O(2) as obtained in the products of a fuel-lean CH(4) air flame. The results indicate that the fluorescence signals in a typical predissociated PLIF imaging experiment are subject to significant amounts of photobleaching. In an effort to provide a convenient way to account for photobleaching, a simple three-level model is developed. This model provides an analytic solution that describes satisfactorily the time-integrated fluorescence signal when compared with both the five-level model and the measurements. The results also indicate that at the low laser irradiances required to minimize the effects of photobleaching, the correspondingly low fluorescence signal levels make the acquisition of single-shot PLIF images a challenge to currently available camera systems.

Brefeldin A-induced alterations in processing of MHC class II-Ii complex depend upon microtubular function.

The role of microtubules in the brefeldin A (BFA)-associated relocation of major histocompatibility complex (MHC) class II alphabeta chains (alphabeta) and the invariant chain (Ii) was characterized in Raji cells by the use of nocodazole (ND). BFA blocked the transport of alphabeta Ii proteins through the Golgi and redistributed them to the endoplasmic reticulum (ER) along with Golgi-resident enzymes. The result of the colocalization of processing enzymes and newly synthesized proteins was a downshift of alphabeta Ii molecular weight (MW) of 2 kDa, and their resistance to endoglycosidase H (endo H) after 6 hr of chase. ND by itself had no effect on the processing and transport of alphabeta to the cell surface. The addition of ND to BFA-treated cells downshifted alphabeta Ii by 4 kDa. Additionally, alphabeta Ii proteins remained sensitive to neuraminidase after 16 hr of chase. In vitro alpha-mannosidase treatment of immunoprecipitated alphabeta Ii generated a similar 4-kDa downshift of MW. Either 1-deoxymannojirimycin (DJN) or swainsonine (SWN) blocked the MW downshift caused by BFA + ND treatment. These observations indicated that in Raji cells, most of the BFA-associated relocations of cis-, medial Golgi proteins, and the addition of sialic acid from the trans-Golgi were microtubule-independent. The retrograde transport of the medial Golgi enzyme N-acetylglucosamine transferase, however, required microtubular function. Microtubule disrupters could affect BFA treatment of viral infections by further disrupting viral protein processing.

Structural analysis of invariant chain subsets as a function of their association with MHC class II chains.

Respective subsets of human invariant chain (Ii), as identified with antibodies to two different epitopes, were characterized as a function of their associations with major histocompatibility complex (MHC) class II alpha,beta chains and intracellular processing. E1 antiserum to Ii(183-193) and VIC-Y1 monoclonal antibody to an N-terminal determinant identified Ii(E1) and Ii(VIC) populations, respectively. Ii proteins comprise several species which have been defined with either genomic or post-translational processes: Ii itself; IpN and IpO, which represent the glycosylated forms on asparagine or threonine/serine, respectively; gamma 2 and gamma 3, which originate from an alternative initiation site for transcription; and p41, which has a 64-amino-acid insert which originated from an additional exon placed after the sixth exon of Ii. Immunoprecipitates of detergent-solubilized protein complexes from [35S]methionine-labeled Raji cells showed that Ii(E1) consisted of Ii, p41, IpN, and immature alpha chain, while Ii(VIC) consisted of Ii, processed Ii with N- and O-linked glycosylation (IpN,IpO), p41, and associated MHC class II alpha,beta chains. In the MHC class II-deficient P3HR-1 cells, Ii(E1) and Ii(VIC) were virtually identical. Ii(E1) was resistant to cathepsin B digestion while Ii(VIC) was sensitive. In pulse-chase radiolabeling experiments with brefeldin A (BFA)-treated cells, Ii(VIC) progressively became resistant to endoglycosidase H (endo H) and had a longer half-life than that in cells not treated with BFA, but Ii(E1) remained susceptible to endo H and its half-life was unaffected by BFA. Since BFA redistributes Golgi enzymes to the endoplasmic reticulum, this observation suggests that Ii(E1) is protected from processing enzymes while Ii(VIC) is not. These studies define association of Ii with MHC class II molecules when certain epitopes on Ii are exposed or not. These differences relate to intracellular transport of Ii and to its release for the binding of antigenic peptides.

High-resolution oxygen absorption spectrum obtained with an external-cavity continuously tunable diode laser.

We have measured the electronic absorption spectrum of the oxygen (O(2)) A band b(1)Sigma(+)(g)(nu' =0)? X(3)Sigma(-)(g)(nu'' = 0) near 760 nm in room air by using a newly available external-cavity diode laser. The external-cavity diode laser permitted a continuous, single-mode scan of the P branch of ambient oxygen by 2f modulation spectroscopy. The external-cavity diode laser opens up many applications limited in the past by the noncontinuous tunability of standard monolithic Fabry-Perot cavity laser designs. Specifically, absorption transitions that coincide with he gain bandwidth of any available diode laser (635 to 1550 mn) may now be reached without the problems and imitations imposed by mode hops inherent in standard Fabry-Perot cavity designs.

Characterization of the invariant chain C-terminus (Glu183-Glu193) epitope which is obscured in processed Ii, MHC alpha,beta trimers.

The E1 serum was developed against invariant chain peptide Ii (183-193) in order to study the function of the Ii protein which associates with class II MHC alpha,beta chains from time of synthesis until cleavage and release, possibly regulating the binding of antigenic peptides. Subpopulations of Ii, Ii(VIC) and Ii(E1), respectively, were demonstrated by sequential immunodepletions and immunoprecipitations with: (1) VIC-Y1 monoclonal antibody to an N-terminal epitope of Ii, and (2) E1 rabbit antiserum to Ii(183-193). In 3 hr radiolabeled cells, VIC-Y1 recognized Ii, Ii and N- and O-linked glycosylation (IpN, IpO), p41 and co-precipitated class II alpha,beta chains, while E1 recognized Ii, IpN and immature Ii-alpha complex. In 15 min radiolabeled cells, each antibody recognized similar, immature Ii forms without alpha,beta. Urea denaturation of Ii(VIC) rendered the main Ii species but not IpO immunoprecipitable with E1. E1 recognized O-glycanase-treated Ii (VIC). We conclude that the Ii(183-193) epitope was obscured by interactions of Ii with class II alpha,beta chains and by the O-linked glycosylation of Thr187, which may in part regulate association of Ii to class II alpha and beta chains.

Effects of brefeldin A on cleavage of invariant chain to p21 and p10 and the appearance of Ii-freed class II MHC dimers.

Intracellular cleavage of class II MHC-associated Ii to p21 and p10 and the appearance of Ii-freed alpha, beta dimers were concurrent events lasting from 1 to 6 hr after synthesis of alpha, beta, Ii trimers, possibly related to charging of foreign peptides to the class II MHC antigen-binding site. Sequential immunoprecipitations of pulse-chase radiolabeled cells were made four times with anti-Ii monoclonal antibody to remove Ii and alpha, beta, Ii trimers and then with anti-class II antibody to detect the time-dependent appearance of Ii-freed alpha, beta dimers. The cleavage of Ii to p21 and p10 was revealed in leupeptin-treated cells. Cell treatment with Brefeldin A (BFA) was associated with a decrease in Ii-freed alpha, beta dimers, with inhibition of leupeptin-revealed cleavage of Ii to p21 and p10, and with persistence of endoglycosidase H susceptibility of Ii and class II alpha, beta chains. We conclude that in untreated cells, cleavage and release of Ii from class II MHC alpha and beta chains occur after those complexes traverse a BFA-sensitive step in the Golgi apparatus.