Aleutian mink disease parvovirus (AMDV) causes Aleutian mink disease (AMD), which is a serious infectious disease of mink. The aim of this study was to get a better understanding of the molecular epidemiology of AMDV in northeast China to control and prevent AMD from further spreading. This study for the first time isolated AMDV from fecal swab samples of mink in China.A total of 157/291 (54.0%) of the fecal swab samples were positive for AMDV. Of these, 23 AMDV positive samples were randomly selected for sequence alignment and phylogenetic analysis based on the acquired partial fragments of VP2 gene with the hypervariable region. Comparative DNA sequence analysis of 23 AMDV isolates with a reference nonpathogenic (AMDV-G) strain revealed 8.3% difference in partial VP2 nucleotide sequences. Amino acid alignment indicated the presence of several genetic variants, as well as one single amino acid residue deletion. The most concentrated area of variation was located in the hypervariable region of VP2 protein. According to phylogenetic analysis, the Chinese AMDV strains and the other reference AMDV strains from different countries clustered into three groups . Most of the newly sequenced strains were found to form a Chinese-specific group, which solely consisted of Chinese AMDV strains.These findings indicated that a high genetic diversity was found in Chinese AMDV strains and the virus distribution were not dependent on geographical origin. Both local and imported AMDV positive species were prevalent in the Chinese mink farming population. The genetic evidence of AMDV variety and epidemic isolates have importance in mink farming practice. Neovison vison) worldwide. It causes a considerable economic loss to the mink farming industry by reducing reproductive output and decreasing the fur value. Clinical signs of progressive AMD infection in adult mink include the excessive level of plasma and gamma globulins in blood, inflammation of the glomeruli in kidney, decreased fertility, spontaneous abortion, and severe chronic immune dysfunction [Aleutian mink disease (AMD) caused by Aleutian mink disease parvovirus (AMDV) is the most commercially important infectious disease affecting farmed mink  and 70.1% (68/97) of the mink samples were AMDV-positive in Jilin province and Dalian city of Liaoning province, respectively  predicted from the detected fragments of VP2 sequences from 23 newly sequenced AMDV strains and AMDV-G strain. Several variants among the 192 amino acid residues were observed in Fig. 92 aa preTo identify the overall possible evolutionary relationships of AMDV in northeast China, a phylogenetic tree based on 37 detected fragments of VP2 sequences was constructed, including 23 newly sequenced Chinese strains and 14 reference strains from different countries. According to the tree topology, AMDV isolates formed three major groups , maximum likelihood and maximum parsimony methods were also performed to verify the reliability of the neighbor-joining phylogenetic tree. The trees constructed by maximum likelihood and maximum parsimony methods were the same or quite similar as showed in neighbor-joining phylogeny, and the bootstrap trees were relatively consistent, which enhance the reliability of neighbor-joining analysis .All the Dalian samples were collected from one farm of Liaoning province. The phylogenetic analysis found that AMDV-Dalian isolates appeared in all three groups, which showed that different AMDV genotypes could coexist in the same farm. Besides, AMDV-Jilin isolates clustered in clades A and B, with the same as many of the AMDV-Dalian isolates , revealing similar AMDV genotypic distribution in different farms from Jilin and Liaoning provinces, which further indicated that the distribution of AMDV strains was not related to the geographical origin. The AMDV distribution in northeast China probably results from the frequent mink trade among different farms and the introduction of both domestic and foreign minks, which contributes to improving local mink availability, but simultaneously leads to the enhancive virus spreading.The phylogenetic analysis also revealed a regional-specific clade B, which only contained the isolates from China, and the clusters of the Chinese AMDV strains may reflect a long history in the breeding of domestic minks. Furthermore, the Chinese AMDV isolates were distributed in all three groups, indicating that the newly isolated Chinese AMDV strains consisted of both prevalent indigenous strains and isolates imported from abroad, which further implied that both local and imported AMDV species were prevalent in the farmed Chinese minks. Similar findings were found by previous reports , 10.In summary, AMD causes severe economic loss to the fur farming industry. Molecular epidemiological studies are necessary to assess patterns of disease transmission to improve disease detection methods. Our study is the first to report AMDV infection rates using fecal swap samples in China. The evidence of AMDV variety and epidemic isolates have importance in mink farming practice. The present report lays a good foundation for further study of AMDV in China.During August–November 2014, a total of 291 fecal swabs samples from minks were obtained from three different farms in Jilin and Liaoning provinces in northeast China. The 94 and 100 samples were collected from farms I and II of Jilin province, respectively. A total of 97 samples were obtained from farm III of the Dalian city of Liaoning province. During sample collection, precautions were taken to avoid cross-contamination of the samples. All samples were stored at − 80 °C for further analysis.Genomic DNA of AMDV was extracted by an EasyPure Genomic DNA kit  according to the manufacturer’s instructions. A pair of PCR primers was designed based on the previous reports , 15, 16 PCR amplification was performed in a 25-μL reaction mixture containing 5 μL of template DNA, 1 × PCR buffer, 0.25 mM of dNTP, 0.5 μM of each primer, and 1 unit of Taq polymerase . The PCR conditions were as follows: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, and a final extension step at 72 °C for 7 min. Positive (DNA extracted from previously preserved AMDV strain in our laboratory) and blank controls (water) were used in each set of amplification.The amplified products (25 μL) were visualized after staining with ethidium bromide and separated by electrophoresis on 1.0% agarose gels. The PCR products were purified with an EasyPure Gel Extraction Kit , and then cloned into pEASY-T1 vector and transformed into Trans1-T1 Phage Resistant Chemically Competent Cells . The positive clones were sequenced at Invitrogen, Shanghai, China. The sequences were edited using DNAStar software.The partial fragments of VP2 nucleotide and deduced amino acid sequences of our newly sequenced AMDV isolates were assembled and aligned by BioEdit 7.0.5.3  and MegATo reveal a possible evolutionary trend of AMDV, a phylogenetic tree was constructed based on the comparison of our 23 partial VP2 nucleotide sequences with the corresponding sequences of 14 reference strains from China, USA, Russia, Germany, Finland and Ireland. The phylogenetic trees were constructed by the neighbor-joining method, using the bootstrap method with 1000 replicates. The maximum likelihood method and maximum parsimony method were also performed for phylogenetic analysis to assess the reliability of the neighbor-joining phylogenetic analysis. The AMDV strains used in this study were shown in Table Additional file 1: Fig. S1. Phylogenetic tree of 37 AMDV isolates based on alignment of the partial fragments of VP2 gene using maximum parsimony method. Bootstrap values higher than 40% are shown (1000 replications). The reference sequences were obtained from the GenBank database, and are marked by the country origins . The strains were separated into groups A, B and C. One single branch that is a little different from neighbor-joining and maximum likelihood phylogenies, is marked by A′. Scale bar indicates the average number of amino acids substitutions per site.


Ticagrelor is a drug widely used in patients with acute coronary syndromes (ACS) that specifically increases the plasma level of adenosine, which is likely to cause atrial fibrillation (AF). Therefore, in this study we aimed to investigate the electrocardiographic and echocardiographic predictors of AF development after P2Y12 receptor antagonists in ACS patients.This cross-sectional study included 831 patients with ACS . Patients were divided into ticagrelor (n = 410) and clopidogrel (n = 421) groups. P wave properties including P wave dispersion and atrial electromechanical conduction properties were measured as AF predictors with surface ECG and tissue Doppler imaging.Baseline characteristics such as age, sex, heart rate, blood pressure, and laboratory parameters were almost the same in the ticagrelor and clopidogrel groups. The statistical analysis showed no significant difference in P wave dispersion (PWD) between ticagrelor and clopidogrel groups . Subgroups analysis according to ACS types also showed no significant difference in PWD . In addition, we did not find significant difference in atrial electromechanical delay (EMD) with tissue Doppler imaging .In conclusion, we did not find any difference in detailed electrocardiographic and echocardiographic parameters as AF predictors between ticagrelor and clopidogrel groups in patients with ACS. Acute coronary syndromes (ACS) are one of the major causes of mortality and morbidity worldwide. Current guidelines recommend dual antiplatelet therapy in patients with ACS . Ticagrelor, one of the relatively new drugs used in ACS, is a reversible and direct-acting oral antagonist of adenosine diphosphate receptor P2Y12, and it was found superior over clopidogrel in the PLATO trial [3]. Although the benefit of ticagrelor has been attributed mostly to its faster, greater, and more consistent P2Y12 inhibition compared to clopidogrel, continuity of growing benefits of ticagrelor and its effect on reduction of cardiovascular mortality in the PLATO trial make it different from other P2Y12-ADP receptor blockers [3–5]. These differences led to the hypothesis that ticagrelor has pleiotropic properties and nonplatelet directed mechanisms of action. These effects of ticagrelor have been mostly attributed to increased half-life and plasma concentration of adenosine .Adenosine is a purine nucleoside primarily produced by endothelial cells [8] and it has a number of effects, such as coronary vasodilatation [9], inhibition of platelet aggregation [10], modulation of inflammation [11], reduced ischemia/reperfusion injury , and reduced atrioventricular conduction [14]. Besides some positive effects, it is also known that adenosine has the potential to cause atrial fibrillation (AF) [15–17]. In addition, there is a case report in the literature suggesting that ticagrelor could cause AF, a possible mechanism of which is increased plasma adenosine level [18]. However, there are no studies in the literature investigating the risk of AF in patients treated with ticagrelor. In this study, we aimed to determine whether ticagrelor predisposes to AF in ACS patients by using surrogate electro and echocardiographic parameters.This cross-sectional study was conducted between January 2016 and February 2017 on patients diagnosed with ACS, which consists of ST elevated myocardial infarction (STEMI) and non-ST elevated myocardial infarction (NSTEMI). STEMI is defined as having a typical angina that lasts 20 min or longer and with STEMI criteria in ECG [2]. Non-ST-elevation myocardial infarction is defined as a rise in troponin level (troponin-I > 0.06 ng/mL) with typical chest pain without STEMI criteria in ECG [1]. The treatment of the patients was arranged in line with the European Society of Cardiology guidelines. Patients were given 180 mg ticagrelor as the loading dose in the ticagrelor group. Angiotensin converting enzyme inhibitors, beta blockers, and statins were started in all patients without contraindication within the first 24 h after diagnosis. Patients were treated with percutaneous coronary intervention . Patients who needed coronary bypass surgery were not included in the study. The other exclusion criteria were as follows: atrial infarction diagnostic criteria described by Liu et al. [19], a history of AF, use of antiarrhythmic drug other than beta-blockers, renal dysfunction (creatinine >1.5 mg/dL), severe valvular heart disease, permanent pacemaker, cerebrovascular disease, and the need for mechanical ventilation. The study was conducted following the principles of the Declaration of Helsinki for Human Research and approved by the institutional ethics committee.Electro-echocardiographic evaluation was performed on patients who had been treated for a median of 2.5 days. A 12-lead surface ECG was obtained from all study participants nearly 2 h after the last dose of the clopidogrel/ticagrelor in the supine position before discharge and analyses were done with this ECG [20]. The point at which the first atrial deflection crossed the isoelectric line was defined as the beginning of the P wave and the return to baseline was considered as the end of the P wave.  Pmax and Pmin durations were measured for all patients in all 12 leads on the ECG. The difference between Pmax and Pmin durations on the ECG was defined as P wave dispersion (PWD) [21]. The laboratory tests included complete blood count, fasting glucose level, lipid profile, troponin level, liver, kidney, and thyroid function tests. The weight and height of the participants were measured, and the body mass index (BMI) (kg/m2) was calculated using the following formula: BMI = weight/(height)2. Echocardiographic examination and evaluation of patients were performed using Philips Healthcare iE33 xMATRIX Echocardiography  with an S5–1 transducer before discharge. Examinations were performed by a single experienced cardiologist who was blinded to the patients and their characteristics. Evaluation of the patients was performed in the left lateral decubitus position. A continuous one-lead ECG was obtained during all examinations. The average of three consecutive beats was used to calculate the associated parameters. M-mode echocardiography was used in the parasternal long-axis view to measure basic echocardiographic parameters, such as the left atrium (LA), left ventricular (LV) end-systolic and end-diastolic dimensions, LV ejection fraction (EF), and diastolic LV septal and posterior wall thickness. Electromechanical properties of the atria were determined by tissue Doppler imaging. Before the study, the Nyquist limit was adjusted to 15–20 cm/s and the monitor sweep speed was set at 50–100 mm/s to optimize the spectral display myocardial velocities. The pulsed doppler sample volume was placed at the LV lateral and septal mitral annulus, and subsequently at the septal mitral annulus and right ventricular tricuspid annulus in apical four chamber view. Atrial electromechanical delay (EMD) was considered as the time interval from the onset of P wave on ECG to the beginning of “A”-wave in tissue Doppler (PA). PA interval, which was measured from the lateral mitral, septal mitral, and tricuspid annulus was called PA lateral, PA septum, and PA tricuspid, respectively. PA intervals between the lateral and right ventricular annulus were accepted as interatrial EMD, the difference between the septal and tricuspid PA intervals as right intraatrial EMD, and the difference between the lateral and septal PA intervals as left intraatrial EMD. The data were analyzed using SPSS 18.0 statistics package . Normally distributed continuous variables were expressed as means ± standard deviation, while continuous variables with a nonnormal distribution were expressed as median and interquartile ranges (IQR). Student’s t-test or the Mann–Whitney U test were used to compare the means or medians of groups, respectively. Categorical data were expressed as proportions and compared using the chi-square test. The relationship between antiplatelet types and P wave duration, PWD, PR interval, P wave axis, and EMD values was assessed by ANCOVA analysis by removing the effects of confounding factors that were significant in the antiplatelet and ACS types. P-values less than 0.05 were considered statistically significant.A total of 1036 patients were assessed with a diagnosis of ACS. Eighty-eight patients were excluded because of the atrial infarction criteria and 45 patients due to the history of AF. After exclusion of the patients with renal dysfunction (creatinine >1.5 mg/dL), severe valvular heart disease, permanent pacemaker, cerebrovascular disease, and the need for mechanical ventilation, 831 patients remained for further analysis. STEMI was found in 486 (58.5%) and NSTEMI in 345 (41.5%) patients. All patients were divided into two groups according to receiving ticagrelor  or clopidogrel . The rate of patients diagnosed with STEMI in the ticagrelor group was found to be higher than in the clopidogrel group . Table 1 summarizes the patients’ baseline characteristics. The groups had no significant differences in terms of age, sex, BMI, heart rate, blood pressure, creatinine, alanine amino transferase, hemoglobin, thyroid-stimulating hormone levels, LA diameter, and LVEF. As shown in Table 1, LDL, HDL, and 24th hour troponin levels were significantly higher in the ticagrelor group. In the analysis of total population data, there was no statistically significant difference in PWD between ticagrelor and clopidogrel groups  (Table 2). The other electrocardiographic predictors of AF, such as PR interval and P wave axis, also showed no significant difference . Subgroups analysis according to ACS types also showed no significant difference in PWD . Echocardiographic parameters are shown in Table 2. Like electrocardiographic parameters, we did not find any differences in echocardiographic predictors. There was no statistically significant difference in left intraatrial EMD, right intraatrial EMD, and interatrial EMD between the groups . In addition, there was no statistically significant difference in echocardiographic predictors of AF in the subgroups according to ACS types. The mean interatrial EMD values of ticagrelor and clopidogrel groups were found as 24.53 ± 3.74 ms and 24.86 ± 3.33 ms in NSTEMI (P = 0.644) and as 23.98 ± 2.82 ms versus 24.23 ± 3.16 ms (P = 0.493) in STEMI subgroup (P = 0.493).The relationship between antiplatelet types and P wave duration, PWD and EMD values, was evaluated by ANCOVA analysis by removing the effects of confounding factors  that were found to be significant between the antiplatelet groups and the ACS subgroups. When the effects of confounding factors were removed, there was no significant relationship between antiplatelet use and AF predictors (P > 0.05).From the date of treatment initiation, subgroup analysis was performed in 66 (16.09%) patients with side effects  related to ticagrelor. There was no significant difference in terms of AF predictors in patients with side effects related to ticagrelor when compared to the group receiving clopidogrel or ticagrelor without side effects. PWD was 38.79 ± 12.28 ms in patients receiving ticagrelor that had side effects, while this value was 41.44 ± 13.17 ms in patients without side effects (P = 0.279) and 40.34 ± 13.14 ms in clopidogrel group (P = 0.657). Interatrial EMD was 24.23 ± 2.79 ms in patients receiving ticagrelor and had side effects, while this value was 24.18 ± 3.21 ms in patients without side effects (P = 0.998) and 24.35 ± 3.01 ms in clopidogrel group (P = 0.990).Our study was designed to find out whether ticagrelor could change AF predictors. We did not find a statistically significant difference in PWD, interatrial and intraatrial EMD durations between ticagrelor and clopidogrel groups in patients with ACS.IKAdo. In addition to that, adenosine has other effects that may promote arrhythmogenesis. Adenosine has sympathoexcitatory effects mediated through baroreflex activation and chemoreceptor stimulation [30]. Adenosine can also hyperpolarize dormant pulmonary vein myocytes and increase excitability, as well as trigger pulmonary vein ectopy . It has been shown that ticagrelor increases plasma adenosine levels as early as six hours after the loading dose in patients with ACS [33]. Therefore, the main point of our study was to determine whether additional increase in adenosine, which is caused by ticagrelor, would increase the susceptibility to AF. In ACS patients, AF is seen at a high rate (approximately 10%) compared to the normal population, and this increase has been associated with long-term increased morbidity and mortality . AF development in patients with ACS is mostly attributed to ischemia and decreased atrial perfusion, increased left ventricular end-diastolic pressure, increased left atrial pressure, diastolic dysfunction, and autonomic regulatory disturbances [24]. In addition, recently, inflammation and neurohumoral factors have been shown to be associated with AF development in patients with ACS [24]. In our study, there were no significant differences between the groups in parameters that could affect the development of AF such as LA diameter, systolic and diastolic functions of LV. Furthermore, patients with atrial infarction criteria on ECG were excluded from the study to reduce the possible confounding effects of atrial ischemia [19]. Apart from the normal course of ACS, there is a case report indicating that ticagrelor, which increases plasma concentration of adenosine, may cause AF [18]. PLATO trial and some case reports have shown that ticagrelor causes bradyarrhythmias, such as atrioventricular block and sinus node pause . The most probable mechanism for these bradycardic events appears to be increasing plasma concentration of adenosine. Besides the bradycardic effects of adenosine, it is known that intravenous adenosine administration could cause spontaneous AF [15]. Moreover, endogenous production of adenosine during metabolic stress conditions has been suggested as a trigger of AF . Although the mechanism is not clearly understood, this phenomenon is thought to be mediated by adenosine’s effects on shortening atrial action potential duration and refractoriness [30]. Because adenosine has little effect on atrial conduction velocity, the net effect of adenosine, therefore, is to shorten the wavelength of activation, thereby potentiating AF. This cellular electrophysiological effect is mediated by its specific G protein-coupled adenosine A1 receptor and this ligand activates the heterotrimeric protein Gi/o, which then activates the inward rectifying K+ current, It has been shown that PWD and Pmax are predictors of AF [34]. Normal value for PWD is defined as 29 ± 9 ms in the literature, while a PWD > 40 ms leads to atrial tachyarrhythmias . Rosiak et al. also demonstrated that PWD is predictive of AF in STEMI patients and that a value of >25 ms is associated with increased risk for AF [36]. In our study, PWD was 40.98 ± 12 in the ticagrelor group and 40.06 ± 12 in the clopidogrel group, and no statistical difference was found between the two groups. PWD value determined in our study is higher than the PWD values in non-ACS patients in the literature, but the patient groups show some differences. In particular, the high value of PWD may explain the fact that since our patient group included ACS patients, additional comorbidities were common and LVEF was lower than other studies in the literature. Thus, Ding et al. found that patients with cardiac resynchronization therapy had high PWD values and showed a decrease in PWD when EF was improved [37]. Besides electrocardiographic parameters, we also looked at echocardiographic parameters, which were shown to predict AF . Likewise, in electrocardiographic analysis, there was no statistical difference between the interatrial, right and left intraatrial electro-mechanic delays that were measured by TDI between ticagrelor and clopidogrel groups.  Although recent investigations have shown that dyspnea is caused by direct P2Y12-inhibitory effect on the central nervous system, in our study patients with side effects due to ticagrelor were also considered as subgroups due to probably increased adenosine levels than those without side effects [40]. However, subgroup analyses for AF predictors showed no significant difference in patients with or without side effects [20]. To the best of our knowledge, this is the first study investigating the association between ticagrelor and AF predictors. Nevertheless, the lack of follow-up in terms of future arrhythmic episodes is among the major limitations of our study. Reliable methods for detecting AF could be used, such as long-term rhythm monitoring. In addition, the lack of difference between the ticagrelor and clopidogrel groups in terms of AF predictors may be attributed to several causes. For example, possible atrial structural disturbances due to ACS and having multiple risk factors may have masked potential effects of adenosine on AF development. There were also some differences in frequency of ACS types and troponin levels in the two study groups. Furthermore, the increase in adenosine level caused by ticagrelor might not be sufficient to alter the AF predictors we have investigated. It has also been shown in the literature that adenosine causes AF in a dose-dependent AF [14].In conclusion, in this study, we found that there was no significant difference in electro-echocardiographic AF predictors such as PWD and EMD in ACS patients who received ticagrelor or clopidogrel. In addition, there was no difference even in patients with side effects, suggesting that the possible increase in adenosine level did not lead to AF susceptibility.


In some cases, these cues are sufficient to accelerate or inhibit terminal differentiation of murine oligodendrocyte progenitors. However, our understanding of oligodendrocyte lineage mechanobiology has been restricted primarily to animal models to date, due to the inaccessibility and challenges of human oligodendrocyte cell culture. Here, we probe the mechanosensitivity of human oligodendrocyte lineage cells derived from human induced pluripotent stem cells. We target phenotypically distinct stages of the human oligodendrocyte lineage and quantify the effect of substratum stiffness on cell migration and differentiation, within the range documented in vivo. We find that human oligodendrocyte lineage cells exhibit mechanosensitive migration and differentiation. Further, we identify two patterns of human donor line-dependent mechanosensitive differentiation. Our findings illustrate the variation among human oligodendrocyte responses, otherwise not captured by animal models, that are important for translational research. Moreover, these findings highlight the importance of studying glia under conditions that better approximate in vivo mechanical cues. Despite significant progress in human oligodendrocyte derivation methodology, the extended duration, low yield, and low selectivity of human-induced pluripotent stem cell-derived oligodendrocyte protocols significantly limit the scale-up and implementation of these cells and protocols for in vivo and in vitro applications. We propose that mechanical modulation, in combination with traditional soluble and insoluble factors, provides a key avenue to address these challenges in cell production and in vitro analysis.Oligodendrocytes produce and repair myelin, which is critical for the integrity and function of the central nervous system (CNS). Oligodendrocyte and oligodendrocyte progenitor cell (OPC) biology is modulated  Glial cells play fundamental roles in homeostasis, pathology, and repair of the central nervous system (CNS). Among glia, oligodendrocytes primarily produce, maintain, and repair the myelin sheaths surrounding neuronal axons, which enable cognitive and motor abilities in vertebrate animals Bunge, . These cin vitro murine models almost exclusively, due to the practical limitations of obtaining glial cells from human CNS tissue  and time-varying (dynamic) mechanical cues. This mechanosensitivity has been established in the last decade using Recent sequencing and transcriptomics studies have revealed species-specific features that highlight the importance of studying human cells to recapitulate the pathology of CNS disorders  reprogrammed from epidermal fibroblasts . Lines were thawed, cultured and expanded on 6-well plates coated with Matrigel  in mTeSR1 medium  or StemFlex medium . Cells were passaged enzymatically every 3-4 days using with Stempro Accutase  for 5 min and re-plated in mTeSR1 medium with 10 μM ROCK Inhibitor  for 24 h.A total of five hiPSC lines were used in this study, derived from skin biopsies of apparently healthy, deidentified donors upon specific institutional review board approvals and informed consent . Four hiTM EZPassageTM Disposable Stem Cell Passaging Tool , or the Corning cell lifter . Each well was split across two wells of a Corning Costar ultra-low attachment 6-well plate  in N2B27 medium , and the slides were incubated for 10 min. PFOCTS-functionalized slides were stored for up to a month.Polyacrylamide (PAAm) hydrogel disks were prepared on glass coverslips, and assembled in 24-well plates for spheroid culture experiments. The protocol was developed from a combination of previously published methods  stock solutions were prepared fresh every day. Hydrogel disks were prepared up to four at a time to reduce the risk of polymerization in the preparation tube. APS (0.5% w/v) and TMED (0.05% v/v) were gently mixed with acrylamide pre-mixes with a pipette tip avoiding bubbles, as oxygen inhibits the polymerization. A 22 μl drop of the pre-polymer mixture was pipetted unto a superhydrophobic glass slide, and covered with a functionalized coverslip (treated side down). After the corresponding reaction time  and bis-acrylamide  mixtures were prepared by mixing the two components with 1× phosphate buffered saline (PBS) according to the recipes in ion time , the hydTM 184 PDMS base and crosslinker were vigorously mixed with a stand mixer in 1:9 ratio for 5 min and degassed in a vacuum chamber until all visible bubbles disappeared. A small drop (~10 μl) of PDMS mixture was placed in the center of each well of a 24-well plate. A hydrogel-coverslip was placed on top of each PDMS drop allowing it to spread (gel side up). Enough PBS was placed on each hydrogel to completely cover it without spilling over the sides. The plates were wrapped in parafilm and left at room temperature overnight to allow the PDMS to polymerize and solidify. The plates were flooded with PBS and stored at 4°C for up to 2 weeks.Polydimethylsiloxane (PDMS) was used to immobilize hydrogel-coverslips unto multi-well plates to ease the transportation of plates across laboratories and the plating of spheroids. Dow SYLGARD2, measured near the hydrogel surface. Excess solution was removed and the hydrogels were rinsed twice with 20 mM HEPES (pH 8.0). The hydrogels were subsequently shaken in an orbital shaker at 70 rpm for 1 min , followed by one additional wash and incubation in a solution of 50 μg/ml PO in 20 mM HEPES buffer (pH 8.0). The hydrogels were incubated in PO overnight at 4°C and washed three times with PBS. At this point functionalization was either continued in the same laboratory or in a second laboratory, where cell experiments would be carried out. For the latter, washed plates were carefully wrapped and shipped on ice overnight. PO-coated and washed plates were incubated overnight in 12 μg/ml mouse laminin in PBS at 4°C. The hydrogels were washed 3 times with PBS and incubated with DMEM/F12 at 37°C at least 30 min before plating.PAAm hydrogels were allowed to equilibrate in PBS for at least 24 h before functionalization. The surface of PAAm hydrogels were covered with sulfo-SANPAH  at 0.5 mg/ml in 20 mM HEPES buffer (pH 8.0). Each hydrogel was immediately exposed to UV light  for 3 min at an approximate intensity of 30 mW/cmTissue culture polystyrene control plates were incubated with 50 μg/ml PO in distilled water overnight at 37°C. The PO solution was aspirated and plates were allowed to dry. Plates were subsequently incubated in 12 μg/ml mouse laminin in DMEM/F12 for 4 h. Laminin solution was aspirated and plates were washed twice with DMEM/F12 before cell plating.E of PAAm hydrogels was measured via load-depth indentation experiments using an atomic force microscope (AFM) . Hydrogel disks were equilibrated in 1× PBS for at least 24 h at room temperature before measurements. All hydrogels were indented while fully immersed in 1× PBS. Hydrogels with E below 10 kPa were indented with a spherical borosilicate probe of diameter 20 μm mounted on a cantilever of nominal spring constant k = 0.03 N/m (Novascan); cantilever base velocity was 6 μm/s, and probe retraction was triggered after reaching a maximum deflection of 100–200 nm. Hydrogels with E above 10 kPa were indented with a spherical polystyrene probe of diameter 25 μm mounted on a cantilever of nominal spring constant k = 0.6 N/m (Novascan); cantilever base velocity was 6 μm/s, and probe retraction was triggered after reaching a maximum deflection of 100–200 nm. Spring constants were calibrated using the thermal noise method  adaptive thresholding based on mean and standard deviation of channel intensities; (3) superposition of region positive for the marker of interest with DAPI-stained nuclei; and (4) and counting of cells with overlapping channels. The percentage of O4+ cells was quantified as the number of O4+ cells with DAPI-stained nuclei, relative to the total number of DAPI-stained cells. Mechanosensitivity trends across hydrogel substrata were evaluated with the non-parametric Spearman’s rank-order correlation coefficient, which does not assume linearity. Trends with non-zero Spearman coefficients and p-value < 0.05 were deemed statistically significant. The results for a given line were considered reproducible if for each independent experiment the Spearman correlation coefficient was moderate to strong (absolute magnitude higher than 0.5), had the same sign, and had a p-value < 0.05. t-test was used to compare each hydrogel with its corresponding TCP controls, on spheres pooled from two replicate experiments per line. Data normality was determined with Shapiro-Wilk normality test. Statistics were performed with Prism 7 software.Images for quantification of population composition at day 60 and 68 were acquired at 10× or 20× magnification. These images were put through a custom Fiji pipeline consisting of four general steps: (1) focus correction E (of magnitude 0.1–70 kPa) that approximates and exceeds the stiffness of the human CNS environment . We quantified the propensity for the outward migration of cells from the spheroids as the total area enclosed by the outer perimeter of cells further away from the spheroid, and an inner perimeter demarcated by the edge of the spheroid  compared to TCP  for each hiPSC donor line. The area of migration was similar or smaller on the most compliant hydrogels . In contrast, in type B response the fraction of O4+ cells increased with substratum stiffness (donor lines 2 and 4). The association between expression of transcription factor OLIG2 with substratum stiffness at the end of the differentiation protocol (d68) was inconsistent  and polymer chemistry (polyacrylamide), but of different ligand coating , suggesting the involvement of more than one mechanism.During mammalian development, OPCs migrate along neuronal axons and blood vessels from the neural tube to the brain and spinal cord , for consistency. For this substratum, we previously observed increasing differentiation of primary rat oligodendrocytes (defined as % of MBP+ cells) with substratum stiffness  compared to TCP (E = 70 kPa instead of standard TCP. It is important to note that while stiffness is one material property that distinguishes these hydrogels from TCP, it is certainly not the only differing characteristic of these two material types that affects cell adhesion, proliferation, migration, and differentiation. Thus, while we do not claim substratum mechanics as the only explanatory variable for the larger number of oligodendrocytes obtained on this hydrogel compared with on standard TCP, we conclude that mechanotransduction may be used in concert with other biochemical factors to modulate oligodendrocyte biology to improve processes in vitro such as migration or proliferation. That increased efficiency of cell proliferation may be advantageous, for example, in the advancement and viability of cell therapies.Beyond these intriguing differences in mechanosensitivity of hiPSC-derived OPCs, we briefly note the increased proliferation observed on one of these hydrogels, relative to current practice. Production of human oligodendrocytes for basic and applied studies requires a stage of cell proliferation. In the present study, the extent of migration of cells was higher, or at least similar, on stiffer PAAm hydrogels (d to TCP . Furtherd to TCP . Becaused to TCP , migratiin vitro conditions that more closely approximate in vivo cues and environments. Understanding what makes cells from healthy or diseased cohorts respond poorly in terms of migration or differentiation in a CNS tissue lesion environment can offer new directions for treatment. Additionally, studying the pathways affected by such mechanical stimulation may offer new targets for intervention, including for approaches to cell therapy and personalized medicine.Overall, our results show that human oligodendrocyte biology is affected by extracellular mechanics, and emphasizes the role of mechanosensing in phenotypic maturation and migration of cells comprising the CNS. This work highlights the importance of studying CNS stem and glial cells under All datasets presented in this study are included in the article/DE-H co-designed the study, carried out material synthesis and characterization, cell experiments and analysis, drafted and edited the manuscript, and created and edited the figures. SRB carried out cell experiments, reviewed all results, and edited the manuscript and figures. JC carried out cell experiments and analysis. TJ co-designed the study, carried out cell experiments and edited the manuscript and figures. MN carried out cell experiments and edited the manuscript and figures. AJ co-designed the study, reviewed all results and edited the manuscript and figures. VF and KJVV co-led the research team, co-designed the study, reviewed all results, and edited the manuscript and figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proximal gastrectomy (PG) is one of the function‐preserving surgical methods for the treatment of upper gastric cancer. Favorable postoperative results have been reported in comparison with total gastrectomy. However, because there are challenges, such as postoperative reflux esophagitis, anastomotic stenosis, and residual food, appropriate selection of a reconstruction method is crucial. Some methods include esophagogastric anastomosis, including simple esophagogastrostomy, tube‐like stomach esophagogastrostomy, side overlap with fundoplication by Yamashita, and double‐flap technique, and reconstruction using the small intestine, including double‐tract methods, jejunal interposition, and jejunal pouch interposition. However, standard reconstruction methods are yet to be established. PG has also been employed in early gastric cancer of the upper third of the stomach, and indications have also been extended to esophagogastric junction cancer, which has shown an increase in recent years. Although many retrospective studies have revealed the functional benefits or oncological safety of PG, the characteristics of each surgical procedure should be understood so that an appropriate reconstruction method, with a reflux prevention mechanism and minimal postoperative injury, can be selected. These include double‐tract methods, jejunal interposition, and esophagogastric anastomosis (among others), and the choice must be made in consideration of the prevention of gastroesophageal reflux and ensuring a good dietary intake.Proximal gastrectomy is also indicated for EGJ cancer, which has shown a recent increase in Japan. The Japanese Gastric Cancer Treatment Guidelines published the algorithm for the surgical treatment for EGJ cancer <4 cm in diameter, including early and advanced cancer, where the extent of the lymph node dissection falls within the range of PG.PG, which was mainly adapted for EGC patients, was shown to achieve favorable prognosis with reduced surgical invasiveness, and its indications overlap those of laparoscopic gastrectomy, a form of minimally invasive surgery.In this review, we summarize the current landscape of the PG procedure, including indications, lymph node dissection, reconstruction methods, and remnant stomach cancer, for upper gastric and EGJ cancer.2With the recently increasing incidence of proximal gastric cancer in Asian countries, PG is widely accepted as a FPG in EGC.The guidelines recommend that the size of the remnant stomach after PG should be half or more than the original size. Furthermore, because the size of the remnant stomach is closely related to the tumor location and the surgical margin, it is an indication itself for this procedure. Fundamentally, PG is a function‐preserving operation, and it is considered that the size and function of the residual stomach would be related. Nomura et alAnother interesting decision‐making question is whether to choose PG or subtotal gastrectomy for EGC of the upper stomach.The guidelines defined an algorithm for the dissection range of early and advanced EGJ cancers of ≤4 cm. In this category, lymph node dissection on the pyloric side is unnecessary, and PG is theoretically possible. Large phase II studies of EGJ cancers deeper than T2 have shown similar results, regardless of tumor size.3The Japanese Gastric Cancer Treatment Guideline defines PG as a function‐preserving operation for cT1N0 ECG of the upper stomach, with the extent of lymph node dissection as D1 or D1+. Lymph node dissection in the distal part of the pancreas is unnecessary in this adaption. If PG is indicated for advanced gastric cancer, D2 lymph node dissection will be defined, and lymph node dissection distal to the pancreas will be required. Yura et alFirst reported by Uyama et al in 1995, laparoscopic PG has been increasingly performed, and several technical reports and case studies with small sample sizes have been published.As described above, dissection of the lymph nodes on the pyloric side may become unnecessary in cases of EGJ cancer.4There are various postgastrectomy reconstruction methods, and the method chosen must prevent reflux esophagitis and ensure a good dietary intake. As well as the short‐term results of the reconstruction method, the extent of long‐term nutritional effects, state of anemia, and frequency of gastric cancer in the remnant stomach are important points to consider.There are two major methods of reconstruction after PG; one uses esophagogastric anastomosis, and one uses the small intestine. Esophagogastric anastomosis includes simple esophagogastrostomy, tube‐like stomach esophagogastrostomy, side overlap with fundoplication by Yamashita (SOFY), and the double‐flap technique. Reconstruction methods using the small intestine include the double‐tract, jejunal interposition, and jejunal pouch interposition methods. Because EGJ cancer requires a higher level of anastomosis than typical gastric cancer, a more careful choice of reconstruction is essential.In particular, a high level of anastomosis would be required depending on the advanced stage of EGJ cancers. Reconstruction with a gastric tube is stable for high level of anastomosis, but reflux is a complication after surgery. Although there are restrictions on raising the small intestinal mesentery, it would be better to select this method if safe reconstruction is possible. Kurokawa et al conducted a large‐scale phase II study of EGJ cancer deeper than T2, in which 180 (49.6%) of 363 enrolled cases underwent PG.In recent years, PG by laparoscopic procedure has been widely used; however, reconstruction may be a major technical complication. A prospective phase II study (JCOG1401) was conducted to confirm the safety of esophagogastric anastomosis or esophagojejunostomy after TG or PG. This study reported that esophageal anastomotic failure was 2.5% and confirmed the safety of laparoscopic reconstruction.The exclusion criteria of the JCOG1401 study included “no esophageal invasion.” Therefore, there was no clear evidence regarding the safety of laparoscopic PG for EGJ cancer. However, many institutes have extensively performed laparoscopic PG for EGJ cancer, possibly favoring the stability of lymph node dissection by a trans‐hiatal approach.However, determining which reconstruction method after PG would be the best is challenging. Another PGSAS‐45 study is currently underway on the reconstruction method after PG for upper gastric cancer. At present, there is no standard reconstruction method after PG; however, when the results of this research are obtained, a guideline for a reconstruction method with high QOL could be obtained. It would be possible to perform randomized controlled trials if the facility is accustomed to the surgical methods with respect to the high level of reconstruction methods from the results of this study.We describe some common methods of reconstruction following PG below. Tables 4.1Esophagogastrostomy, also called simple esophagogastrostomy, was used as the reconstruction method after Mikulicz's first PG in 1897.4.2The postoperative condition of patients who underwent a tube‐like stomach esophagogastrostomy has been reported in several studies.4.3Side overlap with fundoplication by Yamashita is a recently developed method of esophagogastric anastomosis with a unique anti‐reflux mechanism and can be easily performed laparoscopically.4.4The double‐flap technique involves esophagogastric anastomosis after PG to which a fundoplication based on valvuloplasty was added by Kamikawa et alAlthough the double‐flap technique after PG is an excellent reconstruction method, the complications associated with the laparoscopic suturing technique must be solved for its widespread use. Stress‐free laparoscopic suturing, which is indispensable for this surgical procedure, may be performed using robotic surgery, which has become increasingly popular in recent years.4.5A retrospective study reported the results of surgical outcomes after using the double‐tract method for PG.4.6The success of the jejunum interposition reconstruction method was announced to the world first by Prof. Merendino, who reported an animal experiment and its clinical application in jejunal interposition after PG.Eleven studies have reported results of jejunal interposition, with morbidity, stenosis, reflux esophagitis, and residual food occurring in 0%‐31.6%, 3.1%‐31.8%, 0%‐33.3%, and 8.5%‐31.8% of cases, respectively. 4.7Residual food is the biggest problem in jejunal pouch reconstruction, and Nakamura et al. have reported food residues in >90% of cases.5Comparisons between jejunal interposition and other reconstruction methods have been reported as follows: jejunal interposition vs esophagogastrostomy, n = 3Of the four retrospective studies comparing the outcomes of jejunal interposition and esophagogastrostomy, one study found increased early postoperative complications of jejunal interposition (20.0% vs 3.1%),Of the three studies comparing the outcomes of jejunal interposition and jejunal pouch interposition, one retrospective study found an increased risk of reflux esophagitis in the jejunal interposition group (33.3% vs 11.3%),One retrospective study reported the outcomes of jejunal interposition and tube‐like stomach esophagogastrostomy.In the one retrospective study comparing the outcomes of tube‐like stomach esophagogastrostomy and esophagogastrostomy, the tube‐like stomach procedure showed a decreased incidence of reflux esophagitis (5.7% vs 22.0%) and a similar incidence of stenosis and emptying dysfunction.With respect to the nutritional status, although a large retrospective study using the postgastrectomy syndrome assessment scale reported that PG reduces symptoms after gastrectomy syndrome more than TG,6An important consideration in PG is the increased risk for remnant gastric cancer. The rate of remnant gastric cancer has been reported as higher after PG (3.6%‐9.1%) than after distal gastrectomy (0.4%‐2.5%). 7Regarding the reconstruction method after PG, the characteristics of each surgical procedure should be understood so that an appropriate reconstruction method, with a reflux prevention mechanism and minimal postoperative injury, can be selected.Conflict of interests: There are no conflicts of interest to disclose.: (I) conception and design: S Nunobe; (II) administrative support: S Nunobe; (III) provision of study materials or patients: all authors; (IV) collection and assembly of data: S Nunobe; (V) data analysis and interpretation: S Nunobe; (VI) manuscript writing: all authors; (VII) final approval of manuscript: all authors.Author Contributions


For each tumor type, a panel of miRNAs was defined that provided cancer-detection accuracies of 91%, 94%, 89%, and 77%, respectively. In conclusion, we have described a series of circulating miRNAs that define different tumor types with a very high diagnostic performance. These panels of miRNAs would constitute the basis of different approaches of cancer-detection systems for which clinical utility should be validated in prospective cohorts.The purpose of this study is to clinically validate a series of circulating miRNAs that distinguish between the 4 most prevalent tumor types (lung cancer (LC); breast cancer (BC); colorectal cancer (CRC); and prostate cancer (PCa)) and healthy donors (HDs). A total of 18 miRNAs and 3 housekeeping miRNA genes were evaluated by qRT-PCR on RNA extracted from serum of cancer patients, 44 LC, 45 BC, 27 CRC, and 40 PCa, and on 45 HDs. The cancer detection performance of the miRNA expression levels was evaluated by studying the area under the curve (AUC) of receiver operating characteristic (ROC) curves at univariate and multivariate levels. miR-21 was significantly overexpressed in all cancer types compared with HDs, with accuracy of 67.5% ( Nowadays, cancer is expected to rank as the leading cause of death in every country of the Western world in the 21st century. The Global Cancer Observatory (GLOBOCAN) 2018 estimated that there would be 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018. Globally, the incidence rate for all cancers combined was about 20% higher in men than in women, with the incidence rates varying across regions in both males and females, indicating that 1 in 8 men and 1 in 10 women will develop cancer along their life. For both sexes combined, lung cancer (LC) is the most commonly diagnosed cancer , followed by female breast cancer (BC) (11.6%), colorectal cancer (CRC) (10.2%), and prostate cancer (PCa) (7.1%) [Despite the high mortality of this disease, it has been demonstrated that the chance of curing cancer is very high when identified early. In this sense, cancer screening programs (CSPs) play a critical role in identifying cancer before symptoms appear, and their impact on cancer-specific and overall survival has been well documented . Many ofMany of these analytical tests are based on the identification of blood-based tumor biomarkers which constitute a readily available, inexpensive, and minimally invasively tool which also allows for repeated sampling .Particularly, microRNAs (miRNAs) are 19–25 nucleotide noncoding RNA molecules that regulate a variety of cellular processes including cell differentiation, cell cycle progression, and apoptosis . Some sthttps://saphely.eu/) is focused on developing a nanophotonic-based handheld point of care nanophotonic device for detecting specific circulating miRNAs for the four major cancer types: LC, BC, CRC, and PCa.To take advantage of miRNAs as biomarkers for the early identification of cancer, the H2020 European project SAPHELY (Self-amplified photonic biosensing platform for microRNA-based early diagnosis of diseases)  (p < 0.0001)  A. Intere 0.0001) B.As for miR-21, the diagnostic performance of the other circulating miRNAs was evaluated with ROC curves for each tumor type. The results are summarized in The analysis was performed separately for each tumor type considering those tumor-specific miRNAs . For eacp-value < 0.05)  . ROC cur < 0.05) . Therefon with n being the sample size of each of the four datasets). Every subset is randomly resampled in each iteration up to 1000. These subsamples show that stabilization of the AUC along with standard deviation shrinkage is achieved before reaching the sample size studied in this work, showing an asymptotic pattern and suggesting that an increase of the number of cases for each tumor type would not significantly change the observed AUC  in the probability of being classified as cancer cases as tumor stage rises, corroborating the performance of miRNAs selected as diagnostic and prognostic tools   and prostate specific antigen (PSA) values. In this case, despite the fact that the median values of the probabilities are higher in GS, ≥7, and serum PSA, ≥4.33 ng/mL, the differences were not statistically significant ( > 0.05) , mainly Cancer represents the set of diseases with more incidences and mortality in Western countries, and its numbers are increasing every year with the consequent social and economic impacts . For thaMore than one decade ago, it was reported that miRNAs are also present in blood . Circulahttps://saphely.eu) intends to break into the field of screening tests for early diagnosis of the four major cancer types, PCa, BC, LC, and CRC, by using a novel ultrahigh sensitivity nanophotonic-based sensing technique for the direct detection of circulating miRNA biomarkers through a combination of molecular beacon probes with an attached high index nanoparticle so that the hybridization events are translated into the displacement of these nanoparticles from the sensor surface [https://youtu.be/6ZAuSkfJrB8?list=PL8qM5Jl41EI7iW2a57QEyaCvRFHn-ix95.Taking all these premises into consideration, the H2020-SAPHELY project  significantly increases the performance of the PCa miRNA panel.For PCa, the diagnostic accuracy defined by the miRNA panel  was 94%, the highest of our series and similar to other clinico-molecular multi-biomarker panels focused on detection of high-grade PCa . WhereasFinally, CRC miRNA panel  achieves an 89% diagnostic performance. These miRNAs have been already described by other authors ,38,39, aAdditionally, due the role of miRNA as key regulators of virtually every biological process, it is interesting to study the impact of miRNA in phenotypes by studying the role of its effector mRNAs. In this sense, there is a great corpus of evidence from both functional studies and computational predictions. This data is organized in public databases on the Internet along which one of the most important is miRBase .An in silico analysis reveals a dense network of gene effectors of the miRNA in every one of the four tumor types studied. Gene Set Enrichment Analysis (GSEA) highlighted the three top overrepresented pathways. In all four datasets, the main Kyoto Encyclopedia of Genes and Genomes (KEGG) category is pathways related to cancer. In LC, this category is accompanied by ErbB signaling and PCa pathways, whereas PCa showed altered pancreatic and PCa pathways. CRC and finally the BC series have overrepresented pancreatic and PCa pathways. There is difficulty drawing direct conclusions of such a dense network of genes; thus, the network was previously pruned according the degree of the nodes in every one of the disease set. The top overrepresented KEGG pathways are closely related with cancer in all the cases with interesting potential mRNA biomarkers which deserve to be explored .Moreover, we have demonstrated that the tumor-selected circulating-miRNA are associated to different tumor stages but not to GS and serum PSA values in the case of PCa. This observation gives robust evidence of circulating-miRNA utility as a diagnostic and prognostic tool in these four cancer types.However, this present study has some limitations. In the first place, we only have analyzed some miRNAs considered tumor specific for each cancer type, but there are more miRNAs that have not been investigated that could be relevant for cancer diagnosis. Moreover, we need an amplification step in order to increase the miRNA concentration to being able to detect them, but the SAPHELY device is not be able to perform this step, a point that could be crucial for the success of the device. Finally, we have defined a panel for each cancer type, but they are not always represented, with all altered miRNAs in all cases hindering the disease identification.Because of these limitations, this present study must be continued by analyzing more miRNAs that can be completely tumor-specific and by improving the detection system to offset the low concentration of miRNAs in bloodstream.This study was approved by the Clinical Research Ethics Committee (CREC) of the Fundación Instituto Valenciano de Oncología of Valencia (FIVO) at the meeting celebrated on 30 December 2014. Written informed consent was obtained from each participant prior to sample collection.Patients with newly diagnosed LC, BC, CRC, and PCa were considered eligible for this study. Within the period from October 2011 to March 2017, a total of 156 cancer patients  was finally selected. Patients’ clinical and histopathological information are summarized in In view of the role of miRNAs as diagnostic biomarkers, a literature search was performed to identify those miRNA candidates to be implemented as biomarkers. A list of potential miRNAs was selected for each tumor type by applying a decision-making algorithm using PubMed as source of information. The search criteria used were (“microRNA” OR “miRNA” OR “miRNAs”) AND  AND (“serum” OR “plasma”) AND (“diagnosis”). All the papers selected met the following criteria: expression of miRNAs in serum; papers written in English; and clinical setting: diagnosis, including control samples.The results of the literature search are summarized in g at RT and stored at FIVO Biobank at −80 °C in 1-mL aliquots until RNA could be extracted and purified.Seven milliliters of peripheral blood from cancer patients and HDs were collected at the time of diagnosis in SARSTEDT Monovette Serum gel tubes  and left to clot at room temperature (RT) for 30 min. The serum was separated by centrifugation for 10 min 3000× Total RNA was isolated using the miRNeasy Serum/Plasma Kit  following the manufacturer’s instructions, starting with 200 µL of serum. The concentration of total RNA in each sample was measured using a NanoDrop 1000 spectrophotometer .One-hundred and fifty ng of total RNA were reverse-transcribed using the TaqMan MicroRNA Reverse Transcription kit  in a total volume reaction of 15 µL containing 2 mM dNTPs, 3.3 U/mL MultiScribe reverse transcriptase, 1× reverse transcription buffer, 0.25 U/mL RNase inhibitor, and 0.02× of each specific miRNA primer , that weFive µL of cDNA was pre-amplified using the TaqMan PreAmp Master Mix (2×) according to the manufacturer’s instructions (Applied Biosystems). For this reaction, 5 µL of cDNA sample was added to a total volume of 25 µL containing 12.5 µL of TaqMan PreAmp Master Mix (2×), 7.25 µL of polled assay mix (0.2×), and nuclease-free water. The reaction was performed using a Veriti Thermal Cycler (Applied Biosystems) during 14 cycles at 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 4 min.cDNA, previously pre-amplified, was diluted 1:10 in Tris-EDTA (TE) buffer 1× and added to a final qRT-PCR reaction volume of 20 µL, which contained TaqMan MicroRNA assay primers (Applied Biosystems) for each miRNA , TaqMan -∆Ct, where ∆Ct = mean CtmiRNA-mean Ct, and Ct = threshold cycle.After validating the Ct mean of the housekeeping genes, U6snRNA, miR-16, and miR-1228, as reference miRNAs, the relative levels of each specific miRNA was calculated using the equation 2The differences in circulating miRNA levels and the ROC curves were evaluated using IBM SPSS Statistics V22.0 . Kruskal-Wallis nonparametric test was used to perform a statistical analysis of serum miRNA levels; for post hoc pairwise comparison, U Mann-Whitney test was used. The median expression level of each miRNA between different groups was compared (cancer vs. HDs).For the multivariate analysis, Fisher linear discriminant analysis was employed. In order to establish the feasibility of this kind of analysis, assumptions of the model in terms of inequality of covariances and variances were checked with M-Box and Lambda of Wilks tests, respectively. The obtained discriminant function was used to classify the samples as tumor or normal. Performances of univariate and multivariate analysis were studied with the AUC of ROC curves.https://www.mirnet.ca/miRNet/home.xhtml) [In this analysis, miRNet tool was used (url: e.xhtml) .Briefly, this resource implements algorithms for differential expression and mining in miRNA databases in order to create a graph representing the relationships between miRNAs and its targets. Networks can be manually amplified and pruned. Finally, it implements hypergeometric tests to perform a gene set enrichment analysis with KEGG pathways .In conclusion, with this study, we have defined a series of circulating miRNAs that define different tumor types with a very high diagnostic performance. In addition, these miRNAs would constitute the basis of a multianalyte blood test through the SAPHELY detection system; however, their clinical utility should be demonstrated in a prospective study in the context of a CSP.


This study aimed to evaluate three cases of ocular SCC in additional breeds and determine if DNA testing for the DDB2 variant in warmblood horses and Connemara ponies is warranted. Histopathology confirmed ocular SCC in all three cases and DNA testing confirmed each horse was homozygous for the DDB2 risk factor. The DDB2 risk allele frequency was estimated to be 0.0043 for Holsteiners (N = 115), 0.014 for Belgian Warmbloods (N = 71), and 0.22 for Connemara Ponies (N = 86). Taken together these data support using DNA testing for DDB2 in Connemara Ponies to assist in mate selection and clinical management. Given the low observed allele frequencies in both the Holsteiner and Belgian Warmblood breeds and that the case under investigation was a warmblood cross-bred, evaluating additional SCC affected warmbloods is warranted to fully determine the importance of DDB2 genotyping as a risk factor in warmblood breeds.Squamous cell carcinoma (SCC) is the most common cancer affecting the equine eye. A missense variant within the gene damage-specific DNA binding protein 2  DDB2 c.1013C>T, p.Thr338Met) . ,16. DDB2MATP) and is thought to disrupt pigmentation by impairing normal trafficking of pigment granules within the developing melanocyte [MATP variant, in combination with their extent of UV exposure, would help to better understand the relationship between DDB2 and variants that dilute pigment, and how this impacts SCC risk and disease progression. Case 3, who was 17 when diagnosed, was older than the reported mean age, but also displayed a gray coat color, which is the progressive loss of pigment caused by a 4.6 kilobase duplication in an intron of the syntaxin 17 (STX17) gene [Other potential risk factors for SCC development have been studied previously, including age, sex, coat color, and equine papilloma virus type 2 infection . While rlanocyte . Horses lanocyte . Thus, a17) gene . Previou17) gene , yet howPrevious research has also implicated hormones as a risk factor for ocular SCC with geldings being overrepresented compared to stallions and mares in retrospective studies ,23,24,30DDB2 c.1013C>T variant for Holsteiner and Belgian Warmblood horses was determined to be relatively low (f(A) = 0.0043 and 0.014 respectively), these sample sets of horses were not available to be phenotyped for ocular SCC. While the likelihood of a purebred Holsteiner or Belgian Warmblood being T/T is low, as estimated by the allele frequency of DDB2 c.1013 C>T, the potential of a Holsteiner being a carrier was approximately 9 horses in a 1000 and was greater (approximately 3 in a 100) for the Belgian Warmblood. Due to the presence of the risk allele in Holsteiner and Belgian Warmblood breeds, this variant may be an important genetic risk factor for ocular SCC in multiple warmblood breeds. As the most common breed examined for ocular issues in France, retrospective data also support the evaluation of the Selle Français for genetic risk factors for ocular SCC. Clinical phenotyping of warmblood horses from diverse breeds, including Selle Français, along with DDB2 DNA testing, should be performed to further evaluate if loss of function mutations in DDB2 and UV-induced damage also explain increased risk for ocular SCC in warmblood horse breeds.While the allele frequency for the DDB2 c.1013C>T variant within the Connemara Pony was calculated to be 0.22. This is similar to that of the Haflingers, Belgian Drafts, and Rocky Mountain Horse  [The allele frequency for the ctively) ,16,17. Fctively) . While oThe DDB2 risk variant has now been identified and connected with ocular SCC in six distantly related horse breeds. Clinically evaluating horses for ocular SCC by examination and histopathology from breeds commonly presenting with ocular SCC, including the Selle Français and the American Paint Horse identified from the retrospective data presented in this report, in addition to genotyping the DDB2 variant in a diverse breed sample set phenotyped for ocular SCC, would assist in a better understanding of the breed distribution and connection to ocular SCC across equine breeds. Establishing breed-specific recommendations with regard to genetic testing would allow for the best use of this genetic test for mate selection in order to decrease the incidence of disease and identify which horses are at highest risk of developing ocular SCC so they can be monitored for early detection, increasing the likelihood of a more favorable outcome.


Hepatitis B virus (HBV) infection is a leading cause of liver disease worldwide. Hepatitis B surface antigen (HBsAg) rapid diagnostic tests (RDTs) could be an ideal tool for a large-scale HBV screening in settings with high endemicity but limited infrastructure. The aim of this study was to evaluate the diagnosis performance of such RDTs for screening HBV infection in Ivory Coast.  From September 2018 to January 2019, a cross-sectional phase I evaluation study of RDTs was conducted in three laboratories of Abidjan , on a panel of 405 whole blood samples and 699 plasmas. Four HBsAg RDTs  were evaluated. The diagnostic performance (sensitivity and specificity) was calculated in comparison to the reference sequential algorithms of two EIA tests (Dia.Pro HBsAg® one version ULTRA and Monolisa™ HBsAg ULTRA).  The Determine™ HBsAg and Vikia HBsAg® tests performed well, with 100% of sensitivity, specificity both on plasma and on whole blood. For SD Bioline HBsAg WB® and Standard Q HBsAg®, the specificities were 99.8% and the sensitivities 99.3% and 97.1% respectively. Finally, there were a total of 19 false negative results: 3 with SD Bioline HBsAg WB® and 16 with Standard Q HBsAg®.  Determine HBsAg® from Alere and Vikia HBsAg® from Biomérieux are the most suitable RDTs for screening for HBV in Ivory Coast. A phase II evaluation must be initiated. Hepatitis B virus (HBV) is a serious public health problem worldwide and a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). The World Health Organization (WHO) estimated that in 2015, 257 million persons, were living with chronic HBV infection in the world with 890 000 deaths from cirrhosis, and HCC , 8.48% iEarly diagnosis is critical in reducing hepatitis-related morbidity and mortality, as well as identifying candidates for HBV vaccination. HBsAg is used as the marker of infection for both HBV screening and to detect suspected acute cases or chronic hepatitis B in any community . HBsAg iAlthough some evaluations have been carried out in others countries , 10, no From September 2018 to January 2019, a cross-sectional phase I evaluation study of RDTs was conducted at the Institut Pasteur of Ivory Coast (IPCI), the Center for Diagnosis and Research on AIDS and other infectious diseases (CeDRes) and the National Blood Transfusion Center (NBTC). It was initiated by the Direction of Pharmacy, Drug and Laboratory (DPML) in association with the National Program of fight against the viral hepatitis according to the procedures used at national level. The selection criteria of tests were : colorimetric reading between one and two steps; performing tests on serum / plasma and whole blood obtained by venous or capillary sampling; sensitivity and specificity known in other regions of the world on plasma / serum / whole blood; test life greater than 12 months; storage and stability at room temperature (20-30°C).This study was carried out on serum / plasma and whole blood samples. According to WHO RDTs evaluation guide , six hunWe evaluated four HBsAg RDTs: DetermineTM HBsAg , SD Bioline HBsAg WB® , Standard Q HBsAg®  and Vikia HBsAg® . These qualitative tests are based on immunochromatographic techniques for lateral association of monoclonal and polyclonal antibodies specific for HBsAg. We performed the test according to manufacturer's procedure. All these tests give visual readout.OD) to the threshold value (TV). Samples with OD/TV values higher than or equal to 1.00 are considered reactive. Positive results obtained with Dia.Pro Ag were confirmed with the Monolisa AgHBs ULTRA®.HBsAg status of the panel serum/plasma samples has been determined using a sequential algorithm consisted of two commercially available enzyme-linked immuno-assay (ELISA) tests: Dia.Pro HBsAg® one version ULTRA  and Monolisa™ HBsAg ULTRA . The result unit used is a ratio of the sample optical density ,negative predictive value (NPV) and accuracy of these four kits were calculated with 95% confidence interval according to the existing formula . Kappa cThe performance of each of the four RDTs evaluated in this study is shown in Globally, there were a total of 19 false negative results: 3 with SD Bioline HBsAg WB® and 16 with Standard Q HBsAg® for a discordance rate of 0.36% and 1.54% respectively. Details are resumed in Many HBsAg rapid tests using immunochromatographic assays have been commercialized worldwide. The major challenge for these tests is to detect the low levels of the target antigen that are present in a relatively high proportion of asymptomatic carriers , 15. TheWe found that Determine™ HBsAg and Vikia HBsAg® tests had best technical performance. Global specificities obtained with Determine™ HBsAg and Vikia HBsAg® were similar to those reported by previous studies 100%) , 10, 16.0% , 10, Many studies revealed that the sensitivity and specificity of the SD Bioline WB kits were 94.1 to 100% and 99.3 to 100% for the HBsAg kit , 19, valThe disparity of results on the sensitivity and specificity of rapid tests could be explained by the existence of mutant viruses which have modified surface antigens (HBsAg), thus making their detection by routine immunological techniques impossible , 21. TheAlso lower HBsAg concentration and viral load could lead to a false negative reaction , 20, 21.Regarding the Standard Q HBsAg, no previous study, apart from the one reported by the manufacturer, has been documented, not allowing us to make comparison.According to current WHO procurement eligibility for HBsAg assays that require that RDTs assays might have a diagnostic sensitivity and specificity of > 99% and > 98% respectively , three oWe did not observe any significant difference in the performance of the tests evaluated with whole blood and serum/plasma. Njai et al. reported higher sensitivity in serum (88.5% vs 95.3%) but higher specificity in whole blood (100% vs 93.3%) [An important consideration of definitive laboratory diagnosis also relates to controlling for false-positive and false negative results. We noticed a low rate of false negative results only with SD Bioline WB® (0.5%) and Standard Q® (2.8%) in this study. On the contrary, other studies have reported false-negative results of Determine and Vikia HBsAg tests. They associated this with a low HBsAg concentration, HBsAg mutants, low viral load, and certain viral genotypes , 10, 21.In our study, the inter-reader variability and variability of results over time were not investigated. These parameters would have allowed us to appreciate the stability of the test results over time.Among the four RDTs evaluated in this study, Determine™ HBsAg and Vikia HBsAg® tests should be appropriate for HBV screening and marketed in Ivory Coast. These two tests are rapid, simple and very suitable for peripheral laboratories and provided excellent performances both on serum / plasma and on whole blood. As recommended by the World Health Organization, Ivory Coast must now start the phase II of evaluation of these two RDTs to validate these performances under field conditions in an environment characterized by limited resources and multiple public health priorities.


Nasal High Flow (NHF) therapy delivers flows of heated humidified gases up to 60 LPM (litres per minute) via a nasal cannula. Particles of oral/nasal fluid released by patients undergoing NHF therapy may pose a cross-infection risk, which is a potential concern for treating COVID-19 patients.Liquid particles within the exhaled breath of healthy participants were measured with two protocols: (1) high speed camera imaging and counting exhaled particles under high magnification (6 participants) and (2) measuring the deposition of a chemical marker (riboflavin-5-monophosphate) at a distance of 100 and 500 mm on filter papers through which air was drawn (10 participants). The filter papers were assayed with HPLC. Breathing conditions tested included quiet (resting) breathing and vigorous breathing . Unsupported  breathing and NHF at 30 and 60 LPM were compared.Imaging: During quiet breathing, no particles were recorded with unsupported breathing or 30 LPM NHF (detection limit for single particles 33 μm). Particles were detected from 2 of 6 participants at 60 LPM quiet breathing at approximately 10% of the rate caused by unsupported vigorous breathing. Unsupported vigorous breathing released the greatest numbers of particles. Vigorous breathing with NHF at 60 LPM, released half the number of particles compared to vigorous breathing without NHF.3). In quiet breathing with NHF at 60 LPM, small quantities were detected in 4 out of 29 quiet breathing tests, not exceeding 17 μL/m3. Vigorous breathing released 200–1000 times more fluid than the quiet breathing with NHF. The quantities detected in vigorous breathing were similar whether using NHF or not.Chemical marker tests: No oral/nasal fluid was detected in quiet breathing without NHF  releases 200 to 1000 times more oral/nasal fluid than quiet breathing . During vigorous breathing, 60 LPM NHF therapy caused no statistically significant difference in the quantity of oral/nasal fluid released compared to unsupported breathing. NHF use does not increase the risk of dispersing infectious aerosols above the risk of unsupported vigorous breathing. Standard infection prevention and control measures should apply when dealing with a patient who has an acute respiratory infection, independent of which, if any, respiratory support is being used.ACTRN12614000924651 Nasal high flow (NHF) has been increasingly used as an intervention for type 1 respiratory failure. Appropriate use of NHF has been shown to reduce intubation rates, which carries a risk of infection transmission for both patients , and heaConcerns have been raised about a potential risk of NHF spreading infection by generating aerosols and droplets when used to support patients with acute respiratory infections. NHF therapy is classed by some countries as an aerosol generating procedure (AGP). A systematic review of AGP and risk of transmission of acute respiratory infections (ARI) to healthcare workers (HCW) reported that more invasive respiratory procedures such as tracheal intubation, tracheotomy and manual ventilation were a significant risk factors for SARS transmission . This waSome countries or healthcare systems have guidance against using NHF for patients with COVID-19 infection, while others are using NHF as a first line therapy. Interestingly, there does not appear any substantive evidence of NHF causing large dispersions of infectious aerosols and infecting heathcare workers. In fact, the low number of relevant dispersion studies and lack of understanding of infection transmission has been highlighted in three recent publications –7.This paper compares the release of particles of oral/nasal fluids during quiet resting breathing, snorting, voluntary coughing and voluntary sneezing, both in the absence of respiratory therapy and when receiving NHF at 30 and 60 LPM (litres per minute). Two techniques are used to determine volumes of oral/nasal fluid released: high speed optical video microscopy and air sampling with a chemical marker instilled into the nose and mouth. It is our hope that this data will inform evidence-based decisions about using NHF, particularly for patients with ARI.Ethical approval was obtained from the Upper South B Regional Ethics Committee, Ministry of Health, New Zealand URB/09/12/064 and the University of Canterbury Human Ethics Committee (refs. 2009/173 and HEC 2017/105). Written, informed consent was obtained from all subjects.In the terminology commonly accepted in healthcare, aerosols are suspensions of small particles, including droplet nuclei, with an aerodynamic diameter of 10 μm or less. Droplets are particles with an aerodynamic diameter of > 10 μm. Airborne transmission in a healthcare setting refers to transmission by aerosols of < 10 μm . Particl™  NHF cannula. Air flows conditioned to 37°C and 100% relative humidity (44mg/L) were generated with an AIRVO™ 2 humidifier-flow generator . Participants were instructed to breathe with their mouth closed to create higher nasal velocities more likely to release particles. All participants spent at least five minutes acclimatizing to NHF at 30 LPM. Prior to each measurement, 1 mL of 0.9% NaCl solution was instilled into each nostril to artificially moisten the nasal mucosa, as the healthy volunteers do not have the increased mucus production common to viral respiratory infections. Saline has a lower viscosity than nasal mucous. Exposed to the same forces, a lower viscosity fluid will yield a greater mass of droplets [Six healthy volunteers participated: three females aged 20 to 27 years, and three males aged 26 to 59 years. Participants wore a medium size Optiflow™, 1040 frames per second, shutter speed of 40 μs and resolution 1280×1024 pixels, Redlake Imaging Corp.). The region was backlit such that particles and the outline of the nose cast a shadow onto the camera  for particle frequency, diameter and velocity. The number of particles were recorded in bins with diameter of 0–50, 50–100, 100–150, 200–500, 500–1000, 1000–2000 and 2000–5000 μm. The smallest detectable particle occupies one pixel i.e. has a diameter of 33.0 μm.The six experimental conditions, each conducted in triplicate on each nostril, were:Quiet breathing (at rest) with no therapy, with 30LPM NHF, and with 60LPM NHF.Voluntary snort  with no therapy, with 30LPM NHF, and with 60LPM NHF.™+ cannula  was selected, and was used with an Airvo™2 humidifier set to 37°C and 100% relative humidity (44 mg/L). Immediately before each measurement, the participant instilled 0.5 mL of marker solution into each nostril. 0.5 mL was assumed sufficient as excess ran out of the nose. For coughing and sneezing, the participant placed an additional 1 mL onto their tongue, via pipette. The marker solution was 2.8 grams of riboflavin 5’ monophosphate sodium salt hydrate  per litre of deionized water.Ten healthy volunteers  participated. Participants sat in a chair with a headrest. Each participant carried out the sequence of actions described in Air was sampled at a distance of either 100 mm or 500 mm from the participant’s nose, using 125 mm diameter qualitative filter paper  supported on an acrylic grille through which air was drawn at 2.2–18.1 LPM with a vacuum pump. The air flow rate was measured with a TSI 4040 meter . This sampling system was placed to intercept the maximum exhaled nasal velocity, determined for each test after the participant found a position they could maintain comfortably for the duration. The filter papers were stored in Petri dishes (used as delivered in clean sterile packaging) and frozen for later analysis. A clean grille was used after each block of quiet breathing tests, and after each snort, cough or sneeze.Background measurements (7 min duration) were run before and after each participant. No riboflavin was detected in any of these, indicating the room ventilation was adequate to prevent contamination between tests.® 3000 pump, and Ultimate® 3000 fluorescence detector. 20 μL samples were injected into a Kinetex 5 μm C18 100 Å, 100 x 2.1 mm LC Column . The mobile phase was 95% NH4HCO3 and 5% methanol. Samples were run at a column temperature of 35°C, pressure of 62 bar and an isocratic flow rate of 0.2 mLPM for a total run time of 36 minutes.For quantitative analysis, the samples were brought to room temperature and riboflavin was extracted from the filter paper using 3.5 ml of Milli-Q water  with 5 minutes of gentle agitation. 1.5 mL of the supernatant was drawn off and expelled through a 0.45 μm filter into HPLC vials. These were analyzed with a Dionex UHPLC Focused  fitted with an autosampler, UltimateA six-point external standard calibration curve  of 95% purity riboflavin 5’ monophosphate  was used for the quantification of samples and the calibration standards were analyzed prior to starting the sample sequence. Milli-Q blanks were analyzed after the calibration standards and after every 14 samples to ensure there was no carry-over from the previous runs. A 100 ng/mL check standard was run after every sample sequence to confirm the validity of the calibration curve. Calibration standards and samples were analyzed in duplicate aliquots. Chromeleon (c) Dionex (Version 7.2.7.10369) software was used for peak fitting and calibration.The concentration of oral/nasal fluid emitted by the participant and collected by the sampling system was calculated using the concentration of the riboflavin, the air flow rate through the filter paper, the duration of the test, and the volume of Milli-Q water used to extract the riboflavin.The duplicate aliquots agreed within 4% except for two instances in which four overlapping peaks were fitted at the riboflavin retention time.The detection limit for riboflavin, estimated based on the signal to noise ratio in the chromatogram (S/N = 3) was 2.75 ng/mL. The concentration of oral/nasal fluid in air required to meet this detection limit is given in (1) Imaging. During quiet breathing with no therapy and with 30 LPM NHF, no particles were detected for any of the participants tested. Particles were detected during quiet breathing with 60 LPM NHF, for two of the six participants.When snorting, five out of six participants produced visible particles in all conditions. An example image is shown in The number of particles detected , are given in (2) Chemical marker. The calculated quantities of oral/nasal fluid in the air captured by the sampler are given in Only the obvious difference in quiet versus vigorous breathing was significant (p < 0.001). There were no significant differences due to therapy, nor between types of vigorous breathing, nor due to distance (quiet only), nor due to therapy order, and there were no two-way interactions between these factors.Oral/nasal fluid was not detected in quiet resting breathing without NHF therapy. Oral/nasal fluid was detected 4 times in 29 tests in quiet breathing with NHF therapy. It never exceeded 17 μL per cubic metre of sampled air, and counting only those instances where it was detected, it was at a mean level of 6.3 μL per cubic metre of sampled air. This was 1/200 th of the average level detected in snort, cough or sneeze. It was detected in two cases during quiet breathing where this was the first action the participant did, and in two cases where they completed no-therapy actions first.With no therapy, oral/nasal fluid was detected in quantities exceeding 633 μL per cubic metre in all snorts, nine out of ten coughs, and nine out of ten sneezes.With NHF (60 LPM), oral/nasal fluid was detected in quantities exceeding 483 μL per cubic metre in all snorts, 9 out of 10 coughs, and all sneezes.Snort, cough and sneeze all release the same order of magnitude of oral/nasal fluid. The quantities detected in with-therapy and no-therapy cases are of similar order of magnitude.To our knowledge this is the first study to compare the optical video-microscopy and chemical marker methods for oral/nasal fluid dispersion in the presence and absence of NHF. It was evident from both measurement modalities that high energy respiratory maneuvers like coughing, sneezing and snorting generated high volumes of particles, either with or without NHF, compared to quiet breathing. Higher expired flow velocities are expected to generate stronger shear forces over the mucosa, generating more airborne particles .During vigorous breathing there were fewer particles produced with NHF than without NHF. This was true at both flow rates in the imaging tests, and true when averaged over snort, cough and sneeze in the marker tests. NHF’s apparent mitigation of particle generation during vigorous breathing may be explained by particles impacting on the cannula interface. Another possible explanation is that NHF imposes an expiratory flow resistance that increases expiratory time  and reduOn the points on which they are directly comparable, the results of the two methods agree qualitatively that (1) quiet breathing with no therapy did not produce detectable aerosol, (2) some individuals produced aerosol during quiet breathing with 60 LPM therapy, (3) snorting usually produced aerosol, at much greater masses than quiet breathing, and (4) snorting with and without therapy produced particles at the same order of magnitude of mass, but lower mass with therapy.3 in half a minute i.e. . Quiet breathing with NHF at 60 LPM produced on average 1.72 μL/m3 in 7 minutes (0.246 μL/m3/min). It would take 86 hours of quiet breathing with NHF at 60 LPM to release the same quantity of oral/nasal fluid as a minute of coughing with no therapy . Cough is a common symptom for coronavirus, with a recent study reporting an average cough frequency of 17 events in a 30 min test [For the oral/nasal fluid collection, it should be noted that with quiet breathing alone there were no detectable levels in the twenty nine measurements, while the addition of 60 LPM NHF increased this to four out of twenty nine tests with detectable volumes. However, these quiet breathing measurements were taken over a seven-minute period and produced 200 to 1000 times lower volumes of oral/nasal fluid than the higher energy respiratory maneuvers of coughing, sneezing and snorting achieved in a 30 second period. Looking at this another way, coughing with no therapy produced 633 μL/mmin test .3 of air. For particles of 1.75 μm, this detection limit is 10 particles per cm3. This is greater than the concentrations produced by breathing and speaking, though in breathing the particles are thought to be produced in the bronchioles [SARS-CoV-2 RNA has been detected in particles of 0.25–4 μm diameter , 15. At nchioles , and theIt has been postulated that a higher clinical acuity Acute Respiratory Distress Syndrome (ARDS) patient will generate more particles due to higher work of breathing, increased closing capacity and altered respiratory tract fluid . In a reOne study had five healthy participants sit in a chair, gargle 10 ml of food coloring then cough, and measure how far the visible droplets went on paper on the floor. They reported that the discernable particles traveled on average 2.48 m with no therapy and 2.91 m with NHF at 60 LPM . While tThis study’s findings are in general concordance with these other published studies of particle dispersion with NHF. However there is still a dearth of high quality data regarding aerosol and droplet generation, and this should be resolved to improve the understanding of the possible mechanism of transmission, and to inform evidence based guidelines for health care workers treating patients infected with Covid-19 and related diseases .Many clinical interventions and therapies are considered to have the potential to generate aerosols, including standard oxygen therapy, non-invasive ventilation, intubation, nebulisation and NHF therapy. It is important to gain a full understanding of the potential benefits of using any of these therapies in the context of any associated risks. The mechanism of droplet and aerosol virus transmission is also not fully understood. The emerging evidence suggests the generation of numbers of particles increases with the vigor of the respiratory activity.The potential benefits of using NHF therapy should be weighed against the risks. One of the primary indications for use of NHF is treating type 1 respiratory failure. In a large RCT conducted in 23 ICUs in France and Belgium involving 310 participants, NHF was reported to reduce intubation rates compared to non-rebreather face mask or NIV for a subgroup of those with Acute Hypoxic Respiratory Failure (AHRF) and a PaO2:FIO2 of 200 mm Hg or less, of whom the majority had community acquired pneumonia [A systematic review of SARS-related literature conducted to understand the risk of transmission to healthcare workers from aerosol-generating procedures , reporteThere is speculation that NHF may increase the chances of aerosol generation and COVID-19 transmission. However, there is a lack of evidence to support that NHF is any worse than other low risk respiratory therapy such as oxygen therapy or NIV, and its use may avoid the need for high risk procedures such as intubation.This study had numerous limitations: the sample size is small, all participants were healthy; there was a narrow age distribution  releases 200 to 1000 times more oral/nasal fluid than quiet breathing.60 LPM NHF does not make the levels of oral/nasal fluid emitted during cough, snort or sneeze greater compared to unsupported breathing.We do not find evidence for large numbers of particles being dispersed by NHF, compared to cough or sneeze. NHF is a useful therapy for Type 1 Respiratory failure and reducing escalation to high infection risk procedures like intubation.Vigorous breathing without NHF is the worst-case scenario that should drive infection prevention and control measures. The use of NHF, per se, does not increase the risk of generation of infectious airborne aerosols above the risk of patient-generated aerosols. Adherence to standard contact and droplet precautions are likely to be sufficient when NHF is applied.S1 Data(XLSX)Click here for additional data file.


Salmonella enterica lytic bacteriophages, NBSal006 and NBSal007, candidates for Salmonella biocontrol.The use of bacteriophages as antimicrobial agents represents a promising alternative for the control of pathogenic bacteria. Here, we present the complete genome sequences of two novel  Salmonella enterica lytic bacteriophages, NBSal006 and NBSal007, candidates for Salmonella biocontrol.The use of bacteriophages as antimicrobial agents represents a promising alternative for the control of pathogenic bacteria. Here, we present the complete genome sequences of two novel  Salmonella enterica is a Gram-negative pathogen recognized as one of the most common causal agents of foodborne infections  as the host; 3 passages were required to identify single plaques. Isolated phages were amplified by incubating phage and Salmonella cultures for 18 h at 37°C in tryptic soy broth or agar.To isolate bacteriophages, 50 g of waste samples was collected from Peruvian poultry ; these samples were stored at 4°C until their use in phage isolation. For phage isolation, the soft agar overlay method was performed as described by Adams , using SPhage genomic DNA was extracted using a modified phenol-chloroform method, including a DNase step to limit bacterial DNA contamination . The QIASiphoviridae family, particularly NBSal006 with the previously described phage Shelanagig  and phage NBSal007 with the phage vB_StyS-sam  The numbers of sequencing reads generated for NBSal006 and NBSal007 were 5,008,144 and 5,111,521, respectively; phage NBSal006 had a genome of 41,850 bp with 3,915× coverage, and NBSal007 had a genome of 43,747 bp with 4,241× coverage. Both phage genomes had a G+C content of 49.8%. The genomic annotation for the phages presented 56 coding sequences with assigned function out of 66 annotated coding sequences for NBSal006 and 54 out of 57 for NBSal007. Moreover, no tRNA coding sequences were identified in the phages. Both phages presented circularly permutated genomes with redundant ends and a headful packaging mechanism. The genomic relation to other phages was evaluated with a nucleotide BLAST search, which showed notable similarity across the entire phage genome with phages from the Salmonella enterica lytic phages with the potential to be used in the development of a phage-based antimicrobial for the biocontrol of Salmonella enterica in poultry.The discovery of new phages with strong lytic activity is an essential step in the development of phage-based antimicrobial products. The study of the new phages’ genomes enables us to have a better understanding of their biology and have a solid foundation to conduct further functional studies on them. Here, we present the complete genome sequences of 2 MT677933 and SRR12133959  and MT677934 and SRR12127194 .The genome sequences and raw data have been deposited in GenBank under the accession numbers