Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 μg/L)/linuron (9 or 45 μg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological  and physiological  features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained  One of the most critical wildlife conservation issues today is the global amphibian decline. In the last comprehensive global assessment 2004), one third of amphibian species were reported as threatened with extinction and more than 40% were experiencing population declines, a level greater than for any other vertebrate taxa . Further004, one in situ  has sometimes been used to infer negative impacts on male reproductive fitness, however, evidence suggests they may not necessarily be linked (for review see:  and gr [glucocorticoid receptor] in brain/forelimb/testis) and hormone levels (androgens/corticosterone) with reproductive success in males, as defined by males gaining amplexus with a female under competition and the fertility rate of winning frogs. We conducted these assessments within groups of unexposed animals and within groups of frogs exposed to anti-androgenic chemicals (flutamide (50 μg/L), linuron (9 or 45 μg/L)). The driving aim of this work was to investigate whether features or morphology/physiology in adult frogs could predict reproductive outcomes in those frogs, including under exposure conditions. Anti-androgenic chemicals are particularly relevant for effects on male reproductive success ) and the right testis and right forelimb were dissected and fixed in neutral buffered formalin (4%) for histological analysis. The physiological endpoints analysed were nuptial pad histomorphology , testis weight, testicular histomorphology , gene expression  and plasma hormones .Frogs were euthanised after the second breeding trial, they were weighed and their SVL measured. The frogs were then sacrificed by decapitation under anaesthesia , blood was collected for hormone analyses, the left forelimb/brain/gonad were dissected and placed in RNAlater for analysis of gene expression , sectioned (at 2 μm) and stained with haematoxylin-eosin for analysis using light microscopy  and samples were sectioned (5 μm) and stained with haematoxylin-eosin for analysis using light microscopy (Novex B-range). Transverse sections were cut along the entire length of the arm and three sections, at one quarter (25%), halfway (50%), and three quarters (75%) of the way through the tissue were selected for analysis. The total number of special mucous glands/breeding glands , the number and size of normal mucous glands/serous glands  and keratinised hooks (protruding hook features) from the 3 selected sections were counted. See S3 Fig in Gene expression analysis. Tissue samples  were homogenized and lysed, RNA extracted/isolated, and cDNA synthesised. cDNA samples were tested in two independent PCR runs. Expression levels of genes related to reproductive behaviour were analysed (ar and gr) using RT-qPCR, and with efficiency-corrected normalisation to the rpl8 housekeeping gene. Each sample was run in duplicate and a no-template control (NTC) sample with water instead of cDNA was included throughout. qPCR primers were optimised using standard curves and had efficiency of 85–115 and r2 > 0.9. Melt curve analysis was used to determine amplicon specificity and the absence of amplification in the NTCs (4 runs/plates were excluded). See S5 Methods and S1 Table in Plasma hormone analysis. Following euthanasia, blood was collected in heparinised capillary tubes by cardiac puncture, centrifuged  and the plasma withdrawn and snap frozen in liquid nitrogen. Upon thawing, plasma was extracted twice with ethyl acetate , evaporated under nitrogen gas, and resuspended in 2:1 volume ethanol:plasma ratio, for use in hormone assays . The final dilution for assays was x200 for both corticosterone and for testosterone/dihydrotestosterone. Corticosterone was quantified using radioimmunoassay  and testosterone/dihydrotestosterone  with enzyme-linked immunoassay . All samples were run in duplicate in each assay. The intra-assay variability for corticosterone was 9.3% (2 assays) and for dihydrotestosterone/ testosterone was 6.9% (4 assays).ar/gr, plasma testosterone /corticosterone, testis weight and testis histomorphology ) on winning the competition for amplexus with the female  and for fertility rate (percentage fertilised eggs–Gamma distribution). For the amplexus models, data from both frogs in each pair (winning and losing frogs) were included as these frogs were in competition with each other. For fertility models, only data from the winning frog was included using the assumption that only the winning frog contributed to the fertility observed (see p > 0.17) and therefore breeding trial data from the within group and between group (unexposed versus exposed) conditions were combined. This was done to increase the n number, which was needed to be able to run the models, however, ‘treatment’–defined as belonging to linuron low, linuron high or flutamide treated groups–was included as a covariate in order to control for any non-significant subtle effects of treatment on model outcomes. Due to the large number of endpoints, in order to be able to run the models, variables were grouped for analysis within categories . See S2, S3 Tables in p-value of 0.05, for both frogs (amplexus success models) or for the winning frog only (fertility models). Finally, a simple GLM (Gaussian) was used to investigate associations between nuptial pad colour and other measured endpoints by combining all measured endpoints from other categories  and then sequentially removing variables which contributed least to the data variability until the best fit for the remaining data was achieved .Generalised linear regression modelling (GLM) implemented in the Bayesian framework was used to analyse output from the breeding trials, with data from the first and second breeding trials analysed separately. GLM was run to analyse the effects of morphology , forelimb width/size (length x width)) and physiology  and the covariate “treatment” was not associated with reproductive success (amplexus/fertility) in any models run. No associations for the covariate ‘interval’, which were included in amplexus success models for the second breeding trial, were observed. The covariate ‘egg number’  was not associated with fertility rate in unexposed frogs but was positively associated for some of the models run in both the first and second breeding trials for linuron low/high exposed frogs ) and egg laying occurred by the time of the fourth observation (195 minutes (60 + 4 5+ 45 + 45)). In 7 out of the 148 trials there was no spawning event and these samples were excluded from the analyses. In 118 (84%) of the remaining 141 trials, only one male was observed in amplexus during spawning and this ‘winning’ male was attributed the observed fertility. For the remaining 16% (23/141) of the trials, it was unclear which male won the competition for the female and these data were also excluded from the subsequent analyses. Amplectant frogs naturally separated 6 hours after initiation of amplexus in 82% of cases. There was no effect of treatment on the amplexus success when in competition with unexposed control frogs , positively associated with forelimb width/size (linuron low) and positively associated with nuptial pad proportion . In the ar (The number of keratinised hooks  and forear  were botar  and no car .ar (linuron low) and testis ar (linuron high) were negatively associated with amplexus success . The positive relationships between these endpoints and amplexus success are not surprising, albeit we could find only one previous study in frogs showing a link between these features and reproductive success. Specifically, this was the case where wild male Columbia spotted frogs (Rana luteiventris) in amplexus were shown to have larger nuptial pads than unpaired males [Much research has established that androgens are elevated in reproductively active wild male amphibians  and hereed males . Althouged males  and the ed males , 17. Boded males , howeverar, lower SVL). This was surprising and we cannot currently hypothesise possible explanations for these findings since to the authors’ knowledge, this is the first study to investigate associations between morphology/physiology and amplexus success in laboratory exposed anurans. For the darkened nuptial pad, a plausible explanation for why we only observed this in the second breeding trial could be that the forelimb tissue had become sensitised because of the injection of hCG to induce breeding in the first trial leading to darkened nuptial pads in the second breeding trial. Although the mechanism driving nuptial pad colouration is not known, and proposed sensitisation is a hypothesis only, it is borne out by the fact that nuptial pads in the breeding males were overall significantly darker in the second breeding trial compared to the first breeding trial . As this feature was consistent in predicting amplexus success in male frogs in the second breeding trial and thus with potential as a biomarker of reproductive success in non-naïve wild male frogs, we further investigated whether nuptial pad colour was associated with other measured endpoints . We found that a darker nuptial pad was associated with greater numbers of spermatogonia in the testis and higher plasma androgen levels, whereas a lighter nuptial pad was associated with more spermatocytes and more spermatozoa  and incalamita: ) in popuS. tropicalis exposed to ethinylestradiol in the laboratory [ar/gr mRNA levels) concentration exposures. This indicates that these features may have potential as biomarkers of fertility rate in wild anurans exposed to pollutants, particularly for forelimb/nuptial pad morphology, which could be applied in vulnerable populations since they can be measured non-destructively. However, similarly to the conclusions related to nuptial pad colour, at the present time the utility, or not, of these features are not known.None of the measured morphological/physiological features were associated with fertility rate in unexposed frogs, indicating that amongst the winning frogs, measured endpoints did not influence fertility rate. To the authors’ knowledge, there are no previous studies that have specifically investigated relationships between morphology/physiology and fertility in wild or laboratory amphibians, though reduced spermatozoa was found in parallel with reduced fertility in boratory . In the boratory , 14, we The findings from this study demonstrate some of the challenges of developing a biomarker of reproductive success in anurans. There are clear indications that androgen sensitive features are linked with individual male reproductive success. However, at present it is not possible to ascertain how these markers could be used for field studies where the influence of pollutants is either a likely causative factor in declines or for which levels are unknown. It would be useful to analyse for correlations between nuptial pad colour/forelimb width with fertility rate in amplectant and lone males in wild anuran populations in unpolluted/polluted regions to gain more clarity on the functional relationships between these factors and how they are altered by pollutants. Since these features can be measured non-destructively, these investigations could also be carried out in threatened/declining populations. Other useful developments for better establishing links between the role of androgens in fertility rate of wild frogs include the application of urine collection methods for measuring androgen levels non-destructively . We suggS1 File(PDF)Click here for additional data file.


We report the case of a 28‐year‐old woman with history of lupus treated by Mycophenolate Mofetil and admitted for febrile meningeal syndrome associated with diplopia. Both initial and control brain MRI showed pseudo‐meningiomatous lesion while second CSF microscopy revealed characterizing images of Cryptococcus neoformans. Our case report emphasizes that there is a rare case of cryptococcal meningitis presenting with meningioma‐like lesion especially in the immunocompromised population. Figure .Mycobacterium tuberculosis returned negative. A treatment consisting of a combination of ceftriaxone (6g/day) and aciclovir (10 mg/kg every 8 hours) was initiated.Cerebrospinal fluid (CSF) microscopy performed without Indian ink preparation revealed a lymphocytic meningitis with hyperproteinorachia (2.74 g/L), hypoglycorrhachia (0.19 g/L) but no organisms were seen. CSF culture was sterile and polymerase chain reaction for Cryptococcus neoformans.One week later, the patient's status worsened with deterioration of consciousness associated with generalized seizures of frontal lobe onset. Second Brain MRI highlighted a right frontal extra‐axial annular lesion with peripheral enhancement associated with hydrocephalus Figure . CSF micAfter 10 days of fluconazole (400 mg/day)None declared.AH: Design and conceptualization, data collection and analysis, wrote and drafted the manuscript. OA: analyzed the data and revised the manuscript.


P < 0.05) after the 3 min TT compared with either the 10 or 40 min TT when measured at 1 or 2 min of recovery. MVC force was not different between trials. The results demonstrate that recovery of peripheral fatigue including low-frequency fatigue depends on the duration and intensity of the preceding self-paced exercise. These differences in recovery probably indicate differences in the mechanisms of fatigue for these different TTs. Because recovery is faster after a 3 min TT than a 40 min TT, delayed assessment of fatigue will detect a difference in peripheral fatigue between trials that was not present at exercise cessation.The kinetics of recovery from neuromuscular fatigue resulting from exercise time trials (TTs) of different durations are not well-known. The aim of this study was to determine if TTs of three different durations would result in different short-term recovery in maximal voluntary contraction (MVC) and evoked peak forces. Twelve trained subjects performed repetitive concentric right knee extensions on an isokinetic dynamometer self-paced to last 3, 10, and 40 min (TTs). Neuromuscular function was assessed immediately (<2 s) and 1, 2, 4, and 8 min after completion of each TT using MVCs and electrical stimulation. Electrical stimulations consisted of single stimulus (SS), paired stimuli at 10 Hz (PS10), and paired stimuli at 100 Hz (PS100). Electrically evoked forces including the ratio of low- to high-frequency doublets were similar between trials at exercise cessation but subsequently increased more ( Self-paced endurance exercises of various intensities and durations lead to fatigue . AlreadyThe interaction between performance and perceived fatigability was evident in our pacing study, in which self-paced force, neuromuscular activation, and perceived exertion increased in the end-spurt despite decreasing levels of evoked forces  and possibly increasing levels of metabolites . Hence, A reduction in maximal voluntary contraction (MVC) force is often termed neuromuscular fatigue . Neuromu2+ release from the sarcoplasmic reticulum   and duri2+-related mechanisms. [Ca2+] in myoplasm is decreased, probably because of impairment of Ca2+ release from the sarcoplasmic reticulum. In addition, submaximal force production is influenced by phosphorylation of the regulatory light chains of myosin (RLC). The increase in RLC phosphorylation during exercise, causing increased sensitivity to Ca2+, enhances submaximal contractile response  and 1, 2, 4, and 8 min after completion of the TTs. We hypothesized that evoked peak force would increase more, relative to pre-exercise, for short compared with long TT and that this difference would be apparent within the first 1–2 min following exercise cessation.The present study is the second part of an experiment investigating neuromuscular fatigue and self-paced TTs of different durations. Detailed description of the procedures for warm-up and the experimental TTs are described in full in Twelve subjects  trained in both endurance and strength (training > 7 times a week) participated in the study. Subjects with previous or current injuries of the right leg were excluded. The study was approved by the Regional Ethics Committee Vest in Norway (2012/1486) and the Research and Ethics Committee of the Faculty of Health Science of the University of Cape Town, and the experiments were performed according to the latest (2013) revision of the Declaration of Helsinki. Subjects were instructed to refrain from high-intensity exercise on the day prior to testing and to refrain from alcohol during the last 24 h before testing. Subjects were also instructed to eat a light meal 2–4 h before arrival at the laboratory. Written informed consent was obtained from all subjects who participated in the study. Subjects were given a full explanation of the details and rationale of the study and were informed that they were free to withdraw at any time. The possibility that electrical stimulation might cause discomfort was fully explained, as was the nature of the risks involved.Subjects made nine visits to the laboratory, including six familiarization and three experimental trials separated by 2–3 days. During all familiarization trials, the participants were subjected to neuromuscular function (NMF) assessment and performed a self-paced TT with the right leg, which was always the dominant leg on the dynamometer  . During Subjects were secured to the dynamometer by chest and hip straps to avoid excessive movements . The seaTwo minutes following concentric and isometric warm-up, and five maximal concentric contractions, NMF assessment  started.Pre-TT NMF consisted of three MVCs with a 1-min break between MVCs. SS, PS100, and PS10 were performed after the first two MVCs. PS100 (superimposed doublet) was also applied when the subject produced maximal force during the third MVC. Owing to this sequence of activations, it is assumed that SS, PS100, and PS10 were similarly potentiated for all measures (pre-TT and post-TT).Post-TT NMF  , and each contraction consisted of a knee extension phase at 60 deg⋅smulation  followedA high-voltage  constant current stimulator  triggered by a Power Lab, and Lab Chart software  was used to deliver square-wave stimuli of 1-ms duration. The femoral nerve was stimulated percutaneously via a 10-mm-diameter self-adhesive cathode electrode  pressed manually by the investigator onto the skin at the femoral triangle. The triangle is a subfacial space, which appears as a depression when the thigh is flexed. The anode, a 130 × 80-mm self-adhesive electrode  was applied to the gluteal fold. The stimulation intensity for one SS was determined by increasing the current gradually from 5 mA until a plateau in force (15–35 mA) and maximal M-wave amplitudes were reached. The current was then increased by 30% to ensure supramaximal stimulation. The stimulation current was kept constant for the same subject for all types of electrical stimulation.Force in response to electrical stimulation, as well as voluntary force (isometric and concentric), was detected by a load cell mounted on the lever arm of the dynamometer. The reliability of the dynamometer has been described elsewhere . Force fEMG signals from the vastus lateralis (VL) and vastus medialis (VM) of the right leg were recorded using DE-2.1 single differential surface sensors with 10-mm inter-electrode distance . Delsys recommendations were used for the placement of the sensors on the skin. Sensors were placed in a direction parallel to the general direction of the skeletal muscle fibers. The reference electrode was applied to the patella. EMG signals were sampled at 2,000 Hz, amplified  using Bagnoli-8 (Delsys Inc.), and filtered using a band-pass filter with a bandwidth of 15–500 Hz in Lab Chart Pro software, version 7.3.8 (ADInstruments).MVC force was measured for 1 s during the period of peak force development, that is, 500 ms before and after peak force. The largest MVC force measured before the trials was taken as the pre-TT MVC. The peak force responses to electrical stimulation are reported as evoked peak force, and a reduction in evoked peak force is a measure of peripheral fatigue. Evoked peak force for PS10/PS100 was calculated as an index of low-frequency fatigue .–1)  of the EMG data of VL and VM was calculated for 1 s during the period of peak force of the MVC, that is, 500 ms before and after peak force. M-wave peak-to-peak amplitude (PPA) in response to SS was assessed. RMS during the MVCs was normalized by dividing it with the M-wave PPA of the following SS to estimate neuromuscular activation (RMS⋅M–1) . Because VM data . In sevetivation , whereastivation .post-hoc assessments were used to detect differences over time  and between distances , [time × condition]. Where the assumption of sphericity (Mauchly’s test) was violated, the Greenhouse–Geisser epsilon correction was applied to the degrees of freedom. Statistical tests for the different parameters over the recovery period were performed with values expressed as a function of baseline or end-exercise values. Comparisons of absolute values were performed only between baseline and end-exercise data . Effect sizes for the different ANOVA effects were calculated as eta squared, representing the proportion of total variance explained by the factor (range 0.0–1.0). The statistical significance was defined at P < 0.05.All analyses were performed using GraphPad Prism, v8.02 . The results are presented as mean ± SD unless otherwise noted. After the normality of data distribution was checked using the Shapiro–Wilk test, two-way repeated-measures ANOVAs with Tukey P < 0.0001) without time × trial interactions  = 6.147, P = 0.02, η2 = 0.064], when it was lower than the values following the 3-min TT (P = 0.02). M-wave PPA decreased following the 40-min TT (P = 0.0003), whereas it increased after the 3-min TT (P = 0.02), so that values at cessation of exercise were different between all trials .Absolute values pre-exercise and at exercise cessation, and after 1–8 min of rest are presented in ractions . In contP < 0.001 vs. end-TT). MVC force recovery was not different between the TTs during the 8-min rest period and increased at each subsequent interval (P < 0.001) during the first minute after exercise cessation for all TTs relative to values measured at end of TT (P < 0.05) for the 3 min TT compared with either the 10-min or 40-min TT after 2 and up to 8 min of exercise cessation.The different fatigue-related parameters expressed as% change from the end-TT values are presented in interval . Evoked nd of TT . Evoked P = 0.018; PS10 11.8 ± 2.7 percent points, P = 0.0026; PS100 5.8 ± 1.9 percent points, P = 0.0247; P = 0.0007; PS10 13.2 ± 2.1 percent points, P = 0.0001; PS100 6.3 ± 1.5 percent points, P = 0.0247). These differences persisted until 8 min after the TT. The force ratio for PS10/PS100, a measure of low-frequency fatigue, was also not different between the trials at exercise cessation, but already 1 min after exercise cessation, the ratio had increased more after the 3-min TT compared with both the 10-min  and 40-min TT , and the difference was also sustained throughout the evaluated recovery period.Evoked peak force responses to all types of electrical stimulation  in percentage of pre-exercise were not different between the trials at exercise cessation, but 1 min after exercise cessation, evoked peak forces had already increased more after the 3 min TT compared with after the 40-min TT  = 3.637, P = 0.0085, η2 = 0.052] after the 40-min TT than after 10-min  and 3-min TT , but this difference was not significant after 1-min recovery  = 6.604, P = 0.0009, η2 = 0.013] for the 40-min compared with 10-min TT , which in turn was also lower than the 3-min TT . These differences persisted until the 8th minute of recovery, when the 40-min TT was no longer different from the 10-min TT .MVC RMS⋅Mrecovery . M-wave The main aim of the present study was to compare the short-term recovery in MVC and evoked peak forces between self-paced TTs of three different durations. The most important findings of this study were as follows:i.Evoked peak forces  recovered more after the 3-min TT compared with the 40-min TT.ii.The ratio for force for PS10/PS100, an index of low-frequency fatigue, recovered more after the 3-min TT compared with the 40 min TT.iii.MVC force continued to improve throughout recovery whereas evoked responses improved only for the first 1–2 min after exercise cessation.At the cessation of exercise, the similar evoked peak force between the TTs can be explained by a relatively similar end-spurt in force. Unpublished data collected during the TTs showed that self-paced force during the last four sets (3 min) of the TTs were 64, 61, and 59% of the pre-exercise values for 3, 10, and 40 min TT, respectively. Additionally, M-wave PPA, an index of neuromuscular propagation , was morDespite similar values at exercise cessation, evoked peak forces recovered more, within the first minute after exercise cessation, following the short compared with the longer TT, providing strong evidence that the rate of the recovery processes is influenced by the duration–intensity characteristics of the preceding self-paced exercises. This is in accordance with what we hypothesized based on comparison of previous studies, specifically that peripheral fatigue persists for a long time after long distance endurance exercise , whereasP < 0.001) in 1 min than the response to high-frequency doublets (100 Hz) (21 ± 20%) (The recovery of SS-evoked force (~80%) was greater than that of the recovery of PS100-evoked force (~35%) 1 min after exercise cessation . Therefo1 ± 20%) . This ob2+]i relationship than is the case with high-frequency stimulation. Thus, all situations in which [Ca2+]i and/or the myofibrillar Ca2+ sensitivity are reduced have the potential of showing low-frequency fatigue   . Cytoplacidosis)  and, thecidosis) , which icidosis) . Increas release . There m release , whereas release . SR Ca2+e fibers , and thie fibers , recoverexercise  similar It is also possible that recovery of evoked forces could be masked by different degrees of potentiation at different time points. At cessation of all TTs, it is probable that phosphorylation of the myosin RLC was maximal or close to maximal in the muscles that were important for this exercise. When stimulating before and after the MVC at exercise cessation , evoked –1) during the MVC performed after the 40 min TT , but not after the 3-min TT , a poten0 min TT  – could,0 min TT .Although many studies have assessed peripheral fatigue two or more minutes after termination of a TT , we haveWe have reported rapid recovery in peripheral fatigue after a 6-min TT , 2014. H–1 to calculate neuromuscular activation. RMS⋅M–1 is not considered as valid as voluntary activation to describe changes in central drive  and the Research and Ethics Committee of the Faculty of Health Science of the University of Cape Town, and the experiments were performed according to the latest (2013) revision of the Declaration of Helsinki. The subjects were not allowed to consume alcohol or stimulants during the 24 h preceding the exercise tests. Written informed consent was obtained from all subjects who participated in the study.CF and TN conceptualized and designed the study. CF collected the data and analyzed the data. CF, FB, GM, BM, and TN interpreted the data, revised the manuscript, approved the final version of the manuscript, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. CF drafted the manuscript.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.


Our study aimed to: 1)investigate the diagnostic utility of CSF Aβ42, t-tau, and p-tau to differentiate normal-pressure-hydrocephalus(NPH) from Alzheimer’s-disease(AD) and normal-controls; and 2)investigate if age and ventricular size affect the levels of CSF biomarkers in NPH patients. We recruited 131 participants: (a)Suspected-NPH: 72 with ventriculomegaly and clinical symptoms of NPH. These participants were then divided into two groups of 1)Probable-NPH (N = 38) and 2)Unlikely-NPH (N = 34) based on whether participants experienced gait improvement after removal of a large amount of CSF; (b)AD group: 30 participants with CSF biomarkers and cognitive symptoms consistent with AD; (c)Control-group: 29 participants who were cognitively and functionally normal. Lower levels of CSF Aβ42 and p-tau were observed in the probable-NPH compared to the normal controls. Lower levels of CSF p-tau and t-tau were found in the probable-NPH compared to the AD but the CSF-Aβ42 was low in both the probable-NPH and AD. CSF-Aβ42 correlated with age and Evans-index only in the probable-NPH. Our study supports the hypothesis that age-related atrophy results in better Aβ42 clearance in the CSF because of the increase in the interstitial space. Clinical symptoms include gait and balance impairment, cognitive deficits, and urinary urgency/incontinence1. In both NPH and Alzheimer’s disease (AD) decreased CSF levels of the amyloid β−42 (Aβ42) have been found; however, in contrast to AD, total tau (t-tau) and phospho-tau (p-tau) levels are not increased in NPH cases3. It has been hypothesized that the low Aβ42 in the presence of low t-tau and p-tau is due to the decrease in interstitial space that precludes amyloid precursor protein (APP) fragments and tau proteins from being effectively cleared by CSF and consequently the levels of these proteins decrease in the CSF4. This hypothesis was based on two major observations: 1) low levels of all APP fragments  in CSF obtained both by lumbar and ventricular methods of patients with NPH compared to health controls, which all increased back to normal after shunting1 and 2) Aβ clearance from the interstitial fluid is increased during sleep when the size of interstitial space increases by 60%5.Normal pressure hydrocephalus (NPH) is a syndrome associated with enlarged ventricles without marked elevation in cerebrospinal fluid (CSF) pressureThe aims of our study were to: 1) investigate the diagnostic utility of CSF Aβ42, t-tau, and p-tau to differentiate NPH from AD and normal controls; and 2) investigate if age and ventricular size affect the levels of these CSF biomarkers in NPH patients. Answers to these questions would improve understanding of CSF Aβ42, t-tau, and p-tau production/clearance and facilitate relating CSF biomarkers to patient characteristics.Average age of the patients in the probable and unlikely NPH groups was not significantly different . CSF levels of Aβ42, t-tau, and p-tau were not significantly different between the probable and unlikely NPH groups . However, significantly lower levels of the CSF Aβ42 and p-tau were observed in the probable NPH group compared to the normal controls . When CSF Aβ42, t-tau and p-tau were compared between the probable NPH group and AD group, lower levels of CSF p-tau and t-tau were found in the probable NPH group compared to the AD group  but the mean CSF Aβ42 was low in both the probable NPH and AD group . Figure Although the CSF biomarkers could not differentiate probable NPH from unlikely NPH, PCA was able to differentiate the probable NPH group from the AD and normal controls groups Fig. . InitialAge was significantly correlated with the levels of CSF Aβ42 and p-tau in the probable NPH group  but not in the unlikely NPH group  or in the normal controls   in the probable NPH group  and showed that Evans index is contributing to the levels of CSF Aβ42 independent of age  in the probable NPH group.The results of this study revealed lower levels of CSF Aβ42 and p-tau in the probable NPH group compared to the controls. The mean level of CSF Aβ42 was similar between the probable NPH and AD groups. As our PCA results showed, including CSF Aβ42, p-tau, and t-tau allowed differentiation of probable NPH from controls and patients with AD  and more studies are required to investigate this further. Also, it should be noted that as mentioned above, approximately 40 mL of CSF was collected from each NPH participant. This is a relatively large amount of CSF and may potentially affect the biomarker concentrations. However, large amount CSF collection is common in NPH assessmentIn conclusion, our study showed that the mean level of CSF Aβ42 was similar between the probable NPH and AD groups and lower than that of normal controls while both p-tau and t-tau levels were significantly lower in probable NPH compared to AD. Also, probable NPH patients with higher Aβ42 values tended to be older in our study which supports the hypothesis that age-related atrophy results in better Aβ42 clearance in the CSF because of the increase in the interstitial space. Finally, given the association between age and levels of CSF Aβ42 in the probable NPH group, we proposed a hypothesis that following up on the levels of CSF biomarkers longitudinally is a stronger biomarker for the diagnosis of probable NPH compared to a single measurement of these biomarkers.A total of 131 subjects were included in this study:6. Patients with suspected NPH group were divided into two groups based on the criteria previously described by Relkin et al.6: 1) Probable NPH: 38 patients with clinical symptoms and radiological findings consistent with NPH who experienced an improvement in their gait after removal of a large amount of CSF (35–40 ml). Large amount of CSF removal caused clinically significant improvement in this group’s patients’ gait. Patients in this group were not suffering from any other neurological, psychiatric, or medical conditions that could potentially explain their presenting symptoms; 2) Unlikely NPH: 34 patients with clinical symptoms similar to the previous group but large amount of CSF removal did not significantly improve their gait impairment, i.e. little or no change in speed or stride length.(a) Suspected NPH group: 72 patients  with ventriculomegaly on MRI brain or CT head with clinical symptoms of NPH  were recruited by three neurologists  through Toronto Western Hospital, University Health Network (UHN), Toronto, Canada. All patients underwent lumbar puncture (LP) and all with a normal opening pressure10.(b) AD group: included 30 patients  with CSF biomarkers consistent with AD(c) Control group: 29 subjects who were cognitively and functionally normal including gait and neuroimaging but some had a history of depression but no evidence of AD on CSF biomarkers.11, Toronto Cognitive Assessment (TorCA)12) were used to assess the cognitive status.As part of the clinical evaluation all participants underwent a cognitive assessment. However, different tests The University Health Network’s Research Ethics Board approved the study. Written informed consent was obtained from all participants or their legally authorized representatives, in case the participant was not competent to consent, before participating in the study. All methods were carried out in accordance with relevant guidelines and regulations.13. Approximately, 40 mL of CSF was collected from each NPH participant by allowing CSF to drip into the collecting tubes (gravity drip). After CSF was collected in polypropylene tubes, it was transported to the adjacent laboratory within 30 minutes, aliquoted, and stored at -80 °C. A sandwich ELISA was used to measure concentrations of Aβ42 (Innotest β-amyloid (1–42), Fujirebio), p-tau (Innotest phospho-tau (181p), Fujirebio), and t-tau  following the manufacturer’s instruction15. All analyses were performed at the Tanz Centre for Research in Neurodegenerative Diseases  by one operator (FT). All samples were measured in duplicate and repeated if the difference between individual optic density (OD) values was greater than 20%. In addition to the ready-to-use calibrators (CAL) and Run Validation Controls (RVC) which were part of the Fujirebio Innotest assay kits, internal controls were also included in each run. After calculating the mean absorbance for the CAL, RVC, and unknown CSF samples, a sigmoidal 4-parameter curve fitting was used to determine the corresponding concentrations. CSF biomarkers were considered consistent with AD diagnosis if p-tau > 68 pg/ml and Aβ42 to t-tau index (ATI) < 0.810.All subjects underwent a lumbar puncture (LP) that was performed according to Alzheimer’s Disease Neuroimaging Initiative (ADNI) protocol16.Evans index was calculated for the subjects in the suspected NPH group using their axial Fluid-attenuated inversion recovery (FLAIR) magnetic resonance images (MRI) or axial computed tomography (CT) images when MRI was not available. The ratio of the maximum width of the frontal horns of the lateral ventricles to the maximal internal diameter of the skull at the same slice of the axial MRI or CT images was used to calculate this index17.We constructed a Support Vector Machine (SVM) to separate the probable NPH patients from AD and controls based on individual levels of CSF Aβ42, t-tau and p-tau. SVM is a powerful supervised learning algorithm that can be trained on a subset of data and test its predictions on the rest of the testing data. First, a Principle Component Analysis (PCA) was used to visualize the data and compute composite scores for Aβ42, t-tau and p-tau for each patient. The first two components of PCA were then used in the SVM for determining whether the linear combination of CSF Aβ42, t-tau, p-tau levels separated probable NPH patient from the other two groups . The SVM was designed with a linear kernel for the classification, and we used 8-fold cross validation that was implemented in the function. PCA in this process was not used to select the significant factors, but rather utilized to create composite variables as an input for SVM. For this step, all data was scaled and processed using R studio18 was performed to compare the probable NPH group with each of the 3 other groups. The Pearson correlation coefficient was used for correlations. Statistical significance level was set at p < 0.05.Statistical analyses were conducted using SPSS software (SPSS Inc. v. 24). One-way analysis of variance (ANOVA) was used to compare the CSF biomarkers  among the groups . Dunnett-T3 post hoc analysis


Induction of immune tolerance for solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. This would obviate the need for life-long immunosuppression which is associated with serious adverse outcomes, such as infections, cancers, and renal failure. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of living donor renal transplantation, the mixed chimerism approach has not achieved durable immune tolerance on a large scale in preclinical or clinical trials with other solid organs or vascular composite allotransplants (VCA). Ossium Health has established a bank of cryopreserved bone marrow (BM), termed “hematopoietic progenitor cell (HPC), Marrow,” recovered from deceased organ donor vertebral bodies. This new source for hematopoietic cell transplant will be a valuable resource for treating hematological malignancies as well as for inducing transplant tolerance. In addition, we have discovered and developed a large source of mesenchymal stem  cells (MSC) tightly associated with the vertebral body bone fragment byproduct of the HPC, Marrow recovery process. Thus, these vertebral bone adherent MSC (vBA-MSC) are matched to the banked BM obtained from each donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than traditional BM-MSC; however, their abundance is >1,000-fold higher than obtainable from living donor BM aspirates. Based on our own unpublished data as well as reports published by others, MSC facilitate chimerism, especially at limiting hematopoietic stem and progenitor cell (HSPC) numbers and increase safety by controlling and/or preventing graft-vs.-host-disease (GvHD). Thus, vBA-MSC have the potential to facilitate mixed chimerism, promote complementary peripheral immunomodulatory functions and increase safety of BM infusions. Both HPC, Marrow and vBA-MSC have potential use in current VCA and solid organ transplant (SOT) tolerance clinical protocols that are amenable to “delayed tolerance.” Current trials with HPC, Marrow are planned with subsequent phases to include vBA-MSC for tolerance of both VCA and SOT. SOT has become standard of care over the last half century, resulting in not only a significant life extension but also an enhancement of quality of life (QOL) for end-stage organ failure patients , 2. MoreThe continuing negative impact of chronic rejection, combined with the severe adverse effects of conventional immunosuppressive regimens, has spurred intense research into new and safer strategies to prevent allograft rejection. While chronic allograft failure is associated with more frequent hospitalization, higher morbidity and increased health care costs, chronic immunosuppression (IS) is linked to side effects that range from malignancy, infection, toxicities  to cardiovascular and metabolic diseases –15. MediBesides tolerance regimens, various alternative strategies to inhibit rejection are in development to replace or reduce the need for current mainstay IS drugs. These regimens seek to shift the balance of lymphocytes in favor of regulatory T (Treg) cells over effector/memory T (Tem) cells, as opposed to pan-T cell inactivation with calcineurin or mTOR inhibition , 28. Of The vascularized BM component of VCA has innate immunomodulatory properties; however, this is not sufficient to fully tolerize recipients to the highly immunogenic skin component of the composite tissue , 36, 40.Although superficially similar in that both approaches administer hematopoietic cells, there are fundamental differences with respect to safety and mechanisms between the mixed chimerism-based approaches that are currently used in clinical trials to promote tolerance in SOT and the immunomodulatory approach in clinical use for VCA. Induction of tolerance through mixed chimerism necessitates non-myeloablative conditioning in the form of irradiation , cytotoxic agents  and cell-depleting agents . LaCurrently, there are three U.S. centers , Stanford University, and Northwestern University) that are investigating clinical protocols for inducing SOT tolerance . The proVCA grafts are invariably from deceased donors, which are also a source of high quality BM obtained from the donor vertebrae that can be cryopreserved for subsequent infusion , 53, 54.The achievement of immunomodulation with BM infusion that clinically translates into significantly reduced need for IS in VCA demonstrates that (1) harvesting and cryostorage of deceased donor BM is feasible, (2) cryopreserved deceased donor BM can be safely infused, and (3) delaying infusion of previously cryopreserved deceased donor BM over a significant period following VCA still achieves desirable biological effects. This suggests that delayed tolerance with deceased donor SOT may be possible.Feasibility of delayed BMT for tolerance in SOT in fact has been demonstrated in rodent and non-human primate models of solid organ and vascularized composite allotransplantation . As has also been demonstrated over the last few decades with cryopreservation of cord blood, it is well-established that BM remains biologically active following long-term cryopreservation –81. The Efforts are currently underway in collaboration with the national Organ Procurement Organization (OPO) network in the U.S. to build the first bank of cryopreserved deceased donor BM. The U.S. OPO network provides an existing refined infrastructure for procuring and transporting bone tissue recovered from deceased donors. Approximately 10,000 deceased donor organs are recovered each year in the U.S., with a further 40,000 donations, yielding approximately 30,000 organs and over a million tissues recovered annuallyProtocols for isolation of HPC, Marrow from deceased donor vertebral bodies were based on original work at University of Pittsburgh and optimized at Johns Hopkins University , 53, 54.Our team has identified an abundant population of MSC associated with the vertebral body (VB) bone fragment byproduct of HPC, Marrow recovery. These MSC remain tightly adhered to cancellous bone fragments and can only be released by enzymatic treatment. We have determined that these vertebral bone adherent MSC (vBA-MSC) are identical to BM-MSC when cultured . The vBAThe potent immunoregulatory properties of MSC comprise a spectrum of secreted and cell-bound molecules that modulate a wide array of innate and adaptive immune responses. The multifaceted mechanisms of MSC immunomodulation have been detailed in numerous reviews and, therefore, will only be briefly introduced here. The MSC secretome includes both freely soluble factors as well as those encapsulated by extracellular vesicles. Mechanisms include metabolic inhibition , immunomodulatory cytokines , and checkpoint inhibitors . These myriad factors inhibit T cell activation and proliferation, as well as enhance proliferation of regulatory cells –88.Preclinical studies have demonstrated the therapeutic potential of MSC for inducing operational tolerance of SOT and VCA –102, proIn addition to potentially facilitating graft survival through ameliorating alloreactivity, MSC have demonstrated considerable potential for suppressing GvHD which could be an unintended consequence of SCT to induce tolerance , 113–126Another mechanism to minimize the risk of GvHD is titrating down the HSPC graft to a minimal efficacious dose, which correspondingly reduces the donor T cell load. MSCs have been reported to facilitate and enhance engraftment of allogeneic HSPC clinically, even after initial graft failure and rejection of conventional stem-cell grafts . PreclinBesides GvHD, engraftment syndrome (ES) that occurs in 7–90% of cases during neutrophil recovery after autologous and allogeneic HSCT poses a potential limitation . It is aEach of the current protocols for inducing tolerance in VCA and SOT lend themselves to deceased donor BM augmentation and BMT, respectively, with modification to accommodate donor availability. Inclusion of vBA-MSC either prophylactically or to treat GvHD is possible. Following BM isolation and quality control testing, HPC, Marrow would be cryogenically preserved until shipping under the same conditions for infusion into the patient 14 days following surgery, as described previously . SimultaIn regard to SOT, the MGH delayed tolerance protocol appears to be the most easily adaptable to HPC, Marrow, providing that encouraging results in NHP and early clinical trials in humans are repeated in future larger clinical trials , 146–1486 CD34+ cells from HPC, Marrow recovered from three donors. Importantly, these methods can be used on either freshly isolated or previously cryopreserved HPC, Marrow; thus, providing flexibility in cell production. The Stanford protocol infuses cryopreserved selected cells at 11 days after kidney transplant which would provide sufficient time to prepare HPC, Marrow as well as over a billion very early passage GMP vBA-MSC  or durable  mixed chimerism –154. TheDespite clinical realization of tolerance and preclinical evidence supporting the feasibility of delayed tolerance protocols as outlined above, tolerance induction is still limited to a few highly specialized centers . WidesprFuture broad success with BM and MSC induction of tolerance and potent immunoregulation will have profound effects on transplant patients. Achieving immune tolerance, in particular, will alleviate the burden of life-long IS and associated morbidity, avoid chronic rejection, and significantly improve overall outcomes. Furthermore, it will overcome current compliance and adherence-based limitations that negatively impact graft survival due to subsequent subliminal rejection and the development of donor-specific antibodies. Tolerance and immunoregulation will be especially impactful for patients by increasing accessibility to transplantation and through positively shifting the risk:benefit ratio by reducing associated long-term risk.In view of these potential opportunities and substantial benefits, the establishment of a bone marrow bank for delayed tolerance protocols marks a crucial step in making this resource available for present as well as future transplant patients. The complementary treatment with vBA-MSC could further increase safety with the added potential of enhanced efficacy. Furthermore, the ability of vBA-MSC to promote HSPC BM engraftment would allow lowering of HPC, Marrow doses which effectively extends the number of patients who receive organs from a single donor that are able to benefit from this procedure.All authors contributed to writing and editing the article, and approved the submitted version.BJ, GB, and EW have equity ownership in Ossium Health. BJ and EW are inventors on patents pending relevant to the subject matter. EW is a co-founder of Ossium Health. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hantaviridae, order Bunyavirales) can cause two serious syndromes in humans: hemorrhagic fever with renal syndrome (HFRS), associated with the Old World orthohantaviruses, and hantavirus cardiopulmonary syndrome (HCPS), associated with orthohantaviruses in the Americas. In Europe, four different orthohantaviruses  are associated with human disease. As disease severity and zoonotic source differ between orthohantavirus species, conclusive determination of the infecting species by either RT-PCR or comparative virus neutralization test (VNT) is of importance. Currently, the focus reduction neutralization test (FRNT) is considered the ‘Gold Standard’ for orthohantavirus VNTs, however this test is laborious and time-consuming. Consequently, more high-throughput alternatives are needed. In this study, we developed a comparative orthohantavirus microneutralization test (MNT) including all four human pathogenic orthohantavirus species circulating in Europe. The assay was validated using RT-PCR-confirmed rodent (n=17) and human sera (n=17), DOBV-suspected human sera (n=3) and cohorts of orthohantavirus-negative rodent (n=3) and human sera (n=85). 16/17 RT-PCR-confirmed rodent sera and 18/20 of the RT-PCR-confirmed and DOBV-suspected human sera were serotyped successfully, while for the remaining rodent (n=1) and human sera (n=2) no neutralizing titers could be detected. All negative control sera tested negative in the MNT. The assay was subsequently evaluated using a clinical cohort of 50 orthohantavirus patients. Orthohantavirus infection was confirmed in all 50 patients, and 47/50 (94%) sera were serotyped successfully, confirming PUUV as the major cause of orthohantavirus infections in Netherlands. Notably, two previously unrecognized SEOV cases from 2013 were diagnosed using the MNT, underlining the added value of the MNT in a diagnostic setting. In conclusion, we demonstrate the successful development and clinical implementation of a comparative European orthohantavirus MNT to determine the infecting virus species in European HFRS patients. Identification of the causative species is needed for an adequate Public Health response and can support individual patient care. For many labs, the implementation of orthohantavirus neutralization tests has not been a straightforward procedure. This issue will be addressed by the rollout of the comparative MNT to multiple European laboratories to support patient diagnostics, surveillance and Public Health responses.Orthohantaviruses (family  Hantaviridae, order Bunyavirales  carried by the bank vole (Myodes glareolus); Seoul virus (SEOV) carried by different species of rats (Rattus spp.); and Tula virus (TULV) carried by the common vole  ((A. agrarius), is generally believed to be only present in Asia, some studies suggested that it may also be present in eastern parts of Europe (Four different orthohantavirus species are associated with human disease in Europe: Dobrava/Belgrade virus (DOBV) carried by three closely related species of the genus arvalis) . Additiof Europe .Of the orthohantaviruses with confirmed presence in Europe, DOBV is associated with the most severe form of HFRS with case fatality ratio’s (CFR) up to 12%, depending on the genotype . SEOV isArvicolinae-borne orthohantaviruses, while DOBV and SEOV (together with HNTV) are part of the serogroup of Murinae-borne orthohantaviruses. Unfortunately, however, as high levels of cross-reactive antibodies are observed within but also between the different serogroups , the infecting virus species cannot be determined conclusively using routine serological assays . To date, multiple variations of orthohantavirus neutralization assays have been described , howeverGreen monkey kidney Vero-E6 cells (ATCC C1008 CRL1586) were maintained in Dulbecco’s Modified Eagle’s MEM  supplemented with 5% heat-inactivated fetal bovine serum , 100 U/ml penicillin and 100 U/ml streptomycin . Cells were cultured at 37°C under 5% CO2.via the European Virus Archive (EVAg). Stocks were created by passaging twice on Vero-E6 cells using a low multiplicities of infection (MOI 0.001-0.1), and harvesting 7–14 days post infection.DOBV strain SK/Aa , PUUV strain Sotkamo , SEOV strain 80–39 , and TULV strain Moravia (no longer available from EVAg) were all acquired 50) assay adapted for indirect detection of virus-infected cells. Briefly, tenfold viral dilutions were inoculated on Vero-E6 cells and incubated for 7  or 12 days (PUUV) at 37°C, which corresponded to peak titers during virus culture (data not shown). Subsequently, cells were fixed using 4% PFA and permeabilized using 0.02% Triton X-100 in PBS. Subsequently, cells were washed with PBS and stained using mouse anti-hanta nucleoprotein   or mouse anti-hantavirus Dobrava strain nucleoprotein  (for DOBV) as primary antibodies and goat anti-mouse-AlexaFluor488  as a secondary antibody. Positive wells were visualized using a Leica DMIL LED fluorescence microscope and scored by eye. TCID50 titers were calculated using the Spearman and Kärber algorithm  or 12 (PUUV) days at 37°C, before cells were fixed, permeabilized and stained as described above. Virus-positive cells were visualized using a Leica DMIL LED fluorescence microscope. The neutralization titer was determined by scoring the wells in which infection was inhibited ≥ 95% compared to the positive control (MNT95).The orthohantavirus neutralization test was designed as a microneutralization tests (MNT) as previously described for the Influenza A virus and the orthohantaviruses HTNV and SEOV  with an R. norvegicus sera (n=3) that previously tested negative in both RT-PCR and the orthohantavirus FRNT in Uppsala, Sweden  originated from Dutch patients and were diagnosed using the genotype-specific RT-qPCRs described by , while sMurinae-borne) orthohantaviruses. Two of three sera were known to be convalescent (both samples were taken more than one year after onset of symptoms), while for the third serum the status was unknown. None of these sera were confirmed by RT-PCR.In addition, a small serum panel from DOBV-suspected human patients (n=3) kindly provided by Sabine Lederer from Euroimmun  was tested in the orthohantavirus MNT. All samples showed the highest IgG titers against DOBV in the Euroimmun mosaic immunofluorescent assay (IFA) , with a Finally, 85 randomly selected sera collected from healthy Dutch blood donors in 2016 were included as negative controls.Sera of 50 Dutch patients clinically suspected of an orthohantavirus infection, collected in the period 2013–2017, and diagnosed with an orthohantavirus infection based on routine serology were selected for testing in the orthohantavirus MNT. Initial routine orthohantavirus diagnostics were performed using the Euroimmun mosaic IFA , in whic50), it was not possible to determine levels of neutralization of PUUV and TULV infection robustly using this approach (data not shown).The aim of this work was to set up a MNT including all human pathogenic orthohantaviruses circulating in Europe, namely DOBV, PUUV, SEOV, and TULV. Initially, we designed the European orthohantavirus MNT according to the approach of , in whic95 titer, corresponding to the highest antibody dilution that inhibited infection ≥ 95%, was determined by eye. As shown in 95 titer was preferred over an end-point neutralization titer (complete inhibition of infection), as at times small breakthrough infections could be observed that prevented the determination of an end-point neutralization titer, even though clear neutralization was observed.Consequently, the MNT was designed with a fluorescence-based read-out, in which the MNT95 titers of the rodent serum validation panel. Neutralization titers of ≥ 40 were considered positive  from multiple rodent species, previously determined to be serologically (IgG) and RT-PCR positive for either DOBV, PUUV, SEOV or TULV, was used. positive . Additiorotyping .A. agrarius), no neutralization was observed. As this serum was only weakly positive in the IFT, it is possible that the serum was taken early after infection of the respective rodent and neutralizing antibodies were not yet present at the time of sampling.Close to all rodent sera (16/17) had distinct neutralizing titers toward the infecting virus species and could be serotyped as expected Table 3.For most sera (n=11/17), some degree of cross-neutralization toward the non-infecting other orthohantaviruses was observed, which was most prominent toward the orthohantaviruses from the same serogroup (PUUV ↔ TULV and SEOV → DOBV) . Cross-nR. norvegicus sera that were found to be positive in a genus-wide orthohantavirus ELISA but were previously determined to be negative in both RT-PCR and an orthohantavirus FRNT (95 (data not shown).As a negative control, wild-caught rus FRNT , were teNext, we aimed to validate the orthohantavirus MNT using sera of RT-qPCR-confirmed human orthohantavirus cases . For this purpose, we used sera from 11 Dutch and six Slovenian patients with a RT-qPCR-confirmed orthohantavirus infection diagnosed in the period from 2011 to 2017. Of these patients, 10 were RT-qPCR positive for PUUV (9 Dutch patients and 1 Slovenian patient), two were positive for SEOV (both Dutch patients) and five were positive for DOBV . As human TULV infections are rare, no sera from RT-qPCR confirmed TULV patients were available.Neutralizing titers for the RT-qPCR-confirmed patient sera are shown in In addition to the RT-PCR-confirmed rodent and human sera, a small serum panel  from patients with a suspected DOBV infection was tested in the MNT. As expected, sera of all three patients were serotyped as DOBV infections Table 5.As the incidence of orthohantavirus infections in Netherlands is relatively low , 85 randTo evaluate the MNT in a clinical diagnostic setting, a cohort consisting of 50 Dutch patients diagnosed with an orthohantavirus infection based on routine serology was tested in the comparative MNT. All sera (100%) showed neutralizing titers against at least one of the orthohantaviruses, and could thus be confirmed as orthohantavirus infections based on Gold Standard serology Table 6.95 titers of 80 and 40, respectively) while no cross-neutralization within the serogroups was observed (both DOBV and PUUV were negative). Most likely this patient was infected with SEOV, as the patient owned SEOV-positive rats.Of the sera that could not be serotyped, two sera (15-03 and 16-12) showed a neutralizing titer for both PUUV and TULV with only a two-fold difference, and were thus considered inconclusive. A third inconclusive serum (17-09b) showed a rather atypical neutralization profile of cross-neutralization between the different serogroups  for serotyping orthohantavirus infections. This is in line with two previous studies  that shoA. agrarius serum remained negative in the MNT . For both PUUV and DOBV neutralizing antibodies could already be detected in the first few days after infection . These 95 of 640, the highest neutralizing titer measured against DOBV in this study), which might explain the low levels of cross-neutralization toward PUUV and TULV. This explanation is supported by the notion that also R. norvegicus sera with high neutralizing titers against SEOV (≥1280) showed some degree of cross-neutralization toward PUUV. Interestingly, however, cross-neutralization from the Arvicolinae-borne orthohantaviruses (PUUV and TULV) toward the Murinae-borne orthohantaviruses (DOBV and SEOV) was not observed during validation, even though some rodent and human sera had neutralizing titers of ≥5120 toward either PUUV or TULV. It was, however, observed once in the diagnostic cohort  might be difficult to serotype due to high cross-reactivity in the VNT . Here, rc cohort .The vast majority of cases (44/50) in our clinical cohort of orthohantavirus infections were identified as caused by PUUV, which confirms the assumption that PUUV is the major cause of orthohantavirus infections in Netherlands . Three hA. flavicollis) has only limited presence in Netherlands and is considered not to carry the virus. TULV, on the other hand, is circulating among the common vole populations at multiple locations in Netherlands  and Slovenian Animal Ethics Committee .TH, JR, and CR contributed to the design of the study. TH, IZ, MH, and AV performed the experiments. JR, MM, TA-Ž, AV, and MK provided critical samples for validation and assessment of the assay. TH and CR wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


R)-N2-ATTAF-1 and (S)-N1-ATTAF-2 complexes is obtained 6.36 and 4.19 Å, respectively and about 2 Å in the other tautomer and enantiomer complexes, reflecting the lower basicity of the N-4 atom in the 1,2,4-triazole ring of (R)-N2-ATTAF-1 and (S)-N1-ATTAF-2 in comparison with the N-4 atom in the 1,2,4-triazole ring in other tautomers and enantiomers and supporting higher selectivity of (R)-N2-ATTAF-1 and (S)-N1-ATTAF-2 towards the target CYP51 enzymes vs. human. Interestingly, we have investigated unfavorable interactions (donor-donor) with TRP239 and MET378 for (R)-N2-ATTAF-1 and (S)-N1-ATTAF-2, respectively. These unfavorable interactions also have been seen in case of posaconazole and isavuconazole. The data presented in this article are related to the research paper entitled "In silico prediction of ATTAF-1 and ATTAF-2 selectivity towards human/fungal lanosterol 14α-demethylase using molecular dynamic simulation and docking approaches".The data have been obtained for tautomers and enantiomers of ATTAF-1 and ATTAF-2 that were developed based on antifungal standard drugs with triazole scaffold. These compounds were docked into the human and fungal lanosterol-14α-demethylase. In order to validate the data, 8 standard triazole antifungal drugs  were also docked into the human and fungal lanosterol-14α-demethylase. The binding conformations of these molecules and their interactions with lanosterol-14α-demethylase may inform the development of further small molecule lanosterol-14α-demethylase inhibitors with significant selectivity toward this enzyme. The analysis has done on the basis of type of interactions (bond type and distance). The length of the Fe-N coordination bond for ( Specifications table•The lanosterol 14α-demethylase has been identified as a molecular target for the treatment of fungal diseases.•The modeling data was produced to rationalize the structural necessities for lanosterol 14α-demethylase inhibition.•The binding conformations of the ATTAF-1 and ATTAF-2, their interactions with human and fungal lanosterol 14α-demethylase and coordination bond distance may inform further studies focused on the development of lanosterol 14α-demethylase inhibitors.•in silico docking data and research time can be minimized significantly.Novel synthetic analogues with improved bioactivity and minimized side effects can be expanded against this target by using this •This dataset can be useful to model other potent antifungal agents in future and researchers in pharmaceutical chemistry can gain from the data.1Lanosterol-14α-demethylase (CYP51) is found in mycobacteria, fungi, plants, animals and humans. This enzyme is required for biosynthesis of sterol in eukaryotes and is the major target for azole antifungal agents . In mamm22.1www.rcsb.org). Protein preparation was done by preprocessing the structures by removing water molecules, ions and cocrystallized ligands, polar hydrogens addition and assigning Gasteiger-Marsili partial charges, adjusting bonds and formal charges for metals, and removing unwanted chains. In order to rmsd validation, the co-crystallized ligand was re-docked. The target input files were converted to PDBQT format for AutoDock by using the AutoDockTools-1. 5. 4.The crystal structures of the selected proteins were retrieved from protein data bank. (PDB database, 2.2Ligands 3D structures were sketched by using ChemDraw Ultra 8.0 and energy minimized using PM3 force field. For all ligands, the nonpolar hydrogen atoms were merged and the Gasteiger charges were assigned. Then set number of torsion with detect root and choose torsion in Autodock program. Later, ligand input files were also saved as PDBQT format utilizing the AutoDock Tools. The minimized structures were docked on the prepared protein.Discovery Studio Client 2016 and Molegro Molecular Viewer were used for further analysis.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


COVID-19 pandemic currently affects nearly all countries and regions in the world. Washing hands, together with other preventive measures, to be considered one of the most important measures to prevent the disease. This study aimed to characterize reported handwashing practices of Vietnamese people during the COVID-19 pandemic and associated factors. Kobo Toolbox platform was used to design the online survey. There were 837 people participating in this survey. All independent variables were described by calculating frequencies and percentages. Univariate linear regression was used with a significant level of 0.05. Multiple linear regression was conducted to provide a theoretical model with collected predictors. Seventy-nine percent of the respondents used soap as the primary choice when washing their hands. Sixty percent of the participants washed their hands at all essential times, however, only 26.3% practiced washing their hands correctly, and only 28.4% washed their hands for at least 20 seconds. Although 92.1% washed hands after contacting with surfaces at public places , only 66.3% practiced handwashing after removing masks. Females had better reported handwashing practices than male participants . Better knowledge of handwashing contributed to improving reported handwashing practice . Poorer handwashing practices were likely due, at least in part, to the COVID-19 pandemic information on the internet, social media, newspapers, and television. Although the number of people reported practicing their handwashing was rather high, only a quarter of them had corrected reported handwashing practices. Communication strategy on handwashing should emphasize on the minimum time required for handwashing as well as the six handwashing steps. As of 12th April 2020 there were 258 infections, 144 of the affected patients have recovered and 0 death Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus. Since its emergence in Wuhan, China in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had rapidly spread throughout the world following an exponential growth curve prompting it to be characterized as a pandemic by the World Health Organization on March 11, 2020 Hand hygiene is considered as one of the most fundamental preventive measures against many infectious diseases. Several studies have indicated that the efficacy of keeping hands clean reduced significantly the rates of infectious illnesses in the community, including diarrhea World Health Organization (WHO) and the Vietnam Ministry of Health (MOH) has repeatedly emphasized that regularly and thoroughly performing hand hygiene with soap and water or alcohol-based hand rub is an effective way to prevent the spread of the new coronavirus SARS-CoV-2 Widely reporting of statistics and facts about the COVID-19 pandemic by the global media may lead to confusion and anxiety about the disease along with the preventive measures, including handwashing recommended by many organizations including WHO and MOH. This study describes the knowledge and practices of people in Vietnam about handwashing with soap and water or the alcohol-based hand rub, and the associated factors during the COVID-19 pandemic in March 2020.2.2.1.This was a cross-sectional study.2.2.Participants were eligible to participate if they were adults, currently lived in Vietnam, had internet connection, able to read, and understand the provided questions. Participants voluntarily participated in an online questionnaire administered through email and social networks.2.3.d = 0.05, design effect DE = 2, and p was the proportion of handwashing people using soap at essential times . The calculated sample size was 277 people.The study sample was calculated using the one-proportion sample size formula with an absolute precision Participants were selected using non-probability, self-nominated sampling. The researchers sent the link contained online questionnaire to respondents through email, social networks . The online questionnaire was available from 0.00 am 25 March to 11.59 pm, 01 April 2020. At the time of the survey closing, 870 submissions were recorded. Among the submissions, 859 agreed to participate in the survey, making the response rate of 98.74%. Among those who agreed, 837 participants were included in the analysis, 22-excluded submissions were participants who had answered only 10% of total questions or fewer.2.4.https://ee.kobotoolbox.org/x/#Juy6CuzP). To prevent duplication, an email address was required for each time respondents submitting their responses. The average time to complete the questionnaire was approximately 10 minutes.We used Kobo Toolbox (kf.kobotoolbox.com) to develop the self-administered questionnaire and to collect data. The questionnaire could be easily accessed on any devices  with an internet connection through a link .2. Could the respondent list ALL the 5 necessary times for handwashing? (2 available choices: Yes/No). Five mentioned necessary times were: 1) after going to the toilet, 2) after touching surfaces at public places , 3) before having meals, 4) before preparing meals for family and 5) after removing face mask.3. Did the respondent have handwashing with 6 steps? (2 available choices: Yes/No).4. Did the respondent have handwashing in at least 20 seconds? (2 available choices: Yes/No). , age (in years), the highest educational level , occupation (student/employee/other), marital status ), a source where respondents get information about handwashing , and knowledge of handwashing (in points). Knowledge variable was created through a combination of nine questions and the highest score would be 33, details were described as below:2.6.Both descriptive and inferential statistics were performed in this study. All independent variables were described under the main outcome by calculating frequencies and percentages. Univariate linear regression was used for each independent variable with the main outcome with a significant level of 0.05. Multiple linear regression was conducted to provide a theoretical model with collected predictors.2.7.This study was approved by the Institutional Review Board of the Hanoi University of Public Health under the Decision No. 105/2020/YTCC-HD3 dated 20 March 2020. The study information about objectives, selection criteria, personal information privacy, data protection, advantages, and potential harm, were all provided to participants before doing the online questionnaire. Each participant was assigned a study number, no private data was collected, and all collected data was coded.3.3.1.The responses of 837 participants were included in the analysis. Out of 837 participants, 633 (75.7%) were female. The average age in years of the respondents was 33.3 (SD = 10.9), male respondents were older than females . Most respondents were employed, which accounted for about 60.5% while 25% of the respondents were students, and the rest of them were self-employed (4.4%), business/small business (3.8%), retired (2.8%), and other (2.3%). At the time of data collection, 23 of the63 provinces in Vietnam reported to have COVID-19 patients. In our survey, the proportion of respondents from the provinces with COVID-19 patients was about 72%. There was 64% of the respondents completing vocational/college/university as their highest educational level, while about 28% of total participants had completed postgraduate education. About half of the respondents were living with their spouse. The proportion of single respondents was 39.2%, and 3.8% of the respondents were separated/divorced/widowed.3.2.Overall, the rate of respondents with correct reported practices of handwashing was 26.3%. When looking into four aspects that assessed respondents correct reported practices, using water and soap as a primary choice for hand hygiene was the aspect with the highest correct proportion, which accounted for almost 80% of the total respondents. In this survey, only 60.6% of the respondents had a correct answer for the all necessary times for handwashing, and 42.8% of the respondents could list all six steps of proper hand hygiene practices. The aspect in which the respondents had the lowest correct level was the adequate handwashing time. Only 28.4% of participants reported washing their hands in at least 20 seconds as recommended by WHO.Regarding the necessary time for handwashing, the popular times at which most respondents knew that they need to perform handwashing were: after going to the toilet (96.6%), after touching surfaces at places such as elevators, doorknobs, etc. (92.1%), and before having meals (92.1%). The proportion of respondents knew that they needed to wash hands before preparing meals for their families was 84.5% and only 69.3% of respondents answered that they needed to wash hands after removing their face masks.3.3.Some characteristics were found to be statistically significant with reported practices of handwashing among the participants in the univariate analysis including age, gender, occupational status, marital status, getting COVID-19 information from friends, health staff, government/MOH, and knowledge of handwashing (in points). When adjusting all variables in multivariate analysis, female respondents were found likely to be 1.88 times (95% CI: 1.15–3.09) higher to have correct reported practices of handwashing than males. We also found that respondents who received information regarding handwashing from health staff had the odds of reported handwashing correctly 2.96 times (95% CI: 1.70–5.13) higher than those who did not. This survey also revealed the negative impacts of internet/social networks and newspaper/television on the reported practice of handwashing among the respondents. In detail, the participants who received hand hygiene information from the internet/social networks had 56% (95% CI: 4–79%) chances lower to wash hands correctly than those who did not. The same trend was observed among the respondents getting information from newspaper/television with a proportion of 59% (95% CI: 13–80%). At last, respondents who had more knowledge of handwashing correctly could have correct reported practices of this behavior, for each point increased in knowledge, the odds of reported practices correctly increased 1.3 times (95% CI: 1.2–1.41).Our multivariate analysis did not found characteristics including age, occupational status, original provinces, educational level, marital status, getting information from friends, relatives, government/MOH to be associated with correct reported practices of handwashing among participants.4.4.1.th April 2020 The study results showed that the prevalence of people reported washing their hands with clean water and soap as their primary choice was remarkably high (79.3%). This result was relevant with the recommendation from the Vietnamese MOH that people should perform handwashing with soap and clean water. This method is considered as the priority measure for handwashing and the sterilized sanitizer is recommended only when soap and water are not available However, the high frequencies of reported handwashing at essential times in this study might not have implied that the actual handwashing practices of the participants were that high, as many of the studies around the world have shown that the reported handwashing practices were much higher than observational handwashing behaviors st April 2020 The study revealed that only 42.8% of the participants reported practicing all six steps during handwashing and only 28.4% performed hand hygiene for at least 20 seconds recommended by WHO 4.2.It revealed from our study that female participants had better reported practices on handwashing than male participants , which wMany international studies Besides, various studies showed that better knowledge associated with better reported handwashing practices One interesting finding from this study was that participants who obtained knowledge on handwashing from the internet sources  or newspapers/television had poorer handwashing practices that those who did not receive knowledge from these sources. The participants who gained knowledge from healthcare staff had better reported handwashing practices than those who did not get information from this staff. As suggested by Sandman (2012) above, due to the uncertain of the COVID-19 disease, the community's outrage for this disease increased and therefore contributed to the increased perception on the risk of the disease 5.st of April 2020 Although certain interesting results were found in this study, our study is subject to several limitations. First, our sample was not representative because we only collected data online, which might have resulted in most of the respondents were at higher educational levels (more than 90% had the college/university levels or higher). Other studies found that users of the internet were not representative of the general population, and not all felt familiar with the online protocol 6.Although handwashing practices at all essential times recorded in this study was rather high, the prevalence of practiced washing hand correctly was low, mainly due to the incorrect practice on the six handwashing steps and the minimum required time for handwashing. The Government should pay attention to the communication strategy on COVID-19 prevention measures to the citizens, with a focus on the six steps of handwashing and the minimum required time for handwashing. Also, the Government should remind the Vietnamese people to select appropriate and reliable information on COVID-19 pandemic for references, especially information on the internet, social media, newspapers, etc.


Streptococcus mutans, a bacterium that causes dental plaques, forms a biofilm on tooth surfaces. This biofilm can cause gingivitis by stimulating the gingival margin. However, there is no established treatment for biofilm removal. Hainosan (Painongsan), a traditional Japanese Kampo formula, has been used to treat gingivitis. Therefore, we investigated the biofilm suppressive effects of the hainosan extract (HNS) and its components on S. mutans. We conducted scanning electron microscopy and confocal laser microscopy analyses to clarify the anti-biofilm activities of HNS and its crude drugs. We also performed a quantitative RT-PCR assay to assess the biofilm-related gene expression. HNS showed a significant dose-dependent suppressive effect on biofilm formation. Both the scanning electron microscopy and confocal laser microscopy analyses also revealed the significant inhibitory effects of the extract on biofilm formation. Transmission electron microscopy analysis showed that HNS disrupted the surface of the bacterial wall. Furthermore, HNS reduced the hydrophobicity of the bacteria, and suppressed the mRNA expression of β-glucosyltransferase (gtfB), glucosyltransferase-SI (gtfC), and fructosyltransferase (ftf). Among the constituents of hainosan, the extract of the root of Platycodon grandiflorum (PG) showed the strongest biofilm suppression effect. Platycodin D, one of the constituent natural compounds of PG, inhibited S. mutans-associated biofilm. These findings indicate that hainosan eliminates dental plaques by suppressing biofilm formation by S. mutans. Streptococcus mutans [Dental plaques induce dental caries and periodontal disorders . The pros mutans .S. mutans is regarded as a primal dental cariogenic bacterium [S. mutans is its capacity to metabolize various sugar carbohydrates [S. mutans uses this ability to form biofilms on the enamel surface of the tooth [acterium . It has acterium . The cenhydrates . S. mutahe tooth . Bacterihe tooth . This mahe tooth ,6,7.S. mutans plays a major role in dental decay, it should be removed as much as possible [S. mutans is valuable for maintaining a healthy oral cavity [S. mutans [S. mutans [Among the most successful strategies for the discovery of novel agents is the usage of natural products derived from medicinal plants . Becausepossible . Thus, sl cavity . It has . mutans . Several. mutans .Citrus aurantium , the dried root of Paeonialactiflora , and the dried root of Platycodon grandiflorum  [Hainosan (Painongsan) is a formula used in traditional Chinese medicine and traditional Japanese Kampo medicine. The prescription of hainosan was described in Jingui Yaolüe by Zhang Zhongjing in the Later Han Dynasty. This formula has been explained for the treatment of acute or chronic purulent diseases, including periodontal disease. Hainosan contains three kinds of herbs: the dried immature fruit of dix, PG) .Porphyromonas gingivalis [S. mutans. Thus, our objective was to investigate the antibacterial effects of HNS against S. mutans in this study.While we found that the hainosan extract (HNS) may suppress the bacterial growth and biofilm formation of ngivalis ,12, it r2O and stored at −20 °C until use. The fingerprint image of HNS was presented elsewhere [Hainosan consists of 2.25 g of CA, 2.25 g of PL, 1.125 g of PG in a daily dose for humans. Each crude drug was supplied from JPS Pharmaceutical. Co. Ltd. . These crude drugs were under the grade of Japanese Pharmacopoeia 17th Edition . A four-lsewhere .S. mutans UA159  was used in this study [2. The bacteria were suspended in sterile PBS again. The bacterial density was defined by quantifying the absorbance at 600 nm (A600). The bacterial suspension was diluted with PBS to 108 CFU (colony forming unit)/mL.S. mutans (1 × 106 CFU) were cultured in trypticase soy broth (Becton Dickinson) with 0.25% sucrose  (TSB) with the HNS extract at 37 °C under 5% CO2 for 24 h. After that, the aliquot was cultured on trypticase soy agar with 5% sheep blood agar at 37 °C for 24 h under 5% CO2.S. mutans (1 × 106 CFU) were cultured TSB in 96-well polystyrene plates  with or without HNS, the extract, or platycodin D (Fujifilm Wako) for 48 h at 37 °C. After the removal of the medium, we washed the plates three times with PBS. After that, the attached bacteria were stained with 0.2% safranin red (Fujifilm Wako) for 10 min at room temperature and gently washed three times with PBS. Each biofilm was evaluated by quantifying the absorbance at 490 nm. Wells incubated without bacteria were used as blanks. Absorbance of the blank wells was subtracted from the test values.We performed the biofilm assay with some modification . S. mutaS. mutans was cultured in TSB on glass coverslips placed in 24-well polystyrene plates (Thermo Fisher) for 48 h at 37 °C under 5% CO2. After the removal of the medium, we washed the wells three times with PBS. We stained the glass coverslips with fluorescein isothiocyanate isomer (FITC) (Fujifilm Wako). The biofilm images were evaluated utilizing an LSM 510 confocal laser microscope . We also created three-dimensional images from Z-stack images by utilizing Imaris software (Carl Zeiss).We performed a confocal microscopic analysis with minor modifications . S. mutaS. mutans treated with HNS and those not treated with HNS were incubated in TSB on glass coverslips in 24-well polystyrene plates (Thermo Fisher) for 48 h at 37 °C under 5% CO2. Subsequently, we placed the coverslips in 2.5% glutaraldehyde  prepared in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 24 h. After the coverslips were rinsed 2 times with 0.1 M phosphate buffer (pH 7.4), we post-fixed the coverslips utilizing 2% osmium tetroxide (Nisshin EM) at 25 °C for 2 h. We rinsed with distilled water finally. Then, we dehydrated the samples utilizing graduated concentrations of ethyl alcohol for 30 min each. The samples were dried using a critical point dryer . We utilized the carbon conductive paint for mounting. After that, the osmium coating was completed utilizing an Osmium Coater . At last, we examined each sample using a microscope .We carried out the preparation for the scanning electron microscopic analysis as described earlier . S. mutaS. mutans strains treated with HNS were cultured in TSB at 37 °C for 24 h under 5% CO2. We prefixed the samples with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C. After that, we post-fixed the samples with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 45 min. Subsequently, we dehydrated the samples in a graded series of ethanol and embedded them in epoxy resin. We cut ultrathin sections utilizing an ULTRACUT-S (Leica) with a diamond knife and stained them with 2% uranyl acetate in distilled water for 15 min. Then, we stained them with a lead staining solution for 5 min. At last, we evaluated the sections using a JEM-1400 plus electron microscope  at 100 kV. The samples were placed on a 300-mesh carbon formvar copper grid (Nisshin EM), and observed by TEM. We captured the digital images with a Mega View Slow-scan camera (JEOL).Bacterial morphological analysis by transmission electron microscopy (TEM) was performed as described below. First, S. mutans treated with HNS and those not treated were grown until the exponential phase and adjusted in PBS to 1.0 (A600). After we added the 200 μL of n-hexadecane (Fujifilm Wako) to 2 mL of bacterial suspensions in glass tubes, we measured the A600 value of the lower aqueous phase. Then, the tubes were vortexed for 2 min vigorously, followed by 10 min of incubation at 25 °C for phase separation. After that, we measured the A600 value of the lower aqueous phase. We calculated the hydrophobicity utilizing the following equation: percent of hydrophobicity = (1 − (A600 after vortex/A600 before vortex)) × 100.We evaluated the hydrophobicity of the cell surfaces by utilizing the hexadecane method with some modifications . S. muta2, the S. mutans colonies were disrupted using 300 μL of ISOGEN (Nippon Gene) with glass beads . RNA-containing supernatant was treated with 700 μL of ISOGEN for 30 min at 55 °C. After centrifugation, the total RNA was recovered by using chloroform (Fujifilm Wako), isopropanol (Fujifilm Wako), and 70% EtOH (Fujifilm Wako) and dried under appropriate sterile conditions. We quantitated the RNA samples with the Nano Drop ND-1000 (Thermo Fisher). We measured 260/280 nm on this machine to evaluate the RNA. We confirmed that the evaluation value of RNA was all included in 1.8 to 2.0. We synthesized the cDNA with ReverTra Ace qPCR RT Master Mix  according to instructions. We used a total RNA amount of 0.5 μg in this reaction. We used the qPCR for the quantification of cDNA, executed with the Thunderbird qPCR Mix (Toyobo) and Applied Biosystems 7900HT Fast Real Time PCR System (Thermo Fisher) according to the manufacturer’s instructions. The relative quantifications of β-glucosyltransferase (gtfB), glucosyltransferase-SI (gtfC), and fructosyltransferase (ftf) mRNA expression were performed using 16S rRNA as a reference gene. Gene-specific primers were described elsewhere [We used RNA extraction by utilizing ISOGEN  according to the manufacturer’s instructions. Briefly, after culture in TSB with or without HNS at 37 °C for 12 h under 5% COt-test for 2 groups, and Tukey’s multiple comparison t-tests for the differences among multiple groups. The p-values < 0.05 indicated statistical significance.We expressed the study data as the means ± standard deviation (SD). We also conducted the statistical analysis using the Student’s S. mutans was investigated. Although we observed significant bacterial reduction by the treatment of HNS in a concentration-dependent manner, HNS did not show a marked bacteriostatic effect as would an antibiotic  (S. mutans was remarkably inhibited (p < 0.01). Thus, we confirmed that the anti-biofilm activity of HNS occurred in a concentration-dependent manner.Then, to evaluate whether HNS could suppress biofilm formation,  < 0.01) . With anS. mutans treated with HNS and those not treated were stained with FITC dye. We analyzed by confocal laser scanning microscopy  were significantly reduced.Furthermore, we tried to evaluate another assay using SEM . SEM scaS. mutans by morphological analysis. TEM demonstrated that there was no remarkable difference in the bacterial size between HNS-untreated and -treated groups. However, S. mutans treated with HNS were significantly thinner than those of the untreated S. mutans (p < 0.01)To clarify the reason for the reduction of biofilm, we performed the analysis using TEM a. We triS. mutans treated with HNS in comparison with that in the S. mutans of the untreated group (p < 0.01)  .gtfB, gtfC, and ftf in S. mutans UA159 was measured using RT-PCR system. p < 0.01, respectively). It is indicative of the high efficiency of HNS in the diminution of these adhesive accelerating mRNA expressions in S. mutans.The mRNA expression of S. mutans, we evaluated the effect of the samples of HNS by removing one component each, using safranin red staining. We found that with removal of PG, HNS did not exhibit any significant suppressive effect on biofilm formation by S. mutans. However, significant suppressive effects on biofilm formation by S. mutans were exhibited by HNS on removal of the other two crude drugs (p < 0.01) . The extract of PL also showed significant anti-biofilm effects even if weaker (To assess the contribution of each crude drug component of HNS to the inhibitory effect on biofilm formation by  < 0.01) . Thus, wf weaker .S. mutans biofilm formation by safranin red staining. As expected, the remarkable suppressive effect of platycodin D on S. mutans biofilm formation was verified in a concentration-dependent manner, and 10 μg/mL of platycodin D remarkably suppressed the biofilm formation by S. mutans (p < 0.01) (Platycodin D is known as one of the constituents in PG . We asse < 0.01) .S. mutans. We validated that HNS had a biofilm-suppressive effect against S. mutans by triple kinds of assays; absorbance, confocal laser microscopy, and scanning electron microscopy. We revealed that this effect of HNS was related to both the disruption of the surface of bacteria and the reduction of hydrophobicity of the surface of bacteria. We also found that the extract of PG, which is a component of HNS, had a biofilm-suppressive effect on S. mutans, and that one of its components, platycodin D, also exhibited this effect.Here, we clarified the mechanism of the anti-biofilm effect of HNS and its crude drugs on gtfB, gtfC, and ftf mRNAs. A fundamental factor of S. mutans is its glucan-mediated and sucrose-dependent colonization of tooth surfaces [S. mutans synthesize extracellular glucans from the glucose moiety of sucrose by gtfB, gtfC, and gtfD and the fructans from the fructose moiety of sucrose by ftf [S. mutans-secreted glucosyltransferases make specific binding sites useful for the bacterial colonization of the tooth surface as the precursor of dental caries [gtfB, gtfC, and ftf genes are critical for the sucrose-dependent adhesion of S. mutans to hard surfaces, gtfD is not essential [gtfB, gtfC, and ftf genes have become attractive targets for protection against tooth decay and we focused on these three biofilm-associated genes in this investigation. As S. mutans produce extracellular polysaccharides from dietary sucrose, this bacterial virulent factor remarkably augments the cariogenicity [S. mutans [S. mutans biofilm, to control tooth decay [S. mutans including the suppression of mRNA expression except for those constituting hainosan. We reported the anti-biofilm effect of HNS on Porphyromonas gingivalis because of the injury of bacterial cell surface [P. gingivalis but also S. mutans, will advance our therapeutic selection of anti-biofilm effects.We found that HNS suppressed the expression of surfaces . Extracesurfaces . As bothsurfaces ,20. The surfaces . S. mutae by ftf . These Sl caries . As the l caries . Thus, tl caries . As prevssential . Hence, genicity . The les. mutans . Thus, mth decay ,24. Alth surface . AlthougAlthough our results only showed a direct effect on bacteria in vitro, which is not a direct reflection of the effect in humans, it is necessary to evaluate how much time and in what concentration the drug can contact bacteria by the oral administration of the drug in the oral cavity. This needs to be confirmed in the future animal studies and human clinical trials.Platycodon grandiflorum, had a suppressive effect on S. mutans-induced biofilm. This effect was related to the natural compound, platycodin D, contained in Platycodon grandiflorum. Furthermore, the addition of HNS suppressed the biofilm formation of bacteria by reducing the expression of glucosyltransferases and fructosyltransferase of S. mutans. We suggested HNS as a promising agent for the elimination of dental plaques.Our results showed that the extracts of HNS, especially