The present study is the second part of an extensive research conducted by our group.The goal of the present work was to provide a large set of detector‐specific output correction factors for seven small volume ionization chambers on two linear accelerators in four megavoltage photon beams utilizing perpendicular and parallel orientation of ionization chambers in the beam for nominal field sizes ranging from 0.5 cmkQclin,Qreffclin,fref were experimentally determined on two linacs, Elekta Versa HD and Varian TrueBeam for 6 and 10 MV beams with and without flattening filter for nine square fields ranging from 0.5 cm2 × 0.5 cm2 to 10 cm2 × 10 cm2, for seven mini and micro ionization chambers, IBA CC04, IBA Razor, PTW 31016 3D PinPoint, PTW 31021 3D Semiflex, PTW 31022 3D PinPoint, PTW 31023 PinPoint, and SI Exradin A16. An Exradin W1 plastic scintillator and EBT3 radiochromic films were used as the reference detectors.Output correction factors kQclin,Qreffclin,fref were lower for parallel orientation compared to those obtained in the perpendicular orientation. Five ionization chambers from our study set, IBA Razor, PTW 31016 3D PinPoint, PTW 31022 3D PinPoint, PTW 31023 PinPoint, and SI Exradin A16, fulfill the requirement recommended in the TRS‐483 Code of Practice, that is, 0.95<kQclin,Qreffclin,fref<1.05, down to the field size 0.8 cm2 × 0.8 cm2, when they are positioned in parallel orientation; two of the ionization chambers, IBA Razor and PTW 31023 PinPoint, satisfy this condition down to the field size of 0.5 cm2 × 0.5 cm2.For all ionization chambers, values of output correction factors 2 × 0.5 cm2 for two orientations of ionization chambers — perpendicular and parallel. Our main finding is that output correction factors are smaller if they are determined in a parallel orientation compared to those obtained in a perpendicular orientation for all ionization chambers regardless of the photon beam energy, filtration, or linear accelerator being used. Based on our findings, we recommend using ionization chambers in parallel orientation, to minimize corrections in the experimental determination of field output factors. Latter holds even for field sizes below 1.0 cm2 × 1.0 cm2, whenever necessary corrections remain within 5%, which was the case for several ionization chambers from our set.The present paper provides experimental results of detector‐specific output correction factors for seven small volume ionization chambers. Output correction factors were determined in 6 and 10 MV photon beams with and without flattening filter down to the square field size of 0.5 cmTRS‐483 recommended perpendicular orientation of ionization chambers for the determination of field output factors. The present study presents results for both perpendicular and parallel orientation of ionization chambers. When validated by other researchers, the present results for parallel orientation can be considered as a complementary dataset to those given in TRS‐483. The notation fref can refer to either a machine‐specific reference (msr) field or in the case of conventional linear accelerators where a 10 cm2 × 10 cm2 field can be set, the reference field size fref is equal to 10 cm2 × 10 cm2. In the present study, fref=10 cm2 × 10 cm2.A new international Code of Practice (CoP) for reference and relative dosimetry in high energy small static photon fields, TRS‐483, has recently been published jointly by the International Atomic Energy Agency (IAEA) and the American Association of Physicists in Medicine (AAPM).MQclinfclin and MQreffref asFor large clinical fields, a quotient of the absorbed doses in clinical and reference fields can be approximated by the ratio of detector readings kQclin,Qreffclin,fref for the determination of field output factors. Therefore, in the case of small fields, the correct expression for field output factors is given by Eq. ΩQclinCombining Eqs.  and 3),,3), we gIn addition to the new formalism for dosimetry in small static photon fields, TRS‐483 provided a large set of data for detector‐specific output correction factors for solid‐state detectors and cylindrical ionization chambers determined in 6 and 10 MV photon beams from various types of linear accelerators.kQclin,Qreffclin,fref for a few small volume ionization chambersTRS‐483 recommends that the determination of field output factors using cylindrical ionization chambers should be made from measurements made in the perpendicular orientation, that is, the longest axis of the chamber should be oriented perpendicular to the beam central axis as shown in Fig. The orientation of the ionization chamber may not be crucial for the accuracy of relative measurements in large fields where lateral charged particle equilibrium (LCPE) exists on the beam central axis. However, in small fields below a size of about 2.0 cm, the selection of ionization chambers and choice of orientation of the longest axis of the chamber with respect to the beam central axis is important for performing field output factor measurements because of the onset of lack of LCPE, where the dimensions of ionization chambers, even small ones, become comparable to the field sizes.Many air‐filled ionization chambers are designed such that they have larger cavity lengths along the direction of the chamber’s long axis compared to the cavity dimension in the radial direction. Depending on chamber design and orientation used for measurements of field output factors, the volume averaging effect can be significant if a chamber is placed with its stem perpendicular to the beam central axis. Furthermore, some ionization chambers show considerable stem effect, which can be minimized if the chamber is oriented with its stem parallel to the beam central axis, that is, in parallel orientation.The goal of the present work was twofold. First, we aimed to provide a large set of detector‐specific output correction factors for seven small volume ionization chambers/two linear accelerators/four photon beams combinations, utilizing perpendicular orientation as shown in Fig. Secondly, output correction factors for all seven small volume ionization chambers were also determined in the parallel orientation [Fig. It should be noted that the present work is the second part of an extensive research conducted by our group. In the first part, we discussed the determination of field output factors and output correction factors for seven solid‐state detectors using two reference detectors.2The experimental equipment and methodology used in the present study are the same as those used in the first part of our study, where we gave detailed discussion about the determination of field output factors using plastic scintillator Exradin W1  and radiochromic films EBT3 , as reference detectors and output correction factors for six diodes and a microdiamond detector.Output correction factors for ionization chambers determined in this study represent the “total” correction factors for a particular detector and comprise individual correction factors for all contributing effects including volume averaging, polarity, and recombination.2.13, while the rest of the chambers belong to the group of small, sometimes named as mini ionization chambers having active volumes ranging from 0.01 cm3 < V < 0.3 cm3. Table Seven mini and micro ionization chambers were selected for the determination of detector‐specific output correction factors in small static fields in megavoltage photon beams: IBA CC04 and IBA Razor , PTW 31016 3D PinPoint, PTW 31021 3D Semiflex, PTW 31022 3D PinPoint, PTW 31023 PinPoint , and SI Exradin A16 . The selection was based on their physical dimensions, characteristics, and suitability for clinical use, following the recommendations given in TRS‐483. Two ionization chambers included in the study, IBA Razor and SI Exradin A16, are classified as micro ionization chambers with active volume V ≤ 0.01 cmIt should be noted that PTW 31022 3D PinPoint and PTW 31023 PinPoint chambers superseded previous models PTW 31016 3D PinPoint and PTW 31014 PinPoint, respectively. The newer models are geometrically and constructionally almost identical to their predecessors with the exception of the diameter of the central electrode which has been increased from 0.3 mm (old models) to 0.6 mm (new models).Similarly, IBA Razor ionization chamber superseded previous model IBA CC01. However, in this case, there is significant constructional difference between both models: the new model has been designed with a graphite electrode while the old model is built with a steel electrode. The rest of the characteristics of the new model, that is, cavity dimensions, wall material, and wall thickness, remained the same as the old one.2.2TM  equipped with Agility™ MLC system and Varian TrueBeamTM  equipped with Millenium™ MLC system, using photon beams of nominal energies 6 and 10 MV. Both MLC systems have leaves with 0.5 cm width in the central part of the radiation fields. Beams with flattening filters (WFF) as well as flattening filter free (FFF) beams were used for all measurements, and are denoted hereafter as 6 MV WFF, 6 MV FFF, 10 MV WFF, and 10 MV FFF. The measurement geometry consisted of an isocentric setup with a source‐to‐surface (SSD) distance of 90 cm, a depth of 10 cm, and gantry at 0°. For each point measurement, 100 MU was delivered to nine square fields with nominal side lengths of 0.5, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, and 10.0 cm. On Elekta linac, radiation fields were shaped with MLC in cross‐line (x) direction and with jaws in the in‐line (y) direction. On Varian TrueBeam linac, radiation fields were collimated using the linac jaws in both axes, x and y. A conventional reference field reff = 10 × 10 cm2 was used as the reference field for the calculation of detector‐specific output correction factors. At least three measurements of collected charge were taken for each setup, using a reference class PTW Unidoswebline  electrometer. In the case of low signals, the range of the electrometer was adjusted appropriately. Measured raw signals were corrected for environmental conditions (temperature and pressure), whenever necessary. No other corrections  were applied to the measured data obtained with ionization chambers.Dosimetry measurements for the determination of output correction factors were performed at two hospitals on two different linear accelerators, Elekta Versa HDA 3D water phantom  was used for the measurements on the Elekta Versa HD linac, while an MP3‐M water phantom  was used for measurements in the Varian TrueBeam linac.2 (for PTW MP3‐M water phantom) and myQA Accept (for IBA Blue Phantom 2);CAX alignment, following the procedure within the integrated softwares Mephysto mcrepositioning (centering) of the ionization chamber after acquiring lateral beam profiles along cross‐line and in‐line directions;finally, each detector was moved using manual mode in 0.2 mm (0.1 mm if necessary) steps along both, x and y axes, and irradiated every time with 100 MU to verify the position where the collected charge was maximal.Before measurements, each ionization chamber was positioned with its effective point of measurement at the reference depth of 10 cm. Two sets of measurements were performed: (a) with the chamber’s stem perpendicular to the beam axis as shown in Fig. The position where the collected charge reached the highest value was assumed to lie at the peak of the beam profile along the beam central axis, that is, the center of the radiation field. Thus, obtained position of the ionization chamber was considered as the correct one and was used for the subsequent measurements of output factors. The above procedure for lateral alignment of detectors was done separately for each photon beam and for the two smallest fields (0.5 and 0.8 cm). Similar alignment procedure is also recommended in the ICRU Report 91.Sclin was calculated according toA and B correspond to the radiation field width at half maximum (FWHM) in the in‐line and cross‐line directions, respectively, which were determined using EBT3 film measurements in the present work.As suggested by Cranmer‐Sargison et al.,2.3Sclin, discrete values of output correction factors kQclin,Qreffclin,frefSclin were calculated using the following equationMQclinfclin and MQreffref denote chamber readings in clinical and reference fields, respectively. Discrete values of field output factors ΩQclin,Qreffclin,fref were obtained from the analytical function ΩSclin proposed by Sauer and Wilbert,For every ionization chamber and each measured equivalent square small field size kQclin,Qreffclin,frefSclin determined by Eq. , ratios kperp/kpara were calculated using Eq.  equal cavity volumes can have significantly different kvol, since their cavity dimensions in longitudinal and radial axes, dL and dR, differ. Therefore, differences in kvol will be reflected also in differences of total output correction factors.The primary reason for the differences in output correction factors measured with different ionization chambers is the volume averaging effect of the different detectors. It is apparent that chambers with larger cavity volumes will have larger volume averaging correction factors dL/dR, ratios of kperp/kpara was analyzed in more detail for two small volume ionization chambers, PTW 31022 3D PinPoint and PTW 31023 PinPoint. These two chambers have almost equal cavity volumes, however, they differ considerably in the ratios dL/dR; PTW 31022 3D PinPoint chamber has equal cavity dimensions in its longitudinal and radial axis, dL=dR=2.9mm, whereas PTW 31023 PinPoint chamber is more elongated, having dL=5.0mm and dR=2.0mm  have approximately normal distribution with 95% of values inside limits Sclin.The combination of both uncertainties yielded a total uncertainty of 0.09 mm for the clinical field sizes 2.5.2For the determination of field output factors, signals measured with EBT3 films and W1 plastic scintillator were used jointly after normalization and rescaling. Detailed methodology is described in the first part of our study.For EBT3 films, the total uncertainty budget includes several sources of uncertainty: intra‐fragment uncertainty, inter‐fragment uncertainty, and intra‐lot difference with respect to the calibration. Estimated total relative uncertainty was 1.7% for measurements made on the Elekta linac and 2.2% for measurements made on the Varian linac.Dispersion of measured signals and uncertainties associated with the calibration procedure were considered as type A uncertainty for measurements with W1 plastic scintillator. Total uncertainties ranged from 0.3% to 1.1% and from 0.8% to 1.3% for measurements made on the Elekta and Varian linacs, respectively.Mean values of the signals measured with both detectors and associated uncertainties were employed in the fitting to the analytical function from Eq. . Field o2.5.3ΩSclin obtained from the analytical function from Eq.  to the uncertainty of the measured signal MQclinfclin (y‐axis) has been done asFor the determination of output correction factors, field output factors from Eq.  were usen in Eq. . In addiuC of the mean value of measured signals for particular clinical field Sclin was finally estimated as the combination in quadrature of both types of uncertainties. No other influencing quantities were considered as contributing factors to the type B uncertainty including possible field size‐dependent effects such as polarity, recombination, or stem effect. In our opinion, inclusion of additional uncertainties of type B could lead to an overestimation of the overall uncertainty budget, since our approach was to determine total output correction factors, which contain all individual correction factors.Combined uncertainty uB,x=L/2=0.05mm(L=±0.1mm), yielded the overall relative uncertainties uCk=1 around 1.0%, ranging from 0.7 to 1.3% for different MQclinfclin/MQreffref ratios.A combination of the uncertainty of type A and estimated uncertainty of type B, 2.5.4Values of output correction factors provided in TRS‐483 were compared with the data obtained in our study for three ionization chambers: IBA CC04, PTW 31016 PinPoint 3D, and SI Exradin A16. Comparison was done only for perpendicular orientation of ionization chambers since the TRS‐483 does not give data for parallel orientation and for field sizes for which detector‐specific output corrections do not exceed 5%.Sclin from our study. Compatibility of both sets was evaluated using the difference between corresponding values of output correction factors from both datasets, that is, kTRS-483-kperp, where kTRS-483 denotes output correction factors from TRS‐483, and kperp stands for the output correction factors obtained in our study in perpendicular orientation of ionization chambers. We assumed that the output correction factors between values in the TRS‐483 and our data would differ significantly (within 95% confidence limits), if the relationFor TRS‐483, values for output correction factors and their estimated uncertainties were taken from tables 26, 27, and 37 . Before comparison with our results, those values were interpolated to match clinical field sizes 2.5.5kperp/kpara from Eq. , while the other corresponds to the PTW 31023 Pinpoint chamber (dL/dR=2.5). Ratios kperp/kpara were determined as average values of output correction factors determined in all megavoltage beams on both linear accelerators using the analytical function from Eq. . It should be noted that output correction factors were compared for field sizes given in TRS‐483 — for IBA CC04 and PTW 31016 PinPoint 3D chambers only down to 1.0 cm2 × 1.0 cm2, whereas for SI Exradin A16 chamber, the smallest field in the comparison was 0.8 cm2 × 0.8 cm2.Among the seven ionization chambers from our study set, data for only three chambers are available in TRS‐483 for comparison: IBA CC04, PTW 31016 PinPoint 3D, and SI Exradin A16. To determine the statistical significance of differences between corresponding sets of output correction factors, we have examined fulfillment of the condition from Eq. . Data frfrom Eq.  was founWe can conclude that with the possible exception stated above, detector‐specific output correction factors obtained in our study for three ionization chambers, IBA CC04, PTW 31016 PinPoint 3D, and SI Exradin A16, confirm the corresponding data published in the TRS‐483 using the 95% confidence limits. The data for output correction factors determined in this experimental study for the remaining four ionization chambers, IBA Razor IC, PTW 31021 3D Semiflex, PTW 31022 PinPoint 3D, and PTW 31023 PinPoint, are considered a valuable supplement to the literature and to the TRS‐483 dataset.4.2kQclin,Qreffclin,fref in small fields both orientations for only a few small or micro ionization chambers.The rationale for recommending perpendicular orientation of ionization chambers in TRS‐483 for the determination of field output factors in small fields has recently been clarified by the authors of TRS‐483.kQclin,Qreffclin,fref are found to be lower for the parallel orientation than the corresponding values determined for the perpendicular orientation. This observation holds for field sizes below 1.0 cm2 × 1.0 cm2, regardless of the energy or linac being used. The significance of these differences was tested using the condition from Eq. . Latter characteristic also indicates that there should be only small (negligible) difference in their response regardless of the orientation, perpendicular or parallel, in the beam. Both chambers have a cavity length of 2.9 mm, which is identical to their cavity diameters. However, in this case, differences between the two orientations were found to be more prominent than in the case of previous two examples. Namely, for the PTW 31016 chamber, significant differences are observed for all small fields up to the 1.5 cm2 × 1.0 cm2 field size in all photon beams on both linacs, while for the PTW 31023 chamber, significant differences in the response were found for four smallest investigated field sizes (up to 1.5 cm2 × 1.5 cm2) on both linacs. The most pronounced differences, concerning the orientation, were found for the PTW 31023 PinPoint chamber, which was expected, since this chamber has the most elongated cavity geometry among all chambers included in the study, and thus the most notably expressed volume averaging effect in the perpendicular orientation. For IBA Razor chamber, we noticed a slight overresponse for smallest field sizes in particular in 10 MV FFF beam on Elekta Versa HD  larger than the diameter of the cavity. These two chambers are IBA Razor and PTW 31023 PinPoint. Both chambers have a cavity diameter of 2.0 mm. However, they have different cavity lengths: IBA Razor has a cavity length of 3.6 mm, while the PTW 31023 PinPoint chamber has a cavity of length 5.0 mm  in perpendicular orientation, confirm the corresponding data published in the TRS‐483 using the 95% confidence limits.This large set of output correction factors for seven ionization chambers determined in perpendicular orientation is considered a valuable supplement to the TRS‐483 dataset, in particular for four ionization chambers IBA Razor IC, PTW 31021 3D Semiflex, PTW 31022 PinPoint 3D, and PTW 31023 PinPoint for which TRS‐483 did not provide kQclin,Qreffclin,fref values for the same set of small volume ionization chambers in parallel orientation [Fig. 0.95<kQclin,Qreffclin,fref<1.05 is not violated even for field sizes below 1.0 cm2 × 1.0 cm2 for five ionization chambers used in the present study. Additionally, output correction factors kQclin,Qreffclin,fref determined with IBA Razor and PTW 31023 PinPoint ionization chambers in a parallel orientation were always within the interval 0.95–1.05 regardless of the photon beam energy or linac used down to the smallest investigated field size of 0.5 cm2 × 0.5 cm2.In addition to the output correction factors determined in perpendicular orientation, we have also provided a large set of To conclude, for minimizing corrections in the experimental determination of field output factors, parallel orientation of ionization chambers is advantageous over the perpendicular. This latter outcome of our study is considered as a valuable contribution to the discussion on the orientation of ionization chambers in small MV beams. While the results from the present study justify our final statement, further investigations and confirmation of our findings regarding the orientation of ionization chambers in small fields, from other research groups, are necessary for an eventual update of the TRS‐483 in the future. Until that happens, the recommendations from TRS‐483 should be followed.


Paenibacillus polymyxa SC2, a bacterium isolated from the rhizosphere soil of pepper (Capsicum annuum L.), promotes growth and biocontrol of pepper. However, the mechanisms of interaction between P. polymyxa SC2 and pepper have not yet been elucidated. This study aimed to investigate the interactional relationship of P. polymyxa SC2 and pepper using transcriptomics.P. polymyxa SC2 promotes growth of pepper stems and leaves in pot experiments in the greenhouse. Under interaction conditions, peppers stimulate the expression of genes related to quorum sensing, chemotaxis, and biofilm formation in P. polymyxa SC2. Peppers induced the expression of polymyxin and fusaricidin biosynthesis genes in P. polymyxa SC2, and these genes were up-regulated 2.93- to 6.13-fold and 2.77- to 7.88-fold, respectively. Under the stimulation of medium which has been used to culture pepper, the bacteriostatic diameter of P. polymyxa SC2 against Xanthomonas citri increased significantly. Concurrently, under the stimulation of P. polymyxa SC2, expression of transcription factor genes WRKY2 and WRKY40 in pepper was up-regulated 1.17-fold and 3.5-fold, respectively.P. polymyxa SC2 to inhibit pathogens was enhanced. P. polymyxa SC2 also induces systemic resistance in pepper by stimulating expression of corresponding transcription regulators. Furthermore, pepper has effects on chemotaxis and biofilm formation of P. polymyxa SC2. This study provides a basis for studying interactional mechanisms of P. polymyxa SC2 and pepper.Through the interaction with pepper, the ability of The online version contains supplementary material available at 10.1186/s12866-021-02132-2. Plant growth-promoting rhizobacteria (PGPR) inhabit the rhizosphere soil and can effectively promote plant growth through a variety of direct and indirect mechanisms . PGPR diPaenibacillus polymyxa, an important member of the PGPR, is widely distributed in the rhizosphere soil of wheat, maize . The plant negative control comprised pepper seedlings treated with 1 mL of 1× PBS buffer; roots of the pepper seedlings were collected after 20 h and marked as [P]. The P. polymyxa SC2-pepper co-cultured group comprised peppers treated with 1 mL P. polymyxa SC2; bacterial cells and pepper roots were collected after 20 h and named as [SH] and [PH], respectively. Each treatment was designed with three biological replicates. The collected bacteria and pepper roots were frozen in liquid nitrogen and stored at − 80 °C until further processing.To better analyze the interaction mechanisms between s method . CultureP. polymyxa SC2 and peppers was extracted and purified using the TRIzol (Invitrogen) method. Total RNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer. RNA integrity number (RIN) was investigated using an Agilent 2100 Bioanalyzer. RNA quality is an essential factor in RNA-seq, therefore only RNA samples with RIN > 6, 230/260 and 260/280 ratios> 2 were used. The mRNA of pepper was isolated from the crude RNA via Oligo (dT) according to the manufacturer’s (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®) instructions. The mRNA of P. polymyxa SC2 was isolated by removing rRNA. The mRNA was randomly fractured into small fragments of approximately 200 bp, and was reversed to a single strand of cDNA by random primers. Two-strand cDNA was further synthesized to form a stable double-strand structure. End Repair Mix was used to complement double-stranded cDNA into flat ends, and then an A base was added at the 3′ end. The library was enriched and sequencing of cDNA fragments was conducted using an Illumina Hiseq4000 platform.Total RNA of https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle). In this stage, the reads without inserting fragments due to the self-connection of connectors, and the reads with N ratio over 10%, and the reads less than 20 bp were removed. Then the high-quality clean data were assembled and aligned. Clean reads of P. polymyxa SC2 were mapped to the reference genome of P. polymyxa SC2 (https://www.ncbi.nlm.nih.gov/nuccore/NC_014622.2). Bowtie2 was used to align the clean reads and reference genome [http://trinityrnaseq.sourceforge.net/, vision:trinityrnaseq-r2013-02-25) was used to assemble the short fragment sequences of pepper after obtaining high-quality sequencing data from RNA-seq [− 5). The software edgeR was used to analyze differentially expressed genes [https://github.com/tanghaibao/GOatools) [p-value (p_fdr) ≤ 0.05 indicated that the GO/KEGG function was enriched significantly.Raw RNA-seq data were stored in a fastq file format. To ensure the accuracy of subsequent bioinformatics analysis, raw reads were filtered by SeqPrep (e genome . Since ted genes . GO enriOatools) . KEGG paOatools) . Fisher’Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biotechnology (Hunan) Co., Ltd). Selected genes for each treatment were amplified to validate the RNA-seq results. These genes were selected from the DEG lists obtained for each condition. Primer sequences were designed using Beacon Designer 7 and are listed in Additional File Table S5. The gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of pepper was used as the reference gene and GAPDH of P. polymyxa SC2 was used as an endogenous control. Relative expression levels were calculated using the ΔΔCt method [One microgram of purified total RNA was used as a template for first-strand cDNA synthesis using an t method . Three bP. polymyxa SC2. There were three treatment groups. Control: fermentation medium 2SO4 6.66 g/L, CaCO3 6.26 g/L, KH2PO4 0.2 g/L, NaCl 0.2 g/L, MgSO4 0.2 g/L) inoculated with P. polymyxa SC2. Treated group 1 (MS): fermentation medium supplemented with 1 mL MS medium and then inoculated with P. polymyxa SC2. Treated group 2 (PM): fermentation medium supplemented with 1 mL MS medium (which has been used for culturing peppers) and then inoculated with P. polymyxa SC2.An experiment was designed to test the effects of pepper root exudates on antibiotic production by P. polymyxa SC2 was inoculated into 5 mL LB liquid medium and cultured at 37 °C, 180 rpm for 8–12 h, before being subcultured into the appropriate fermentation medium at 2% inoculation volume and incubated at 37 °C, 180 rpm for 72 h. For the antagonistic test, the P. polymyxa SC2 cultures were centrifuged at 12000 rpm for 10 min to remove cells, and the supernatants were used. Sterile water with 2% agar was added into Petri dishes and allowed to coagulate, then Oxford cups were placed in the Petri dishes. Next, 2 mL Xanthomonas citri cells were mixed with 200 mL LB medium containing 1% agar and cooled below 55 °C to prepare the plates. A total of 100 μL P. polymyxa SC2 culture supernatant was loaded into the well of the Oxford cup and incubated at 37 °C for 24 h to observe the growth inhibition effect on X. citri. All treatments had three replicates.t-test in SPSS 19.0. Columns were drawn using GraphPad Prism 7. P < 0.05 (*) in columns means there was a significant difference, and P < 0.01 (**) means there was an extremely significant difference.Statistical analyses were performed using the Student’s Additional file 1. Detailed statistics of reads mapping: Table S1 Mapping proportion statistics in RNA-seq of strain SC2, Table S2 Mapping proportion statistics in RNA-seq of peppers.Additional file 2: Fig. S1 PCA analysis of samples based on the gene expression level (FPKM) in RNA-seq.Additional file 3: Table S3 Annotation of differentially expressed genes in P. polymyxa SC2.Additional file 4: Table S4 Annotation of differentially expressed genes in pepper.Additional file 5: Fig. S2 Heatmap of DEGs in P. polymyxa SC2.Additional file 6: Fig. S3 Heatmap of DEGs in pepper.Additional file 7: Table S5 Primers for RT-qPCR.


KMT2B) gene have recently been reported to be associated with childhood‐onset generalized dystonia. There have been no studies investigating ablative treatments for the management of this disorder. Three patients underwent either a staged unilateral pallidotomy and contralateral pallidothalamic tractotomy , a unilateral pallidothalamic tractotomy  or a simultaneous unilateral pallidothalamic tractotomy and ventro‐oral thalamotomy . The average total patient score on the Burke‐Fahn‐Marsden Dystonia Rating Scale‐Movement Scale improved from 39.5 to 13.2 (66.6%) after the procedures. No significant complications were identified. Ablative treatments appear to be a promising alternative surgical option for generalized dystonia with KMT2B mutation.Mutations in the lysine methyltransferase 2B ( KMT2B) gene have been recently reported to be associated with childhood‐onset generalized dystonia (DYT‐28 dystonia), a movement disorder that is characterized by limb onset and progression to cranio‐cervico‐axial involvement.Mutations in the lysine methyltransferase 2B  of the globus pallidus internus (GPi).The ethics committee of the Tokyo Women’s Medical University approved the study. Written informed consent was obtained from all patients.Detailed patient characteristics and surgical outcomes are shown in Tables KMT2B mutation. His development slowly decelerated after birth, resulting in microcephaly, short stature, cognitive decline, and dysarthria. At 6 years of age, left foot inversion developed. Two years later, myoclonic movements in bilateral arms and truncal bending developed. At 15 years of age, his truncal bending worsened, resulting in wheelchair dependence, and dysphonic symptoms developed. His disease progressed with time, and treatment with several oral medications and botulinum toxin injections was ineffective. As a result, he was referred to our hospital for surgical treatment. At presentation, his most significant symptom was truncal bending while walking or standing. His preoperative Burke‐Fahn‐Marsden Dystonia Rating Scale‐Movement Scale (BFM‐MS) score was 66 (Video Patient No. 1 was a 19‐year‐old man with generalized dystonia and a confirmed 66 Video . Because66 Video . His tru66 Video . Six mon66 Video . At the KMT2B mutation. Bilateral jerky hand movements initially developed at 10 years of age. These jerky movements remained stable for approximately 20 years, and the patient was employed without significant difficulty. At 32 years of age, however, the patient developed neck and trunk tilting to the left, resulting in unemployment. Oral medications, including anticholinergics and clonazepam, as well as botulinum toxin injections, were not effective. After 1 year, his axial symptoms suddenly deteriorated, and he was referred to our hospital for surgical treatment. His preoperative BFM‐MS score was 32. Due to his predominantly left‐sided dystonic symptoms (Video Patient No. 2 was a 34‐year‐old man with generalized dystonia and a ms Video , the patms Video . At the KMT2B mutation. At 14 years of age, he developed difficulty in writing with his right hand and progressive difficulty in extending his right elbow. He was subsequently diagnosed with dystonia. Treatment with anticholinergics, levodopa, clonazepam, and botulinum toxin injections were attempted, without significant symptomatic improvement. Twelve years after onset, neck tilting, trunk bending, and right shoulder elevation developed (Video Patient No. 3 was a 29‐year‐old right‐handed man with generalized dystonia and a ed Video , and he ed Video . After tThis report is the first to describe the use of ablative surgeries in patients with DYT‐28 dystonia, and these cases demonstrate three important findings. First, limb symptoms responded very well to contralateral ablations. Second, unilateral ablations were not sufficient to successfully improve axial symptoms. Third, bilateral ablations greatly improved axial dystonic symptoms, as previous studies using GPi‐DBS have also reported.KMT2B gene have been reported to be associated with the development of generalized DYT‐28 dystonia.Various mutations in the Previous studies have shown significant improvements in patients with DYT‐28 dystonia after bilateral GPi‐DBS, with 5 to 10 years of improvement after the initial procedure.Patients who develop generalized dystonia at a young age are likely to require neurosurgical treatments, including DBS or ablative procedures. The reported mean age of onset for DYT‐28 is 6 years, and disease progression to generalized dystonia generally requires 2 to 10 years.The safety of bilateral ablative treatment has not yet been elucidated, and bilateral pallidotomies may lead to unpredictable and poor outcomes, including parkinsonism and delayed cerebral infarctions. Therefore, we utilized a staged one‐sided pallidotomy and then contralateral pallidothalamic tractotomy in patients requiring bilateral ablative surgeries for dystonia. We have recently reported the preliminary outcomes of this strategy.The present study demonstrated that these three patients with DYT‐28 dystonia responded well to ablative treatments, which suggests that ablative treatments appear to be a promising alternative surgical option for generalized dystonia with KMT2B mutation. Due to the limitation of this study including small number of patients and short follow‐up period, further studies with larger sample sizes and longer follow‐up period are needed to confirm the safety and long‐term efficacy of ablative treatments for DYT‐28 dystonia.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.SH acquired and analyzed the data and drafted the manuscript. KA and HA collected and analyzed the data. TN acquired the data. TK supervised this study. TT acquired and analyzed the data and supervised this study.Video S1. Pre‐ and post‐operative condition after surgery in Case 1Click here for additional data file.Video S2. Pre‐ and post‐operative condition after surgery in Case 2.Click here for additional data file.Video S3. Pre‐ and post‐operative condition after surgery in Case 3.Click here for additional data file.


Support walkers are a type of assistive device that may enable non-ambulant children with disabilities to walk independently and promote improvements in bowel function, bone mineral density (BMD), mobility, independence, participation and social function. However, there is little evidence to support these benefits and there is a lack of research describing the use of support walkers in clinical practice. This study aimed to examine the use of support walkers for children with disabilities in clinical practice.A survey was distributed via professional organisations, charities and schools associated with paediatric disabilities in the UK. Participants were recruited between January and March 2018. Populations of interest were those who prescribe support walkers to children with disabilities and those who work with children who use them.In total, 125 people were included in the analysis; 107 responders prescribed support walkers and 18 responders worked with children who used support walkers. The population of children who use support walkers ranged from 6 months to 18 years and included children with cerebral palsy, chromosomal abnormalities and other medical conditions. Use of these devices was also reported in schools, at home and in the community for varying lengths of time. Numerous perceived benefits were noted, most frequent of which were increases in physical activity and enjoyment. By comparison, fewer perceived problems were identified but centred on lack of space and difficulty with transfers.This study provides insight into the use of support walkers in the UK, particularly surrounding current practices, which may help to improve consistency in clinical settings. Perceived benefits and problems may provide a basis for identification of appropriate outcome measures to monitor effectiveness. These results should also provide a basis for designing future studies to examine effectiveness of support walkers for paediatric disabilities.The online version contains supplementary material available at 10.1186/s12887-020-02401-5. Children with disabilities, particularly those with severe motor impairments, participate in less physical activity and exhibit more sedentary behaviour than their typically developing peers , 2. BarrHandheld walkers are commonly used to promote independent locomotion. However, these may not be feasible for children with severe mobility impairments who lack the motor control or strength to stand upright . SupportSupport walkers may enable non-ambulant children the opportunity to walk independently. In a systematic review, several studies found positive changes across all dimensions of the International Classification of Functioning, Disability and Health , followiWhile some studies have investigated the effectiveness of support walkers on a variety of outcomes, there is a lack of research examining how support walkers are currently used for children with disabilities in clinical practice. While one study has described the use of support walkers among children with disabilities in the United States , the stuA cross-sectional survey design was used. Approval for the current research study was granted by Brunel University London Research Ethics Committee . Completion of the survey indicated consent. All responses were anonymous.Participants were recruited over 10 weeks between January and March 2018. Professionals who prescribe support walkers, such as physiotherapists , and those who work with children who use support walkers but do not prescribe them, such as teachers  were invited to complete a survey. To be included in the study, prescribers must have prescribed a support walker to at least one child, aged 0–18 years. To be included in the study, non-prescribers must have worked with at least one child, aged 0–18 years who used, or currently uses a support walker. A support walker was defined as any device that permits mobility for children who require more support than provided by handheld walkers, including some level of trunk, pelvic and/or head support . No restInformation about the study was shared with 33 Special Educational Needs (SEN) schools, 9 professional organisations and 10 charities for young people with disabilities in the UK. These were identified via an internet search. Emails were sent and replies were received from eight UK organisations and charities for children with disabilities  and eight SEN schools. Gatekeepers who worked in each organisation  distributed a brief description of the study and link to the information sheet and survey via email or social media. One school was provided with paper versions of the survey and information sheet to distribute at their request.Separate surveys were developed for prescribers and non-prescribers following a literature review , 6, 9–12The surveys contained 28 and 29 questions for the non-prescriber and prescriber surveys respectively, taking approximately 10–15 min to complete. The terms ‘Please tick all that apply’ or ‘Please select only one response’ followed the questions to maximise clarity. Most questions were close-ended, although there were three open-ended questions to permit greater exploration of opinions and depth of understanding. The open-ended questions were: “What, in your opinion, would be the ideal age to begin use of a support walker?”; “Please identify if there are any precautions for use of support walkers (e.g. supervision)” (Only present in the prescribers survey); “Any other comments?”Prior to data analysis, surveys in which responders reported working exclusively with adults (over 18 years old) were excluded. Descriptive statistics  were used to report data. If five or more participants provided the same response to a question where there was an option to select “other”, these responses formed a new category. Where a range of ages was given in response to the ‘age of introduction’ questions, the mean of this range was calculated and subsequently included in the calculation for mean of all responders. Responses to open questions were analysed using a simplistic form of content analysis, whereby similar responses were assigned a code and percentages of each code were calculated. Responses to the ‘any other comments’ question underwent a more detailed process of content analysis as described by Elo and Kyngäs , wherebyOne hundred twenty-six surveys were returned; 107 from prescribers and 19 from non-prescribers. One non-prescriber survey was excluded as the responder reported only working with adults; thus, 18 were included in analysis. No response rates could be calculated as it was unknown how many eligible individuals received the survey.Most prescribers were female (97.2%) and physiotherapists (98.1%). One occupational therapist and one therapy assistant also reported prescribing support walkers. Prescribers reported working across multiple settings including the community setting (80.4%), SEN schools (57.9%), mainstream schools (42.1%), outpatient settings (28.0%), inpatient settings (6.5%) and ‘other’ areas (3.7%). The occupation of non-prescribing professionals was more heterogeneous, comprising of physiotherapists (33.3%), occupational therapists (33.3%), teachers (22.2%), classroom assistants (5.6%) and other (5.6%). Prescribers and non-prescribers had worked with children using support walkers for a mean of 16.1 (range 2–35 years), and 11.7 (range 1–25 years) years, respectively.n = 20, 66.7%), more specifically their stage of development, cognition, motivation and motor ability. Five responders (16.7%) reported support walkers were often prescribed following recognition that a child was attempting to weight bear and step.Table All prescribers reported prescribing support walkers to children with spastic cerebral palsy. Many prescribers also reported prescribing support walkers to children with dyskinetic CP, ataxic CP, chromosomal abnormalities and spina bifida Table . SimilarApproximately 90% of prescribers and non-prescribers reported that children using support walkers also used a wheelchair Table . HoweverA child’s level of mobility was the most common factor affecting prescription of support walkers, as reported by 97.2% of prescribers Table .Table 2In total, 21 support walkers were identified by prescribers , in schools 94.4%) and in the community (69.2%) . The second most frequently reported precaution was the need for a suitable environment including a suitable surface, adequate space and distance from other children . Other precautions to consider were the need for competent or trained staff and parents  and the presence of other medical conditions, particularly those with epilepsy .Ninety prescribers (84.1%) provided a response to question “Please identify if there are any precautions for use of support walkers”. The requirement for supervision was most commonly reported  provided a response to the question ‘any other comments’. Where quotations are reported below, the responder’s individual identification (ID) number follows.“we strive to get children walking as soon as they tolerate supported standing or start nursery when peers are exploring areas in standing … many go on to independent walking without aids. An early approach to walking puts steps in place for the child to progress quicker and reduced frustration at not being able to get about.” (Participant 38)“[I] generally don’t prescribe them until later when it becomes evident that the child’s walking potential has been met and only then for enjoyment and [the] other factors mentioned” (Participant 17)This category details two conflicting points of view regarding the age that support walkers should be introduced. The first is that support walkers should be introduced early as this may precipitate independent walking. Conversely, others believed that support walkers should only be introduced after no further gain in walking ability is expected. The contrasting points of view are exemplified below.“it is frustrating that they mostly have to be privately funded or by seeking charity funds” (Participant 103)Some prescribers also noted that even when funding was available certain criteria must be met, such that they may only be prescribed if the child is expected to gain the ability to walk, as in the next quote.“our manager will not allow NHS funding for a supportive walker unless it is likely to lead to that child becoming an independent walker” (Participant 99)Comments highlighting issues acquiring funding for support walkers were common. For example,“In my experience walkers give children with otherwise no independent mobility a change of position, exercise and mobility which they love” (Participant 57)“Some early [developmental] delay children we also put in as a way of gaining weight bearing but they will not be long term users and progress to independent walking.” (Participant 89)Several prescribers remarked that support walkers are primarily for non-ambulant children, although a wide range of children may use them. The quotations below exemplify this:This study aimed to examine the use of support walkers for children with disabilities in clinical practice in the UK. The findings highlight that support walkers are prescribed to children with a variety of medical conditions including cerebral palsy, chromosomal abnormalities and spina bifida. Mobility level, tolerance and activity level of the child were the most reported factors affecting prescription of support walkers which were subsequently used for differing lengths of time per day. Many perceived benefits, including increasing physical activity, enjoyment and participation were reported. A substantial gap in the literature on support walkers exists, making comparisons to previous research difficult.Below is a discussion of the findings in relation to each research question.While prescribers and non-prescribers report that most children who use support walkers have cerebral palsy, these findings highlight that support walkers are prescribed to children with a substantial diversity of conditions. Children with chromosomal abnormalities were the third most frequently reported group that support walkers were prescribed to, even though no previous research examining the effects of support walkers have included this population.This study found that some children are prescribed support walkers from as young as one-year-old. However, children below the age of 3½ years old are frequently excluded from participating in studies examining effectiveness of support walkers , 10. AltSeveral types of support walkers were reportedly used, most of which currently have no research investigating their effectiveness. However, approximately a quarter of responders suggested that the available evidence influenced their decision. A child’s mobility level and motor control were commonly reported to influence prescription of support walkers. Previous research examining effectiveness has commonly excluded children of higher motor function , 11. HowMost responders reported that children continue using support walkers for 5–10 years, far exceeding the duration of follow-up in previous research, which ranged from 3 months to 3 years , 9–11. ADeterioration of a child’s condition was identified in this study as the most reported reason for discontinuation. The progressive nature of some conditions (e.g. muscular dystrophy) or negative changes that occur during growth spurts (e.g. scoliosis and hip problems) will likely make walking difficult or impossible for these children . The resAlmost half of prescribers reported support walkers should be used as much as able, suggesting that longer use is encouraged. However, both prescribers and non-prescribers also noted that a lack of staff limited the duration of use. Huang et al.  remarkedResponders perceived that support walkers have numerous benefits. The most commonly reported perceived benefits were increased physical activity, participation and enjoyment. Fewer prescribers and non-prescribers reported physiological changes associated with use of support walkers, including bone mineral density and bladder and bowel function. Despite never being examined as an outcome in studies of effectiveness of support walkers, increasing the amount of time a child is physically active was the most reported benefit by prescribers and non-prescribers. For a group that commonly exhibits less physical activity, support walkers may therefore promote participation in physical activity and associated health benefits, such as reduced pain, fatigue and osteoporosis among children with disabilities . In addiThe perception that support walkers increased participation in everyday life for children with disabilities was commonly reported. This may relate to many tasks encountered during the typical day, from education to play. Being upright affords children different opportunities to experience their surroundings, which was frequently reported in this research. McKeever et al.  found thLack of space was perceived as a problem by many prescribers and non-prescribers in this study. Huang et al.  reportedInstability and reports of support walkers tipping over were noted in previous literature when using a particular support walker , particularly when on uneven surfaces outside . Almost A relatively small sample size and the inability to calculate response rates are perhaps the most important limitations associated with this study, especially for the non-prescribing group. The sample of convenience may have also introduced selection bias. In addition, despite the use of open-ended questions, the survey design prevented the ability to probe responders and thereby lack the conceptual richness and depth of knowledge. This study is also limited in that these results only provide a snapshot and do not adequately describe the process of prescription.This study describes the paediatric populations who use support walkers, which may enable clinicians to consider their current practice, especially regarding the use of support walkers among children with medical conditions, such as chromosomal abnormalities, where no previous research exists. Identification of common contraindications or precautions for use may also support the clinical decision process when prescribing support walkers. Additionally, identification of common problems may enable individuals to put in place strategies to overcome these common barriers before prescribing support walkers. Prescribers should also take note of the perceived benefits, as they may be used to support an individual’s decision to use support walkers as well as provide a list from which appropriate outcome measures may be selected to monitor progress. The findings from this study may also provide a basis for future research. Randomised controlled trials are needed to establish the effectiveness of support walkers. These findings may support researchers to define the population, identify appropriate outcomes, and determine an appropriate duration of follow-up in future trials. Investigation of differences in outcomes between age groups or conditions may also promote clearer decisions surrounding the prescription of support walkers.In summary, this study provides an insight into the current use and perception of support walkers for children with disabilities in the UK. Findings suggest that the population of children that use support walkers is far more diverse than previously included in studies of effectiveness. There is large variation in current clinical practice, including the age at which support walkers are prescribed, the functional mobility of children who are prescribed support walkers, and the duration for which they are used. Numerous perceived benefits were reported, including increased participation, physical activity and enjoyment. The effectiveness of support walkers on many of these outcomes has not been previously examined. These findings should represent the first step to improve consistency in clinical practice and aid the clinical decision-making process. However, further research, with these findings in mind, must be conducted so that clinicians may deliver evidence-based practice.Additional file 1: Supplemental Table 1. Prescribers reasoning behind a given duration of use. Supplemental Table 2. Types of support walkers used among prescribers and non-prescribers. Supplemental Table 3. Responses from prescribers regarding the factors that influence which support walker is prescribed and the individuals involved in this decision. Supplemental Table 4. The environments in which support walkers are used, as reported by prescribers and non-prescribers. Supplemental Table 5. Prescribed and actual daily duration of support walker use, as reported by prescribers and non-prescribers. Supplemental Table 6. The duration support walkers are used for within a child’s lifespan, as reported by prescribers and non-prescribers. Supplemental Table 7. Percentage of children for whom their walking ability progresses  and the factors which affect this.Additional file 2. Example of a support walker


Clostridium sp. strain P21, isolated from old hay from Stillwater, Oklahoma. This announcement describes the generation and annotation of the 5.6-Mb genomic sequence of strain P21, which will aid in studies targeting genes involved in the enhancement of acid-alcohol production.Here, we report the genome sequence of  Clostridium sp. strain P21, isolated from old hay from Stillwater, Oklahoma. This announcement describes the generation and annotation of the 5.6-Mb genomic sequence of strain P21, which will aid in studies targeting genes involved in the enhancement of acid-alcohol production.Here, we report the genome sequence of  Clostridium sp. strain P21 (DSM 111390) is a Gram-positive, strictly anaerobic rod-shaped bacterium isolated from old hay from Stillwater, Oklahoma. Strain P21 was isolated in parallel with Clostridium carboxidivorans P7T (DSM 15243) and “Clostridium ragsdalei” P11 (DSM 15248) and enriched with CO at 38°C  was added. DNA extraction was performed using the automated Maxwell 16 tissue LEV total RNA purification kit (Promega) purification system v4.90 modified from the manufacturer’s instructions as described by Oldham et al. , and raw sequencing reads for strain P21 were deposited in GenBank and the Sequence Read Archive (SRA) under the accession numbers


View Supplementary Video S1View Supplementary Video S2View Supplementary Video S3 The merits of exercise for persons with Parkinson's disease (PD) are increasingly recognized.For 3 years, a 56‐year‐old proficient swimmer experienced a decline in his swimming abilities. Specifically, he noticed his left leg was progressively lagging during the crawl. Over the course of 2 years, this escalated until he could no longer swim independently, because of a drowning risk. By then, he had also developed a mild resting tremor of his left hand, hyposmia, and hypokinetic gait. He was diagnosed with PD (H & Y stage II). Video 1 shows his swimming pattern before the start of dopaminergic treatment. Bradykinesia and poor interlimb coordination result in decreased propulsive force and failure to maintain a horizontal body position.The importance of this observation is three fold. First, we illustrate that near‐drowning in persons with PD is probably caused by both bradykinesia and impaired interlimb coordination. The underlying mechanism likely involves impaired corticostriatal input, as suggested by a recent report of PD patients losing their swimming ability after STN‐DBS.Regardless of the exact underlying mechanisms, the clinical implications are evident. Persons with PD should be well informed about the potential drowning hazard. And, importantly, swimming capacity can be improved through a complementary approach of pharmacological and nonpharmacological interventions.(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Manuscript: A. Writing of the First Draft, B. Review and Critique.A.T.: 1A, 1B, 1C, 2A, 2BM.N.: 1B, 2BN.V.: 1B, 2BB.B.: 1A, 1B, 2BJ.N.: 1A, 1B, 2BEthical Compliance Statement: This study was performed in accordance with the guidelines proposed in the Declaration of Helsinki. IRB approval was not necessary for this work. Written consent was obtained from the patient involved. The corresponding author (A.T.) confirms that authorization signed by the depicted patient has been obtained in compliance with the applicable regulations regarding patient authorizations relating to the use or disclosure of protected health information. The 3 nonpatients visible in the video have all agreed that the figure with their image might be published. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.Funding Sources and Conflicts of Interest: The authors report no sources of funding and no conflicts of interest.Financial Disclosures for previous 12 months: J.N. was supported by ZonMw and the Michael J. Fox Foundation. B.B. has received honoraria from serving on the scientific advisory board for AbbVie, Biogen, UCB, and Walk with Path; has received fees for speaking at conferences from AbbVie, Zambon, Roche, GE Healthcare, and Bial; and has received research support from The Netherlands Organisation for Scientific Research, the Michael J. Fox Foundation, UCB, AbbVie, the Stichting Parkinson Fonds, the Hersenstichting Nederland, the Parkinson Foundation, Verily Life Sciences, Horizon 2020, the Topsector Life Sciences and Health, and the Parkinson Vereniging.Video 1. Swimming before the start of dopaminergic treatment (l‐dopa naïve). Marked bradykinesia—characterized by slowness, reduced movement amplitude, and sequence effect—is present during both crawl and breaststroke. Bradykinesia is most pronounced on the left side. Additionally, interlimb coordination is affected during the crawl: Normally, the frequency of leg movements during the crawl is higher than the frequency of arm movements, but the arm‐to‐leg movement ratio is 1/1 in this patient.Click here for additional data file.Video 2. Swimming in the on dopaminergic state. Bradykinesia is reduced after dopaminergic treatment. However, the impaired interlimb coordination does not seem to be affected by l‐dopa: The arm‐to‐leg movement ratio remains 1/1.Click here for additional data file.Video 3. Swimming in the on dopaminergic state, with additional use of compensation strategies. A more effective leg stroke (in terms of both frequency and force) is achieved by using swim fins, as well as by holding a floating board. Use of internal cueing (counting and making a leg stroke at every count) appears to improve the amplitude of the stroke during both crawl and breaststroke, but does not improve leg stroke frequency.Click here for additional data file.


Each patient received CRP for BPPV involving the hypoactive canal or, in case of normal VOR-gain, the assumed affected canal. Each subject exhibiting VOR-gain reduction for the involved canal developed normalization of vHIT data after proper repositioning (p < 0.001), proving a close relationship with otoliths altering high-frequency cupular responses. According to our results, overall vHIT sensitivity in detecting the affected SC was 72.9%, increasing up to 88.6% when considering only cases with persistent pDBN where an incomplete canal plug is more likely to occur. vHIT should be routinely used in patients with pDBN as it may enable to localize otoconia within the labyrinth, providing further insights to the pathophysiology of peripheral pDBN.Positional downbeat nystagmus (pDBN) represents a relatively frequent finding. Its possible peripheral origin has been widely ascertained. Nevertheless, distinguishing features of peripheral positional nystagmus, including latency, paroxysm and torsional components, may be missing, resulting in challenging differential diagnosis with central pDBN. Moreover, in case of benign paroxysmal positional vertigo (BPPV), detection of the affected canal may be challenging as involvement of the non-ampullary arm of posterior semicircular canal (PSC) results in the same oculomotor responses generated by contralateral anterior canal (ASC)-canalolithiasis. Recent acquisitions suggest that patients with persistent pDBN due to vertical canal-BPPV may exhibit impaired vestibulo-ocular reflex (VOR) for the involved canal on video-head impulse test (vHIT). Since canal hypofunction normalizes following proper canalith repositioning procedures (CRP), an incomplete canalith jam acting as a “low-pass filter” for the affected ampullary receptor has been hypothesized. This study aims to determine the sensitivity of vHIT in detecting canal involvement in patients presenting with pDBN due to vertical canal-BPPV. We retrospectively reviewed the clinical records of 59 consecutive subjects presenting with peripheral pDBN. All patients were tested with video-Frenzel examination and vHIT at presentation and after resolution of symptoms or transformation in typical BPPV-variant. BPPV involving non-ampullary tract of PSC was diagnosed in 78%, ASC-BPPV in 11.9% whereas in 6 cases the involved canal remained unidentified. Presenting VOR-gain values for the affected canal were greatly impaired in cases with persistent pDBN compared to subjects with paroxysmal/transitory nystagmus ( Positional downbeat nystagmus (pDBN) represents one of the most common findings related to central nervous system (CNS) disorders involving brainstem and cerebellum. As the main function of central vestibular system is to estimate the angular velocity, gravity orientation, and inertia processing peripheral vestibular afferents within the velocity-storage circuit, any lesions disrupting this network can generate pDBN , 2. ThouThis study was approved by our Institutional Review Boards  and was conducted according to the tenets of the Declaration of Helsinki. We performed a retrospective review of clinical-instrumental data of a cohort of 93 patients presenting with pDBN who were evaluated at our centers between June 2019 and May 2020. Overall subjects were admitted either to the outpatient units or to the emergency units. In order to select only patients with peripheral pDBN due to vertical SC-BPPV, subjects exhibiting oculomotor central signs  or abnormal findings on gadolinium-enhanced magnetic resonance imaging (MRI) were excluded from the analysis. Likewise, patients with past history of vestibular pathologies potentially resulting in pDBN or possible VOR-gain abnormalities for vertical SCs [i.e., Meniere's disease , vestibuAny of the following strategies were used for the identification of the SC involved by BPPV:Detection of the SC with impaired VOR-gain values on vHIT with either covert or overt saccades.In case of pDBN with torsional components, recent history of BPPV with paroxysmal positional nystagmus documented with Video-Frenzel goggles addressed the diagnosis toward a specific SC .In cases lacking of the above-mentioned findings, detection of the affected canal could only be provided after physical treatment, basing on the following findings:Resolution of pDBN after proper CRP designed to release a specific SC from debris, as therapeutic maneuvers, though effective in moving debris, would not result in restoring BPPV affecting other SCs.Conversion of pDBN in classical paroxysmal positional nystagmus involving either ipsilateral PSC  or horizontal SC (HSC)  after any CRP performed, consistently with otoconial switch from the affected canal either to other ipsilateral SCs or to another tract of the same affected SC .All patients underwent specific physical therapy aimed to move debris back to the utricle from the assumed affected vertical SC. In cases with BPPV involving the non-ampullary tract of PSC, DS maneuver was mainly used, followed by the 45°-FPP in case of persistence of symptoms following DS . WhereasIn cases with ASC involvement, patients were mostly treated with Yacovino's technique , followeIn cases where affected SC could not be ascertained due to the lack of the aforementioned findings, several CRP were pursued according to examiner's experience to obtain a canal switch, to move otoconia toward another tract of the involved SC or to directly free the affected canal.Each subject was checked within 3–4 days. In case of persistence of pDBN, additional CRP were pursued with following check within further 3–4 days, and so on until a complete recovery or a canal switch was achieved. Physical therapy outcome was considered as successful either if patients were free from symptom and signs or if they exhibited a conversion into a typical form of BPPV. In case debris moved to the ampullary tract of PSC, Epley's or Semont's maneuvers were performed according to examiner's preference or patient's compliance, whereas proper CRP for geotropic and apogeotropic variants of HSC-BPPV were used , 48 in cEye movements were analyzed with video-Frenzel goggles or video-oculography (VOG). Horizontal, vertical and torsional nystagmus were qualitatively assessed. Horizontal (right/leftbeating), vertical (up/downbeating) directions of nystagmus and torsional components  were described from the patient's point of view. Bedside-examination included assessment of spontaneous and positional nystagmus evoked by both DH and SHH positionings. Once evaluated any spontaneous DBN , positional nystagmus was checked for latency (with/without), direction , inhibition with visual fixation (yes/no), duration and temporal trend  and reversal when returning upright following positionings (with/without).Vestibulo-ocular reflex (VOR) gains for all three SCs were tested on both sides in response to high-frequency head stimuli on vHIT, an ICS video-oculographic system . At least 15 impulses were delivered for stimulating each SC and averaged to get corresponding mean VOR-gains. Vertical SC were considered hypoactive if VOR-gains were <0.7 with at least either covert or overt saccades , 30. AllCervical and ocular vestibular-evoked myogenic potentials  for air-conducted sounds were recorded using 2-channel evoked potential acquisition systems  with surface electrodes placed according to standardized criteria . PotentiU-test was employed for pairwise comparisons between subgroups. Results were considered statistically significant if p < 0.05. Statistical analyses were performed using IBM SPSS ver. 20.0 .Quantitative variables were checked for normal distribution using both Kolmogorov-Smirnov and Shapiro-Wilk tests. Continuous variables were described by mean ± 1 standard deviation for normally distributed variables or by median, interquartile range and range for non-normally distributed variables. Diagnostic sensitivity of vHIT in detecting the involved SC in BPPV with pDBN was calculated as the ratio of cases with hypoactive SC to overall patients. Conversely, diagnostic sensitivity of vHIT for persistent pDBN was calculated as the ratio of cases with persistent pDBN exhibiting a deficient SC to overall cases with persistent pDBN, whereas vHIT sensitivity for transitory/paroxysmal pDBN was derived dividing the number of cases with transitory/paroxysmal pDBN presenting with a hypoactive SC for overall cases exhibiting transitory/paroxysmal pDBN. Fisher's exact test was used for categorical comparisons. Spearman's rank correlation was used to correlate patient's age with SCs VOR-gains. Wilcoxon signed-rank test was used to compare pre- and post-treatment vHIT data for all six SCs. Mann-Whitney Fifty-four patients  with pDBN due to vertical SC-BPPV were included in the study. Recurrence of pDBN due to BPPV involving the same SC was recorded in a case and it was considered twice in the analysis. Similarly, four patients  were considered twice as they exhibited either simultaneous or newly sequential BPPV involving other vertical SCs with pDBN. Therefore, clinical and instrumental data concerning 59 cases with pDBN due to vertical SC-BPPV were finally analyzed. Detailed information about overall 59 cases included in the study can be found in p = 0.039) compared to ASC involvement. Conversely, it exhibited transitory and typical paroxysmal crescendo-decrescendo course in most cases with ASC-BPPV  and in half of patients where the affected SC was not identified , ASC-BPPV in 11.9% of cases  whereas in 6 cases (10.1%) neither the involved SC nor the pathologic side could be ascertained . Recent entified . The vasentified . No signentified . Despiten. 53) was performed prior to investigating VOR-gain behavior among different subgroups, to ensure lack of consistent age-related bias involving canal activity (p = 0.043) and for the contralateral SC other than the canal coupled with the affected SC  was found  in subjects with defined affected SC (activity . Only a as found .In 43/59 cases (72.9%) an isolated vertical SC hypofunction could be identified without statistically significant difference between apogeotropic PSC  and ASC-BPPV  . In all p < 0.001; When dividing overall cohort of patients according to pDBN duration, subgroup of patients presenting with persistent positional nystagmus (44/59) exhibited a significantly higher rate of cases with VOR-gain impairment (88.6%) compared to patients in which positionings elicited a transient/paroxysmal pDBN (26.7%) (p < 0.001), for its coupled contralateral canal (p < 0.001) and for the other contralateral vertical SC  and with spontaneous DBN (p = 0.002) than subjects exhibiting transient/paroxysmal pDBN and lacking of spontaneous nystagmus, respectively , a significant functional improvement could be found for the affected SC  and with pDBN resolution after CRP (p = 0.035), respectively (Both cVEMPs and oVEMPs to air-conducted sounds were performed only in 26/59 patients (44%) to test saccular and utricular function, respectively. Whereas, no significant difference in utricular function could be found among subgroups , cases wectively .canalolithiasis) or adhere to the cupula (cupulolithiasis)   , 54, it ith jam) , 56. Sinith jam) , 58, otoThe distinguishing feature of the typical variant of PSC-canalolithiasis (involving its ampullary arm) is represented by paroxysmal upbeat nystagmus with torsional components beating toward the undermost ear, referring to the upper corneal poles, evoked by ipsilateral DH maneuver. Geotropic upbeat nystagmus is disconjugated, with a weaker downward and stronger intorsional slow component in the eye of the pathologic side and a stronger downward and weaker extorsional slow component in the opposite eye, resulting from the transitory activation of ipsilateral PSC-ampulla, since debris within the ampullary arm move away from the cupula during diagnostic maneuvers . ResultiNevertheless, most authors have advocated BPPV involving ASC ampullary arm as underlying mechanism for pDBN . In thisAccording to our original series of 93 patients, most pDBN recognize a peripheral origin confirming previous studies , 9, 15. Despite specific rehabilitative treatments for these BPPV-variants have been designed reporting good results , 68, 69,The importance of a precise detection of the affected SC is reflected in higher number of CRP needed to restore patients with undefined affected SC compared to cases with identified pathologic canal in our series , with obincomplete canalith jam  while impeding the ampullary receptor to respond to high-frequency inputs (head impulses leading to impaired vHIT data). Partial embedment of debris within narrower portion of membranous duct may also account for the usual persistent course of pDBN with smaller frequency compared to typical PSC-canalolithiasis. In fact, hypothesizing that otoconia could remain incompletely entrapped in these canal tracts, a small amount of endolymphatic reflux is expected and fluid column may continue to press against ampullary receptor, resulting in a slower return of the ampullary crest to the resting position than typical BPPV  could likely account for different behavior of pDBN and affected SC activity in these BPPV variants, explaining also how vertical SCs function could have been normal in a series of patient diagnosed with AC-BPPV presenting with mainly transitory pDBN  evoked in DH or SHH positions has been related to PSC-cupulolithiasis –62 or toregular canalolithiasis vs. incomplete jam vs. complete canalith jam) accounting for different patterns of pDBN due to BPPV and vHIT measurements with corresponding assumed pathomechanism is summarized.Finally, when considering different VOR behavior for the affected canal between cases exhibiting spontaneous DBN or not, the former group presented with highly reduced function compared to the latter . This daDespite our instrumental assessment included both cervical and ocular-VEMPs to air-conducted sounds, they were neither routinely performed to search for possible AR differences among patients nor they were routinely tested both before and after CRP to look for amplitudes changes following proper repositioning. Moreover, due to the lack of bone-conducted stimuli in our equipment, a reliable measurement of both saccular and utricular function could not be obtained. On the other and, analysis of variations in VEMPs amplitudes among BPPV patients would have gone beyond the aim of our investigations. These methodological bias could likely account for the lack of statistically significant results among VEMPs data , 12.Our investigation presents some other limitations. First of all, being a multicentre investigation, each involved otoneurologist collected data by him/herself, and an inter-observer agreement for ambiguous cases  was never used. Then, despite corrective saccades were always checked to avoid artifacts inclusion in hypoactive VOR-gain plots, our analysis on vHIT data focused almost solely on SC VOR-gain values, whereas morphological study of saccades  was not pursued, being beyond the aims of this study. Moreover, we considered as deficient only VOR-gains below normative data without considering gain asymmetry between coupled pairs of SC. In addition, the subgroup of patients with ASC involvement was significantly smaller than population with apogeotropic PSC-BPPV, leading to possible misleading conclusions when comparing subsamples data. The inclusion in the analysis of 6 subject where the affected SC could not be identified could also have altered final results of our investigation. Though they presented with pDBN exhibiting the same characteristics of the remainder of cases with BPPV and restored with positionings, it could not be excluded they were not affected by vertical SC-BPPV. Finally, despite our cohort size with pDBN collected in a 1-year period was similar to others series, the small-sized sample analyzed does not permit to achieve definitive conclusions on the sensitivity of vHIT in detecting the affected canal in these patients. Further prospective investigations with a more significant number of subjects with pDBN will be needed to better determine the role of vHIT in vertical canals BPPV presenting with pDBN.incomplete jam is likely to occur. Unlike complete canalith jam, where otoconia are thought to plug the entire canal lumen impeding endolymphatic flows for both high- and low-frequency domains due to a continuous pressure exerted by the clot on a persistently displaced cupula, in these conditions particles partially entrapped within a narrow portion of the canal likely behave as a “low-pass filter” for the ampullary receptor. This phenomenon may actually impair high-frequency dynamic responses for the affected canal while allowing low-frequency endolymphatic movements, thus explaining reduced VOR-gain for the affected SC on vHIT despite pDBN, respectively, and normalization of head impulse data following symptoms resolution or pDBN conversion into a typical paroxysmal nystagmus. These findings should encourage clinicians to routinely use the vHIT in case of pDBN, including high-frequency testing of canal function in the test battery for these patients, particularly in cases lacking of torsional components, where detection of the affected canal and differential diagnosis with CNS disorders may be challenging.According to our data, vHIT may play a key role in the diagnosis of the affected canal in BPPV involving vertical SC presenting with pDBN. In fact, conversely to typical BPPV with paroxysmal positional nystagmus, where particles are free to move along the membranous ducts, in case of persistent pDBN an All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Area Vasta Nord Emilia Romagna Institutional Review Committee. The patients/participants provided their written informed consent to participate in this study.AC, PM, and SM led the conception of the study and conducted most data acquisition, interpretation and made significant contributions to the writing, and editing of the manuscript. AC conducted data analysis and creation of figures. CB and MR were involved in project conception and manuscript editing. SD, SQ, ER, and EA contributed to data acquisition and manuscript review. EA, MM, AG, and GL were involved in manuscript review. All authors approved the final version of 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.


Nominal condition) and their active state . The results demonstrated that the HR and EBR estimated through the facial—video technique or the laboratory equipment did not statistically differ (p > 0.1), and that these neurophysiological parameters allowed to discriminate between the Nominal and Non-nominal states (p < 0.02).Current telemedicine and remote healthcare applications foresee different interactions between the doctor and the patient relying on the use of commercial and medical wearable sensors and internet-based video conferencing platforms. Nevertheless, the existing applications necessarily require a contact between the patient and sensors for an objective evaluation of the patient’s state. The proposed study explored an innovative video-based solution for monitoring neurophysiological parameters of potential patients and assessing their mental state. In particular, we investigated the possibility to estimate the heart rate (HR) and eye blinks rate (EBR) of participants while performing laboratory tasks by mean of facial—video analysis. The objectives of the study were focused on: (i) assessing the effectiveness of the proposed technique in estimating the HR and EBR by comparing them with laboratory sensor-based measures and (ii) assessing the capability of the video—based technique in discriminating between the participant’s resting state ( Nowadays telemedicine platforms are employed in a wide range of medical and clinical applications, such as the diabetes management , asthma However, all the above mentioned telemedicine and remote healthcare concepts require physical contact between the patient and sensors, and the need of high—qualified personnel to set up the entire equipment and provide technical assistance to the patients. The internet-based video-conferencing telemedicine platforms do not require physical contact between patients and providers, although they imply a large limitation due to the lack of sensors to evaluate the neurophysiological parameters of the patients.nominal condition) could play a crucial role in several telemedicine application, such as the sleep apnea remote monitoring  : the actIn (i) the Eyes Open condition was used to identify a threshold that when exceeded identified a potential blink. The threshold was calculated as follows, according with the BLINKER algorithm :(1)ThresRegarding the EBR estimation through the video-based technique, a PC webcam  was used for facial video recording during the experimental protocol .Dlib [adaBoost classifier [The RGB camera was set to a resolution of 640 × 480 (pixel) at a frame rate of 30 (fps). The camera was placed in front of the participant. Subsequently, the recorded video was analyzed offline. The participant’s face was automatically identified using a specific Python library named Dlib  coupled assifier . In partassifier . Then, sassifier . Each evThe ECG signal was gathered by means of gel-based Ag/AgCl electrode fixed on the participant’s chest , connectDlib Python library [adaBoost classifier [sklearn.decomposition.PCA Python library included in the Scikit-Learn Python library [Regarding the HR estimation by means of the video–based technique, the same participants’ facial video was analyzed. As described for the EBR estimation, the 68 visual feature required for the facial recognition were selected using the  library  in conjuassifier . This classifier ,50, in o library . The con library . Then, t library  based on library  by the fThe HR values were computed with a 60 s time resolution for each experimental condition, considering a sliding time window of 100 image frames for each HR value. The processing time for computing one HR value was 0.041 s. The main steps of the described video—signal processing for HR estimation are presented in All the considered neurophysiological parameters, i.e., the EBR and HR, were normalized to obtain comparable distributions related to each sensor technology employed in the study. The normalization consisted in the subtraction of the baselines from the respective values estimated during each experimental condition. The statistical analysis was performed on the normalized parameters. The Shapiro–Wilk test was performed to determine the normality of each distribution involved in the analyses. The Student’s t-test was used to compare normal pairs, while the Wilcoxon signed-rank test was performed if the normality was not confirmed. For all tests, the statistical significance was set at α = 0.05.The results related to the DG task will not be reported because almost all the participants got too close to the game board to accurately extract the objects causing face-video signal loss therefore the impossibility to acquire and consequently analyze their facial videos.EBR’) evaluated during the NB and WEB tasks did not show any significant difference . The percentage difference between the EBR estimated through the video—based technique and the laboratory equipment was 4.5% during the NB task and 4.8% during the WEB task.Regarding the EBR estimation, the paired Wilcoxon signed-rank test performed on the normalized EBR (fference  between HR’) estimated during the NB and WEB tasks . The percentage difference between the HR estimated through the video–based technique and the laboratory equipment was 9.3% within the NB task and 3.3% within the WEB task.The same result  was obsep < 10−27) correlation, as demonstration of how the two technology provided similar EBR estimations. Similarly, p < 10−18) correlation between the HR values estimated every 60 s by means of the laboratory and the video-based technique.Furthermore, to investigate the reliability of the video-based technique with respect to the laboratory technology, the repeated measure correlation (rmcorr) analysis was performed. As reported in p = 0.0005) differences among the nominal and the non-nominal conditions of the NB task in terms of perceived mental demand (p = 0.02) increase of the perceived arousal and control between the Nominal and the Non-nominal conditions of the WEB task  or in an active state . Regarding the NB task, the paired Wilcoxon signed-rank test performed on the normalized EBR and HR estimations provided by the video-based technique .As mentioned in the Introduction, the second objective of the present study consisted in assessing the capability of the video-based technique in discriminating the participants’ state while they were in a resting state .Similarly, the paired Wilcoxon signed-rank test performed on the normalized EBR and HR evaluated by the video-based technique during the WEB task  showed anominal and non-nominal participant’s mental states. In particular, the normalized EBR (EBR’) estimated within the NB and WEB tasks significantly decreased during the non-nominal condition, while the normalized HR (HR’) significantly increased during the non-nominal condition within both tasks. These evidences are consistent with prior related works. In fact, Aricò and colleagues demonstrated the link between the EBR decrease and the visual attention increase [nominal and non-nominal conditions were actually different in terms of mental demand, therefore validating the experimental hypothesis at the basis of the presented analysis. Such evidences open the path to apply video–based techniques for healthcare monitoring of patients in remote locations. In fact, such a technique does not require any physical contact between the patient and the sensor, nor the presence of a doctor or a facilitator for the sensors setting. In addition, the video-based technique implies very limited costs, since it needs only a commercial webcam, compared to the existing telemedicine platforms, which include commercial and medical wearable devices. Beyond the telemedicine and remote healthcare applications, the explored video–based technique could provide a valuable contribution in operative and industrial applications. To this regard, different works [The present study aimed at investigating the reliability of an innovative video—based technique in estimating neurophysiological parameters  while dealing with different activities to find out if it could be a potential solution for healthcare telemonitoring of patients. Regarding the NB and WEB tasks, the results demonstrated the reliability of the video—based technique compared with the laboratory technology, generally considered as the gold—standard in scientific literature . Moreoveincrease , while wincrease . With rent works ,59 alreant works , in air nt works  and in cnt works .Despite the promising results we should highlight some limitations. The proposed video–based technique implies a direct visual contact between the subject and the video recorder, a condition that could not be easy to achieve in specific context as the telemedicine one, where the patient could not stand in front of a camera for long time period. In fact, during the execution of the DG task the posture of almost all the participants did not allow to acquire the participant’s face, hence to neither estimate the neurophysiological parameters considered nor assess the participants’ mental states. Therefore this aspect should be carefully considered when a video–based solution would be employed. Moreover, the investigated video–based technique could likely be sensible to illumination variations , a paramnominal) and active state . Such evidences positively answer to the two initial experimental questions, and they pave the path to apply video—based approaches for estimating neurophysiological parameters not only for the telemedicine and remote healthcare, where it would provide a valuable monitoring tool for early adverse clinical events detection [The proposed study demonstrated the reliability of the innovative video–based technique for computing the EBR and HR neurophysiological parameters. Both parameters evaluated through the video–based technique did not differ by more than 5%, except for the HR evaluated during the NB task which differed by 9.3%, from the measurements provided by laboratory equipment. In addition, the results revealed its capability in discriminating between the participants’ resting state (etection  especialetection  and operetection . To thisetection ,67.


Scientific Reportshttps://doi.org/10.1038/s41598-020-59293-9, published online 13 February 2020.Correction to: The Supplementary Information published with this Article contains errors.The footage contained in Supplementary Video S2 is a duplicate of Supplementary Video S1, which shows “Intermale aggression test: intruder mouse swabbed with water” rather than “Intermale aggression test: intruder mouse swabbed with male tears”.The correct Supplementary Video Supplementary Video S2.