Fixed tissue sections of frontal cortex and caudate from 10 of the AD cases were processed for microscopy using fluorescent derivatives of flutemetamol (cyano-flutemetamol) and PiB (cyano-PiB) and compared to Aβ immunohistochemistry and pan-amyloid (X-34) histology. Using epifluorescence microscopy, percent area coverage and fluorescence output values of cyano-PiB- and cyano-flutemetamol-labeled plaques in two-dimensional microscopic fields were then calculated and combined to obtain integrated density measurements. Using confocal microscopy, we analysed total fluorescence output of the entire three-dimensional volume of individual cored plaques and diffuse plaques labeled with cyano-flutemetamol or cyano-PiB. [3H]Flutemetamol and [3H]PiB binding values in tissue homogenates correlated strongly and their binding pattern in tissue sections, as seen on autoradiograms, overlapped the pattern of Aβ-immunoreactive plaques on directly adjacent sections. Cyano-flutemetamol and cyano-PiB fluorescence was prominent in cored plaques and less so in diffuse plaques. Across brain regions and cases, percent area coverage of cyano-flutemetamol-labeled plaques correlated strongly with cyano-PiB-labeled and Aβ-immunoreactive plaques. For both ligands, plaque burden, calculated as percent area coverage of all Aβ plaque types, was similar in frontal cortex and caudate regions, while integrated density values were significantly greater in frontal cortex, which contained both cored plaques and diffuse plaques, compared to the caudate, which contained only diffuse plaques. Three-dimensional analysis of individual plaques labeled with either ligand showed that total fluorescence output of a single cored plaque was equivalent to total fluorescence output of approximately three diffuse plaques of similar volume. Our results indicate that [18F]flutemetamol and [11C]PiB PET signal is influenced by both diffuse plaques and cored plaques, and therefore is likely a function of plaque size and density of Aβ fibrils in plaques. Brain areas with large volumes/frequencies of diffuse plaques could yield [18F]flutemetamol and [11C]PiB PET retention levels comparable to brain regions with a lower volume/frequency of cored plaques.Specificity and sensitivity of positron emission tomography (PET) radiopharmaceuticals targeting fibrillar amyloid-β (Aβ) deposits is high for detection of neuritic Aβ plaques, a mature form of Aβ deposits which often have dense Aβ core . However, imaging-to-autopsy validation studies of amyloid PET radioligands have identified several false positive cases all of which had mainly diffuse Aβ plaques , and high amyloid PET signal was reported in the striatum where diffuse plaques predominate in Alzheimer’s disease (AD). Relative contributions of different plaque types to amyloid PET signal is unclear, particularly in neocortical areas where they are intermixed in AD. In vitro binding assay and autoradiography were performed using [The online version of this article (10.1007/s00401-020-02175-1) contains supplementary material, which is available to authorized users. Development of positron emission tomography (PET) radiopharmaceuticals targeting fibrillar amyloid-β (Aβ) has enabled imaging of Aβ deposits (plaques) in brains of living people clinically suspected of having Alzheimer’s disease (AD). The National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association criteria  from ove11C]PiB PET has been used widely to image Aβ deposition and progression in research settings since 2003 [11C]PiB PET is somewhat more superior to other recently-developed PET ligands for Aβ plaques in its sensitivity to detect most, if not all Aβ plaque forms PiB  [18F]flutemetamol that were encountered using CERAD scoring alone flutemetamol PET detects moderate-to-frequent diffuse Aβ plaques in the striatum and this could be used to improve the accuracy of clinicopathological diagnosis of AD [3H]flutemetamol and [3H]PiB for tissue binding studies, and cyano-labeled flutemetamol (cyano-flutemetamol) and PiB (cyano-PiB) for histofluorescence studies to explore the degree to which diffuse and cored types of Aβ plaques contribute to the total binding of flutemetamol and PiB in autopsy brain tissues.In AD brain, neuritic Aβ plaques are abundant in neocortex and often contain cores of densely-packed Aβ fibrils , 31. Somis of AD . These sStudies were approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents. Brain tissue samples were obtained from 30 autopsy cases in the University of Pittsburgh Alzheimer’s Disease Research Center’s brain bank. Cases identified neuropathologically as AD as well as non-AD controls with none or sparse amyloid pathology were selected from this series based on availability of tissue for studying. Case demographics and neuropathological characteristics are listed in Table At autopsy, the left cerebral hemisphere was placed in ice-cold 4% paraformaldehyde  for 21 days. CERAD-designated brain regions were sampled for diagnostic purposes . Fixed tCyano-PiB (2-(4′-methylaminophenyl)-6-cyanobenzothiazole) is a highly fluorescent derivative of PiB (2-(4′-methylaminophenyl)-6-hydroxybenzothiazole) created by replacing the 6-hydroxy group of PiB with a 6-cyano group (provided by W.E.K. and C.A.M.) . When apCyano-labeled compounds were viewed using an Olympus BX53 microscope with fluorescent attachment (X-cite 120Q) and equipped with an Olympus DP72 digital camera connected to a Dell Precision T5500 computer running Olympus cell Sens Standard 1.6 imaging software. Cyano fluorescence was visualized using a hydroxycoumarin filter  and images were obtained using a 10 × microscope objective . Fluorescence imaging and image acquisition parameters were held constant throughout the experiment. Consistency in these parameters across imaging sessions was confirmed by imaging a slide containing fluorescent microspheres . A total of nine images were obtained from three sections (three images per section) for each case/region, focusing on the gray matter. Each image was saved as a tagged image format file and imported into ImageJ  for analysis. For each microscopic field, plaque load was calculated by measuring the percent area occupied by plaques relative to total area measured to give a value of percent area coverage. Cerebral vascular Aβ deposits were only rarely encountered and were excluded from the analysis. In the caudate and frontal cortex from 10 AD cases, integrated fluorescence intensity values were obtained using the Integrated Density option from the Analyze menu of ImageJ.After epifluorescence microscopic imaging, subsets of cyano-flutemetamol-processed 4% PFA-fixed, 40 µm thick sections were de-coverslipped in tap water, rinsed in potassium phosphate buffer, and photobleached prior to overstaining. Sections were then overstained using cyano-PiB as described above, images were acquired from the same microscopic fields from which cyano-flutemetamol images were obtained, and sections were de-coverslipped and photobleached a second time. Photobleached sections were then overstained a second time using X-34, a highly fluorescent derivative of Congo red and pan-amyloid marker  to detecz axis: 0.11 µm; x and y axis: 0.108 µm), 0.11 µm z axis step size, and 100 ms exposure time. The objective of this experiment was to obtain confocal stacks through the complete volume of cored plaques (including both the core and the corona) and diffuse plaques, thus plaques were sampled from the inner third of the tissue section z axis to avoid clipping of plaques at the tissue surfaces. Obtaining the full volume of plaques was achieved most readily using 160 microns-thick tissue sections thus tissue sections of this thickness were used for the quantitative analysis. Tissue shrinkage during processing was minimal and did not appear to affect the morphology of plaques—for example, the x–y diameter of a cored plaque circular in the x–y plane equaled its z axis diameter. Plaques within confocal stacks were analysed as three-dimensional volumes. Once a three-dimensional stack was created, the analysis methodology consisted of selecting a region outside the plaque diameter (background) in the mid-plane and subtracting this from all voxel values within the plaque. A voxel in the x, y, and z dimensions was positioned at the centre of the plaque and a spherical volume of interest was then grown radially outward to the periphery of each plaque and the net integrated intensity and volume were measured such that the total fluorescent output could be obtained together with the fluorescence intensity per unit volume.Slide-mounted tissue sections (thickness 90–160 µm) from frontal cortex and caudate were rinsed in potassium phosphate buffer and processed using cyano-PiB and cyano-flutemetamol as described above. Image acquisition was performed using an Olympus BX-51WI upright microscope equipped with an Olympus DSU spinning disk confocal, a super-corrected 60 × Olympus Plan Apo N 1.42 numerical aperture oil immersion objective, MBF CX9000 front-mounted digital camera , a BioPrecision2 XYZ motorized stage with linear XYZ encoders , a Sedat Quad 89,000 filter set , and a Lumen 220 metal halide lamp . The microscope was controlled using Stereo Investigator 11 (MBF Bioscience) and SlideBook 6  software. All images were obtained using the acquisition parameters: 1024 × 1024 frame size, near isotropic pixels (3H-labeled compound (3 nM), rinsed in sodium phosphate buffer at 4 °C, air dried, and placed in an exposure cassette  in close apposition to a storage phosphor screen  for six days. The phosphor screen was scanned using a Typhoon FLA7000 imager  equipped with a 390 nm band-pass imaging plate filter and images were converted to tagged image format files for analysis. Sections immediately adjacent to those used for autoradiography were processed for immunofluorescence using anti-Aβ antibody clone 6E10  block of tissue containing the hippocampus was dissected at autopsy and immediately frozen on dry ice and stored at − 80 °C until processing. The block was then cut using a cryostat  at − 20 °C into sequential, 10 µm thick sections. Sections were immediately thaw-mounted onto Histobond + slides  and stored at − 80 °C. Slide-mounted tissue sections were immersed in sodium phosphate buffer (pH 7.4) for 30 min, followed by incubation for 1 h at room temperature in 1198896; ) and an 3H]PiB or [3H]flutemetamol and the unlabeled compound (over a concentration range of 0.2–1000 nM) were prepared in 900 µl sodium phosphate-buffered saline. Binding was initiated by addition of 100 µl of a 1 mg/ml homogenate of frontal cortex tissue from an AD case in sodium phosphate-buffered saline (in triplicate) and the samples were incubated at 22 °C for 60 min. Filtration was performed as previously described except that the filters were washed three times Flutemetamol autoradiography signal matched the distribution of Aβ-immunofluorescent plaques in adjacent sections of AD hippocampus . In frozen AD brain tissue homogenates, there was a strong correlation between [3H]flutemetamol and [3H]PiB binding using an in vitro binding assay  and Aβ-immunoreactive plaques   Fig. . Cyano-f18F]flutemetamol signal. Similar results were obtained by cyano-PiB fluorescence analysis (not shown) in tissue sections adjacent to those used for cyano-flutemetamol analysis.Confocal microscopy stacks of 0.11 µm-thick planes were obtained through entire volumes of individual cored plaques and diffuse plaques labeled using cyano-flutemetamol and cyano-PiB  of plaques and packing densities of Aβ fibrils within plaques. In neocortical regions of AD brains, diffuse and cored plaques are typically intermixed, thus in vitro binding assays with 3H-labeled compounds are limited in their ability to discern ligand binding to each plaque type. To overcome this limitation, we designed a histopathology study to analyze flutemetamol and PiB in diffuse plaques separately from cored plaques in post-mortem brain tissue sections at the high resolution of conventional and confocal fluorescent microscopy. Due to the low endogenous fluorescence of native flutemetamol and PiB compounds, we modified them by replacing the 6-hydroxyl group with a 6-cyano group. This modification only slightly altered each ligand’s binding affinity as determined in AD cortical tissue homogenate  and is not expected to significantly change their interactions with Aβ fibrils.Amyloid PET radiopharmaceuticals flutemetamol and PiB interact with Aβ fibrils in diffuse plaques, cored plaques (with or without neuritic pathology), and cerebral amyloid angiopathy (CAA) and do not bind to neurofibrillary tangles. The relative contributions of diffuse and cored plaques to [3H]flutemetamol and [3H]PiB autoradiography signals corresponded closely to Aβ plaques detected by immunofluorescence. Because autoradiography and Aβ immunofluorescence procedures were performed on separate, adjacent sections, some plaques of smaller size may not have spanned both sections. Additionally, Aβ immunofluorescence is slightly more sensitive than the autoradiography technique and appeared to reveal slightly more pathology than that observed in the autoradiograms Below is the link to the electronic supplementary material.


More rarely, they originate from donor hematopoietic stem cells, developing the so-called donor cell leukemia (DCL) or myelodysplastic syndromes (DC-MDSs). DCL and DC-MDS can be considered as an NRAS, RUNX1, and BCOR mutations. Because of a familiar history of colorectal neoplasia and the variant allele frequency (VAF) of NRAS mutation at the onset, this mutation was searched on germline DNA in both the donor and the recipient, but the result was negative. Moreover, after transplant (+4 months), the patient developed severe and long-lasting chronic graft-versus-host disease (cGVHD), requiring multiple lines of treatments. Because of the severe immunosuppression, recurrent infections occurred and, lately, the patient died due to septic shock.We report a case of DC-MDS diagnosed 5 years after an allo-SCT from a matched related donor (patient’s sister) in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). The sex-mismatch allowed us to identify the donor cell origin. At the onset, the DC-MDS was characterized by chromosome seven monosomy and This case report highlights the need, whenever possible, to evaluate the donor origin of the posttransplant myelodysplasia and acute leukemias. The potential key role of the impaired immune surveillance and of long-lasting immunosuppression appears to be emerging in the development of this case of DC-MDS. Finally, this case reminds the importance to investigate the familiar genetic predisposition in donors with a familiar history of neoplasia. Allogeneic stem cell transplantation  is the most effective treatment for many hematologic diseases, being a curative approach. Unfortunately, posttransplant relapse is the first cause of transplant failure, and it occurs in at least 30–50% of patients with acute leukemia (AL) or myelodysplastic syndrome (MDS) , 2.de novo from donor progenitor cells. Being an extremely rare event, very few cases are described in the literature and most of them are ALs, “donor cell leukemia” (DCL), while sporadically MDSs [“donor cell MDS” (DC-MDS)] ] , 4.in vivo model of leukemogenesis, and even if the pathogenetic mechanisms remain speculative, a genetic predisposition of donor progenitor cells, an altered host microenvironment, and the impairment of immune surveillance are considered the main causes.DCL and DC-MDS can be considered as an Although the historic division of DCL/DC-MDS into two groups remains useful , (1) a mHere, we present a case of DC-MDS diagnosed 5 years after matched related allo-SCT in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL).t translocation . The Ph+ location  resultedlocation  , considering the HR disease relapse. The conditioning regimen was based on cyclophosphamide 60 mg/kg for 2 days  and total body irradiation , and he received methotrexate and cyclosporine-A (CyA) for graft-versus-host disease (GVHD) prophylaxis.In the first 100 days after transplant, the patient developed a WHO grade 4 acute GVHD (aGVHD)  of the sECP, and rituximab. The posttransplant course was also characterized by several CMV reactivations and multiple infections of skin ulcers, complicated by recurrent sepsis and one episode of septic shock caused by Pseudomonas aeruginosa (July 2018).Four months later, he developed severe chronic GVHD (cGVHD)  involvinDuring all the follow–up, the patient remained in continuous complete morphological/molecular remission with 100% CD34+ donor chimerism.9/L], and hyperleukocytosis [white blood cell count (WBC): 20.45 × 109/L] (+ B-ALL was still in complete response (CR) .Five years after allo-SCT (January 2018), the patient presented with anemia [hemoglobin (Hb) 12.2 g/dl], thrombocytopenia [platelet (PLT): 71 × 10× 109/L] . MDS witin situ hybridization (FISH) for sex mismatch (361/361 XX), confirmed the donor cell origin (patient’s sister) of the MDS. At the time of DC-MDS, the patient presented severe lymphopenia . In parallel, the patient’s sister has been periodically evaluated, and her peripheral blood counts have always been in range.The cytogenetic analysis suggested the donor cell origin of MDS due to the female karyotype. Moreover, the karyotype was characterized by the presence of a monosomy of chromosome 7  [26]; 45 XX, −7q [2]). Conversely, no Ph+ metaphases were found. The chimerism on CD34+ cells (100% donor), together with the fluorescent NRAS [variant allele frequency (VAF) 51%], RUNX1 (VAF 62.2%), and BCOR (VAF 44.7%) mutations. Notably, the variants detected were not known to be associated with malignancies or other diseases. Considering the VAF of NRAS and the familial history for neoplasia , we investigated the presence of a familiar genetic predisposition for the development of hematologic and solid malignancies. To this purpose, the patient and the donor were enrolled in the NEXT-FamlY clinical trial (NCT03058588)  . This isUnfortunately, because of the patient’s conditions compromised by the recurrent infections and GVHD complications, any treatments for the MDS could not be given and the patient died of septic shock a few months after the onset of DC-MDS (September 2018).DCL and DC-MDS are very rare events that may occur in transplanted patients , 11–18. The first DCL case was described by Fialkow et al.  in 1971.The umbilical cord seems to bear a potential higher risk for DLC than other stem cell sources . ConversCyclophosphamide with total body irradiation (Cy + TBI) is the conditioning regimen reported to be more frequently associated with DCL development . This isIn this presented case, the sex-mismatch easily allowed the recognition of the donor cell origin—thanks to the karyotype and the FISH analysis for gonosomal. Conversely, the cytogenetic analysis is not helpful for detecting the donor origin of MDS/leukemias in sex-matched transplants, for whom the DCL diagnosis is more difficult and often underestimated. In these patients, variable number of tandem repeats or short tandem repeat (STR) analysis should be used to detect DCL/DC-MDS, together with the study of chimerism on CD34+ cells in peripheral blood.At DC-MDS onset, the patient presented with chromosome seven monosomy, which is the most common cytogenetic alteration observed in DC-MDS and DCL . DespiteNRAS, RUNX1, and BCOR genes were mutated as shown by NGS analysis. NRAS and RUNX1 are among the most frequently mutated genes in patients with MDS and monosomy 7/deletion 7q , and the so-called “blend pedigree” have been described in literature in the last years (etion 7q , and theetion 7q , 27. Intst years , 28.NRAS on germline DNA. Nevertheless, the evaluation of the donors’ clinical history investigating the presence of a possible pedigree suspicious for familiar neoplasms should always be strongly recommended. That is in order to avoid the use of donor cells that may already present a genetic familiar predisposition to develop hematological malignancies after allo-SCT.Therefore, germline DNA from epithelial buccal cells of the donor and the patient was sequenced using traditional Sanger methods. However, neither the donor nor the recipient presented the variant of As expected, DCL and DC-MDS prognosis is poor , with a NRAS variants on germline DNA, supports the hypothesis that either the host bone marrow microenvironment or the immunosuppression could play a pathogenetic role in this presented case. On one hand, the primitive Ph+ ALL and the subsequent chemotherapies should have determined an altered microenvironment, which plays a crucial role in leukemogenesis (Although several hypotheses have been made, the underlying pathogenetic mechanisms for DCL/DC-MDS developing remain unknown and speculative , 13. Theogenesis –39. Neveogenesis , 41 and ogenesis . In factogenesis . In addiDespite being rare events, DC-MDS and DCL may occur as late complications after allo-SCT. They are an intriguing phenomenon, and they were probably underdiagnosed for many years. Therefore, searching for the donor cell origin should be performed in all patients relapsing after allo-SCT. The new molecular technologies for chimerism monitoring made easier and more reliable the DCL diagnosis even when the donor origin could not be easily detected, such as in sex-matched cases.in vivo leukemogenesis model to investigate the underlying pathogenetic mechanisms, which, so far, remain speculative. Beyond the hypothetical pathogenetic mechanisms, the setting of cGVHD and related immunosuppressive treatments may allow DCL development through an impaired immunosurveillance. Conventional and new interesting approaches are available and should be considered to monitor the immune reconstitution and the development of different competent lymphocyte populations (Donor cell-myelodysplastic syndrome and DCL represent an ulations –46.Finally, considering that the DCL and DC-MDS may be a consequence of a genetic predisposition, the familiar history for neoplasia of the donors has to be investigated. When it is present, searching for DNA germline mutations both in the recipient and in the donor is mandatory to avoid the use of donor cells that may have a genetic predisposition to develop DCL/DC-MDS after allo-SCT.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comitato Etico di Brescia. The patient provided their written informed consent to participate in this study and for the publication of this case report.MF, SB, MM, and DR designed the study. MF, SB, MM, TZ, LG, DG, EM, NP, AT, CZ, and BR collected and analyzed the data. SB and CZ performed the laboratory NGS analysis. MF, SB, MM, and DR wrote the manuscript. All authors gave their final approval before submission and 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.


Enterobacteriaceae for differences in conjugation at different time points with five ESBL-producing Escherichia coli strains.Extended spectrum beta-lactamase (ESBL)-producing enterobacteria pose a major hazard to public health. Due to the possibility of genetic transfer, ESBL genes might spread to pathogenic enterobacterial strains. Thus, information on possible genetic transfer between enterobacteria is of high interest. It was therefore the aim of this in vitro study to screen the capacity of a wide range of E. coli donor strains producing the enzymes CTX-M-1, CTX-M-15, SHV-12, TEM-1, TEM-52 and CMY-2, and six potential recipient strains commonly detected in the gastrointestinal tract of poultry  enterica serovar Typhimurium and Proteus mirabilis) were obtained. Different combinations of donor and recipient strains were co-incubated for between 0 and 22 h and spread on selective agar. Conjugation frequencies were calculated as transconjugants per donor.Conjugation frequencies for five potential Proteus mirabilis did not accept plasmids from any of the given donors and the E. coli ESBL10716 donor was unable to transfer its plasmid to any recipient. Enterobacter cloacae only accepted the plasmids from the donors E. coli ESBL10708 and E. coli ESBL10716 while E. coli ESBL10708 did not transfer its plasmid to Serratia marcescens subsp. marcescens. E. coli IMT11716 on the other hand did not perform conjugation with the donor E. coli ESBL10689. The remaining mating pairs differed in conjugation frequency, ranging from 10− 5 to 10− 9 transconjugants/donor. The earliest conjugation events were detected after 4 h. However, some mating pairs turned positive only after 22 h of coincubation.Some donor and recipient strain combinations did not perform plasmid transfer within 22 h. Hence, the recipient A suitable mating pair for future in vivo studies to combat transfer of antibiotic resistance to pathogenic bacteria in broiler chicken was determined. The results of this study also suggest that the kinetic of conjugation differs between mating pairs and is independent of species origin. This should be considered when performing conjugation experiments. Enterobacteriaceae were identified as one of the antibiotic resistant bacterial groups currently posing the highest threat to public health [Escherichia coli and Salmonella spp. as the most common bacterial hosts [Consequential to the global increase of multidrug resistant bacteria, severe economic and public health related costs have been predicted to rise significantly in the near future . In thisc health . These bc health . ESBL-prc health , 5. CTX-al hosts . Often, al hosts .ESBL encoding genes are generally located on plasmids, which can be transferred between bacterial strains and species, including pathogenic strains , 6, 7. TE. coli from poultry to the microbial community of a human stool sample resulted in the establishment of the strain and plasmid transfer to an E. coli of human origin [A transmission of ESBL-carrying bacteria from animals to humans, were the animals constitute a reservoir for human infections, has been suggested –13. Antin origin . Both don origin . The afon origin .Enterobacteriaceae strains commonly detected in poultry were obtained in vitro within a 22 h timeframe.The present study was undertaken to investigate the changes of conjugation events at different time points as a first step to estimate the possible transfer frequencies of ESBL genes in the intestinal tract and in the environment. Conjugation kinetics of ESBL-carrying The results obtained from the agar disc diffusion tests  suggests this disc type for screening for ESBL-producing bacteria or 1 μg CTX/mL for broth microdilution [The present in vitro study investigated the conjugation kinetics between ESBL-producing dilution . Hence, E. coli ESBL10716 did not transfer its plasmid to any of the recipients provided and the Proteus mirabilis recipient did not mate with any of the given donors. Plausible reasons for the absence of conjugation events are that the recipients may already harbor plasmids with the same replicon [bla genes were located on the chromosome or a non-conjugative plasmid in this donor [5 cfu/mL is high compared to other studies [The propensity of ESBL-producing donors to transfer their plasmids to various recipients differed significantly between strains from the same species in the present study. replicon  or that is donor . Furtheris donor . The lat studies  and the  studies , 24. HowE. coli ESBL10689 donor, which showed the highest conjugation frequency with a Salmonella recipient. This study cannot confirm a general conclusion in either direction, but rather suggests strain specific differences.Conjugation frequencies differed between various donor and recipient strains in the employed in vitro assay. Genera and strain depending variations in conjugation frequency have been described previously , 25. SomE. coli recipient [Citrobacter freundii, a strain not investigated in the present study, revealed high conjugation frequencies, while S. marcescens donors led to comparatively low conjugation frequencies, similar to the results of this study. In another study, Enterobacter spp. donors reached an average of 10− 5 transconjugants/donor when co-cultivated with E. coli recipient strains [− 6 transconjugants/donor when used as a recipient for E. coli donors in the present study. No information on incubation time or cell concentrations was provided. In this study, the highest conjugation frequency of 1.04 × 10− 5 transconjugants/donor occurred when E. coli 10,682 was co-incubated for 4 h with the pathogen S. enterica serovar Typhimurium L1219-R32. This frequency corresponds with results obtained from conjugation trials with Klebsiella spp. donors and Salmonella spp. recipients with 24 h coincubation [The relatively high conjugation frequencies reported in the literature compared to the results reported in the present study may depend on the usage of different strains. While some studies used different strains as donors and a consistent ecipient , 26, the strains , comparecubation .9 cells/mL compared to 105 cells/mL in this study) may explain the differences in conjugation frequencies. It must be mentioned that high conjugation frequencies may also be observed in experiments with low initial concentrations [Higher initial concentrations of donors and recipients used in studies such as Franiczek et al.  or Franitrations  and thattrations –30. The trations .The time period until detection of transconjugants differed significantly between donors with the same recipients. Conjugation kinetics for ESBL-carrying plasmids have previously been studied, at similar time points , but maiS. marcescens and S. enterica serovar Typhimurium revealed lower growth rates than the donor strains   with or without antibiotic supplementation, depending on the experiment. The pH value of the medium was 7.5 at room temperature. MacConkey agar  was applied for all plates, except for an agar disc diffusion assay, where Müller Hinton agar  was applied, and incubated aerobically overnight at 37 °C.n = 32) and the potential donors (n = 5) by agar disc diffusion tests  were obtained for the relevant antibiotics by broth microdilution in duplicates. Turbidity was measured at 690 nm every 10 min for 24 h in a microtiter plate reader . Non-inoculated media served as negative control, while inoculated MHB without antibiotics provided the positive control. The MIC was defined as the concentration, at which no growth was observed within the 24 h measurement period. According to the MIC and growth curves, antibiotic concentrations for agar plates for the conjugation trials were chosen.In short, strains were pre-cultured from cryo stocks overnight in MHB without antibiotics and subsequently washed twice in phosphate-buffered saline . The cells were resuspended in MHB without antibiotics and diluted to 106 cells/mL. Equal volumes (100 μL) of donor and recipient suspensions were added to 800 μL MHB without antibiotics. Thus, in the coincubation suspensions, both donors and recipients were initially present at a concentration of 105 cells/mL and the initial total bacterial concentration was 2 × 105 cells/mL. Single strain donor and recipient dilutions served as control. The screening was implemented in duplicates.In order to identify mating pairs, the potential donor and recipient strains were co-cultivated in duplicates for 22 h  with only one bacterial strain (donor or recipient) served as controls.A kinetic assay was designed to obtain conjugation frequencies for the different mating pairs at 6 time points (supplementary data: Fig. SImmediately after the inoculation, 300 μL of the suspension were plated on two double antibiotic agar plates (supplementary data: Table SAdditional file 1.Additional file 2.


The second protocol comprised the 1,000-m TT.This study aimed to develop a predictive explanatory model for the 1,000-m time-trial (TT) performance in young national-level kayakers, from biomechanical and physiological parameters assessed in a maximal graded exercise test (GXT). Twelve young male flat-water kayakers (age 16.1 ± 1.1 years) participated in the study. The design consisted of 2 exercise protocols, separated by 48 h, on a kayak ergometer. The first protocol consisted of a GXT starting at 8 km.hResults: In the GXT, they reached an absolute Omax2 of 3.5 ± 0.7 (L.min−1), a maximum aerobic power (MAP) of 138.5 ± 24.5 watts (W) and a maximum aerobic speed (MAS) of 12.8 ± 0.5 km/h. The TT had a mean duration of 292.3 ± 15 s, a power output of 132.6 ± 22.0 W and a Omax2 of 3.5 ± 0.6 (L.min−1). The regression model [TT (s) = 413.378–0.433 × (MAP)−0.554 × (stroke rate at MAP)] presented an R2 = 84.5%.Conclusion: It was found that Omax2, stroke distance and stroke rate during the GXT were not different from the corresponding variables  observed during the TT. The MAP and the corresponding stroke rate were strong predicting factors of 1,000 m TT performance. In conclusion, the TT can be useful for quantifying biomechanical parameters (stroke distance and stroke rate) and to monitor training induced changes in the cardiorespiratory fitness (Omax2). Olympic male flat-water kayak competitions comprise 200 and 1,000 m distances. At Rio 2016, medals won within 35.195 to 35.662 s and 211.447 to 213.363 s, respectively, for 200 and 1,000 m. Performance in different competitive distances requires a distinct combination of physiological and anthropometric factors . The knowledge of the most influential variables affecting the 1,000 m TT in kayak ergometer in young kayakers, could help coaches to interpret relevant physiological and biomechanical variables, assisting in the selection for competitive proposes.The study aims first to verify the predictive power of biomechanical, physiological, maturational and the morphological characteristics on a 1,000 m time-trial performance (TT), assessed by a maximal graded exercise test (GXT) in young kayakers. Second, compare biomechanical and physiological parameters between both tests (TT and GXT–n = 12) Portuguese national-level male young sprint kayakers volunteered to take part in this study . The same experienced researcher performed the anthropometric measurements. The athletes visited the laboratory on two occasions during which they accomplished two experimental protocols (GXT and TT). The athletes were familiar with the kayak ergometer and testing procedures applied. On the first visit, upon arrival at the laboratory, the athletes completed a pre-test questionnaire to characterize the training and food intake in the last 24 h. The questionnaire was adapted from Tanner and Gore (Twelve n =  Portugueand Gore . The athThe athletes were familiar with the kayak ergometer and testing procedures applied. The testing situations were conducted at least 24 h apart and completed within 10 days. All testing sessions were held in the same period of the day (17:00–19:00 h). The day before testing, athletes were required to avoid fatigue accumulation; all participants were asked to abstain from intense physical exertion and only a light intensity workout 24 h prior to each testing session was allowed.The anthropometric and body composition assessments were done during the first visit to the laboratory. Body mass was measured using a calibrated scale  and stature and sitting height were measured using a standard stadiometer . Body composition was assessed through plethysmography . Both protocols were performed in an air-braked, drag adjustable ergometer designed to assess flat-water kayakers .www.dansprint.com). The same warm-up procedures were implemented before both tests. Briefly, after 10′ of freely chosen light stretching exercises, they paddled for 15′ at an intensity lower than 70% of their estimated HRmax, during which athletes were asked to perform 5 short sprints of 3″ , ambient humidity (44.3 ± 2.1%) and pressure (763.7 ± 1.0 mmHg) were maintained constant during the tests (Oregon Instruments USA). After both tests, blood lactate (La) was determined at 1, 3, 5, and 7 min of recovery from capillary blood samples collected using heparinized ear-lobe tubes for determination of peak lactate value (Lactate Pro®). Ventilatory parameters were monitored breath by breath with a Quark CPET COSMED® gas analyser  calibrated before each testing session according to manufacturer's instructions.Maximal graded test: Speed started with paddling at 8 km.h−1, with increments of 1 km.h−1 each 2 min, until exhaustion. The data was averaged in 30 s segments. Additionally, maximum aerobic power (MAP), maximum aerobic speed (MAS), power output, stroke rate and distance per stroke at each intensity were calculated. Before testing, the gas analyser system  was calibrated for room and gas with known O2 and CO2 concentrations following the manufacturer's recommendations. The determination of ventilatory thresholds (VT1 and VT2) and 2max followed the criteria proposed by Howley et al.  between researchers ranged from 0.3 to 2.7%. The determined intraclass-correlations (ICC) were 0.93, 0.90 and 1 for VT1, VT2, and 2max, respectively, for the values reported by the three researchers. Accordingly, the mean of each of the value were used in the ventilatory data analysis.y et al. . Briefly1,000 m time-trial: The athletes were required to perform the 1,000 m time-trial (TT) in the shortest time, while receiving verbal encouragement to maintain speed as high as possible. The athletes had full access to a visual display of TT variables, such as distance traveled, time, and stroke rate. During the 1,000 m time-trial the same gas analyser system  was used and the peak oxygen uptake (2peak) was determined. The 2peak was defined as the highest continuous 30 s during the exercise bout.t-test for pairwise comparison was used between the variables obtained in the GXT and TT. Effect sized was computed to ascertain magnitude of the difference of the mean trivial (0–0.19), small (0.20–0.49), medium (0.50–0.79), and large (0.80 and greater)  . If normp > 0.05), correlations between VO2peak, stroke rate and stroke distance obtained during the TT and the corresponding in the GXT were found. Between protocols (GXT and TT) correlation of the mean speed and mean power output also founded  and a medium mean difference  and MAP  with a small mean difference . Higher blood lactate values were observed at the end of the TT when compared to that recorded after finishing the GXT  and a high mean difference .The results of the GXT and the physiological, metabolic and biomechanical variables observed during the 1,000 m time-trial are presented in nded see . Additio2max, MAS, VT1, and VT2 velocities, stroke rate and MAP displayed significant and negative correlations with time during TT. No morphological and maturational variables were significantly correlated with TT performance.p = 0.001) and the corresponding stroke rate  were strong predictors of TT performance. The regression model explained (adjusted R2) 84.5% of the 1,000 m TT performance [F = 30.985 p < 0.000].The multiple regressions enter method analysis revealed that MAP . It was found that 2peak, the stroke distance and stroke rate during the GXT were not different from the corresponding variables observed during the TT. Besides, the MAP and the corresponding stroke rate were strong predictive factors of 1,000 m TT performance.This study aimed first to analyse the predictive value of biomechanical, maturational, morphological characteristics, and physiological parameters determined through a GXT on the 1,000 m TT performance in kayak-ergometer in young national-level kayakers. Second, compare biomechanical and physiological parameters between both tests  can also be monitored using the former. The agreement between the GXT and TT founded in our study agrees with that obtained in 2peak reached during the incremental test and time-trial in cycling athletes , and HR (6.7%). In another study, the model regression analysis showed that the 2max was the best predictor of performance in 1,000 m TT, which is in line with our results  performance influences the all-out time-trial performance.Comparing the results of the GXT and TT, we found differences on the percentage of MAP 4.2%), MAV (3.2%), absolute %, MAV  observed during the TT, and that MAP and the corresponding stroke rate were strong predictors of 1,000 m TT performance. Our results confirmed that 2max, chronological age, the speed at VT1 and VT2, stroke rate and power output are significantly correlated with the performance in a 1,000 m time-trial. This study demonstrated that in well-trained young kayakers, cardiorespiratory fitness could be assessed during a time-trial test that mimics the actual performance attained in flat water. The final lactate confirmed the high contribution of the lactic anaerobic pathway  as performance markers when monitoring their athletes with regards to predictive factors of sport-specific performance. Programming training to improve these variables may therefore also improve 1,000-m kayaking performance.In conclusion, we found that  pathway . Therefo2max, the stroke distance and stroke rate among young, national-level kayakers due to its practicality and the ability to mimic actual performance. Findings from the current study suggest that 2max, chronological age, the speed at VT1 and VT2, stroke rate and power output are significant predictors of 1,000 m kayaking performance. Therefore, coaches can use these variables as performance indicators when monitoring their athletes with regards to predictive factors of sport-specific performance. Programming training to improve these variables may hence also improve 1,000 m kayaking performance.TT testing on a kayak ergometer may be a suitable and preferable alternative to graded exercise testing for determining The main limitation of the study was the small sample size. However, the athletes in the sample, are representative of the Portuguese national age group in flatwater. Unfortunately, we are not possible to research the ecological environment due to the equipment limitations. Nevertheless, the laboratory equivalent conditions in the applied protocols seem to be an appropriate research option with some advantages.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Local Ethics Committee of the Faculty of Sports Science and Physical Education, University of Coimbra (CE/FCDEF-UC/00292019) approved the procedures. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.AC and LR conceived the experiment. AC, MM, FN, and LR designed and conducted the experiment. AC, FN, FA, AF, AD, MM, and LR analyzed the data, performed statistical analysis, and primary responsibility for the final content. All authors have read and approved 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.


Spatial filtering of multi-channel signals is considered to be an effective pre-processing approach for improving signal-to-noise ratio. The use of spatial filtering for preprocessing high-density (HD) surface electromyogram (sEMG) helps to extract critical spatial information, but its application to non-invasive examination of neuromuscular changes have not been well investigated.Aimed at evaluating how spatial filtering can facilitate examination of muscle paralysis, three different spatial filtering methods are presented using principle component analysis (PCA) algorithm, non-negative matrix factorization (NMF) algorithm, and both combination, respectively. Their performance was evaluated in terms of diagnostic power, through HD-sEMG clustering index (CI) analysis of neuromuscular changes in paralyzed muscles following spinal cord injury (SCI).The experimental results showed that: (1) The CI analysis of conventional single-channel sEMG can reveal complex neuromuscular changes in paralyzed muscles following SCI, and its diagnostic power has been confirmed to be characterized by the variance of Z scores; (2) the diagnostic power was highly dependent on the location of sEMG recording channel. Directly averaging the CI diagnostic indicators over channels just reached a medium level of the diagnostic power; (3) the use of either PCA-based or NMF-based filtering method yielded a greater diagnostic power, and their combination could even enhance the diagnostic power significantly.This study not only presents an essential preprocessing approach for improving diagnostic power of HD-sEMG, but also helps to develop a standard sEMG preprocessing pipeline, thus promoting its widespread application. Spinal cord injury (SCI) is a leading cause of adult disability worldwide . The disIn clinical routine, an invasive approach using concentric needle is applied to electrophysiological examination of MU properties , 5. The Surface EMG (sEMG) is a technique to record the electrical activity of muscle using electrodes placed over skin surface. Due to the benefits of non-invasive and low-cost properties, sEMG recording has been used in examining neuromuscular activities –10. HoweThe spatial filtering technique can be employed to remove artifacts of HD-sEMG data and to retain useful information given the muscular activation heterogeneity. Its basic principle is to preserve the sources of interest and suppress unwanted components from signals –26. VariIt is hypothesized that spatial filtering can help to enhance and mine useful diagnostic-related information of HD-sEMG, thus improving diagnostic power of the sEMG examination. The purpose of this study is to verify this hypothesis by the means of testing it on the SCI data. Both the PCA and the NMF algorithms were selected for performing the spatial filtering due to their artifact removal and task-related region localization capabilities, respectively. Their combination was used as well by considering their functional complementarity. Clustering index (CI) analysis  was adopNine subjects with incomplete cervical SCI  were recruited from the Clinical Neuroscience Research Registry at the Chicago Rehabilitation Institute . Demographic and clinical measures for the subjects with SCI are summarized in Table The abductor pollicis brevis (APB) muscle was examined in this study. It is the largest and superficial muscle within the thenar muscle group on the palm with a distinct and simple function of thumb abduction. This distal muscle on the hand is representative for reflecting motor impairments , 41, 43.The experiment was carried out in a quiet room in order to reduce the impact of the environmental noises. During the experiment, subjects were seated in a comfortable mobile chair. Their tested arm was bent approximately 90° and was placed on a height-adjustable desk. In the beginning of the experiment, the subject was encouraged to perform three maximal voluntary contractions (MVCs). The maximum value of these trials determined by monitoring the EMG amplitude was taken as a valid MVC. Then the subject was asked to generate an isometric contraction by abducting the thumb with increasingly graded force levels, roughly corresponding to 10%, 30%, 50%, 70%, submaximal (90%) and maximal voluntary contraction (MVC) in terms of the MVC percentage via the EMG amplitude. The subject was encouraged to remain at least 3 s as stable as possible for each contraction level. Sufficient rest was also allowed to avoid muscle fatigue between two consecutive trials.The raw HD-sEMG data collected from the APB muscles on both sides of subjects with SCI and on a random side of control subjects were imported to the MATLAB  software for analysis. Figure A fourth-order Butterworth band-pass filter at 20–500 Hz was applied to eliminate potential low-frequency noises  and high-frequency interferences. Then, a set of second-order notch filters were used to remove the 50-Hz power line interference and its harmonics. Subsequently, the spatial filtering methods could be applied to the HD-sEMG data.U are the corresponding eigenvectors, and the m × m orthogonal matrix V satisfies In the PCA algorithm, the calculation of PCs is realized by diagonalization of the covariance matrix of data. The relevance of the PCs can be ranked in terms of the eigenvalues and reflect its contribution to the data in terms of variance for every PC. Suppose that the original signal mentclass2pt{minim signals , 29, 44.The NMF algorithm is formulated as a solution to a minimization problem with nonnegative bound constraints . In thisentclass1pt{minimas of activation patterns, while the matrix C represents their corresponding time-varying activation coefficients. For processing the HD-sEMG data recorded on an entire muscle, each column of W here represents a spatially correlated activation pattern over the m-channel electrode array, anatomically reflecting the localization of a specific signal source. Thus, each row of the matrix C here specifies how an activation pattern is modulated during the task performance. The variable s varies from 1 to m, representing the number of activation patterns. In practice, the average variability accounted for  between the original matrix  presented several segments of sEMG activities according to the experiment protocol. The onset and offset of each sEMG activity burst can be easily determined. For CI analysis, a series of 1-s epochs  need to be segmented from the EMG activity. Since actual muscle force was not recorded in this study, the generally observed sEMG amplitude intensities were used to roughly estimate the muscle force fluctuations. The force was considered to be relatively stable when the sEMG intensity remained at a consistent level. Otherwise, the corresponding epochs would be discarded especially at the switch of two consecutive force levels. Thus, the number of epochs in a multi-channel form ranged from 20 to 40 over all force levels for each of all subjects.CI is a non-invasive quantitative method for analyzing uneven distribution and cluster of the processed signal to different neurogenic and myopathic changes . To calcThe CI has a value between 0 and 1, and a relatively high value represents a highly clustered signal, appearing with isolated large action potentials. If the EMG epoch carries multiple channels , the average CI value was calculated from all chosen channels. While the values of CI depended on the contraction level: the increase in contraction levels resulted in a lower value, the area of each epoch, that is the area of all windows, was used to estimate the muscle contraction level . It had The quantification of the normal data reference in the CI-area plot is the prerequisite to subsequent diagnosis. To establish the distribution of the normal cloud, a linear regression analysis was performed on all analysis epochs . For a specific muscle with certain abnormal changes, the greater Z score dispersion from the normal range was yielded by the examination approach , the higher its diagnostic sensitivity became according to the CI calculation. Given a group of tested muscles expected to have changes in various type and degree, the variance of Z scores over this group could be used to evaluate the diagnostic sensitivity of the CI indicators, after different spatial filtering methods. Suppose the Z score variance was g to Eq. , which rThe ADI values were calculated respectively under different conditions. In this study, the condition was defined by the use of both the channel and the spatial filtering method for data analysis. A special condition was designed as a representative approach without any spatial filtering method for the comparison purpose, which simply averaged CI values over all used channels when HD-sEMG data were used. The higher an ADI value was yielded, the greater diagnostic sensitivity  the corresponding method had.p < 0.05 for all above analyses. All statistical analyses were completed using SPSS software .In order to verify the generally sequential consistency of individual muscles’ diagnostic outcomes, a series of linear regression analyses were performed on the CI Z scores derived from both the SCI-left and SCI-right groups, between any two different conditions. Two separate two-way repeated-measure ANOVAs were performed on the Z score, with the group  considered as the between-subject factor and the condition  considered as the within-subject factor, to simultaneously examine their effect on the Z score group means. Another two-way repeated-measure ANOVA was performed, with both the side/group (two levels: the SCI-left group versus the SCI right group) and the condition (four levels) considered as within-subject factors. The level of statistical significance was set to Figure Figure The PCA-based method yielded varying ADI values when the number of components to be removed corresponding to smallest eigenvalues was set from 0 to 6, respectively, as shown in Table s of activation patterns was set at 1, 2 and 3, respectively. Evidently, the mean VAF exceeded over 95% when the number of patterns was increased from 1 to 2, and the addition of the 3rd activation pattern (the number is three) did not help to increase the VAF much. Therefore, the variable s was set to 2 in the NMF algorithm implementation.Figure Another sensitivity analysis was also performed to determine the number of selected channels optimally for implementing the NMF-based spatial filtering method, according to the diagnostic power. The ADI results were shown in Table Figure p < 0.001). The ANOVAs revealed no significant main effect of the group and no significant difference in group means of the CI Z score between any muscle group following SCI and the control muscle group, or no significant main effect of the spatial filtering method (p = 0.855) on the CI Z score. However, significant difference was found between the SCI-left muscle group and the SCI-right muscle groups (p < 0.05).The linear regression analyses reported strong correlations (R2 from 0.80 to 0.93) between the CI Z scores made by any spatial filtering method and the simply averaging approach , and estimates of coefficients were all statistically significant  the complex neuromuscular changes following SCI were revealed by the CI analysis of conventional single-channel sEMG, and the diagnostic power could be characterized by the variance of Z scores derived from a group of subjects  used for analysis, it can be observed from Fig. Four muscles of the SCI subjects had an abnormal CI Z score increase indicating neurogenic changes. These changes can be attributed to loss of MUs and subsequent reinnervation of denervated muscle fibers. The MU loss may take place after gray matter is destroyed at and near the lesion epicenter and it can lead to a decrease in the number of activable MUs and denervation of muscle fibers . CompletEleven muscles had abnormally lower Z scores indicating myopathic changes, which could be related to muscle fiber disuse atrophy. Atrophied and angular muscle fibers could lead to partial denervation, which can be indicated by intramuscular motor axon sprouting, an important compensatory mechanism for recovery of muscle innervation after death of some motoneurons . A selecThe resultant Z scores of remaining muscles were located within the normal range. However, substantial muscle weakness was also found in these muscles. Their paralyses are likely to be attributed to a deficit of descending central drive as a result of the severance of central nervous system axons and demyelination of central or peripheral axons, while the affected muscles still function more or less normally . AlthougAs a result, the experimentally observed CI abnormality consists of two patterns, which lead to CI deviation in two different directions, respectively. Therefore, a pooled analysis of a group of paralyzed muscles following SCI can allow their CI indicators to spread from the centered normal range, indicating that there are complex neuromuscular changes following SCI. This phenomenon explains why there was no significant difference in group means of CI Z scores in the ANOVAs, and therefore the ADI was more appropriate to characterize the diagnostic power of the CI method in this study.Given the HD-sEMG recording, varied distributions of the CI Z scores were observed and thus different diagnostic decisions were made when data from different channels were used see Fig. . Thus, iTaking the averaged CI value over HD-sEMG channels is the most straightforward method to extract the global information of muscle activity and eliminate the influence of the electrode position. However, unsurprisingly, we found that the ADI diagnostic power obtained in this way was only at the median level of those using individual channels. This shows that simply averaging may smooth or cancel the useful diagnostic information present in the local channels, which is detrimental to revealing specific abnormalities in individual muscles. In this case, the resultant CI indicator reflected poor understanding of underlying pathological muscle changes.After the determination of appropriate settings towards performance optimization see Fig. , 3, the The PCA-based filtering method was designed to deliberately remove PCs representing homogeneously changing and common features, and detail PCs representing high-frequency noise and cross-talk , 44. SucAccording to their calculation principles, both algorithms were regarded to emphasize different aspects of information convoyed in the raw HD-sEMG data. As a result of their complementary effect, it is easy to explain that their combination can further improve the diagnostic power in comparison to sole use of the PCA-based or the NMF-based method.The HD electrode array physically covers the examined muscles, providing a wealth of spatial information in a large area. Direct averaging these channels is not satisfactory for diagnostic improvement. By contrast, spatial filtering methods evidently improve the performance of HD-sEMG examination, approximating to the maximal level of diagnostic powers when individual channels are used. Thus, the use of spatial filtering helps to highlight and refine useful diagnostic information associated with heterogeneity of the muscle activation, and provides a necessary and convenient approach to pre-process HD-sEMG data for further examination of neuromuscular changes. In addition to enhancing diagnostic power, this can also minimize the potential influence of electrode placement. The spatial filtering of HD-sEMG can facilitate sEMG examination, indicating the ease of reflecting potential abnormality in certain muscles by the means of a noninvasive approach.It is worth noting that the presented spatial filtering methods involve an unsupervised matrix factorization algorithm without any association with the diseased or the diagnostic information (no label is required). The filtering approach was conducted independently on the data recording trial or the examined muscle. Thus, it is straightforward to apply the proposed method to data from any given muscle to be examined, and the filtering outcome is supposed to rely on the structural nature of its HD-sEMG data. All these features confirm a good generalization of the proposed spatial filtering method. This is also a prerequisite to its involvement in a standard pipeline for preprocessing the HD-sEMG signals towards various applications.Since the CI analysis performed the examination of individual muscles, it is straightforward to compare the neuromuscular changes in muscles on both side muscles of a subject. It was observed that the two side muscles of some SCI subjects showed different CI decisions. For example, the left muscles of S1, S3 and S5 were diagnosed as being ‘myopathy’ while their contralateral muscles were diagnosed as being ‘neurogenic’ changes. This may be attributed to the asynchrony of the left and right muscles. When the physiological balance is broken following SCI, it always overcorrects so that the left and right muscles may present different types of neuromuscular changes at different degrees.This paper just focuses on the application of spatial filtering and presents only three types of common spatial filtering methods. The use of the CI method has also limited performance in examining specific MU property alterations. More advanced methods including sophisticated spatial filtering methods and more sufficient diagnostic indicators can be developed for improved performance. In addition, the sample size used in this study is relatively small. It is sufficient only for technically confirming the benefit of using the spatial filtering methods in HD-sEMG diagnosis. Although the CI method would be applicable for examining muscles with diverse impairments at any degrees, the sEMG diagnostic approach requires more or less voluntary contraction ability of the examined muscle to emit sufficient sEMG activities. This is the main reason for recruiting subjects with incomplete SCI preserving certain hand functions in this study. The small sample size limits clinical significance of the current study. In order to establish diagnostic criteria and reveal neural or muscular pathology, a big sample size from a large population of subjects with different impairments is demanded. These will remain our future work.This paper examined the feasibility of performing spatial filtering methods using the PCA algorithm, the NMF algorithm and their combination, for enhancing HD-sEMG examination of neuromuscular changes. The experimental results demonstrated that spatial filtering of HD-sEMG can help to improve diagnostic power of CI method with respect to that with no spatial filtering, and that the combined PCA-NMF-based spatial filtering method yielded the highest diagnostic power in identifying complex neuromuscular diseases following SCI. The proposed method facilitates HD-sEMG examination of neuromuscular changes and it helps to develop a standard pipeline for pre-processing the HD-sEMG data towards practical and meaningful applications.


Dynamic assessment of cerebrospinal fluid (CSF) is essential for diagnosis, treatment, and prognosis of tuberculous meningitis, one of the most severe forms of central nervous system (CNS) infection.Mycobacterium tuberculosis. Later, the dose of dexamethasone was reduced, large number of neutrophils (57%) was present and protein level was immediately elevated in CSF specimen, indicating a possible relapse of tuberculous meningitis. Since the clinical condition of the patient was not worsening, the patient was stick to reduced dose of dexamethasone and standard anti‐tuberculosis agents. He was discharged from the hospital on day 34, with 1‐year continuation standard anti‐tuberculosis therapy, and was clinically resolved from tuberculous meningitis.A 45‐year‐old man sought care as he developed confusion, clonic convulsion, and coma. Longitudinal, comprehensive analyses of cytological, biochemical, and microbial changes in CSF specimen were assessed for this patient. On day 1 of hospitalization, modified Ziehl‐Neelsen staining of CSF identified positive acid‐fast bacilli, cytological analysis revealed neutrophilic‐predominant pleocytosis (neutrophils 77%), and adenosine deaminase (ADA) was substantially elevated. Therefore, tuberculous meningitis was diagnosed and first‐line standard anti‐tuberculosis treatment was initiated. Interestingly, after 7‐day treatment, the patient was greatly improved, and CSF disclosed a dominant percentage of lymphocytes (82%) as well as macrophages engulfing Detailed analyses of cellular composition, biochemical results, and microbial tests of CSF specimen provide the physician direct evidence of the immune surveillance status during tuberculous meningitis, which facilitates early diagnosis, optimal treatment, and improved prognosis. AFBacid‐fast bacilliCNScentral nervous systemCSFcerebrospinal fluidMRImagnetic resonance imagingMTB/RIFMycobacterium tuberculosis/rifampicinWBCwhite blood cell count1Tuberculous meningitis is the most severe form of tuberculosis, leading to devastating clinical outcome. Timely diagnosis and immediate anti‐tuberculosis treatment are vital to reduce morbidity and mortality. For central nervous system (CNS) infection, dynamic assessment of cerebrospinal fluid (CSF) is essential for diagnosis, treatment, and prognosis of tuberculous meningitis. In this case, we monitored cytological, biochemical, and microbial changes of CSF from a patient with tuberculous meningitis, which highlight the clinical significance of longitudinal CSF analysis during tuberculous meningitis.22O. Analysis of CSF showed a neutrocytic pleocytosis   Figure . Further) Figure , while G2 of 6.8 kPa. The patient still presented substantial hypoxemia (SpO2 78%), necessitating oral incubation and mechanical ventilation. A repeat lumbar puncture showed an increased opening pressure (40 cm H2O), pleocytosis   assay. On day 8, the patient's mental status progressively improved. He was able to blink, and no neck stiffness was present. CSF analysis also revealed almost disappearance of neutrophils. However, AFB staining was still positive , with a respiratory rate of 35 breaths/min. The patient had an excessive sputum but with difficulty to cough up. He needed a 10 L/min oxygen via a face mask, resulting in an arterial PaO) Figure . During e Figure .Mycobacterium tuberculosis. Elevated ADA level and AFB were detected again  were found and protein was dramatically increased, indicating a relapse of tuberculous meningitis. However, clinical assessment of the patients was not worsening. Therefore, the patient was stick to 6 mg of dexamethasone and standard first‐line anti‐tuberculosis drugs. He was discharged from the hospital on day 34, with 1‐year continuation standard anti‐tuberculosis therapy, and was clinically resolved from tuberculous meningitis.3Mycobacterium tuberculosis is a cytozoic pathogen and its numbers are very few in cerebrospinal fluid.M tuberculosis in CSF from tuberculous meningitis patients is still a challenge for clinicians.Meningitis is the most severe form of tuberculosis, result in mortality or neurological disability in 50% of patients.Mycobacterium tuberculosis, suggesting the activation of immune surveillance in CNS.Longitudinal evaluation of CSF specimen is critical to monitor disease progression of tuberculous meningitis. A compressive CSF assessment includes biochemical, cytological, and microbiological results. We closely and comprehensively analyzed CSF specimen from day 1 until day 45 since hospitalization of the patient. First, in our case, the microbiological test revealed by modified Ziehl‐Neelsen stain results in prompt diagnosis and treatment. AFB was not detected cleared until day 27 post‐treatment, along with the alleviation of cytological and biochemical laboratory parameters. Furthermore, since CSF is an immunologically active body fluid, cytological evaluation of CSF specimen provides important information over the disease progression. On CSF examination at day 1, we found dominant presence of neutrophil granulocytes, indicating the patient underwent acute phase of tuberculous meningitis.The role of neutrophils remains controversial in pathogenesis of tuberculous meningitis.Mycobacterium tuberculosis. Despite ADA test has high sensitivity and specificity, the diagnostic value of CSF ADA remains to be explored. It is necessary to interpret the ADA result with the combination of clinical symptoms and laboratory results.Traditional laboratory tests including ADA of CSF are also helpful for diagnosis and prognosis. As an enzyme wildly presented in tissues and body fluid, ADA has been routinely used in To summarize, detailed analyses of cellular composition, biochemical results, and microbial tests of CSF specimen provide the physician direct evidence of the immune surveillance status during tuberculous meningitis, which facilitates early diagnosis, optimal treatment, and improved prognosis.Not applicable.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images.All relevant data and materials are included in the manuscript.


Midazolam significantly reduced connectivity in the DMN . The effect sizes for ketamine for resting networks showed a positive correlation  with the effect sizes for schizophrenia‐related deficits derived from ENIGMA's study of 261 patients and 327 controls. Effect sizes for midazolam were not correlated with the schizophrenia pattern . The subtraction of ketamine and midazolam patterns showed a significant positive correlation with the pattern of schizophrenia deficits . RsPhfMRI reliably detected the shared and divergent pharmacological actions of ketamine and midazolam on cerebral networks. The pattern of disconnectivity produced by ketamine was positively correlated with the pattern of connectivity deficits observed in schizophrenia, suggesting a brain functional basis for previously poorly understood effects of the drug.Subanesthetic administration of ketamine is a pharmacological model to elicit positive and negative symptoms of psychosis in healthy volunteers. We used resting‐state pharmacological functional MRI (rsPhfMRI) to identify cerebral networks affected by ketamine and compared them to the functional connectivity (FC) in schizophrenia. Ketamine can produce sedation and we contrasted its effects with the effects of the anxiolytic drug midazolam. Thirty healthy male volunteers (age = 19–37 years) underwent a randomized, three‐way, cross‐over study consisting of three imaging sessions, with 48 hr between sessions. A session consisted of a control period followed by infusion of placebo or ketamine or midazolam. The ENIGMA rsfMRI pipeline was used to derive two long‐distance  and one local  FC measures. Ketamine induced significant reductions in the connectivity of the salience network (Cohen's  A placebo‐controlled, randomized, three‐way cross‐over design was used where participants were blind to the drug they were receiving. Each subject participated in all three sessions. Individual sessions were separated by 48 hr to allow for a wash‐out of drug effects. Each session collected resting‐state imaging data. Participants were instructed to have their eyes open and fixated on a small cross on a projection screen during infusion. Written informed consent was obtained from all participants and the local Ethics Committee approved the study. The data analyzed here have been previously reported in . For the resting‐state data reported here, 437 volumes were acquired .All experiments were conducted using a 3 T MRI scanner  equipped with a 20‐channel head coil. BOLD fMRI data were acquired using a T2*‐weighted echo‐planar imaging (EPI) sequence (repetition time (TR) 2,200 ms, echo time (TE) 27 ms, flip angle 79°, 30 interleaved 3‐mm slices, field of view 215 mm, voxel size 3 Drugs were administered to a subanesthetic level through an intravenous line controlled by an infusion pump , programmed by a supervising anesthesiologist, located in the MR control room. Ketamine was administered with a 0.25 mg/kg bolus dose, followed by a 0.25 mg/kg/hr infusion. Doses were similar to those used in previous literature , resulting in predrug and postdrug administration datasets and removal of the 2‐min bolus period. The ENIGMA resting‐state analysis pipeline implemented in the Analysis of Functional NeuroImages (AFNI) software Cox,  was usedResting‐state network templates were defined based on the probabilistic regions of interest (ROIs) from an independent components analysis of the BrainMap activation database and resting‐state fMRI dataset  was designed to investigate changes in local spontaneous brain activity by performing a nearest neighbor analysis of similarity of the BOLD time series and assigning a Kendall's coefficient of concordance (KCC) score /2 is the mean of the Ri's; K is the number of time series within a measured cluster , that is, one given voxel plus a number of its neighbors, and n is the number of ranks  the nearest neighboring nodes can be defined spatially, which usually reflects the anatomical, morphological, and geometric features in a local brain structure; (ii) the rank‐based computation is efficient in the time domain; and (iii) this score is robust against noise by integrating noise‐filtering operations across both the spatial domain and the temporal domain. Of note, the ReHo values of the voxels near the border of the brain mask need to be discussed with caution. The ReHo map, the collection of all voxels' KCC score of each voxel with  26 nearest neighboring voxels, for each subject each condition, was calculated using the MATLAB function “y_reho.m” available in the DPABI_V3.0_171210 package. The ReHo calculation was performed in three‐dimensional volumetric space, and still the calculation on the two‐dimensional cortical surface is preferred for the better representation of the neural neighborhood across the heavily folded cortex  and predrug (preinfusion) rsFC measures from both the seed‐based and dual regression analysis approach for the ketamine, midazolam, and placebo conditions. We compared KCC scores from each of the resting state template ROIs between postinfusion and preinfusion of the drugs and the placebo.Paired‐sample d‐values)—for ketamine, midazolam and the placebo—was calculated for each of the resting‐state functional connections; and for seed‐based and dual regression analysis approaches, separately. Similarly, Cohen's d‐values (postinfusion relative to preinfusion) were calculated using KCC scores for each of the resting state template ROIs.The difference between postinfusion and preinfusion measures , keeping the ROIs and the functional connections within the same hemisphere. The reasons behind this are that the FPN exhibited strong within‐hemisphere interactions but with distinct patterns in each hemisphere and preferentially coupled to the DMN and language‐related regions in the left hemisphere but to AttN in the right hemisphere  the rsFC measures derived from RSNs, and KCC scores from ReHo methods between the postinfusion minus preinfusion for the drugs and the postinfusion minus preinfusion for the placebo.To determine the drug impacts beyond the placebo effect, we compared  followed by the auditory network  and default mode network  followed by the left attention network  and the left frontoparietal network . Other connections showing significantly reduced FC strength under midazolam were between the left middle frontal area and the left superior parietal area ; and from the left inferior parietal lobule to the left inferior frontal gyrus . The only functional connection that showed a significant increase in FC was from the left motor cortex to right motor cortex in SMN (p < .05)  for ketamine, midazolam, and the placebo  were found between rsFC measures in the seed‐based and dual regression approaches with ReHo KCC scores  and AN connections (p < .01) with ketamine administration. Significant reductions in FC strengths for the DMN and left AttN (lAttN) connections (p < .01) with midazolam  whereas AN, SN, SMN, and VN ROIs showed no significant changes with midazolam  and SN (Cohen's d = 1.13) were roughly twice the significant patient‐control differences observed in rsFC measures for SN (Cohen's d = 0.43), and AN (Cohen's d = 0.53) in schizophrenia  on the DMN connectivity was not significant in the patient‐control differences in schizophrenia (Cohen's d = 0.24). The effect sizes of ketamine were found correlated  with the network‐based patient‐control differences in the FC in schizophrenia, but no association was found for midazolam  on activities of the DMN, SN, and AN were about twice those observed in the patient‐control differences in these networks in schizophrenia and the correlation between the patterns of affected networks approached significance , presumably by reducing the sedation effects that both drugs exerted through DMN. Overall, this study suggests that ketamine challenges can produce the pattern of functional disconnectivity that mimics that observed in schizophrenia with exception of sedative effects of ketamine that can be corrected by contrasting with the effects of the midazolam challenge.A single‐blind, randomized cross‐over approach was used to investigate the shared and distinct effects of ketamine and midazolam on the activity of brain functional networks and compare them with the pattern of cerebral disconnectivity observed in schizophrenia patients. The effects of the two drugs were readily detected by three independent resting‐state analysis approaches. Administration of a placebo produced no significant changes. Both drugs reduced the activity in the default mode network (DMN) that we attributed to sedation. The effect sizes of ketamine  and localized (ReHo) measures of connectivity. Overall, these results are consistent with findings from an fMRI study that used ketamine as one of the functional contrasts . We believe this to be a minor limitation because there were no significant sex differences in the schizophrenia pattern of cerebral disconnectivity in schizophrenia (Adhikari et al., 5In healthy male volunteers, both ketamine and midazolam led to significantly disrupted rsFC on the DMN but actions of two agents on other networks diverged. The effects of two agents were robustly detected by three rsFC measures and the effect sizes were consistent across three analysis approaches. Effects of ketamine were positively correlated with the pattern of disconnectivity observed in schizophrenia patients. This correlation became significant after accounting for sedation effects of ketamine by subtracting effects of the midazolam.The authors declare no conflict of interest. PT and NJ received a research grant from Biogen, Inc., for research unrelated to the topic of this manuscript.Appendix S1 Supporting InformationClick here for additional data file.


BEST1 gene. The phenotype of BD is variable, and there are just a few reports on the histopathology of eyes from donors with BD. Here, we describe the histopathological comparison of donor’s eyes from two patients with BD. Eyes obtained from 85-year-old (donor 1) and 65-year-old (donor 2) donors were fixed within 25 h postmortem. Perifoveal and peripheral retinal regions were processed for histology and immunocytochemistry using retinal-specific and retinal pigment epithelium (RPE)-specific antibodies. Three age-matched normal eyes were used as controls. DNA was obtained from donor blood samples. Sequence analysis of the entire BEST1 coding region was performed and identified a c.886A > C (p.Asn296His) variant in donor 1 and a c.602T > C (p.Ile201Thr) variant in donor 2; both mutations were heterozygous. Fundus examination showed that donor 1 displayed a macular lesion with considerable scarring while donor 2 displayed close to normal macular morphology. Our studies of histology and molecular pathology in the perifovea and periphery of these two BD donor eyes revealed panretinal abnormalities in both photoreceptors and RPE cellular levels in the periphery; donor 1 also displayed macular lesion. Our findings confirm the phenotypic variability of BD associated with BEST1 variants.Best disease (BD), also known as vitelliform macular dystrophy, is an inherited disease of the central retina caused by more than 300 pathogenic variants in the  VMD2 gene, now known as BEST1  cells  cells .BEST1 variants to provide insight into the pathophysiology of the disease. This is the first study of adult postmortem donor eyes from patients with BD due to these specific mutations.The morphological findings described in the eyes of BD patients evaluated with spectral domain optical coherence tomography (SD-OCT) are variable and, in sum, include (1) the accumulation of lipofuscin in the RPE and (2) photoreceptor degeneration over a morphologically intact RPE layer . A limitPostmortem eyes obtained from the Cole Eye Institute Eye Tissue Repository through the Foundation Fighting Blindness (FFB) Eye Donor Program . Eyes from BD donors (FFB# 928 and 458) were enucleated and fixed in 4% paraformaldehyde (PF) and 0.5% glutaraldehyde (GA) in D-PBS 12 and 25 h postmortem. Donors were an 85-year-old female and a 65-year-old male. Normal postmortem donor eyes from an anonymous 65- and 95-year-old woman and an 88-year-old male were fixed similarly within 4 and 18 h postmortem .2 volume scan of the posterior pole @ 500 B-scans/volume × 250 A-scans/B-scan, and (4) 10-mm2 volume scan of the posterior pole @ 500 B-scans/volume × 250 A-scans/B-scan. SLO images were collected using a model HRA2 confocal scanning laser ophthalmoscope . The HRA2 was rotated 90o so that the scan direction was perpendicular to the table surface. The system was operated in high-resolution mode, which provides an image pixel format of 1536 × 1536 when used with a 55° wide-field objective lens. SLO images of the posterior pole were collected using infrared reflectance (SLO-IR), infrared dark field (SLO-IRDF), autofluorescence (SLO-AF), and red-free dark field (SLO-RFDF) imaging modes at field of view (FOV) settings of 55°, 35°, and 25°.Eyes were cut through the ora serrata, transferred to a plexiglass chamber filled with D-PBS, and imaged by Spectral Domain-Optical Coherence Tomography (SD-OCT) and confocal scanning laser ophthalmoscopy (cSLO) as previously described . For theBEST1 coding region was performed and reported by Dr. Edwin Stone (BEST1 pathogenic variant and a c.602T > C (p.Ile201Thr) BEST1 pathogenic variant in donor 2; both mutations were heterozygous. The clinical evaluation of donor 1 was carried out at the University of Illinois with the approval of the Institutional Review Board (IRB) at the University of Illinois Medical Center.Sequence analysis of the entire in Stone . DNA anaFragments of retina–RPE–choroid were cut from the perifovea and periphery. Tissue fragments were further fixed by immersion in 2.5% GA in 0.1 M cacodylate buffer, post-fixed with 1% osmium tetroxide for 45 min on ice, sequentially dehydrated in ethanol, and embedded in Epon as previously described . Toluidi4Cl in D-PBS for 1 h at 4°C followed by changes to 10% (1 h) and 20% sucrose (overnight) made in the same buffer and finally a mix of 20% sucrose and Tissue-Tek “4583” . Finally, samples were transferred to a small cassette filled with the same sucrose and Tissue-Tek mix and frozen. Cryosections (10 μm) were collected on an HM 505E cryostat  equipped with a CryoJane Tape-Transfer system . No perifoveal tissue from donor 2 was available for this analysis.Another set of tissue fragments was fixed by immersion in 4% PF in D-PBS where they remained overnight at 4°C and then quenched with 50 mM NHAutofluorescence of unlabeled cryosections was performed and analyzed using epifluorescence in the green channel (FITC filter: 490 nm excitation/519 nm emission) and red channel (TRITC filter: 550 nm excitation/570 nm emission). Autofluorescence was overlaid on differential interference contrast (DIC) images.®-3 iodide (Thermo Fisher). Sections were also labeled with PNA-Alexa488  and WGA-Alexa594 . Images were acquired using a Leica laser scanning confocal microscope  with a series of 0.33 μm xy (en face) optical sections. Microscopic panels were composed using Adobe Photoshop CC . The perifovea of donor 2 was unavailable for analysis.Cryosections were blocked in D-PBS supplemented with 2% BSA and 0.2% TX100 (D-PBS/BSA/TX) for 30 min and incubated with the following antibodies: GFAP , rhodopsin , MCT3 , EBP50 , bestrophin-1 , and red/green opsin , in PBS/BSA/TX overnight at 4°C. Sections were then labeled with secondary antibodies conjugated with Alexa Fluor 488 and 594  for 45 min at room temperature. Cell nuclei were labeled with TO-PRODonor 1 was last seen for a follow-up eye examination in September 1992 at 65 years of age, 20 years before her death. At that time, her visual acuity was correctable to 20/200 in both eyes. The lenses showed trace nuclear sclerosis. Ocular pressures were 18 mmHg in each eye. The fundus examination showed a hypertrophic scar in the right eye , OD, whiDue to the retrospective nature of this analysis, historical clinical records were unable to be obtained for donor 2.BEST1 affect the function of bestrophin-1 and disrupt the ion transport by the RPE, resulting in the accumulation of fluid between the RPE and the photoreceptors -binding phosphoprotein of 50 kDa (EBP50), a protein that links apical transporters to ezrin and the actin cytoskeleton, was observed at the RPE apical microvilli in both the perifovea and periphery of the control retina; a minor presence was also observed in the basal surface of the cells as previously described , an RPE basolateral transporter . In bothFinally, we investigated bestrophin-1 distribution in control and BD eyes . ImmunolBEST1 pathogenic variants p.Thr6Arg  BEST1 variant] did not display any retinal lesion. However, it did display a substantial fixation artifact in the form of several small areas of retinal detachment. Immunohistological analysis of this tissue revealed that rhodopsin labeling was restricted to the rod outer segments but reduced in expression and substantially disorganized. While cone opsins were distributed throughout the whole cell in the periphery, the distribution of RPE plasma membrane proteins was significantly decreased in the periphery; EBP50 distribution was similar to that observed in control samples, but MCT3 was mostly absent from the RPE.The fundus macroscopy of the retina of donor 2 . The c.704T > C; p.(V235A) BEST1 mutation was previously reported to be mislocalized at least in part to the apical surface of hiPSC-RPEs from an autosomal dominant vitreoretinochoroidopathy patient  and c.602T > C (p.Ile201Thr) proposed .BEST1-related pathogenesis may help to define therapeutic targets. Our results suggest that although BD etiology remains poorly understood, further efforts to understand the unique pathogenesis of each BEST1 mutation are warranted. Only with the full understanding of the cellular and tissue effects on the pathologies is a targeted and efficient therapeutic approach plausible and promises to be successful in the long term. The lower level of bestrophin-1 protein found in both RPE cells does suggest that increasing the levels of this protein, through gene augmentation therapy or by rescuing mutant Best1 from proteasomal degradation, may be a viable means of preventing vision loss in BD. This study resulted in tangible improvements in our understanding of BD pathology. However, it is a snapshot of the BD pathology caused by the BEST variants. Moreover, our study was limited by the lack of clinical data and by the unavailability of perifoveal tissue from donor 2 to be analyzed by immunohistochemistry.Presently, there is no treatment available to treat BD; thus, a better understanding of The datasets generated for this study are available on request to the corresponding author.All procedures in this study adhered to the tenets of the Declaration of Helsinki regarding research involving human tissue and were approved by the Institutional Review Boards of the Cleveland Clinic (IRB14-057).VLB and JGH performed the conceptualization. GAF performed clinical input. VLB, BAB, and MJD performed experimental input. VLB, GAF, SAH, and JGH provided resources. All authors have read, reviewed, edited, and agreed to the published 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.