Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics. Allogeneic hematopoietic stem cells transplantation (SCT) represents an established curative treatment for various malignant and non-malignant hematological disorders such as leukemia, aplastic anemia, multiple myeloma and lymphoma . ReplaceDifferent techniques are available for chimerism analysis, but PCR-based analysis of short tandem repeats (STRs) is most widely used  due to iThe principle of dPCR is based on dividing the whole PCR mix into thousands of identical small “mini-reaction” compartments, before thermocycling takes place. Thus, the template, e.g., genomic DNA, becomes diluted with the consequence that individual compartments may or may not contain (at least) one template. All compartments are amplified in parallel but separately, and successful amplification  is detected by endpoint analysis based on fluorescence detection; individual positive and negative mini-reactions can be counted. The use of Poisson statistics enables quantification of absolute target numbers present in a sample .In 1999, Vogelstein and Kinzler showed that dPCR represents a promising technique for cancer genomic content determination . HoweverIn this work, we aimed at introducing such comprehensive dPCR panel for regular chimerism diagnostics. In order to ensure precise quantification, all individual reactions had to be suitable for duplex PCR against both a reference gene and, in case of sex-mismatched SCT, a Y-chromosome specific PCR. Twenty-nine individual DIP, the reference and Y-chromosome specific assays were optimized and technically validated, before they were tested on artificial serial dilutions. Thereafter, we performed clinical validation on retrospective patient samples in direct comparison with STR analysis. Finally, we designed a screening plate that facilitates identification of suitable markers for a given recipient–donor pair in an easy and comfortable way.The aim of this project was the establishment of a broad panel of dPCR-based assays for chimerism analysis. For convenient use in every-day diagnostics, such panel has to meet several conditions: (i) all reactions should proceed under the same conditions ; (ii) each individual assay should be compatible with each other to allow for duplex-PCRs; and (iii) results should be easily interpretable with clear separation of positive and negative events. However, the clearly most important requirement was the inclusion of multiple alleles at different chromosomes to ensure broad coverage of ideally any donor/recipient constellation, but identical twins.As we have shown previously , both doβ-Globin, can always be applied for duplex-PCR. In contrast, the second, Y-chromosome specific reference  will only be useful if at least one of the two parties, donor or recipient is male, i.e., in approximately 75% of the cases. Importantly, however, after sex-mismatched SCT, inclusion of the SRY assay allows simultaneous quantification of donor vs. patient cells in the duplex PCR.Therefore, we decided to use an alternative strategy. We reasoned that it is much straighter to optimize all individual DIP-directed dPCR assays for duplex PCR with two different reference genes. One “universal” reference (REF) gene, We identified an initial set of 53 human biallelic deletion-insertion polymorphisms (DIPs/Indels) by searching the RefSeq-, UniSTS-, dbSNP- and Online Mendelian Inheritance in Man (OMIM) databases . We applAs stated above, the first task was to establish uniform assay conditions that ensure high specificity and sensitivity of duplex PCRs for all possible combinatory assays, i.e., ideally several dozens of different duplex assays .To this aim, we adjusted a number of parameters to ensure optimal results in duplex PCR. Those included the annealing temperature, primer and probe lengths and concentrations for each single DIP-specific dPCR, but also the use of different numbers and types of quenchers.2. An example for the impact of MgCl2 is provided in Since we realized that the magnesium chloride concentration in the standard Bio-Rad  buffer was not optimal for our duplex assays, we performed titration experiments adding different amounts of MgClNext, we assessed applicability of the 53 DIPs see  for duplFinally, robustness of all individual dPCR assays against variations in several parameters was verified. In particular, we assessed potential deviations in DNA input, applied reaction volumes and PCR cycle numbers, but also the possible impact of the used PCR thermocycler. We found that a minimal amount of 1 ng gDNA still ensured precise and reproducible quantification, albeit, as expected, at lower resolution. Moreover, the assays were very robust with regard to reduced reaction volumes (down to 17 µL instead of 20 µL) that might be, for example, due to not correctly calibrated pipettes. PCR cycle numbers between 35 and 45 warranted essentially identical results; we would suggest applying at least 40 cycles. Finally, we tested six thermocyclers and found excellence data coherence for our assays independent of the used device  for all assays . FurtherWe wanted to test whether dPCR would principally facilitate a sensitivity of 0.01% on a regular basis. To test this, we generated and tested artificial dilutions of 0.01% for 25 DIP + REF/SRY combinations. We submitted 200–600 ng gDNA to single dPCRs. In the representative examples shown in To address reproducibility and precision of dPCR assays, we carried out independent analyses for the same dilution samples using assays based on different DIPs. Data are summarized for one representative example in A further indicator of reliability of an assay is its (intra-assay) reproducibility. We have previously shown excellent repeatability of dPCR in the context of chimerism determination . Here weAs evident in To verify applicability of our panel for diagnostics, we finally assessed chimerism on patient samples post allo-SCT by both STR and dPCR. We analyzed 21 patients , who underwent allogeneic SCT in the Dept. of Stem Cell Transplantation of the University Medical Center Hamburg-Eppendorf between 2008 and 2015. Median patient age at transplantation was 58 years (range from 39 to 74 years). Further patients’ characteristics are summarized in r2 value above 0.98, with only a few divergences in both directions. The only striking differences represent two samples with low, but detectable donor chimerism by STR, which are negative by patient-cell directed dPCR. These can be explained by the technical variations of dPCR in this measurement range  in accordance with the manufacturer’s recommendations. Results were compared with data obtained using the current “gold standard”, i.e., STR analysis, as shown in ange see . It is tTo visualize the degree of agreement between STR and dPCR data, we performed Bland–Altman analysis. The observed mean value of the difference (0.07%) and the agreement range (95% limit of agreement) from −5.94% to 6.07% underline the close correlation of data obtained with the two techniques.Notably, usefulness of the dPCR assays for clinical diagnostics was also proven by successful participation in standardized External Quality Assessment Schemes performed by Instand e.V. .The final dPCR panel that passed all quality checks comprises 30 markers (29 DIPs and the sex-typing locus). In the last step, we developed a screening plate for initial genotyping with DIP-specific duplex dPCR assays in order to facilitate convenient use in routine diagnostics. Using this screening plate, informative DIPs can be easily determined for a given donor/recipient constellation.The loci targeted with the new dPCR panel are distributed over 15 chromosomes, which in theory should warrant a high power of discrimination. To address this question empirically, we investigated all 176 adults transplanted in our department in 2015 for the presence of the respective DIPs. We identified at least one feasible duplex-PCR assay for 174 of the 176 donor/recipient pairs, corresponding to 98.9%, proving the very good discriminative power of the panel.Chimerism monitoring is a crucial diagnostic measure after allogeneic SCT, and STR analysis is the “gold standard” to measure chimerism at present. For quantification, STR relies on capillary electrophoresis broadly used for Sanger sequencing, but nowadays threatened with extinction. Moreover, based on the currently available read-out technique, usefulness of STR is constrained by its low sensitivity of 1% to 5% ,19. ThisMore than a decade ago, real-time quantitative PCR based on a Y-chromosome specific sequences and InDels ,19 was iRecently, others and we have shown that a further development, digital PCR has the potential to overcome limitations of qPCR ,8. IndeeTo address this need, we here aimed at developing a whole panel of assays detecting different DIPs that had to cover multiple regions of the human genome to ensure maximal discriminative power. Obviously, this goal ideally requires a maximal set of assays. At the same time, our idea was to develop an easy-to-use kit, where all individual reactions can be performed with the same buffer and using the same amplification program. This was particularly challenging, since all assays had to be efficient in duplex dPCRs, in order to ensure accurate quantification. Indeed, of the 53 DIP-specific assays initially tested, only 29 successfully underwent all optimization and validation steps. This means that all 29 assays can be performed under the specified standard PCR conditions in conjunction with either the reference assay or the Y-chromosome specific assay that will be particularly useful in the context of sex-mismatched allo-SCT. In addition, the SRY assay was optimized to be carried out in duplex with the REF assay. Notably, for initial genotyping of donor–recipient pairs, we developed a screening plate that allows comfortable and safe identification of suitable dPCR assays in routine laboratories.In our experience, in most cases it will be sufficient to quantify patient cells remaining after allo-SCT for precise chimerism determination. Only in cases of low donor chimerism, it is obligatory to directly measure the contribution of donor cells (see next paragraph). Based on the excellent inter-assay conformity , we suggIt could not be excluded a priori that the used setting (duplex dPCR on relatively high amounts of genomic DNA) might be associated with some technical measuring variability. In fact, despite our optimization strategy, PCRs ran in duplex might still influence each other, and a differential impact of the relatively high amounts of genomic DNA and potential sample contaminations  analyzed on the two individual assays might not be completely excluded. Obviously, sample processing in the used droplet-digital PCR device will further increase the inherent statistical variation in the distribution of the two analyzed alleles. Finally, the emission spectra of the two fluorescence dyes used in the Bio-Rad system show some overlap and therefore require compensation, which obviously might affect results. To address technical accuracy, we simultaneously determined allele numbers in multiple samples using two different DIP-specific assays in a duplex dPCR. Notably, the median difference between the two measured values was below 4%. Thus, the methodological error of our approach cannot be expected to affect validity of data. This underlines usefulness of dPCR for chimerism analysis, particularly for the status of complete or almost complete donor chimerism with only very few remaining cells to be detected. It is important to note, however, that the observed inherent error becomes relevant, if donor chimerism falls under the level of 20% to 30%, and only recipient cells are being detected by dPCR assays , we also tested sensitivity using cell dilutions of 0.01% and detected the low-level cells in all but one cases. It needs to be noted, however, that increased sensitivity can only be accomplished with higher DNA amounts. If too much template is being used, the reference PCR will become saturated , which precludes correct quantification. Moreover, very high sensitivities might not be meaningful for routine chimerism analyses of blood samples, since the latter are usually “contaminated” by recipient cells  . On the r2 > 0.98) indicating that dPCR matches the reliability of STR-PCR. In addition, when we compared data on identical samples independently obtained with either different assays or repetitive measurements using the same assay, we observed excellent accuracy and reproducibility. Importantly, obtained chimerism kinetics were in excellent agreement with the clinical course of the patients included in this study (not shown), which is an essential precondition for using this parameter as a prognostic marker [In order to assess clinical utility of our panel, we analyzed 147 peripheral blood samples from 22 patients post allo-SCT in direct comparison with STR. Notably, we found a very high correlation between chimerism data obtained with the two different methods (c marker  and/or tn = 176). For 174 of the 176 donor–patient pairs (98.9%) we found at least one, in the vast majority several assays informative for chimerism testing. This is conform to the statistical calculations by Dwight et al. [In order to verify whether the number of assays included in our final panel were sufficient to find an informative locus for most if not all donor–recipient pairs, we tested applicability for all consecutive adult patients transplanted in our center in 2015  necessitates relatively long hands-on time per sample, but automated sample-processing devices are available, which significantly reduce the amount of manual work. On the plus side, dPCR is easy to perform and does not require replicate analysis or generation of standard curves for quantification of chimerism samples. Based thereon, chimerism data can be obtained very quickly, usually within 24 h. Moreover, in most cases, existing qPCR protocols can readily be adapted to dPCR conditions.In summary, we have established a very convenient tool for routine chimerism analysis based on digital PCR. We have shown that the panel of dPCR assays introduced here facilitates accurate and high-sensitivity chimerism assessment in patients after allogeneic SCT. Based on theoretical considerations, but also our empirical data, the panel might be applicable for almost 99% of allo-SCT patients. We conclude that our new tool represents a promising alternative technique for molecular chimerism diagnostics.n = 2), MDS (n = 4), AML (n = 8), CML (n = 3), CLL (n = 1) and MF (n = 3). Peripheral blood samples were analyzed for chimerism by STR and dPCR in parallel for at least four time points after SCT. All patients agreed and gave their written informed consent for the acquisition of genetic data in the context of scientific studies.Twenty-one consecutive adult patients  transplanted between 2008 and 2015 in the University Medical Center Hamburg-Eppendorf were included in the clinical follow-up study . Median The testing of human samples for clinical validation of the novel, dPCR-based diagnostic method was approved by the Ethics committee of the Chamber of physicians of the city and state of Hamburg  on 19 May 2015.Genomic DNA was prepared from unseparated peripheral blood samples using the BioRobot M48 workstation . To generate artificial serial dilutions mononuclear cells (MNCs) of two healthy donors were used. DNA was isolated using the QIAamp DNA Blood Mini Kit (QIAGEN).n ± 2 alleles outside stutter region were used for calculation.STRs were PCR-amplified with CE-IVD certified Mentype Chimera PCR Amplification Kit  in accordance with the manufacturer´s recommendations. PCR products were quantified using a 3500 Genetic Analyzer . The ratio was calculated using the software package Chimeris Monitor (Biotype Diagnostic GmbH). Only informative donor and recipient alleles that are separated by at least Digital PCR was carried out with the QX100 and QX200 Droplet Digital PCR System  in accordance with the manufacturer’s recommendations. Briefly a set of 30 duplex assays as dried mixes on a 96-well plate  were available for the initial marker screening. For analysis of chimerism after marker screening, 29 DIPs in combination with a reference gene (β-Globin) or (for sex-mismatched SCT) a Y-chromosome specific locus and additionally the Y-chromosome specific marker with reference gene from the Mentype DigitalQuant Kit (Biotype Diagnostic GmbH) were used. Final dPCR reaction mixtures of 20-µL volume consisted of 10 μL 2× ddPCR Supermix (Bio-Rad), 2 µL 10× DigitalQuant Primermix (after optimization of primer and probe concentrations), 0.5 μL FastDigest EcoRI  and, if not indicated otherwise, about 100 ng DNA. To exclude unwanted restriction, absence of the EcoRI recognition site in all amplicons was verified based on the DNA sequences. The PCR mix was compartmentalized using the QX100/200 Droplet Generator. The produced droplet-containing water-in-oil emulsion (40 µL) was transferred to a 96-well PCR plate , sealed and amplified in a standard thermal cycler : denaturation (95 °C for 10 min), amplification cycles , ramp rate of 1.0 K/s, and final 10 min inactivation step at 98 °C. Afterwards, individual wells were analyzed simultaneously for FAM and HEX fluorescence using the QX100/200 droplet reader following the manufacturer’s instructions. Data were processed with QuantaSoft software (Bio-Rad). The mean concentration of target sequences (copies per microliter) was calculated using Poisson Algorithm incorporated in the software.


Genetic variants in miRNA genes and abnormalities in the concentration of microRNAs (miRNAs) in tissues and biological fluids have recently been associated with a diagnosis of schizophrenia. Most of these studies used post-mortem brain tissue or whole blood as the source of RNA. However, examination of microRNAs in cerebrospinal fluid (CSF) might provide an in vivo biomarker, more accurately reflecting expression level changes in the brain. To date, there are no studies that have investigated miRNA expression in CSF in patients with schizophrenia using small RNA-seq. In the past, our group had the opportunity of investigating the correlation between miRNA profiles in CSF and blood measured using microarray technology. Therefore, to expand our findings and use current cutting-edge technology, we measured miRNA profiles in CSF and plasma using small RNA-seq in a sample of patients with schizophrenia-spectrum disorder (SSD) diagnosis and healthy volunteers.Twenty-two SSD patients and 17 healthy volunteers underwent a lumbar puncture and a blood draw. 15–25 cc of CSF and 5–10 cc of peripheral blood were obtained from each subject. CSF and peripheral blood samples were centrifuged. CSF and plasma samples were aliquoted into 1 mL cryovials, and stored at -80C degrees. Vesicular RNA was extracted from 1 mL of CSF and plasma samples following the protocol from the Qiagen exoRNA easy kit. The BioScientific NextFlex RNA sequencing kit was used for library construction. Sequencing was done on HiSeq2500. Samples that had at least 50,000 reads going to mature miRNA sequences were included in the analysis. Differential expression analyses were conducted in R using the DESeq2 package in Bioconductor.In the overall sample cohort, most subjects were male (66.7%), not Hispanic (81.0%) and black (48.7%). Mean age was 36.8 years (SD=12.3), There were no differences in age, sex, ethnicity or race between the patient and healthy control groups. In the patient group, 16 (72.7%) had schizophrenia, 5 (22.7%) had schizoaffective disorder and 1 (4.5%) had psychosis not otherwise specified. Differential expression (DE) analyses were conducted for 144 miRNAs in CSF and 354 miRNAs in plasma. After adjusting for multiple comparisons, DE analysis between patients and controls in CSF showed statistically significant higher levels in patients of miR-769-5p, miR-99b-3p, miR-107, miR-451a and miR-708-5. Similar analysis in plasma showed statistically significant higher levels in patients for miR-375, miR-204-5p, miR-942-5p, miR-6734-5p, miR-423-5p and miR-144-5p. Principal component analysis showed a clear separation between CSF and peripheral blood samples. Out of 443 miRNAs used to examine the relationship between CSF and plasma, 205 (46.3%) were detected in both plasma and CSF samples, 88 (19.9%) were detected only in CSF samples while 150 (33.9%) were detected only in plasma samples.Five miRNAs were upregulated in CSF samples and six were upregulated in plasma samples of SSD patients compared to healthy volunteers. There was no overlap in the statistically significant upregulated miRNAs between CSF and plasma samples. Therefore, miRNA profiles in CSF and plasma have important quantitative and qualitative differences that may make them excellent, but different, candidate biofluids for biomarker discovery.


Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 μg·mL−1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries.D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development.  The D-Ala dipeptide is synthesized by two enzymes: alanine racemase (Alr), which converts L-enantiomers of alanine to the D-counterparts, and D-Ala-D-Ala ligase, which generates the D-Ala dipeptide.3D-alanine (D-Ala) is an essential substance for the synthesis of peptidoglycan (PG), an important constituent of the cell wall of both Gram-negative and Gram-positive bacteria.4 Previous studies have revealed that Alr is essential for the survival of numerous Gram-positive bacteria, including Bacillus, Lactobacillus, Burkholderia pseudomallei, Burkholderia mallei and Mycobacterium tuberculosis. Knockout of the alr gene in these bacteria resulted in a strict exogenous D-Ala-dependent growth phenotype.8 Similar growth arrest and extensive cell lysis were also observed in the alr mutant of Gram-negative Escherichia coli, which underwent rapid and extensive cell lysis when deprived of D-Ala.9 Further in vitro and in vivo studies of M. tuberculosis have shown that Alr is a primary target of D-cycloserine, and the inhibition of Alr alone could reduce the viability and perseverance of this bacterium.8Alr is a unique enzyme responsible for the racemization of L-Ala to D-Ala.Streptococcus mutans is the major caries-associated bacterium in humans. During cariogenic conditions , S. mutans metabolizes carbohydrates, leading to acid accumulation and subsequent fall in pH in the dental biofilm.10 The acidic micro-environment selectively enriches acidogenic/aciduric species  and suppresses less aciduric commensal residents . This microbial disequilibrium leads to continuous pH decline to reach a critical pH, below which tooth hard-tissue demineralization begins and dental caries gradually occur.12 Although Alr has been well documented as closely associated with the viability and survival of various bacteria,9 its physiological importance in S. mutans has not been explored, particularly in a biofilm context. In the present study, we constructed alr mutant strain and investigated the physiological role of alr in the cell growth, cell wall integrity and interspecies competitiveness of S. mutans. We found that alr is an essential factor to maintain the growth and cell wall integrity of S. mutans. Deletion of alr in S. mutans significantly compromised its competitiveness with other co-residents  in the oral biofilm. We concluded that Alr might represent a promising drug target to modulate the cariogenicity of oral biofilm and to benefit the management of dental caries.S. mutans UA159 was obtained from the Dental Research Institute, University of Toronto13 and was routinely anaerobically  or aerobically  incubated at 37 °C in brain heart infusion (BHI) broth . For the transformation experiments, the cells were maintained in Todd-Hewitt medium  supplemented with 3 ġL−1 yeast extract . The competence-stimulating peptide used for S. mutans transformation was custom-synthesized by Sangon Biotech . For the selection of antibiotic-resistant colonies, BHI plates were supplemented with erythromycin . D-Ala (150 μg·mL−1) was added to the BHI broth to promote the growth of the S. mutans alr mutant strain. The optical density (OD) of the cell culture was measured at 600 nm (OD600).4 DNA ligase were all purchased from New England Biolabs . Taq DNA polymerase was used for overlapping polymerase chain reaction (PCR).Taq DNA polymerase, restriction enzymes and Talr) were generated through PCR using the primer pair alr-up-f/ldh and alr-down-f/ldh. The erm fragment (876 bp) was amplified with primer pair ldh-f/erm-r by PCR.14 The three pairs of primers were specifically designed for subsequent ligation based on the sequences adjacent to mutagenesis site. The restriction enzymes AscI  and FseI were used to digest the up- and down-stream fragments, respectively, and the aforementioned enzymes were also used to digest the purified erm segment. The three digested fragments were subsequently mixed, and T4 DNA ligase was added to generate the proposed segment , washed twice in 200 mmol̇L−1 sodium cacodylate buffer, pre-fixed with 2.5 ġL−1 glutaraldehyde and fixed with 10 ġL−1 OsO4. Samples were embedded in Epon resin, and thin sections (60 nm) were prepared using a microtome. The sections were stained with 40 ġL−1 uranyl acetate and subsequently with 4 ġL−1 lead citrate and were examined using a Tecnai G2 F20 S-TWIN electron microscope .Transmission electron microscopy (TEM) was performed as previously described.S. sanguinis and S. gordonii can support the growth of the alr-deficient mutant under exogenous D-Ala-deprived conditions, we performed a conditioned media assay as previously described.19 Briefly, the culture supernatants of S. sanguinis and S. gordonii at the mid-exponential phase were collected and filter sterilized as a conditioned medium for the growth of the alr mutant. After aerobic incubation (5% CO2) for 24 h, the OD600 values of the bacterial cultures were determined to evaluate the effect of conditioned medium on the growth of the alr mutant. We also diluted the conditioned medium 1:2 with fresh BHI broth medium to evaluate the dose-dependent effects of conditioned medium. In addition, an exogenous D-amino acid oxidase  was added to the conditioned medium to investigate whether the growth compensatory effects of conditioned medium on the alr mutant could be attenuated.To investigate whether other commensal colonizers such as in situ hybridization (FISH) was performed according to Zheng et al.19 Biofilm specimens established on saliva-coated glass coverslips were rinsed with distilled water and dried for 10 min at 46 °C. To enhance probe penetration, the specimens were treated with 1 mL of lysis buffer (100 mmol̇L−1 tris(hydroxymethyl)aminomethane (Tris)-HCl, 50 mmol̇L−1 ethylene diamine tetraacetic acid, and 30 mg·mL−1 lysozyme (Sigma), pH 8.0) for 20 min at 37 °C. The specimens were subsequently rinsed with distilled water, serially dehydrated in ethanol , dried for 10 min at 46 °C, exposed to 20 μL of hybridization buffer  and 20% formamide) containing the designated oligonucleotide probes (2 nmol̇L−1), and incubated at 46 °C for 90 min. After hybridization, the specimens were washed in buffer  for 15 min at 48 °C in a water bath and were subsequently rinsed in ice-cold nuclease-free water.Fluorescent S. sanguinis was designed using ARB software . The S. mutans-specific probe was synthesized according to the sequence provided in a previous study.20 The probe sequences are listed in S. mutans/S. sanguinis was calculated based on the coverage area of each bacterium analysed using Image Pro Plus 6.0 .The specific probe for P-value was <0.05.All experiments were performed in triplicate. The statistical analysis was performed using SPSS16.0 software . Differences were considered as significant when the two-tailed alr deletion without the addition of exogenous D-Ala was lethal to S. mutans. The optimal growth of the alr mutant strain was observed after adding no less than 150 μg·mL−1 of D-Ala to the BHI medium . Specifically, conspicuous cell lysis was observed in the alr mutant strain relative to the wild-type control  to partially compensate for the scarce endogenous D-Ala, its perseverance in the co-existence of other oral streptococci remained compromised.We further investigated the interspecies competitiveness of the us D-Ala , althougs strain . These dalr in the interspecies competition between S. mutans and S. sanguinis within biofilm, we used species-specific FISH to quantify the bacterial composition in a dual-species biofilm model  reflects the inactivation of dlt operon-stimulated autolysis.28 The removal of D-alanyl residues from TA could result in the formation of cell surface holes, which likely confer a higher affinity to cationic PG hydrolases and thereby facilitating cell lysis.27 The observed cell wall damage and cell lysis of the alr mutant in the current study might also reflect the defective PG peptide stem and the D-alanylation of TA. The glutamate racemase (murl) has also been reported as an essential enzyme in the synthesis of the bacterial cell wall.30 The murl mutant of E. coli lysed in the absence of D-glutamic acid,31 and the murl mutant of Mycobateriumsmegmatis also showed aberrant cell shapes.32 Although the precise mechanisms remain unknown, the deletion of alr not only led to growth arrest but also to cell death, indicating that alr might be a potential bactericidal target for the inhibition of S. mutans.The pronounced cell lysis of the alr mutant of S. mutans could survive in concert with other common oral streptococci, such as S. sanguinis and S. gordonii. The partially recovered growth of the alr-deficient mutant in conditioned medium from either S. sanguinis or S. gordonii could be further attenuated through D-amino acid oxidase. These results and data showing that the alr mutant requires exogenous D-Ala for survival, indicating that although the alr mutant might feed on either exogenous D-Ala or bacterial metabolites from other co-residents, the growth and competitiveness of the alr mutant strain are significantly compromised compared with the parental strain. This finding further supports the hypothesis that alr might be a promising target to control the prevalence of cariogenic S. mutans in a multispecies microbial consortium. Because Alr is universal in all bacteria, the utilization of this enzyme to develop antimicrobial drugs specific for S. mutans in multispecies microbial consortium is worthy of further investigation. Strategies as proposed by Professor Shi's group35 might be used to link Alr small-inhibitors with a targeting peptide  and to achieve S. mutans-specific killing, even in a complex microbial consortium.Interestingly, we also observed that the alr is critical for the viability, growth and interspecies competitiveness of S. mutans. Given the ecological importance of S. mutans in initiating oral microbial disequilibrium and dental caries, Alr could be exploited as an antimicrobial target for S. mutans, thereby contributing to the control of dental caries.Taken together, the results of the present study provide the first evidence that


Previous series have demonstrated that Clomiphene Citrate (CC) is an effective treatment to increase Total Testosterone (TT) in Late Onset Hypogonadism (LOH) patients. However, what happens to TT levels after ending CC treatment is still debatable. The objective of this study is to evaluate TT levels 3 months after the discontinuation of CC in patients with LOH who were previously successfully treated with the same drug.Twenty-seven patients with LOH that were successfully treated (achieved TT levels >11nmol/l) with CC 50mgs daily for 50 days were prospectively recruited in our Andrological outpatient clinic. CC was then stopped for 3 months and TT levels were measured at the end of this period.Mean TT level before discontinuation of CC was 22.7±8.1nmol/L (mean±SD). Three months after discontinuation, mean TT level significantly decreased in all patients, 10.2±3.9nmol/l (p<0.01). Twenty-one patients (78%) decreased TT levels under 11nmol/L. Six patients (22%) had TT levels that remained within the normal recommended range (≥11nmol/l). No statistical significant differences were observed between both groups.In the short term LOH does not seem to be a reversible condition in most patients after CC treatment. More studies with longer follow-up are needed to evaluate the kinetics of TT in LOH. The exact prevalence of Late Onset Hypogonadism (LOH) is a matter of debate –3; howevThirty patients  with LOH were prospectively recruited in our Andrological outpatient clinic. Late Onset Hypogonadism was defined according to previous consensus definition :-Symptoms: decreased sexual interest, decreased morning erections, erectile dysfunction.-Total Testosterone <11nmol/l (in at least two different measurements) Regarding symptoms, 13/27 patients complained of decreased sexual desire, 11/27 with Erectile Dysfunction (ED), and 3/27 both symptoms. The median IIEF-5 score of all patients before treatment was 18 (range 11–24). In the subgroup that complained of exclusively ED or decreased sexual interest plus ED the median score was 15 (range 11–19).To be eligible to be treated with CC, patients had normal FSH and LH, no thyroid function abnormalities neither hyperprolactinemia. After a 50-day treatment with 50mgs of CC daily, twenty-seven patients achieved normal TT levels (>11nmol/l). In this specific sub-group (n=27) CC was stopped for 3 months and TT levels were evaluated at the end of this period. Total testosterone was analyzed using electrochemiluminescence inmuno assay (Roche™). LH and FSH were measured using direct chemiluminimetric inmuno assay (Siemens™). The study was approved by the Research Ethical Committee of Pontificia Universidad Católica de Chile.Data analysis was performed using GraphPad™ software. Differences in TT levels before and after discontinuation of CC were analyzed with paired t-test, considering statistical significance with a p<0.05. Results are shown as mean±SD.Mean TT level at the time of diagnosis (n=30) was 8.5±1.8nmol/L (mean±SD), with normal range gonadotropins: FSH 5.1±4.9mIU/mL and LH 4.3±2.9mIU/mL. After a 50-day CC treatment, 27 patients achieved normal TT level. Mean TT level in this group was significantly higher than pre-treatment state, 22.7±8.1nmol/L, p<0.01 . Three mTestosterone replacement therapy has significantly increased in the last decade , howeverTaking all this information into account, therapy with CC makes sense and evidence supports it. The results of our series demonstrate that 90% of men with LOH increased TT under treatment which agrees with other reports showing that in selected patients, CC is efficacious in a high proportion of cases and has the advantages of not blocking the HPT axis, not affecting fertility, not producing polycythemia, and all with lower costs than TRT , 19–21. Regarding the first question, it seems that one of the concerns would be the safety of the drug in the long term. Evidence suggests that long term CC treatment has no adverse effects and that efficacy is maintained, however, in the longer studies the follow-up does not exceed 46 months . In our The main objective of our study was to elucidate if LOH could be reversed by a 50-day CC treatment. Previous evidences were scarce, since most of the studies did not report the kinetics of TT after stopping CC treatment. We found three studies that reported TT level after ending treatment. Lim and cols. in 1976 reported in five hypogonadal uremic men an increase in TT levels after CC treatment that lasted for 12 months . Normal Considering our results and previous reports we think CC treatment would have three roles: First, it is an excellent alternative when patients are concerned about fertility, second, CC is an extraordinary alternative for a therapeutic individual trial in cases where we are not convinced that low testosterone is really the explanation to all its symptoms, especially when the TT level is very close to the normal ranges. Finally, CC represents a good alternative to TRT with the advantage of not blocking HPT axis, but taking into account our results and previous series , 25, it The main limitations of our study are: the short follow-up and the absence of the evaluation of estradiol and free testosterone. Regarding this last point, all patients had TT values under the normal range associated with symptoms, according to the guidelines in these cases TT would be enough for evaluation . Also noLate Onset Hypogonadism on men who successfully respond to CC therapy in the short term do not seem to reverse the condition after ending treatment. More studies with longer follow-up are needed to evaluate the kinetics of TT in these patients.


TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy.Mutations in genes encoding cardiac troponin I  led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy.A mutation in the gene encoding Lamin A/C (Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways.p.98truncationTNNI3) and cardiac troponin T  and the non‐sarcomeric gene mutation encoding lamin A/C (p.R331QLMNA). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane‐permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The p.98truncTNNI3 and p.K217delTNNT2 mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The p.98truncTNNI3 showed pure haploinsufficiency, increased Ca2+‐sensitivity and impaired length‐dependent activation. The p.K217delTNNT2 mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild‐type troponin complex corrected troponin protein levels to 83% of controls in the p.98truncTNNI3 sample. Moreover, upon exchange all functional deficits in the p.98truncTNNI3 and p.K217delTNNT2 samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The p.R331QLMNA mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non‐sarcomeric genes. In this study we defined the pathogenic effects of three DCM‐causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I ( TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy.Mutations in genes encoding cardiac troponin I  led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy.A mutation in the gene encoding Lamin A/C (Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. No difference in Fmax  was observed in IDCM, p.98truncTNNI3 and p.K217delTNNT2 cardiomyocytes, while Fmax was significantly lower in cardiomyocytes with the p.R331QLMNA mutation . A previous study showed that this decreased Fmax was due to decreased myofibril density  over a range of sarcomere lengths , p.98truncTNNI3  and p.R331QLMNA  showed a comparable Fpass development over the range of sarcomere lengths compared to controls. The p.K217delTNNT2 cardiomyocytes showed a significant increase in Fpass compared to controls, which was most pronounced at longer sarcomere lengths . In addition to changes in Fpass, stretching of cardiomyocytes during filling of the heart increases active force development. This LDA of myofilaments is the cellular basis of the Frank–Starling mechanism . The p.98truncTNNI3 cardiomyocytes showed Ca2+‐sensitivity was increased and LDA was blunted  compared to controls. p.K217delTNNT2 cardiomyocytes showed only a minor and non‐significant decrease in Ca2+‐sensitivity and LDA was preserved . The Ca2+‐sensitivity of p.R331QLMNA cardiomyocytes was significantly increased compared to controls, while LDA was preserved . Overall these data illustrate that changes in passive and active myofilament properties differ between different sarcomeric gene mutations and between sarcomeric and non‐sarcomeric mutations.The 12 control samples used for experiments consisted of four females and eight males with a mean age of 44.5 ± 3.4 years. The patient with p.98truncTNNI3 and p.K217delTNNT2 samples. We observed that cTnI protein level was decreased in both the p.98truncTNNI3 and p.K217delTNNT2 samples compared to controls when normalized to the cytoplasmic housekeeping protein GAPDH . If the truncated cTnI protein is present in the p.98truncTNNI3 sample an antibody raised against the N‐terminus of cTnI is expected to show two bands: the native protein and the truncated protein. However, only one band was visible at the height of the native cTnI protein . This indicates that the mutant protein is either not expressed or efficiently degraded resulting in cTnI haploinsufficiency. The level of cTnI relative to the sarcomeric housekeeping gene α‐actinin was also decreased in both p.98truncTNNI3 and p.K217delTNNT2 samples , which indicates that less cTnI is present within the sarcomeres itself. The reduction in cTnI levels was accompanied by a less pronounced decrease in cTnT  and near normal cTnC levels . This indicates that the p.98truncTNNI3 and p.K217delTNNT2 samples have altered stoichiometry of the three troponin proteins since the decrease in cTnI, cTnT and cTnC is not to the same extent.Since the troponin complex and the interactions between the various troponin proteins are important for adequate contractile behaviour of the myofilaments, we studied the composition of the troponin complex in the p.98truncTNNI3 and p.K217delTNNT2 samples, while cardiomyocytes from these samples showed different myofilament properties. We therefore studied phosphorylation of cTnI using phos‐tag analysis. Control samples showed prominent bis‐ and mono‐phosphorylated cTnI and low non‐phosphorylated cTnI . IDCM samples showed reduced cTnI phosphorylation as is evident by prominent non‐ and mono‐phosphorylated cTnI bands and a weak bis‐phosphorylated cTnI band . In the p.98truncTNNI3 and p.K217delTNNT2 samples cTnI was highly phosphorylated indicated by an intense bis‐phosphorylated cTnI band and very weak non‐ and mono‐phosphorylated cTnI bands . On the contrary, the p.R331QLMNA samples showed decreased cTnI phosphorylation evident from prominent non‐ and mono‐phosphorylated cTnI bands . Low phosphorylation of cTnI has been reported previously in DCM  and in the p.R331QLMNA cardiomyocytes . Incubation with exogenous PKA did not change Ca2+‐sensitivity in controls. Also LDA was restored in IDCM samples compared to controls after incubation with exogenous PKA . However, Ca2+‐sensitivity was still significantly increased compared to controls after incubation with exogenous PKA in the p.98truncTNNI3 cardiomyocytes . These experiments confirm that impaired β‐adrenergic receptor signalling, and subsequent hypophosphorylation of cTnI, is the cause of the increased Ca2+‐sensitivity in IDCM and the p.R331QLMNA cardiomyocytes, while the observed increased Ca2+‐sensitivity and impaired LDA is a direct mutation effect in the p.98truncTNNI3 cardiomyocytes.The troponin complex was reduced in both the TnI Fig. A and B.and Fig. A and B.nds Fig. A and B.nds Fig. A and B.DCM Fig. C and intes Fig. D. IncubPKA Fig. C. Howevtes Fig. E. These2+‐sensitivity and reduced LDA in the p.98truncTNNI3 cardiomyocytes. We exchanged the endogenous troponin complex with the recombinant WT troponin complex in order to restore total troponin levels. The level of cTnI increased to 83% after exchange , relative to the cTnI level in control cells exchanged with exogenous recombinant WT troponin complex. The 83% cTnI in the p.98truncTNNI3 sample after exchange consisted of 46% recombinant troponin complex and 37% native troponin complex, as determined by phos‐tag gel analysis . In the control sample 43% of total cTnI levels was derived from the recombinant troponin complex . This indicates that we exchanged endogenous troponin complex with recombinant WT complex, but also added additional recombinant WT troponin complex in the exchange process thereby largely overcoming the haploinsufficiency in the p.98truncTNNI3 cardiomyocytes. Since the recombinant troponin complex is unphosphorylated we incubated the exchanged cells with exogenous PKA prior to functional cell measurements. Upon exchange with the WT troponin complex both Ca2+‐sensitivity as well as LDA were normalized to control values in the p.98truncTNNI3 cardiomyocytes .Next we aimed to assess whether the observed haploinsufficiency of cTnI, and reduced cTnT and cTnC, caused increased Cange Fig. F and H,Fpass in the p.K217delTNNT2 sample. An important determinant of Fpass is titin isoform composition . The observed increase in N2BA/N2B ratio cannot explain the high Fpass in p.K217delTNNT2. Therefore, we examined phosphorylation of titin at three well‐established phosphorylation sites in the elastic I‐band region. Phosphorylation of Ser4010 on titin, a target of PKA, is known to decrease Fpass . Ser12022  and Ser11878  phosphorylation, which would increase Fpass, was within the 95% CI of controls in IDCM but lower in samples carrying mutations compared to controls. Therefore, the increase in Fpass in the p.K217delTNNT2 cardiomyocytes is not caused by alterations in titin phosphorylation at the investigated sites. The increase in Fpass was not due to impaired PKA‐mediated phosphorylation of titin since Fpass remained significantly higher in p.K217delTNNT2 cardiomyocytes compared to controls after incubation with exogenous PKA . Exchange with the WT troponin complex led to a 59% incorporation of recombinant troponin complex in the p.K217delTNNT2 sample . The exchange normalized Fpass in the p.K217delTNNT2 cardiomyocytes to control level . In addition, after exchange of the mutant p.K217delTNNT2 troponin complex into a healthy control sample we observed that only 34% of total troponin present after exchange was recombinant . However, this was sufficient to cause a significant increase in Fpass . The increase in Fpass upon exchange with p.K217delTNNT2 in a control sample was not due to impaired PKA‐mediated phosphorylation since after incubation with exogenous PKA Fpass remained significantly increased . These results indicate that the p.K217delTNNT2 mutant protein itself increases Fpass.We next set out to determine the cause of the increased ple Fig. C and D.022 Fig. E and F 878 Fig. G and H PKA Fig. A. Exchaple Fig. B and C.vel Fig. D. In adant Fig. B and C.ass Fig. D. The ised Fig. E. Thesep98.truncTNNI3 and p.K217delTNNT2 mutations, and the non‐sarcomeric p.R331QLMNA mutation. Our study provides proof that the two sarcomere mutations cause myofilament dysfunction, while changes in myofilament properties in IDCM and the non‐sarcomeric mutation samples are the result of secondary disease remodelling. One of the p.R331QLMNA samples showed a smaller decrease in cTnI phosphorylation compared to the other two samples. This was the sample obtained from a patient who used a LVAD prior to transplantation. It is therefore possible that the LVAD has partly reversed the secondary remodelling   while the increased Ca2+‐sensitivity and impaired LDA in IDCM could be corrected by exogenous PKA . This is in line with Sequeira et al. who reported that the HCM‐causing p.R145WTNNI3 mutation impaired LDA, which could not be rescued with exogenous PKA  mice are embryonically lethal, while heterozygous TNNT2 KO mice have reduced cTnT mRNA levels, but normal cTnT protein levels, and show no cardiac phenotype  production was maintained in order to compensate for the absence of cTnI . The increase in compliant titin therefore seems to be a general hallmark of DCM and not a specific effect of the mutations studied. Despite the increase in compliant titin, Fpass was similar to controls in IDCM, the p.R331QLMNA and p.98truncTNNI3 cardiomyocytes. Interestingly, PKA‐mediated phosphorylation titin was unaltered in the p.K217delTNNT2 sample. In addition, cTnI phosphorylation was also not affected in the p.K217delTNNT2 and p.98truncTNNI3 samples suggesting that these specific mutations do not lead to defects in β‐adrenergic receptor signalling. This is contrary to what we observed in the IDCM samples and to what has been reported in other DCM samples . Since the troponin levels in the p.98truncTNNI3 and p.K217delTNNT2 sample were reduced in a similar fashion we expect that the poison peptide of p.K217delTNNT2 and not reduced troponin complex was the cause of the high Fpass. We hypothesize that the mutant p.K217delTNNT2 troponin complex is less likely to incorporate in the sarcomeres than the WT troponin complex. Also the observed decreased cTnT levels in p.K217delTNNT2 might indicate the mutant protein is not as stable as healthy cTnT. We observed a low exchange rate of the mutant p.K217delTNNT2 protein complex in a control sample (34%) and a high exchange rate of the WT troponin complex in the p.K217delTNNT2 sample (59%) compared to the exchange rate of WT in a control sample (43%) . Most models have high incorporation of the mutant with values reported to be 79% . Inoue et al. also showed a depressed Frank–Starling mechanism  Fig. C. Most l sample 9% comparp.98truncTNNI3 and p.K217delTNNT2 cause reduced expression of the troponin complex and altered stoichiometry between the troponin subunits. In the p.98truncTNNI3 cardiomyocytes this led to increased Ca2+‐sensitivity, which could not be corrected with exogenous PKA but was normalized to control levels upon exchange with WT troponin complex. The p.K217delTNNT2 mutation caused a mild, non‐significant, reduction in Ca2+‐sensitivity and significantly increased Fpass, which could not be corrected by PKA but was normalized to control levels upon exchange with WT troponin complex. In addition, incorporation of the p.K217delTNNT2 mutant troponin complex in a control sample confirmed the mutant protein itself causes increased Fpass. This implies that even mutations in the genes encoding for the troponin proteins have different effects on myofilament function. In contrast, the p.R331QLMNA mutation caused reduced maximal force development and increased Ca2+‐sensitivity due to secondary disease remodelling. Also IDCM samples showed an increased Ca2+‐sensitivity due to secondary disease remodelling. We show that although DCM patients present general hallmarks, the causative mutations underlie different cellular changes. Based on our studies, we propose that different mutations cause DCM via diverse pathways.Mutations in different sarcomeric and non‐sarcomeric genes lead to DCM and we have shown that these mutations trigger different pathological routes leading to end‐stage dilated hearts Fig. . In thisNone declared.I.A.E.B., D.W.D.K. and J.V.D.V. conceived, designed and coordinated the study and wrote the paper. I.A.E.B. created Figs We acknowledge the support from the Netherlands Cardiovascular Research Initiative, an initiative with the support of the Dutch Heart Foundation, CVON2011‐11 ARENA and CVON2014‐40 DOSIS, and Rembrandt Institute for Cardiovascular Sciences 2013. J.R.P. is supported by the National Heart, Lung and Blood Institute of the National Institutes of Health (Grant HL128683). F.W.A. is supported by a Dekker scholarship (Junior Staff Member 2014T001) of the Dutch Heart Foundation and UCL Hospitals NIHR Biomedical Research Centre.


Purpose. To determine the safety of transarticular surface screw (TASS) insertion and the anatomical location of the common iliac veins (CIVs) at the level of the promontorium. Materials and Methods. The locations of the CIVs on 1 mm computed tomography-myelography slices of 50 patients at the level of the promontorium and 20 human cadavers were investigated. Results. Among the patients, the left CIV was closer to the S1 anterior wall than the right CIV . The level of the inferior vena cava (IVC) formation varied among the cadavers. The mean distance between the IVC formation and promontorium tip was 30.2 ± 12.8 mm. The height of the IVC formation and distance between the right and the left CIVs at the level of the promontorium were significantly correlated (P < 0.001). Conclusion. The TASS trajectory is safe as long as the screw does not penetrate the anterior cortex of S1. The level of the IVC formation can help to predict the distance between the right and the left CIVs at the level of the promontorium. The CIVs do not have a uniform anatomical location; therefore, preoperative computed tomography is necessary to confirm their location.  Placement of screws in the sacrum is essential to ensure rigid fixation during instrumentation of the lumbosacral spine. Despite the availability of various screw implantation techniques for the sacrum, failure to achieve a safe, strong screw trajectory continues to be a significant clinical problem. Our center recently developed and implemented a novel sacral screw trajectory for S1 called transarticular surface screw (TASS) implantation. This technique can be combined with an L5 cortical bone trajectory screw and L5 pedicle screw for L5-S1 instrumentation and fusion .A variety of techniques for sacral screw insertion to provide enhanced screw purchase have been described such as the cortical bone trajectory technique , the penHowever, the safest S1 screw trajectory with which to obtain sacral fixation remains the main concern during screw implantation in the sacrum. Screw insertion toward the tip of the promontorium and S1 endplate to enhance screw strengthening is a cornerstone in many types of S1 screw implantation techniques –8, incluA thorough understanding of the anatomy of the structures at risk is a key to safe, successful implantation of screws in this region. Injury to the common iliac veins (CIVs) during S1 screw insertion is not a common complication, but it is serious and may be fatal , 10. TheAfter obtaining institutional ethics approval for this study, we investigated the anatomical location of the CIVs around the sacrum on 1-mm slices of reconstructed CTM images in 50 patients and 20 human formalin-embalmed cadavers to confirm the safety of TASS insertion .Reconstructed CTM images of 50 patients  who underwent spine surgery in our institution were collected and analyzed by two investigators who used the same method and were blinded to each other's results.Two axial slices were meticulously prepared and printed by an expert spine surgeon; one slice was taken from the tip of the promontorium, and the other was taken 1 cm caudal to the tip of the promontorium, parallel to the S1 endplate (both slices included the TASS entry point). All measurements were performed on the above-mentioned printed axial slices Figures  and 3.The locations of the bilateral CIVs were determined in two planes. One plane included the TASS entry point , and the other plane was parallel to the S1 endplate and included the TASS entry point Figures  and 3.The angles of the CIVs from the insertion point of the TASS were measured.The shortest distance from the anterior wall of the S1 vertebra to the nearest wall of the CIV on both the right and the left sides was measured Figures .The distance from the insertion point of the TASS toward the promontorium and anterior wall of the S1 vertebra was measured in two planes and at three different angles .The height and body weight of each patient were measured to determine the presence of any correlation of the patient's height and weight with the distance of the right and left CIVs or any other parameters.Twenty human embalmed cadavers  that had been donated for medical education and research were used in the present study.Each cadaver was placed in the supine position, and the abdomen was opened with a midline incision. All abdominal and pelvic organs were moved away.To locate the center of the promontorium, we measured the width of the L5 vertebra with a digital caliper and divided it into two . The cenThe distance between the formation of the inferior vena cava (IVC) and the center of the promontorium was measured .The distance between the center of the promontorium and the right and left CIVs was also measured .Upon completion of the measurements, the location of the IVC formation was marked and plain anteroposterior and lateral radiographs were taken of each cadaver to verify the location of the IVC radiographically.The diameter of the median sacral vein was measured to identify cadavers with a dilated median sacral vein. The median sacral vein is an unpaired vein located at the medial aspect of the sacrum and accompanies the middle sacral artery to receive blood from the sacral venous plexus, emptying into the left common iliac vein. The median sacral vein was considered dilated when its diameter measured ≥2 mm .P value of <0.05 was considered statistically significant.The mean and standard deviation were calculated for each measurement. Statistical analysis was performed using computer software . A P = 0.016)  .The average angle of the left and right CIVs ranged from 6° to 22° ± 7.0° and 2° to 20° ± 11.85°, respectively .P = 0.001)  .The mean distance between the right and left CIVs at the level of the promontorium was 43.00 ± 8.74 mm.P > 0.05)  at three different angles  was equal on the right and left sides, and the overall screw length in the TASS plane at three different angles was longer than the trajectory parallel to the S1 endplate. However, the difference was not statistically significant ( > 0.05) .The mean height and weight of the patients were 159 cm and 60 kg, respectively. No correlation of the height and weight of the patients with the distance of the right and left CIVs at the level of the promontorium was found.The CIVs lie within the connective tissues immediately in front of the S1 and L5 vertebral bodies.The left CIV was located closer to the center of the promontorium than was the right CIV. The IVC formation was located cranial to the promontorium in all cadavers.Among the 20 cadavers, the IVC formation was located at the level of the L5-S1 disc space or L5 caudal endplate in 10 cadavers, at the level of the L5 vertebral body in 5 cadavers, and at the level of the L4-L5 intervertebral disc or L4 vertebral body in 5 cadavers Figures .P < 0.005) (P < 0.001)  . The mea< 0.001) .P < 0.05)  .P < 0.018)  .A dilated median sacral vein was observed in 4 of 20 cadavers (20%).The CIVs and internal iliac veins are located posterior and lateral to their corresponding arteries. The veins lie in the connective tissue immediately in front of the sacral ala, at the level of the first and second sacral vertebrae –14. ThisIatrogenic injury to major vascular structures is a recognized complication of spine surgery , 15–18. In this study, the anatomical location of the CIVs, location of IVC formation, and distance between right and left CIVs varied in each individual . AdditioThe area between the right and left CIVs at the level of the promontorium seems to be safe if the tip of the screw penetrates the anterior wall of S1, but this distance is not uniform in all humans. We found a statistically significant correlation of the distance of the right and left CIVs at the level of promontorium with the location of the IVC formation. Therefore, we classified the IVC formation into three main groups: low formation , medium formation , and high formation . In the low IVC formation group, the distance between the right and left CIVs at the level of the promontorium was significantly shorter than that in the high IVC formation group. This indicates that if the tip of the screw penetrates the anterior cortex of the sacrum during screw insertion, the chance of a CIV injury is higher in the presence of a low IVC formation than a high IVC formation.In tricortical S1 pedicle screw purchase, Matsukawa et al.  recommenEsses et al. and Ergur et al. strongly recommended avoiding anterior cortex penetration because of the risk of neurovascular injury , 9. Our Mirkovic et al.  defined Therefore, we strongly recommend avoiding penetration of the anterior wall of the S1 vertebra during S1 screw insertion in any trajectory, even in the center of the promontorium, because of the variable locations of the CIVs and dilated median sacral vein.The risk of CIV injury is higher on the left than on the right side if the anterior cortex of S1 is accidently penetrated during the operation. Moreover, the risk of both right and left CIV injury during S1 screw implantation is higher in females than in males. The risk of CIV injury on either side is greater in patients with a low IVC formation than in those with high IVC formation.The tip of the screw should not penetrate the anterior cortex of the S1 vertebra.The length of the screw should range from 32 to 35 mm.A maximum screw length and low-risk trajectory can be achieved by insertion of screw at −10° toward the tip of the promontorium .CT evaluation before screw insertion is recommended to check the location of the CIVs, the level of the IVC formation, and selection of the appropriate screw length based on the axial and sagittal planes.Based on the findings of the present study, we present the following tips for safe TASS implantation:The information provided in this study will be useful for prevention of iatrogenic CIV injury during S1 screw implantation. Additionally, the mini-open anterior approach for L5-S1 fusion and instrumentation involves implantation of a cage between L5 and S1; therefore, a wide distance between the right and left CIVs is safer than a narrow distance. Spine surgeons can detect high-risk patients by checking the level of the IVC formation and distance of the right and left CIVs at the level of the promontorium based on the preoperative CT.Some limitations of this study require acknowledgment. We used CTM images, which are less sensitive than CT angiography for detection of the CIVs. Additionally, embalmed specimens were used for this study, and we cannot exclude potential artifacts resulting from the embalming processes. The specimens were of relatively advanced age; thus, integrant spine scoliosis, anterior longitudinal ligament ossification, and other degenerative changes may have been present and might have changed some features of the CIV structures. Finally, we used Japanese cadavers, and our results might not be applicable to other races.The TASS trajectory is safe as long as the anterior cortex of the S1 vertebra is not penetrated by the screw. The level of the IVC formation is a good indicator with which to predict the distance between the right and left CIVs at the level of the promontorium. The CIV does not have a uniform anatomical location; therefore, preoperative CT evaluation for confirmation of the CIV location is necessary.


Japanese encephalitis virus was detected by deep sequencing for the first time in urine of a 16-year-old boy with encephalitis. Seroconversion and polymerase chain reaction analysis confirmed the metagenomics finding. Urine is useful for diagnosis of flaviviral encephalitis, whereas deep sequencing can be a panpathogen assay for the diagnosis of life-threatening infectious diseases. He was admitted to our hospital on September 18, 2016 fully conscious but with high fever (39°C), neck stiffness, and flaccid paralysis of all 4 limbs with absent reflexes. Analysis of the admission cerebrospinal fluid (CSF) showed 93 × 10One day after admission, he developed fluctuating heart rate and blood pressure and an intermittent erythematous skin rash, indicating severe autonomic dysfunction. His conscious level dropped, and on the fourth day of admission he had focal convulsions of the left arm and left face. He was intubated and mechanically ventilated, and he received phenobarbital, bisoprolol, and high-dose methylprednisone.All initial microbiological investigations were negative, including a human immunodeficiency virus test, CSF microscopy for bacteria, fungi, and acid-fast bacteria, bacterial culture (including Mycobacterium tuberculosis), serological testing for Japanese encephalitis virus (JEV)-specific immunoglobulin (Ig)M, and herpes simplex virus polymerase chain reaction (PCR) analysis of the admission CSF. Due to ongoing diagnostic uncertainty, we ordered additional investigations of the admission CSF, rectal swabs collected on the second day of admission, and urine and plasma both taken on the third day of admission. The additional analyses included 1) generic enterovirus reverse transcription (RT)-PCR of CSF and rectal swab, 2) RT-PCR of the CSF, urine, and plasma for Zika and dengue viruses, and 3) dengue virus NS1 rapid test of the CSF, urine, and plasma, but all were negative. Therefore, we decided to perform deep sequencing analysis of viral nucleic acids extracted from the CSF, plasma, urine, and rectal swab specimens using an in-house nonribosomal random PCR and Illumina MiSeq-based assay [ generic  RT-PCR o dengue vGiven these results, the second CSF, serum, and urine samples were collected on day 15 of hospitalization. Both CSF and serum samples were JEV IgM positive; urine JEV RT-PCR was now negative. Retrospectively tested, JEV IgM was absent in the plasma sample collected on the third day of admission. Brain magnetic resonance imaging taken on day 28 of hospitalization revealed bilateral lesions of the putamen with high signals on T2-weighted and fluid-attenuated inversion recovery images , whereasDespite the availability of a vaccine, JEV remains the most common cause of encephalitis, especially in children, across the Asia-Pacific region . JapanesFlaviviridae family, such as West Nile, dengue, yellow fever, and, most recently, Zika viruses [The detection of virus in urine has been reported for other members of the  viruses . Japanes viruses , but not viruses . TherefoAs exemplified by the recent Zika virus outbreak, the causes of central nervous system infections can change quickly over time. Rapidly changing epidemiology can challenge diagnostic laboratories offering pathogen-specific assays, especially in resource-limited settings, leaving the cause of many brain infections unknown. In this context, next-generation sequencing technologies have emerged as promising single, panpathogen assays for clinical microbiology and public health, although their clinical utility has yet to be fully defined . These mThe present case highlights 2 important lessons. First, urine can be a valuable diagnostic specimen when determining the viral cause of encephalitis in flavivirus-endemic regions. Second, next-generation sequencing and viral metagenomics can improve the clinical care of patients with brain infections by enabling an unbiased investigation of viral cause. However, the current high cost of next-generation sequencing and the need for advanced bioinformatic expertise are major barriers to widespread clinical implementation, especially in resource-limited settings. Yet, it is in these regions, where the burden of infectious diseases and the risk of emerging pathogens are highest, that the benefit of these new technologies will be felt most. Well designed prospective studies are urgently needed to assess the ability of next-generation sequencing technologies to transform the clinical microbiology laboratory and the diagnosis of life-threatening infectious diseases.Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ofx046_suppl_Supplementary_DataClick here for additional data file.


Scientific Reports6: Article number: 3183010.1038/srep31830; published online: 08232016; updated: 11302016This Article contains errors in Figure 4 where the hyperfine values for the donor separations along the [110] direction were calculated incorrectly. The correct Figure 4 appears below as As a result, in the Results and Discussions section,Aij of a P-donor pair in silicon with one bound electron can vary from ~366.0 MHz to ~48.9 MHz within a 5 nm separation range”.“From Fig. 4 we can see that should read:Aij of a P-donor pair in silicon with one bound electron can vary from ~287.4 MHz to ~48.9 MHz within a 5 nm separation range”.“From Fig. 4 we can see that


Endovascular treatment of thoracic and thoracoabdominal aortic diseases is accompanied by a risk of spinal ischemia in 1–19% of patients, depending on the entity and extent of the disease. The use of perioperative drainage of cerebrospinal fluid is one of the invasive measures to reduce the occurrence of this severe complication. This article reviews the incidence of spinal ischemia, its risk factors, the evidence for carrying out cerebrospinal fluid drainage and its modern use by means of an automated, pressure controlled system (LiquoGuard®7). Spinal ischemia represents one of the most serious complications in the treatment of thoracic and thoracoabdominal aortic pathologies. The neurological deficits in the form of paraparesis, paraplegia, or urine and fecal incontinence that can occur in the course of spinal ischemia dramatically reduce quality of life. In addition, affected patients suffer from a significantly increased mortality in the postoperative course . BesidesWhereas the incidence of spinal ischemia is around 20% in open procedures, endovascular treatment of the same pathologies is associated with a significantly reduced risk for neurological complications . This riA multitude of invasive and noninvasive perioperative measures have been developed for the open surgical management of thoracic and thoracoabdominal aortic disorders in order to reduce the incidence of spinal ischemia and minimize the resultant neurological deficits. Examples include strict avoidance of hypotensive phases during surgery and the postoperative phase, reimplantation of large intercostal arteries, permissive hypothermia, and catheter placement for the perioperative drainage of cerebrospinal fluid (CSF). By consistently implementing these protective measures, it is possible to reduce the rate of neurological complications to 5% at specialized centers .Although many of these measures can only be employed during open procedures, some, including perioperative CSF drainage, can also be performed during endovascular interventions. Table LiquoGuard®7, Möller Medical GmbH, Fulda, Germany).This article provides an overview of the current data on the employment of perioperative CSF drainage in the endovascular treatment of thoracic and thoracoabdominal aortic pathologies. In addition to the current guideline recommendations, the article also discusses the risks, the different implementation protocols, and our center-specific guidelines for the use of an automated, pressure-controlled CSF drainage system  .The position paper published in 2015 by the European Association for Cardio-Thoracic Surgery makes the same recommendation (Class IIa C) .The interdisciplinary guideline issued by several American medical societies recommends perioperative CSF drainage in patients at increased risk for spinal ischemia in both open and endovascular procedures (Class I B), although only references relating to the use of CSF drainage in open repair are cited at the respective section .The 2010 German Society for Vascular Surgery (DGG) guideline makes no recommendation on CSF drainage in endovascular procedures .In the case of routine implementation, all patients undergo preoperative spinal catheter placement prior to the planned endovascular procedure .In a so called selective implementation, only those patients at increased risk for spinal ischemia undergo preoperative spinal catheter placement. This is intended to reduce the incidence of neurological complications in high-risk patients without exposing those patients at low risk to the potential additional complications associated with spinal catheter placement. Selective use currently represents the most widespread implementation protocol for perioperative CSF drainage in endovascular procedures , 46, 47.The third possible is to entirely waive a preoperative placement of a spinal catheter. Only those patients who develop spinal ischemia postoperatively undergo emergency spinal catheter placement. In such cases, a consistent reduction in CSF pressure is able to achieve a significant improvement in neurological complications . HoweverThere are three different feasible protocols for the implementation of perioperative CSF drainage:The most serious complication associated with perioperative CSF drainage is cerebral hemorrhage in the form of intracerebral, subarachnoid, or subdural hemorrhage. While the majority of patients sustain either no or only mild neurological deficits as a result of cerebral hemorrhage, cases of severe neurological complications, as well as fatal outcomes, have also been described , 48. LarIf bloody fluid is seen through the spinal catheter, CSF drainage should be stopped immediately followed by optimization of blood coagulation,removal of the spinal catheter and spinal as well as cerebral imaging. Since patients with pre-existing neurological conditions or coagulation disorders are at greater risk for bleeding, they should undergo cranial CT prior to spinal catheter placement and the indication for the use of CSF drainage should be made with great caution .Due to the large diameter of the spinal catheter used, with an outer diameter of up to 1.6 mm, the rate of postdural puncture headaches and CSF leakage requiring treatment is between 3 and 20% and, as such, relatively high , 48.Intracerebral hemorrhage represents the most serious complicationAt a rate of 1%, infections in the form of meningitis due to spinal catheters being in situ for several days are very rare , 54.A large case series of over 1000 patients who received a spinal catheter for perioperative CSF drainage reported a 99.8% technical success rate for drainage placement. Complications were seen in 1.5% of cases, whereby subdural hematoma was identified in 0.4% of all patients .In summary, the incidence of serious complications due to spinal catheter placement, as well as perioperative CSF drainage itself, is between 1 and 4% , 47, whiAt our clinic, we perform SOP-based selective placement of CSF drainage . The criteria for this include, e. g., long segment of aorta to be treated (>20 cm), prior infrarenal surgery (open or endovascular), occlusion of the internal iliac arteries or the left subclavian artery . PatientSpinal catheters are placed on the day prior to surgery. This has proved practical compared with placement directly prior to of surgery since, particularly in the case of complex endovascular procedures, sufficient heparin administration is required; this, however, should be avoided in the first hour following catheter placement. As in the “Guidelines on neuraxial regional anesthesia and thromboembolism prophylaxis/antithrombotic medication” issued by the German Society for Anesthesiology and Intensive Medicine, antiplatelet and anticoagulation drugs are paused prior to surgery . In the An intraoperative ICP of less than 10 mm Hg is set as a target. In addition, the mean arterial blood pressure is elevated to 90 and 100 mm Hg after the implantation of the endoprothesis. These target parameters are maintained for 3 days postoperatively. CSF drainage is then paused, but the spinal catheter left in place in order to ensure a continuously monitoring of the intracranial pressure and to allow immediate CSF drainage in the case of neurological deficits. The spinal catheter is finally removed after additional 24 h.Should paraplegia be observed postoperatively, CSF drainage is resumed for at least 7 days. The target ICP in this cases is below 7 mm Hg.Initial results with this standardized protocol using an automated, pressure-controlled system to monitor ICP and perform pressure-controlled CSF drainage by means of LiquoGuard®7 Fig.  have beeIn addition to a multitude of invasive and noninvasive measures, perioperative CSF drainage during open surgical repair of the thoracic and thoracoabdominal aorta represents an effective means to reduce the incidence of spinal ischemia.Comparable evidence for its use in endovascular procedures is not yet available.Since CSF drainage itself can be associated with severe complications, its routine implementation in endovascular aortic procedures is not justified.Many centers use selective perioperative CSF drainage in patients at increased risk for spinal ischemia.Immediate CSF drainage combined with elevation of the mean arterial pressure represents an effective treatment for postoperative deficits due to spinal ischemia.The use of modern techniques, e. g., the LiquoGuard®7 system, can facilitate the management of CSF drainage.


Pearson's correlation coefficients were calculated for disease progression. Significance level was defined as p < 0.05. Seventy‐six participants were included in this cross‐sectional multicenter international pilot study. Age‐adjusted CSF tau was significantly elevated in gene expansion carriers compared with healthy controls (p = 0.002). UHDRS total functional capacity was significantly correlated with CSF tau  after adjustment for age, and UHDRS total motor score was significantly correlated with CSF tau after adjustment for age . Several UHDRS cognitive tasks were also significantly correlated with CST total tau after age‐adjustment. This study confirms that CSF tau concentrations in HD gene mutation carriers are increased compared with healthy controls and reports for the first time that CSF tau concentration is associated with phenotypic variability in HD. These conclusions strengthen the case for CSF tau as a biomarker in HD.Huntington's disease (HD) is a hereditary neurodegenerative condition with no therapeutic intervention known to alter disease progression, but several trials are ongoing and biomarkers of disease progression are needed. Tau is an axonal protein, often altered in neurodegeneration, and recent studies pointed out its role on HD neuropathology. Our goal was to study whether cerebrospinal fluid (CSF) tau is a biomarker of disease progression in HD. After informed consent, healthy controls, pre‐symptomatic and symptomatic gene expansion carriers were recruited from two HD clinics. All participants underwent assessment with the Unified HD Rating Scale ’99 (UHDRS). CSF was obtained according to a standardized lumbar puncture protocol. CSF tau was quantified using enzyme‐linked immunosorbent assay. Comparisons between two groups were tested using In the era of novel targeted approaches to Huntington's disease, reliable biomarkers are needed. We quantified Tau protein, a marker of neuronal death, in cerebrospinal fluid and found it was increased in patients with Huntington's disease and predicted motor, cognitive, and functional disability in patients. It is therefore likely to be a biomarker of disease progression, and possibly of therapeutic response.Read the Editorial Highlight for this article on page 9. Huntington's disease (HD) is a hereditary autosomal dominant neurodegenerative condition that affects movement control, behavior, and cognition /UCL Hospitals Joint Research Ethics Committee (UK participants) and the University of British Columbia Clinical Research Ethics Board, as appropriate. All subjects gave informed written consent.et al. Subjects were recruited from two HD multidisciplinary clinics, one in London, United Kingdom and other in Vancouver, Canada. Healthy controls, pre‐symptomatic gene expansion carriers, and symptomatic gene expansion carriers were included. Individual with infectious, inflammatory, or other concomitant CNS disorders or significant comorbidities were excluded. Healthy controls were defined as individuals without family history of HD and without symptoms compatible with HD, or without a cytosine‐adenine‐guanine (CAG)‐expanded allele of the HD gene. Asymptomatic gene expansion carriers were defined as individuals with a CAG‐expanded allele of the HD gene and with a diagnostic confidence level inferior to 4. Symptomatic gene expansion carriers were defined as individuals with a CAG‐expanded allele of the HD gene and with a diagnostic confidence level of 4  , examined by microscopy, and centrifuged to remove cells, and the acellular portion was frozen at −80°C. Further details were as previously published  in one round of experiments using one batch of reagents by board‐certified laboratory technicians who were blinded to clinical data.ancova. To study the association of total tau with disease progression, we calculated Pearson's and partial correlations coefficients. Bootstrapping with 1000 repetitions was applied to non‐normal variables. Significance level was defined as p < 0.05.Statistical analysis was performed with Stata version 14 software . Potentially confounding demographic variables (age and gender) were examined in preliminary analyses. Total tau distribution was tested for normality using skewness and kurtosis, Shapiro–Wilk and Shapiro–Francia tests. Comparisons between two groups adjusted for covariates were tested using r = 0.45, p = 0.006) and with disease burden score (a product of age and CAG repeat length) in gene expansion carriers . No difference was found in CSF total tau concentrations between genders (p = 0.436). Age‐adjusted CSF total tau was significantly elevated in gene expansion carriers compared with healthy controls (p = 0.002)  after adjustment for age, but not after adjustment for disease burden and also after adjustment for disease burden score  and also after adjustment for disease burden score . Stroop interference task was not correlated with CSF total tau . There was no association between CSF total tau and CSF hemoglobin concentration.We conducted a cross‐sectional multicenter international pilot study of 76 participants (mean age 47.9; standard deviation [SD] 13.1; range 23–72 years; Table et al. This study confirms in two independent populations the previous finding that CSF total tau concentrations in HD gene mutation carriers are increased compared with healthy controls (Constantinescu To the best of our knowledge, ours is the first work where CSF total tau concentration has been shown to be associated with phenotypic variability in HD as measured by motor manifestations and cognitive dysfunction. These associations persist even after adjustment for disease burden score – indicating that CSF tau has independent power to predict clinical phenotype, beyond the known effects of age and CAG repeat length.et al. Tau is a microtubule‐associated protein, which under certain pathologic conditions, aggregates abnormally. Still, it is controversial whether these accumulations are cytotoxic and cause cellular dysfunction, contributing to neuronal cell death, or are just an epiphenomenon (Bretteville and Planel Our findings strengthen the case for CSF total tau concentration as a biomarker in HD, reflecting neuronal death and/or secondary tau pathology Fig. . They suAppendix S1. Study methods.Click here for additional data file.


Antibodies to myelin oligodendrocyte glycoprotein (MOG-IgG) have been suggested to play a role in a subset of patients with neuromyelitis optica and related disorders.To assess (i) the frequency of MOG-IgG in a large and predominantly Caucasian cohort of patients with optic neuritis (ON) and/or myelitis; (ii) the frequency of MOG-IgG among AQP4-IgG-positive patients and vice versa; (iii) the origin and frequency of MOG-IgG in the cerebrospinal fluid (CSF); (iv) the presence of MOG-IgG at disease onset; and (v) the influence of disease activity and treatment status on MOG-IgG titers.614 serum samples from patients with ON and/or myelitis and from controls, including 92 follow-up samples from 55 subjects, and 18 CSF samples were tested for MOG-IgG using a live cell-based assay (CBA) employing full-length human MOG-transfected HEK293A cells.p < 0.0001), highest during attacks of simultaneous myelitis and ON, lowest during acute isolated ON, and declined following treatment.MOG-IgG was detected in 95 sera from 50 patients with ON and/or myelitis, including 22/54 (40.7 %) patients with a history of both ON and myelitis, 22/103 (21.4 %) with a history of ON but no myelitis and 6/45 (13.3 %) with a history of longitudinally extensive transverse myelitis but no ON, and in 1 control patient with encephalitis and a connective tissue disorder, all of whom were negative for AQP4-IgG. MOG-IgG was absent in 221 further controls, including 83 patients with AQP4-IgG-seropositive neuromyelitis optica spectrum disorders and 85 with multiple sclerosis (MS). MOG-IgG was found in 12/18 (67 %) CSF samples from MOG-IgG-seropositive patients; the MOG-IgG-specific antibody index was negative in all cases, indicating a predominantly peripheral origin of CSF MOG-IgG. Serum and CSF MOG-IgG belonged to the complement-activating IgG1 subclass. MOG-IgG was present already at disease onset. The antibodies remained detectable in 40/45 (89 %) follow-up samples obtained over a median period of 16.5 months (range 0–123). Serum titers were higher during attacks than during remission (To date, this is the largest cohort studied for IgG to human full-length MOG by means of an up-to-date CBA. MOG-IgG is present in a substantial subset of patients with ON and/or myelitis, but not in classical MS. Co-existence of MOG-IgG and AQP4-IgG is highly uncommon. CSF MOG-IgG is of extrathecal origin. Serum MOG-IgG is present already at disease onset and remains detectable in the long-term course. Serum titers depend on disease activity and treatment status. Neuromyelitis optica (NMO) is a severely disabling autoimmune disorder of the CNS. In the majority of cases, NMO is caused by autoantibodies to aquaporin-4 (AQP4-IgG) –6; howevLater on, in a study published in this journal in 2011, some of us demonstrated antibodies to full-length MOG in patients with NMO for the first time by means of a CBA . In the However, there were some obvious limitations: First, many of the previously investigated cohorts were relatively small. Second, long-term data were often absent, with follow-up samples not being available. Third, some cohorts included no Caucasian patients or were genetically mixed, which may be of relevance since genetic factors are thought to play a role in NMO . Fourth,N = 614). In addition, we evaluated (iii) the prevalence of MOG-IgG and AQP4-IgG double positivity based on a very large number of samples (N = 459); (iv) the presence of MOG-IgG at disease onset; (v) the long-term persistence of MOG-IgG in individual patients; (vi) the influence of disease activity and treatment status on MOG-IgG titers; and (vii) the frequency and origin of MOG-IgG antibodies in the CSF.In the present study we assessed the frequency of MOG-IgG as assessed by means of a live-cell CBA  (i) in aThis study is part of an article series on MOG-IgG in CNS inflammation. In part 2, we systematically evaluate the clinical and paraclinical features present in MOG-IgG-positive ON and/or myelitis as well as treatment responses and long-term outcome . In partIn total, 614 serum samples and 18 CSF samples from 522 subjects were tested for MOG-IgG. Group I comprised 386 serum samples from 300 patients referred for routine MOG-IgG testing by 11 European academic centers, including the departments of neurology at the University of Heidelberg, the Charité-University Medicine Berlin, the University of Düsseldorf, the University of Bochum, Hannover Medical School, the University of Würzburg, the University of Rostock, the University of Freiburg, all in Germany; the University of Southern Denmark, Denmark; the MS Center at the Azienda Ospedaliero Universitaria San Luigi Gonzaga, Orbassano, Italy; and the IRCCS, C. Mondino National Neurological Institute, Pavia, Italy; eight of which are members of the German Neuromyelitis optica Study Group (NEMOS). Samples were taken for routine clinical assessment. Diagnoses at the time of blood sampling as reported by the referring centers, all of which were tertiary care university hospitals with specialized neuroimmunological departments, included “ON and myelitis” in 54 patients , “monophasic ON” in 66 (69 samples), “recurrent ON” in 37 , “longitudinally extensive transverse myelitis” in 45 (57 samples), “relapsing remitting MS” (RRMS) in 50 (54 samples), “secondary progressive MS” (SPMS) in 2 (2 samples), “primary progressive MS” (PPMS) in 2 (2 samples), and “other neurological disorder” (OND) in 44 (47 samples).Group II consisted of 89 anonymized serum samples from 83 control patients with AQP4-IgG-positive ON and/or myelitis. Of those, 56 had a history of ON and myelitis, 22 of myelitis but no ON, and 5 of ON but no myelitis. AQP4-IgG had been previously detected by use of a commercial CBA  in these patients .Group III was made up of 85 anonymized serum samples from 85 control patients with MS according to the McDonald criteria .Group IV comprised 54 anonymized samples from 9 control patients with OND (including 8 with connective tissue disorders and brain involvement ) and froNinety-two follow-up samples  from 55 subjects were tested. The sex ratios (m:f) were 1:2.4 in group I and 1:3 in the control groups II–IV. The median age was 39 years in group I and 38 years among the control patients (groups II–IV). See Table MOG) was determined. Calculation of AIs allows quantification of antigen-specific intrathecal antibody synthesis  if only IS/PEX is considered). Of note, low MOG-IgG remained detectable in all four patients treated with rituximab at the time of blood sampling (titers 1:160–1:640). In a further patient treated with rituximab, a MOG-IgG titer of 1:1280 was documented during a relapse and was associated with recurrence of B cells.In total, 17 CSF samples from 15 MOG-IgG-seropositive patients from group I were available for testing. All but one sample were taken during an acute attack (median time since attack onset 10 days). Ten of those 17 CSF samples were obtained within 30 days after disease onset and 7 - including two follow-up samples - later in the disease course (range 71–5406 days after onset).Twelve out of 17 CSF samples (71 %) were positive for MOG-IgG. The median CSF MOG-IgG titer was 1:4 (range 2–64). Individual CSF and serum results are shown in Table In addition, 17 CSF samples from 17 control patients with RRMS were tested. All of those were negative for CSF MOG-IgG.Follow-up CSF samples were available from two patients. In both cases, MOG-IgG were detectable in the CSF a few days after disease onset, at titers of 1:64 and 1:4, respectively, but not at repeat LP 51 and 21 days, respectively, later. One patient had been treated with IVMP, oral steroids, ten plasma exchanges, and IVIG in the meantime, the other one with IVMP alone. The decline in CSF titers was paralleled by a drop in serum titers from 1:10240 to 1:1280 and from 1:2560 to 1:1280, respectively, in these two patients.IgG was below Qlim in 16/17 cases, while QAlb exceeded the age-specific reference range in 6/17 (35.3 %) samples, indicating disruption of the blood-CSF barrier function.Seventeen paired CSF and serum samples were titrated to calculate the MOG-specific AI. Evidence for intrathecal IgG synthesis was present in none of these 17 samples: in 5 samples no MOG-specific IgG was detectable in the CSF, and in the remaining 12 samples the MOG-specific AI was <4  and 6 control sera from group III patients, were tested for MOG-IgG1 using the fixed-cell CBA (Euroimmun). All 14 group I samples were positive for MOG-IgG1; by contrast, none of 6 control sera contained MOG-IgG1 antibodies  were positive for MOG-IgM and none for MOG-IgA Table .Table 3MAlb was indeed noted in 12/36 (32.4 %) patients in the total cohort   37] and