Cerebrospinal fluid from amyotrophic lateral sclerosis patients (ALS-CSF) induces neurodegenerative changes in motor neurons and gliosis in sporadic ALS models. Search for identification of toxic factor(s) in CSF revealed an enhancement in the level and enzyme activity of chitotriosidase (CHIT-1). Here, we have investigated its upregulation in a large cohort of samples and more importantly its role in ALS pathogenesis in a rat model.n = 158), non-ALS (n = 12) and normal (n = 48) subjects were measured using ELISA. Enzyme activity was also assessed . Recombinant CHIT-1 was intrathecally injected into Wistar rat neonates. Lumbar spinal cord sections were stained for Iba1, glial fibrillary acidic protein and choline acetyl transferase to identify microglia, astrocytes and motor neurons respectively after 48 h of injection. Levels of tumour necrosis factor-α and interleukin-6 were measured by ELISA.CHIT-1 level in CSF samples from ALS (CHIT-1 level in ALS-CSF samples was increased by 20-fold and it can distinguish ALS patients with a sensitivity of 87% and specificity of 83.3% at a cut off level of 1405.43 pg/ml. Enzyme activity of CHIT-1 was also 15-fold higher in ALS-CSF and has a sensitivity of 80.4% and specificity of 80% at cut off value of 0.1077989 μmol/μl/min. Combining CHIT-1 level and activity together gave a positive predictive value of 97.78% and negative predictive value of 100%. Administration of CHIT-1 increased microglial numbers and astrogliosis in the ventral horn with a concomitant increase in the levels of pro-inflammatory cytokines. Amoeboid-shaped microglial and astroglial cells were also present around the central canal. CHIT-1 administration also resulted in the reduction of motor neurons.CHIT-1, an early diagnostic biomarker of sporadic ALS, activates glia priming them to attain a toxic phenotype resulting in neuroinflammation leading to motor neuronal death. Diagnosis of amyotrophic lateral sclerosis (ALS), a non-cell autonomous neurodegenerative disorder marked by degeneration of motor neurons along with muscular atrophy, is still challenging as the symptom development could be delayed during initial phase and may overlap with other neurodegenerative disorders –4. BiomaWe have shown that CHIT-1 is exclusively expressed by microglial cells in the CNS, and its expression is augmented when they are exposed to ALS-CSF . CHIT-1 CHIT-1 is reported to be upregulated in several neurological conditions like acute ischemic stroke, Alzheimer’s disease, cerebral adrenoleukodystrophy and cerebrovascular dementia –12. It iCSF samples were collected after obtaining informed consent as per the institutional human ethics committee guidelines. CSF samples of ALS patients diagnosed using El Escorial criteria  were obt2CO3-NaHCO3 buffer (pH 10.7). The fluorescence was captured at 365-nm excitation and 450-nm emission using Tecan 2500 fluorimeter , and the data was expressed as micromoles of substrate hydrolyzed/μl/min.ELISA for CSF  was performed using commercially available kit for CHIT-1  according to manufacturer’s protocol. The CHIT-1 enzyme activity was measured in 45 N-CSF, 10 NALS-CSF and 56 ALS-CSF samples as described previously [The dosage for intrathecal injection of CHIT-1 was based upon the average amount of CHIT-1 present in 5 μl of CSF (approximately 90 pg of CHIT-1 as determined by ELISA). The following doses of CHIT-1 were used: 50 pg, 100 pg, 200 pg and 500 pg.Neonatal Wistar rat pups were obtained from Central Animal Research Facility (CARF), NIMHANS, Bangalore, after obtaining clearance from Institutional Animal Ethics Committee (IAEC). Rat neonates along with lactating mothers were housed at an ambient temperature of 26 ± 2 °C and subjected to the routine light/dark cycle. Lactating mothers had ad libitum access to food and water. Post-natal day 3 pups were intrathecally injected with 5 μl of buffer, different doses of recombinant human CHIT-1, N-CSF and ALS-CSF –22. BrieAfter 48 h of intrathecal injection, animals were anaesthetized with halothane and perfused transcardially using 4% paraformaldehyde , 23. SpiFor immunostaining, antigen unmasking was performed by incubating in sodium citrate buffer  for 5–10 min at 95 °C and blocked using 3% bovine serum albumin  for 3 h. Sections were rinsed in 0.1 M PBST and then incubated in the first primary antibody. The sections were again washed and incubated with appropriate fluorescent-conjugated secondary antibody. Blocking was done with 3% BSA for 1 h and rinsed and treated subsequently with second primary antibody followed by incubation in second secondary antibody. For choline acetyl transferase (ChAT) staining, spinal cord sections were pretreated with ice cold methanol for 10 min. Lists of antibodies used are given in Table The fluorescence images of the immunolabelled specimens were analysed using a laser confocal scanning microscope  with excitation at 488 and 514 nm for FITC and Cy3 respectively. Emission band width of FITC was maintained at 490–540 nm and 550–620 nm for Cy3 to prevent overlap of emission frequencies.Analysis was performed on 10 sections per spinal cord, and 3 animals per group were used for the study. Number of ChAT positive motor neurons and Iba1 positive microglia in the ventral horn was counted, and the individual means were derived. Area and intensity of GFAP and SMI-31 in white matter was measured and quantified on a scale of 0 (minimum)–255 (maximum), using an in-built software.2 for the analysis [Serial sections of the lumbar region of spinal cords post fixed in paraformaldehyde (40 μm) were taken on a vibratome , and every fourth section was stained with cresyl violet. Briefly, slides were dipped in chloroform for 1 min; passed in succession through 100% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 2 min; and rehydrated in distilled water for 5 min and then in cresyl violet for 2 min. Slides were allowed to differentiate in distilled water for 5 min, passed in succession through 70% ethanol for 20 s (× 2), 90% ethanol for 20 s (× 2), 100% ethanol for 1 min (× 2) and xylene for 1 min. The stained slides were dried and mounted with DPX. Images were captured at × 10 with a Leica BX 51 microscope. The number of motor neurons was quantified by using the particle analysis feature of ImageJ. Briefly, the images were converted to 8-bit binary with Huang thresholding, following noise removal and using a size-based filter above 250 μmanalysis . Data wag, and the supernatant was used for ELISA. Samples were protein normalised, and 450 μg/ml of each sample was used. ELISA for IL-10 and TNFα was performed using commercially available kits  according to manufacturer’s protocol.Lumbar region of spinal cord was dissected out 48 h post intrathecal injection from 3 animals per group. Tissues were snap frozen and stored at −80 °C till use. Samples containing 2% tissue lysate with 1% protease inhibitor were sonicated thrice at 15 Hz for 10 s. The lysates were centrifuged for 10 min at 5000×Data was expressed as mean ± SEM. Statistical analysis was performed using the GraphPad Prism 6 and SPSS software. The data was analysed for significance using one-way ANOVA followed by Tukey’s post hoc test. Receiver operator characteristic (ROC) analysis was performed to assess optimal cutoff value and sensitivity and specificity of ELISA. MedCalc diagnostic test evaluation calculator was used to calculate predictive values. Pearson correlation was used to find possible correlation between CHIT-1 activity and disease severity on one hand and CHIT-1 level on the other hand. Spearman’s correlation was performed to assess correlation between CHIT-1 level and disease duration. It was also performed to correlate enzyme activity of CHIT-1 on one hand and disease severity as well as duration on the other hand. Partial correlation was used to find the relationship between CHIT-I level and disease severity and disease duration.p < 0.0001 vs. N-CSF and $$p < 0.01 vs. NALS-CSF). There was no significant difference in the level of CHIT-1 between NALS-CSF and N-CSF. The mean CHIT-1 level in the N-CSF was 1144 ± 763.30 pg/ml (n = 48) and in NALS-CSF was 909.4 ± 934.43 pg/ml (n = 12) whereas in the ALS-CSF the mean was 22,015 ± 25,804 pg/ml (n = 158)      indicating that patients with high disease severity have high level of CHIT-1  and in NALS-CSF was 0.05149 ± 0.03538 μmol/μl/min (n = 10) whereas in the ALS-CSF the mean was 1.33 ± 0.2334 μmol/μl/min (n = 56). Thus, ALS-CSF samples had approximately 15-fold higher enzymatic activity when compared to N-CSF and 25-fold more when compared to NALS-CSF (***p < 0.001 vs N-CSF and $$p < 0.01 vs NALS-CSF) The CHIT-1 activity did not differ significantly between NALS-CSF and N-CSF   or enzyme activity ALS-FRS score . A positive correlation was observed between CHIT-1 level and enzyme activity . CHIT-1 levels and enzyme activity when used together can diagnose ALS patient with sensitivity of 100%, specificity of 88.89%, positive predictive value of 97.78% and negative predictive value of 100%.The mean CHIT-1 activity in the N-CSF was 0.08537 ± 0.01760 μmol/μl/min     Fig. a–i, j. Ier) Fig. a–g, k. TLS) Fig. a–g, l. TQualitative observations revealed the presence of several amoeboid-shaped Iba1 immunopositive cells around the central canal. A number of GFAP immunopositive astroglia bearing long processes were also seen to infiltrate the central canal following CHIT-1 administration Fig. g–o. Thesp < 0.05 C500 and ALS-CSF vs Buffer)    Fig. a–f, g. Ner) Fig. i–o, p. Ter) Fig. a–f, h.FWe have previously reported that CHIT-1 level was higher in ALS-CSF by using proteomics and ELISA in a small sample size of 16 ALS patients . FollowiWe were also the first to report increased CHIT-1 enzyme activity in ALS-CSF. The 15-fold increase in enzyme activity of CHIT-1 in the current study with larger cohort further proves its use in diagnosis of ALS as it has a sensitivity of 80.4% and specificity of 80%. The increase in activity can be due to high level of protein present as a positive correlation was observed between enzyme activity and the levels of CHIT-1. This is the only study which has integrated both CHIT-1 level and its enzyme activity. The positive and negative predictive values (97.78% and 100% respectively) observed when both CHIT-1 level and enzyme activity were considered together suggest that combining the two parameters can significantly improve diagnostic accuracy. When both ELISA and enzyme activity of CHIT-1 is considered, the sensitivity is 100% and specificity is 88.89%, which can reduce the false positive and false negative results.However, the consequence of CHIT-1’s activity, its presence in the CSF and its exact role in ALS pathogenesis remain to be determined. Accumulation of glucose and glucosamine via impaired glucose metabolism along with activation of hexosamine pathway can lead to formation of chitin-like polymers in the brain which have been detected in AD patients , 26. IntThe increased levels of CHIT-1 in ALS-CSF and its enhanced expression by microglial cells upon exposure to ALS-CSF persuaded us to investigate its non-chitin role in ALS pathogenesis. CHIT-1 was able to activate glial cells in the spinal cord of Wistar rat pups. The presence of reactive microglial cells is reported in the cortex, brain stem and spinal cord of the post-mortem samples of ALS patients . PET imap < 0.05 CHIT-1 vs normal control; data not shown, unpublished observations) also hint towards its possible role in BBB breach as increased serum S100β levels act as marker for the same [Increase in amoeboid-shaped microglial cells observed in the ventral horn of spinal cord in CHIT-1 injected group suggests microglial differentiation and they acquire a phenotype with enlarged soma and retracted processes. The presence of microglial cells in the vicinity of the central canal is intriguing. While we are unable to determine the precise cause for this occurrence, it is possible that it might be the result of blood brain/blood spinal cord barrier (BBB/BSCB) breach induced by CHIT-1. CHIT-1 supplementation in an in vitro model of BBB resulted in an increased migration of peripheral blood mononuclear cells across human brain micro-vascular endothelial cells . Our prethe same , 33. HowThe significant increase in the immunoreactivity for GFAP in the ventral spinal cord in response to CHIT-1 may suggest proliferation and activation of astrocytes. Additionally, the GFAP reactivity was particularly intense in the CHIT-1-exposed group compared to controls which were almost GFAP negative. A similar feature was also observed while comparing the dorsal and ventral horns, wherein the latter area was significantly more gliotic, which suggests selective vulnerability of ventral horn (motor neurons) compared to dorsal horn (sensory neurons). Gliosis appeared prominent, even with the lower doses of CHIT-1, and the effect persisted at higher doses as well. Both CHIT-1 and ALS-CSF appeared to produce similar astroglial responses. This result corroborates with our earlier findings of ALS-CSF-induced enhancement of GFAP expression in both grey and white matter of neonatal rat spinal cord  and elevThe intricate cellular interplay between microglia and astrocytes could be a major factor leading to neurodegeneration. Several studies have shown non-cell autonomous degeneration of motor neurons , 4. The CHIT-1 was able to induce microglial proliferation and release of microvesicular structures while it did not exert any significant effect on primary astrocytic cultures . The indWe hypothesize that CHIT-1-induced microgliosis and astrogliosis might be one of the critical events that lead to the motor neuron degeneration. Our study demonstrates CHIT-1 not only as an early diagnostic biomarker for ALS, but also as a key molecule for therapeutic intervention targeting neuroinflammation at the early stages of this crippling and fatal neurodegenerative disorder.


New-generation, cell-based assays have demonstrated a robust association of serum autoantibodies to full-length human myelin oligodendrocyte glycoprotein (MOG-IgG) with (mostly recurrent) optic neuritis, myelitis, and brainstem encephalitis, as well as with neuromyelitis optica (NMO)-like or acute-disseminated encephalomyelitis (ADEM)-like presentations. However, only limited data are yet available on cerebrospinal fluid (CSF) findings in MOG-IgG-associated encephalomyelitis .To describe systematically the CSF profile in children with MOG-EM.l-lactate) from 108 lumbar punctures in 80 pediatric patients of mainly Caucasian descent with MOG-EM were analyzed retrospectively.Cytological and biochemical findings , and the MRZ reaction, the most specific laboratory marker of MS known so far, in 100% (N = 29). If present at all, intrathecal IgG synthesis was low, often transient and mostly restricted to acute attacks. Intrathecal IgM synthesis was present in 21% and exclusively detectable during acute attacks. CSF WCC were elevated in 54% of samples . Neutrophils were present in 71% of samples; eosinophils, activated lymphocytes, and plasma cells were seen only rarely . Blood–CSF barrier dysfunction  was present in 46% of all samples (N = 79) and at least once in 48% of all patients (N = 67) tested. CSF alterations were significantly more frequent and/or more pronounced in patients with acute spinal cord or brain disease than in patients with acute ON and varied strongly depending on attack severity. CSF l-lactate levels correlated significantly with the spinal cord lesions load  in patients with acute myelitis (p = 0.0099). An analysis of pooled data from the pediatric and the adult cohort showed a significant relationship of QAlb (p < 0.0005), CST TP (p < 0.0001), and CSF l-lactate (p < 0.0003) during acute attacks with age.Most strikingly, CSF-restricted oligoclonal IgG bands, a hallmark of multiple sclerosis (MS), were absent in 89% of samples (MOG-IgG-associated EM in children is characterized by CSF features that are distinct from those in MS. With regard to most parameters, no marked differences between the pediatric cohort and the adult cohort analyzed in Part 1 were noted. Our findings are important for the differential diagnosis of pediatric MS and MOG-EM and add to the understanding of the immunopathogenesis of this newly described autoimmune disease. Over the past few years, several studies using new-generation cell-based assays (CBA) have demonstrated a robust association of immunoglobulin G (IgG) autoantibodies targeting full-length, conformationally intact human myelin oligodendrocyte glycoprotein (MOG) with (mostly recurrent) optic neuritis (ON), myelitis, and brainstem encephalitis, as well as with neuromyelitis optica (NMO)-like and acute disseminated encephalomyelitis (ADEM)-like presentations, rather than with classic multiple sclerosis (MS) –11. The So far, only limited data are available on the cerebrospinal fluid (CSF) profile in MOG-EM in pediatric patients. Previous studies were either based on relatively small patient numbers, included mainly adult patients, and/or did not consider Caucasian patients. Moreover, all investigated only a small number of selected CSF parameters.In Part 1 of this article series, we report on the CSF findings in MOG-EM in adults . For theResults from 108 lumbar punctures (LP) in 80 pediatric patients with MOG-EM were analyzed retrospectively. MOG-EM was defined as monophasic or relapsing acute ON, myelitis, brainstem encephalitis, or encephalitis associated with MRI or (in the case of ON only) electrophysiological findings compatible with CNS demyelination and with MOG-IgG as detected by means of a cell-based assay (CBA) employing human full-length MOG as antigen . LongituQIg = IgCSF[mg/l]/Igserum[g/l]. The upper limits of the respective reference ranges, Qlim(IgG), Qlim(IgM), and Qlim(IgA), were calculated against QAlb according to Reiber’s revised hyperbolic function  = [QIgG – Qlim(Ig)] × Igserum × 100 and Igloc[mg/L] = [QIg – Qlim(Ig)] × Igserum, respectively [Oligoclonal IgG bands were assessed by isoelectric focusing and evaluated according to an international consensus . Immunogfunction . Values ynthesis . The fraectively . CSF andCSF[mg/l]/Albserum[g/l], was used to assess the blood–CSF barrier (BCB) function. As the upper reference limit of QAlb is age dependent, Qlim(Alb) was calculated as 4 + (a / 15) × 10−3 with a representing patient’s age according to Reiber et al. (1994) ) (53) Fig. .r2 = 0.75, p < 0.00001)  concentrations in the CSF were elevated in 22/98 (22.4%) samples  and at least once in 19/75 (25.3%) patients with available data. As in the adult cohort, a significant relationship of QAlb and CSF TP levels was found by regression analysis  , which wr = − 0.308, p = n.s.)   since onset of the last attack, especially in the acute MY subgroup (65) Fig. .p < 0.00001)  CSF samples (and at least once in 21/60 [35%] patients tested), with a median concentration of 2 mmol/l    and with CSF total protein   samples without pleocytosis but in 44.4% (16/36) of samples with pleocytosis, in 61.5% (8/13) of samples if CSF WCC exceeded 50 cells/μl, and in 71.4% (5/7) if CSF WCC exceeded 100 cells/μl. A similar relationship was also found in the adult cohort . The difference was even more pronounced in the “acute MY” subgroup . In patients with pleocytosis, the frequency of samples with elevated CSF l-lactate did not significantly differ between samples with or without neutrophil granulocytes, neither in the total cohort (31.3% [5/16] vs. 44.4% [16/36]) nor in the “acute myelitis” subgroup (33.3% [2/6] vs. 58.8% [10/17]); there was also no significant difference when the pediatric and adult data were pooled . This renders it at least unlikely that granulocytes were the main source of l-lactate in patients with elevated CSF l-lactate levels. When considering not only the presence or absence of neutrophils but absolute neutrophil numbers, a weak trend toward a correlation (p = 0.067) was found between CSF l-lactate levels and neutrophil cell numbers in the small subgroup of samples with available data (N = 19); however, this was neither seen in the adult cohort nor in a pooled analysis of the pediatric and the adult data. CSF lactate levels showed a trend toward lower values with increasing time (in days) since onset of the last attack (p = 0.055)  Fig. . Importal-lactate concentrations during acute attacks were slightly lower in the pediatric cohort than in the adult cohort, if the first LP/event is considered . While CSF l-lactate levels exceed 3 mmol/l in 10/103 (9.7%) patients in the adult cohort  adult patients ); in oneN = 23), whereas others were obtained at follow-up LP (N = 6); however, all were negative, irrespective of disease duration at the time of MRZ testing , just as in the adult cohort. Moreover, in three patients, MRZ was tested more than once. All four retests  in these patients were negative as well ; median CSF WCC in patients with pleocytosis ; proportion of samples with pleocytosis ; QIgG values (p < 0.004); QAlb positivity rate ; median QAlb ; and median CSF TP concentration (p = 0.008). Moreover, a higher proportion of samples from patients with severe disease at the time of LP exhibited a positive QIgG positivity rate (24% vs. 9%), positive OCB (17% vs. 4%), neutrophils (51.6% vs. 33.3%), and a positive Link index  (20% vs. 5%), although the differences did not reach statistical significance. See Table In line with the adult cohort, many CSF parameters assessed were higher and/or more frequently pathologically altered in patients classified as having a severe attack at the time of LP by the treating physician than in patients classified as having mild or moderate disease at the time of LP Table , includil-lactate concentrations were significantly correlated (p < 0.010) with the spinal cord lesion load and while QAlb (p = 0.053) and CSF TP concentrations (p = 0.064) tended to correlate with the spinal cord lesion load, no statistically significant differences regarding the frequency of CSF pleocytosis, CSF-restricted OCB, IF-IgG elevation > 10%, BCB dysfunction, or CSF TP elevation were noted when samples were simply stratified into “acute LETM” and “acute NETM” based on the presence or absence of at least one lesion extending over three or more VS . However, the rate of samples with pleocytosis, WCC > 100 cells/μl, IgG-IF > 10%, disturbed blood–CSF barrier, or TP elevation, respectively, were all more frequent and CSF WCC and TP concentrations higher in samples obtained during acute LETM when compared to samples obtained during acute NETM, suggesting that the lack of statistical significance may well be an effect of the small sample size in the NETM group.While CSF  p = 0.05 and CSF As in the adult cohort , CSF finl-lactate elevation, and CSF TP elevation  and the untreated subgroup (N = 92) with regard to OCB frequency, frequency of QIgG positivity, Link index, QAlb, CSF TP, or CSF l-lactate elevation, possibly due to the low number of samples in the former subgroup. Steroids used included methylprednisolone, prednisolone, and “low dose corticosteroids” (N = 11); immunosuppressive and immunomodulatory drugs (N = 2) used at the time of LP comprised interferon beta (1×) and intravenous immunoglobulins (1×).OCB were negative in 12/12 samples in the treated subgroup, QIgG in 8/8, the IgG index in 8/8, CSF l-lactate elevation [44% vs 35%]. There was no significant difference regarding the median time since onset of the last attack between OCB-positive and OCB-negative samples (8 vs. 2 days). Moreover, as in the adult cohort, the age at the first OCB-positive LP did not differ from that of the first OCB-negative LP in patients who never tested positive for OCB (6.5 years vs. 6 years).Interestingly, OCB were exclusively found during acute attacks (11/11), exclusively during attacks of myelitis or encephalitis , and exclusively in untreated patients (11/11). When comparing the CSF findings in the OCB-positive subgroup to that in a similar group of OCB-negative patients , no statistically significant differences were found between the two groups in respect of pleocytosis [73% vs. 70%], median CSF WCC [52 vs. 23 cells/μl], WCC > 150 cells/μl (18% vs. 14%), presence of granulocytes [80% vs 83%], BCB dysfunction [50% vs. 66%], CSF p < 0.03) and median QIgG (p < 0.03) were significantly higher in the subgroup of samples obtained during acute attacks if all samples are taken into account , absolute QIgG values (p < 0.03), frequency of BCB dysfunction (p < 0.04), possibly owing to the low number of samples in the remission group.When considering only the first LP performed during acute events and the latest LP performed during remission of an event (in order to control for differences in the number of follow-up samples available per patient), some differences were more pronounced Table . Of noteFinally, separate analyses of the acute MY, the acute ON, and the acute BRAIN subgroups revealed more pronounced differences between the acute phase and remission with respect to some parameters than is evident from the unstratified analysis of the total cohort  samples showed exclusively normal values. Of these eight samples, two were taken during remission and six during acute attacks (no data in 0). Two of the 'acute' yet negative samples belonged to the acute ON group, three to the acute BRAIN group, and one to the acute MY group (no data in 0). If only a basic panel consisting of CSF WCC, CSF TP, and CSF l-lactate is considered , 29/108  samples would have been classified as 'normal'. Of those, 21/108  would have been false-negatives (with the full panel serving as gold standard).A clinically relevant number of CSF samples exhibited no pathological changes. If CSF WCC, OCB, QIgG, Link index, QIgM, QIgA, QAlb, CSF TP, and CSF l-lactate, respectively, during acute attacks was neither found in the pediatric cohort nor in the adult cohort (data not shown). However, an analysis of pooled data from the pediatric and the adult cohort showed a significant relationship of age at LP and CST TP (p < 0.0001), QAlb (p < 0.0005), and CSF l-lactate (p < 0.0003) during acute attacks; a less pronounced relationship was seen for QIgG (p < 0.04), whereas no significant relationship was found for CSF WCC  samples showed no signs of intrathecal synthesis (IS) of IgG, as indicated by a lack of CSF-restricted OCB. This is in stark contrast to MS, in which OCB are detectable in ≥ 95% of cases , 49. SimOf note, OCB were only transiently positive in 2/3 OCB-positive patients tested more than once , both at onset and at follow-up. Similarly, the MRZ reaction has also been shown to be typically absent in NMOSD  [As in the adult cohort , the int so far) , 46, 64. so far) . Its abs so far)  are combIt is a limitation that most studies that evaluated the MRZ reaction in pediatric patients with bona fide MS did not test for MOG-IgG and AQP4-IgG, two markers that have become available only relatively recently . In younp = n.s.). The MRZR-tested cohort comprised an approximately equal proportion of female and male patients (55.2%% vs. 44.8%%), and the proportion of female patients among all MRZR-tested samples was virtually identical to that among all samples (55.2% vs. 55.6%). This strongly argues against an effect of age and sex. Finally, MRZR was negative both in all OCB-positive and all OCB-negative samples tested and the proportion of OCB-positive samples tested for MRZR (27%) did not differ from the total proportion of samples tested for MRZR (27%). Similarly, MRZR was also negative in all tested OCB-positive samples in the adult cohort.Due to the retrospective design of this study, we cannot know whether MRZR testing was possibly more frequently considered in patients in whom the differential diagnosis between MOG-EM and MS was particularly difficult. However, as all samples tested were negative for MRZR, the use of the test in such clinically highly relevant subpopulation would only underline its differential diagnostic significance. No other potential biases were identified: MRZ results were provided by 13 different centers , widely ruling out a major center-specific selection bias. MRZ was both negative in samples from the acute MY and BRAIN subgroups  and in samples from patients with acute ON . Median age of the MRZR-tested patients was 8 years (range 1–17), which was very similar to that among all samples and that in the non-tested subgroup  used, if supporting evidence from OCB determination  is lacking. Current guidelines on CSF diagnosis set the upper limit for imprecision at 7–10%, for incorrectness at 10%, and for deviation between single measures at 24–30% . In factN = 16, or 25%, based on Qlim, and N = 13, or 21%, based on IgM-IF > 10%) of pediatric samples showed evidence of possible low intrathecal production of IgM antibodies. Like IgG, IgM synthesis was also found only during acute attacks (just as in the adult cohort [n = 2) or IgA IS (n = 6) or both IgG IS and IgM IS (n = 3). By contrast, isolated IgM IS is atypical in MS and should prompt doubt regarding that diagnosis. The specificity of the IgM antibodies in our MOG-EM patients is unknown. While in the adult cohort, five of the 16 CSF samples with elevated QIgM were available for retrospective testing but were all negative for MOG-IgM . Intere results ), no pai results ). A prev results ) cannot p < 0.0001) and QAlb values exceeding 12 × 10−3, which were found in more than a quarter of the adult patients with elevated QAlb, were present only in a single sample (2.9%) in our pediatric patients (p < 0.002). Nonetheless, brain–CSF barrier dysfunction was not rare. Based on age-related upper reference intervals, QAlb was elevated in almost every second sample.Blood–CSF barrier disturbance as indicated by QAlb elevation was common and more severe than in MS. While QAlb is normal in around 90% of MS patients , 40, it It should be kept in mind, as a general rule, than QAlb rather than CSF TP levels should be used to evaluate blood–CSF barrier function, since CSF TP levels depend on both intrathecal synthesis and serum levels , 43. Howl-lactate and, possibly, QIgG in patients with MOG-EM.Age may also influence the extent of CSF pathology. Regression analyses of the pooled dataset from the pediatric and the adult cohort suggest a relationship of age and QAlb, CSF TP, CSF l-lactate and applied in our study. Age-adapted l-lactate CSF levels were elevated in almost one-third of our pediatric patients  and pediatric patients (p < 0.01)  Figs. . In conte levels , 73. Thil-lactate [l-lactate elevation nor median l-lactate levels differed significantly between samples with and without granulocytes, both in the pediatric and in the adult cohort. Moreover, no statistically significant correlation between CSF granulocyte counts and CSF l-lactate levels could be demonstrated . As a limitation, the number of samples with exact data on CSF granulocyte numbers was small. CSF l-lactate is thought to be produced also by astrocytes following glutamate stimulation [l-lactate levels and the spinal cord lesion load [l-lactate in MOG-EM.Granulocytes are a known source of CSF -lactate –78. Howemulation , 80. In ion load , AQP4-Igion load . Howeverion load , an incrion load . It alsoion load , 83. FinAn elevated WCC was found in about 60% of samples from pediatric patients with active disease at the time of LP, which is identical to the rate found in adult patients . Median While a CSF WCC > 50 cells/μl is rare in MS and should prompt physicians to challenge the diagnosis, white cell numbers > 50 were observed in 22% of all pediatric samples, in 28% of all samples with pleocytosis, and in as many as 48% of those taken during acute myelitis . In the acute MY subgroup, WCC exceed even 100 cells/μl in every third patient; such high white cell numbers are virtually never seen in MS. These numbers are almost identical to those in the adult cohort  .l-CSF lactate levels, two laboratory features of bacterial CNS infection, were frequently observed during acute attacks both in children and adults. Granulocytes are also detectable in the CSF during very early-stage viral encephalomyelitis. Given the fact that MOG-EM attacks  are attacks  which maEosinophilic infiltration is not a typical feature of MOG-EM , 85. In It is of note that CSF pathology was strikingly less severe and less frequent in samples obtained during acute attacks of ON than in acute myelitis Fig. , both inWith the re-integration of OCB in the latest revision of the diagnostic criteria for MS  and the We count among the strengths of this study the high number of pediatric patients included (given the rarity of the disease), the large number of both samples and parameters analyzed, and the stratified analysis taking into account the clinical presentation at the time of LP. It is a potential limitation that our study included a large number of centers. However, the rarity of MOG-IgG-associated EM means that monocenter studies cannot be performed if sufficient sample numbers are to be analyzed. Moreover, the multicenter approach reduces the risk of selection bias. Finally, no standardized MRI protocols were used. Although the correlation of lactate and TP levels with the spinal cord lesions lesion load found in our cohort is intriguing, further studies are needed to confirm this finding in a prospective and more standardized fashion.l-lactate levels as well as CSF albumin and CSF TP levels correlated with the spinal cord lesion load ; (vii.) CSF white cell numbers in MOG-EM may well exceed those typically observed in MS, in particular in acute myelitis (> 50 cells/μl in around 50% during acute LETM); (viii.) a lack of pleocytosis, on the other hand, does not rule out the condition but is a frequent finding (around 80% in acute ON); (ix.) the intrathecal, polyclonal antiviral immune response  discriminates sharply between MOG-EM and MS; and (x.) neutrophilic pleocytosis and elevated l-lactate CSF concentrations render the condition—just like AQP4-IgG-positive NMOSD—a relevant differential laboratory diagnosis of  bacterial infection in a subset of patients. In many respects, CSF findings in MOG-EM share much more similarities with NMOSD than with MS. Our data may help to improve the differential diagnosis of MOG-EM and MS and to extend our understanding of the immunopathology of this newly described entity. Except for lower QAlb values and l-lactate levels during acute attacks, CSF findings in the pediatric cohort did not differ substantially from those in the adult cohort. A detailed analysis of the CSF findings in adult patients with MOG-EM can be found in Part 1 of this article series [In summary, our study, the first to review comprehensively and systematically the CSF findings in children with MOG-EM in a large cohort of patients of mainly Caucasian descent, demonstrates that (i.) in sharp contrast to classic MS, intrathecal IgG synthesis is rare in MOG-IgG-associated EM, as shown both qualitatively and quantitatively; (ii.) if present, intrathecal IgG synthesis is low in most patients, partly transient, and restricted mainly to acute attacks (again in contrast to MS); (iii.) CSF findings in acute myelitis differ substantially and significantly from those in acute ON ; (iv.) CSF findings in “monophasic” MOG-EM are not significantly different from those in relapsing MOG-EM; (v.) different from MS, the degree of CSF alteration depends on disease activity and attack severity ; (vi.) in patients with acute myelitis, CSF e series .Additional file 1: Supplementary Figure 1. CSF white cell counts in the ‘acute MY subgroup’, the ‘acute BRAIN subgroup' and the ‘acute ON subgroup’.Additional file 2: Supplementary Figure 2. No marked differences in serum IgG, IgM, IgA and albumin levels between the ‘acute MY’, the ‘acute ON’ and the ‘acute BRAIN’ (B) subgroup, except for slightly higher median IgM values in the ‘acute BRAIN’ subgroup as compared to the acute ‘ON’ subgroup.Additional file 3: Supplementary Figure 3. Regression analysis of QAlb and CSF total protein, demonstrating a close relationship between the two parameters . Additional file 4: Supplementary Figure 4. Influence of age at LP on CSF parameters as detected by a pooled linear regression analysis of data from the pediatric and the adult cohort . By contrast, no significant relationship was observed when the two cohort were analysed separately (not shown).Additional file 5: Supplementary Table 1. CSF findings during acute attacks and during remission in the ‘acute MY’ subgroup, the ‘acute ON’ subgroup and the ‘acute BRAIN’ subgroup  and MOG-IgG-positive non-longitudinally extensive transverse myelitis (NETM). Additional file 8: Supplementary Table 4. CSF findings in MOG-IgG-positive acute bilateral ON and in MOG-IgG-positive acute unilateral ON.


Spontaneous intracranial hypotension (SIH) is classically characterised by orthostatic headache and is commonly secondary to a spontaneous cerebrospinal fluid (CSF) leak near spinal nerve roots. Risk factors for SIH include connective tissue disorders, bariatric surgery and spinal pathologies such as osteophytes and disc prolapse. The annual incidence is approximately 1 in 20,000 individuals per year and the rarity of this condition has made large-scale studies of SIH difficult resulting in a limited evidence base for clinical decision-making.This month’s journal club examines three papers reporting on the presentation, investigation and treatment of SIH. The first is a large systematic review and meta-analysis of over 2000 SIH cases. The second reports on a case series of 44 patients with SIH secondary to a rare spinal structural abnormality: cerebrospinal fluid–venous fistula. The third provides clinical information on 29 patients with frontotemporal brain-sagging syndrome, a rare, but potentially reversible, complication of SIH.This review analysed over 2000 SIH cases extracted from 144 articles up to April 30th, 2020. The majority of studies (62.5%) were retrospective, with just 14.6% being prospective. The remaining 22.9% did not clearly specify how data were collected. The diagnostic criteria for SIH varied across different studies, reflecting the lack of a universally accepted definition. Onset of symptoms was predominantly in middle age but with a large range . Females were affected twice as often as males. Headache was the most common presenting symptom, occurring in 97% of patients. The headache was usually orthostatic (92%), and most often occipital, frontal or diffuse. A wide range of associated symptoms were reported, the most frequent of which were nausea/vomiting (54%), neck pain/stiffness (43%), hearing disturbances (28%), and dizziness (27%). Brain MRI with contrast revealed a number of characteristic abnormalities including diffuse pachymeningeal enhancement (73%), venous engorgement (57%), brain sagging (43%) and subdural collections (35%). Of note, 19% of cases had normal brain MRI.Although the study reported that extradural CSF was detectable on spinal imaging in 48–76% of cases, it was often difficult for the exact leak site to be located. Imaging techniques such as digital subtraction myelography were most successful at locating leaks, and these were most commonly found in the thoracic spine (41%), although also reported in the cervical and lumbar regions, or at multiple levels (24%). Lumbar puncture, when performed, revealed a low opening pressure (< 60 mm CSF) in 67% of cases, with 32% having normal pressure (60–200 mm CSF). Conservative treatment, usually consisting of a long period of bed rest and hydration (up to 9 weeks), led to resolution of symptoms in 28%. If conservative measures failed, second-line therapy was usually an epidural blood patch (EBP) which was successful in 64% of cases. Larger volume EBPs (> 20 ml) were more effective than smaller volume ones , but there was no significant difference between targeted and non-targeted EBPs. This study reported only small numbers of surgical interventions and was unable to draw detailed conclusions about their efficacy.Comment: This large meta-analysis shows that in most cases of SIH, orthostatic headache and brain MRI findings are diagnostic. However, there are atypical headache presentations and almost a fifth have normal MRI brain imaging. Lumbar puncture can show low CSF pressure, but is often not required diagnostically and may worsen symptoms. Spinal imaging often cannot identify a CSF leak site and non-targeted EBP is as effective as targeted EBP. Limitations of the studies presented include that they were mostly retrospective, based on variable diagnostic criteria, and heterogeneous in their designs.D’Antona, L. et al. (2021) JAMA Neurol https://doi.org/10.1001/jamaneurol.2020.4799One rare cause of SIH is CSF leakage secondary to a cerebrospinal fluid–venous fistula. Such fistulae can be iatrogenic, for example after lumbar puncture or spinal surgery, but can also arise from spontaneous rupture of a perineural cyst into an adjacent vein, usually near a spinal nerve root. CSF–venous fistulae are most common in the thoracolumbar region, with 68% found between T7 and T12.This retrospective study identified 44 patients with CSF–venous fistula at a single tertiary centre between 1994 and 2019. Females were affected more commonly than males (approximately 2:1) and symptom onset was in middle age (mean onset 52.6 years). Over 95% of patients presented with headache, often with a preceding period of episodic or chronic headaches. Interestingly, the Valsalva manoeuvre induced or exacerbated the presenting headache in 88% of cases whereas only 69% reported an orthostatic component. Brain MRI with contrast was abnormal in over 80% but CSF opening pressure, when measured, was commonly normal (7–20 mm CSF in 84%). Digital subtraction or positive pressure myelography were best at identifying fistulae, with conventional CT and MRI myelograms having a poor yield. In contrast with other CSF leaks, extradural CSF is not found on imaging; instead, myelographic contrast can be seen in an epidural vein.Most fistulae in this series were thoracic with 76.7% being right sided for unknown reasons. 40 patients received at least one EBP, but the response was far less effective than for SIH in general: only 10% had a good response lasting at least 3 months. 42 patients had surgical treatment of their CSF–venous fistula, an average of 3 years after onset of symptoms. Surgery usually involved ligation of the involved thoracic nerve root and its associated veins, although the nerve root was preserved if the fistula was at the C7/T1 level. There were few post-operative problems reported—transient rebound intracranial hypertension occurred in 7% and one patient reported new mid-thoracic paraspinal pain. Over 80% of patients treated surgically reported improvement of symptoms, with nearly 50% achieving headache-freedom. In most cases of clinical improvement, the MRI brain abnormalities resolved.Comment: This study highlights CSF–venous fistula as a rare but surgically treatable cause of SIH. In a patient with SIH who is not responding to conservative treatment or EBPs, detailed spinal imaging with contrast could be of benefit. Future prospective studies using advanced imaging techniques will likely lead to increasing diagnosis and understanding of this rare abnormality.Duvall, J.R. et al. (2019) Cephalalgia 39: 1847–1854A spinal CSF leak can induce low CSF pressure and sagging of the brain and brainstem. In rare cases, this can cause a clinical syndrome indistinguishable from behavioural variant frontotemporal dementia (bvFTD), consisting of inappropriate social functioning, altered personality, apathy, hyperorality and cognitive impairment. However, in contrast to bvFTD, there is no frontotemporal atrophy on MRI imaging and the clinical abnormalities are reversible on treatment of the CSF leak.This study reports a case series of 29 patients  meeting diagnostic criteria for SIH and bvFTD at a quaternary SIH centre between 2001 and 2017. Mean age at onset of SIH was 50.6 years. The most common clinical scenario was onset of orthostatic headache followed by bvFTD symptoms an average of 2 years later. Of note, all patients reported daytime hypersomnolence and this mostly coincided with bvFTD symptoms, of which disinhibited behaviour and apathy were most prevalent. MMSE scores had a mean of 22.4/30, indicating cognitive impairment. Interestingly, the bvFTD-like symptoms were often worse towards the end of the day and seemed to have an orthostatic component.MRI brain showed severe brain sagging, involving the midbrain and brainstem, and bilateral temporal lobe herniation in all patients. Pachymeningeal contrast enhancement was common. Extensive spinal imaging was unable to detect a CSF leak in any patient although spinal meningeal diverticula were found in 52%. Opening CSF pressure, when measured, was low (< 60 mm) in approximately half of the patients. Improvement after EBP was noticed in 86% but this was often not sustained, and most patients required further EBP(s) and/or a range of surgical interventions including spinal meningeal diverticular repair. Despite these difficulties with treatment, a good outcome of no/mild disability was reported in 72% (2-year mean follow-up time after last treatment).Comment: The combination of orthostatic headache, hypersomnolence and bvFTD-like symptoms should prompt a clinician to consider brain sagging secondary to SIH. Brain MRI can confirm the diagnosis. It is important to recognise this diagnosis as symptoms are often reversible with EBPs and surgical interventions.Schievink, W.I. et al. (2018) Operative Neurosurgery 15: 505–515SIH is a rare but debilitating condition, most often caused by CSF leaks near the thoracic nerve roots. Large volume non-targeted EBPs are the most effective intervention reported. In a few cases, SIH is caused by a CSF–venous fistula which requires specific surgical treatment. One rare downstream effect of prolonged SIH is brain sagging: a potentially reversible cause of bvFTD-like symptoms.


During the concentric phase, anterior deltoid and posterior deltoid showed greater sEMG RMS in frontal raise (effect size (ES)-range: 1.78/9.25)) and LR-internal (ES-range: 10.79/21.34), respectively, vs. all other exercises. Medial deltoid showed greater sEMG RMS in LR-neutral than LR-external ), frontal raise (ES: 10.28(95% CI: 6.67/13.01)), and LR-flexed (ES: 6.41(95% CI: 4.04/8.23)). Pectoralis major showed greater sEMG RMS in frontal raise vs. all other exercises (ES-range: 17.2/29.5), while upper trapezius (ES-range: 2.66/7.18) and triceps brachii (ES-range: 0.41/3.31) showed greater sEMG RMS in LR-internal vs. all other exercises. Similar recruitment patterns were found during the eccentric phase. When humerus rotates internally, greater activation of posterior deltoid, triceps brachii, and upper trapezius occurs. Humerus external rotation increases the activation of anterior and medial deltoid. Frontal raise mainly activates anterior deltoid and pectoralis major. LR variations and frontal raise activate specifically shoulders muscles and should be proposed accordingly.The present study examined the muscle activation in lateral raise with humerus rotated externally , neutrally , internally , with flexed elbow (LR-flexed) and frontal raise during both the concentric and eccentric phase. Ten competitive bodybuilders performed the exercises. Normalized surface electromyographic root mean square (sEMG RMS) was obtained from  Lastly, triceps brachii is a strong stabilizer of the elbow during all LR variations and frontal raise. However, no study has examined its activation during these exercises. A comprehensive examination of the activation of the muscles surrounding the shoulders during LR variations and frontal raise may help to properly plan resistance training sessions to increase strength and possibly promote hypertrophy [Lateral raise (LR) and frontal raise consist of arm abduction and flexion on the frontal or sagittal plane, respectively, and are largely used to stimulate the shoulders muscles ,8,9,10. al plane . Additio flexors ,11. Inteertrophy . As suchdeltoid, upper trapezius, the clavicular head of pectoralis major, and triceps brachii in a range of LR variations  and frontal raise during both the concentric and eccentric phase in competitive bodybuilders.The previous studies investigating muscle activation in LR variations and frontal raise mainly recruited subjects with no-specified experience in strength training ,8,10,11.The present investigation was designed as a cross-over, repeated-measures, within-subject study. The participants were involved in three different sessions. In the first session, the participants were familiarized with the technique of each selected exercise. In the second session, the load for each exercise was determined. In the third session, the muscles’ maximum activation was measured; thereafter, after a minimum of 30 min of passive recovery, the participants performed each exercise in random order. Each session was separated by at least three days, and the participants were instructed to avoid any further form of resistance training for the entire duration of the investigation.The present procedures were advertised by the operators during some regional and national competitions, and, to be included in the study, the participants had to compete in regional competitions for a minimum of at least 5 years. Additionally, they had to be clinically healthy, without any reported history of upper-limb and trunk muscle injury and neurological or cardiovascular disease in the previous 12 months. To avoid possible confounding factors, the participants competed in the same weight category , according to the International Federation of Body Building Pro-League. The use of drugs or steroids was continuously monitored by a dedicated authority under its regulations, albeit we could have not controlled for it. Thereafter, 10 male competitive bodybuilders  were recruited for the present investigation. The participants were asked to abstain from alcohol, caffeine, or similar beverages in the 24 h preceding the test. After a full explanation of the aims of the study and the experimental procedures, the participants signed written informed consent. They were also free to withdraw at any time. The procedures were approved by the Ethical Committee of the Università degli Studi di Milano (CE 27/17) and performed in accordance with the Declaration of Helsinki (1975) for studies involving human subjects.anterior deltoid, medial deltoid, posterior deltoid, the clavicular head of pectoralis major, upper trapezius, and lateral head of triceps brachii was measured in random order. The electrodes placement followed the SENIAM (surface electromyography for the non-invasive assessment of muscles) recommendations [anterior deltoid was placed over the mid-belly of the muscle approximately 4 cm below the clavicle [medial deltoid had 2 electrodes placed on the lateral aspect of the deltoid, 3 cm below the acromion process [posterior deltoid, the electrodes were placed in the area about two fingerbreadths behind the angle of the acromion [pectoralis major were placed on the midclavicular line, midway between the acromioclavicular joint of the shoulder for the clavicular head [upper trapezius, the electrodes were placed at 50% on the line from the acromion to the spine on vertebra C7 [triceps brachii, the electrodes were placed at 50% on the line between the posterior crista of the acromion and the olecranon at two finger widths lateral to the line [The maximal voluntary isometric activation of ndations . The surclavicle . The med process . For posacromion . The sEMlar head . For upptebra C7 . For thethe line . The elethe line . To checthe line . For exathe line . Brieflythe line . If the the line .anterior deltoid, the participants were instructed to flex the elbow to 90° so that the hand was pointed upwards. Then, they were asked to make a closed fist with the hand of the flexed arm and to provide maximal force to produce shoulder flexion against manual resistance [medial deltoid, the participants were instructed to flex the elbow to 90° and were asked to maximally abduct the flexed arm against manual resistance [posterior deltoid, the participants were asked to abduct the shoulder in a slight extension against manual resistance, with the humerus in slight internal rotation [upper trapezius, the participants were instructed to elevate the acromial end of the clavicula and scapula against unmovable resistance pushing downward [pectoralis major, the participants were instructed to horizontally abduct the arm with the shoulder and elbow flexed at 90°. Then, they provided maximal force while attempting to horizontally adduct the arm against unmovable resistance [triceps brachii, the participants were instructed to extend the elbow against unmovable resistance toward the elbow flexion [To determine the maximal voluntary activation of all selected muscles, the participants were asked to exert their maximal force against unmovable or manual resistance. For the sistance . For thesistance . For therotation . For uppdownward . For thesistance . For the flexion . Each at flexion . The opeThe electrodes were equipped with a probe  that permitted the detection and the transfer of the sEMG signal by wireless modality. sEMG signal was acquired at 1000 Hz, amplified , and driven to a wireless electromyographic system  that digitized (1000 Hz) and filtered  the raw sEMG signals.Given the nature of each exercise and to avoid any possible involvement of muscles not directly involved in the movement during the exercise execution , each exercise was performed seated with a load that allowed eight repetitions . The 8-RM was determined, incrementing the load in accordance with previous procedures ,22. OnceAll exercises were performed avoiding any movement in elbow and wrist flexion/extension or wrist pronation/supination. For LR-external, the participants were required to abduct laterally the humerus up to 90°, with humerus externally rotated . For LR-neutral, the participants were required to abduct laterally the humerus up to 90°, with the humerus neutrally rotated . For LR-internal, the participants were required to abduct laterally the humerus up to 90°, with the humerus internally rotated . For frontal raise, the participants were required to frontally flex the humerus up to 90°, with humerus neutrally rotated . For all these exercises, the elbow was almost fully extended, and the wrist in line with the forearm . For LR-The sEMG signals from both the maximal voluntary isometric activation and separately from the concentric and eccentric phases of each exercise were analyzed in time-domain, using a 25-ms mobile window for the computation of the root mean square (RMS). For the maximal voluntary isometric activation, the average of the RMS corresponding to the central 2 s was considered. During each exercise, the RMS was calculated and averaged separately over the 2 s of the concentric and eccentric phase. Out of the six repetitions, we excluded the first and the last to avoid any possible confounding factor related to the beginning and the end of each set and to further exclude any symptom of fatigue. Thereafter, the sEMG RMS of each muscle during each exercise was normalized for its respective maximal voluntary isometric activation ,14,23,24p < 0.05. Eta squared (η2) and partial η2 were calculated to estimate the degree of variance of the dependent factor due to the independent factors and interpreted as follows: <0.059: small; 0.06 to 0.12: medium; >0.13: large [d effect size (ES) with 95% confidence interval (CI) was reported for significant pairwise comparisons and interpreted according to the Hopkins’ recommendations: 0–0.19: trivial; 0.20–0.59: small; 0.60–1.19: moderate; 1.20–1.99: large; ≥2.00: very large [The statistical analysis was performed using statistical software . The normality of data was checked using the Shapiro–Wilk’s test, and all distributions were normal. Descriptive statistics are reported as mean (SD). The intrasession sEMG RMS reliability was calculated using an intra-class coefficient (ICC), and the standard error of the measurement expressed as percentage variability (SEM%) for each muscle maximum voluntary isometric activation and for the concentric and eccentric phase of each exercise comparing the second and the fifth repetition. The ICC was interpreted as follows: ≥0.90: very high; 0.89–0.70: high; 0.69–0.50: moderate. The differences in the normalized sEMG RMS were separately calculated for each muscle between the exercise  and phase (2 levels: concentric and eccentric) using a two-way ANOVA. Multiple comparisons were adjusted using the Bonferroni’s correction. Significance was set at 3: large . The Cohry large .The intrasession reliability of the sEMG RMS signal for muscle maximum voluntary isometric activation and for the concentric and eccentric phase of each exercise is provided in anterior deltoid is shown in p < 0.001, F = 493.3, η2 = 0.998) was found. With the exception of the LR-neutral, greater  sEMG RMS was found during the concentric vs. eccentric phase in all other exercises. During the concentric phase, sEMG RMS was greater in frontal raise compared with LR-external , LR-neutral , LR-internal , and LR-flexed . LR-external showed greater sEMG RMS than LR-neutral , LR-internal , and LR-flexed . LR-flexed showed greater EMG RMS than LR-neutral  and LR-internal . During the eccentric phase, sEMG RMS in LR-external and LR-neutral was greater than LR-internal , frontal raise , and LR-flexed . Frontal raise and LR-flexed had greater sEMG RMS than LR-internal . No further difference was observed.The sEMG RMS amplitude for medial deltoid is shown in p = 0.002, F = 35.7, η2 = 0.973) was found. With the exception of LR-internal, greater  sEMG RMS was found during the concentric vs. eccentric phase. During the concentric phase, sEMG RMS was greater in LR-neutral than LR-external , frontal raise , and LR-flexed . Both LR-external and LR-internal had greater sEMG RMS than frontal raise  and LR-flexed . LR-flexed showed greater sEMG RMS than frontal raise . During the eccentric phase, LR-internal showed greater sEMG RMS than LR-external , LR-neutral , frontal raise , and LR-flexed . Both LR-neutral and LR-flexed had greater sEMG RMS than LR-external  and frontal raise . LR-external showed greater sEMG RMS than frontal raise . No further difference was observed.The sEMG RMS amplitude for posterior deltoid is shown in p < 0.001, F = 234.4, η2 = 0.996) was found. Whatever the exercise, greater  sEMG RMS was found during the concentric vs. eccentric phase. During both the concentric and eccentric phase, LR-internal showed greater sEMG RMS compared with LR-external , LR-neutral , frontal raise , and LR-flexed . During both concentric and eccentric phase, LR-neutral showed greater sEMG RMS than LR-external , frontal raise , and LR-flexed . During the concentric phase, LR-flexed showed greater sEMG RMS than LR-external  and frontal raise , and LR-external had greater sEMG RMS than frontal raise . During the eccentric phase, both LR-external and LR-flexed showed greater sEMG RMS than frontal raise . No further difference was observed.The sEMG RMS amplitude for upper trapezius is shown in p = 0.002, F = 37.3, η2 = 0.974) was found. Whatever the exercise, greater  sEMG RMS was found during the concentric vs. eccentric phase. During the concentric phase, sEMG RMS was greater in LR-internal compared with LR-external , LR-neutral , frontal raise , and LR-flexed . LR-flexed had greater sEMG RMS than LR-external  and frontal raise . Both LR-external and LR-neutral had greater sEMG RMS than frontal raise . During the eccentric phase, both LR-internal and LR-flexed had greater sEMG RMS compared with LR-external , LR-neutral , and frontal raise . No further difference was observed.The sEMG RMS amplitude for pectoralis major is shown in p < 0.001, F = 111.4, η2 = 0.999) was found. Whatever the exercise, greater  sEMG RMS was found during the concentric vs. eccentric phase. During the concentric phase, sEMG RMS was very largely greater in frontal raise compared with LR-external , LR-neutral , LR-internal , and LR-flexed . During the eccentric phase, sEMG RMS of clavicular was very largely greater in frontal raise compared with LR-external , LR-neutral , LR-internal , and LR-flexed . No further difference was observed.The sEMG RMS amplitude for triceps brachii is shown in p = 0.003, F = 28.2, η2 = 0.966). Whatever the exercise, greater  sEMG RMS was found during the concentric vs. eccentric phase. sEMG RMS was greater in LR-internal during the concentric and eccentric phase compared with LR-external , LR-neutral , frontal raise , and LR-flexed . During the concentric phase, LR-external and LR-flexed showed greater sEMG RMS than LR-external  and frontal raise . LR-external had greater sEMG RMS than frontal raise . During the eccentric phase, sEMG RMS was greater in LR-external , LR-neutral , and LR-flexed  vs. frontal raise. No further difference was observed.The sEMG RMS amplitude for anterior deltoid was greater during frontal raise and LR-external. Medial deltoid was more active during the lateral raises, particularly when the humerus had a neutral or internal rotation. Posterior deltoid had greater sEMG RMS in LR-internal compared with all other exercises. Upper trapezius had greater sEMG RMS during LR-internal compared with all other exercises. The clavicular head of the pectoralis major was more active during frontal raise compared with all other exercises. Lastly, the lateral head of triceps brachii was more active in LR-internal compared with all other exercises. Although this muscle activity pattern was overall similar during the eccentric phase, anterior deltoid had similar activation in LR-external and LR-neutral and medial deltoid in LR-internal compared with all other exercises. The present findings showed unique muscle activation for each LR variations and frontal raise. Practitioners should thus select these exercises depending on the shoulders muscles that need to be activated at each occasion.The present study investigated the sEMG RMS in several agonist muscles in a range of shoulder raise exercises during the concentric and eccentric phase of each exercise. During the concentric phase, sEMG RMS in anterior, > ~48% in medial, > ~36% in the posterior head. For LR-neutral, sEMG RMS was similar in medial and posterior (~55% and ~52%), but lower in anterior deltoid (~36%). For LR-internal, sEMG RMS was ~85% in posterior, ~52% in medial, and ~34% in anterior deltoid, in line with previous results [anterior, medial, and posterior deltoids act synergistically as humerus abductors on the frontal plane, anterior deltoid is an internal, while posterior deltoid is an external rotator of the humerus [posterior and anterior deltoid, respectively, thus increasing their activity [anterior deltoid showed similar sEMG RMS in LR-external vs. LR-neutral, possibly because of the greater control needed to decelerate the humerus, as reported previously [medial deltoid activity was found in LR-internal, which possibly needs greater control [triceps brachii activation was overall greater in LR-internal > LR-neutral > LR-external. The greater the internal rotation of the humerus, the greater the weight of the forearm + wrist + dumbbell system becomes against the gravity. Indeed, the internal rotation of the humerus lets the elbow’s mobility be free against the gravity, whereas the external rotation puts the elbow’s extension in favor of the gravity, fixing the elbow joint. Therefore, because all exercises were performed at a fixed elbow angle, triceps brachii increased its elbow-stabilizer role when rotating internally the humerus. The internal rotation of the humerus also increased the role of upper trapezius in LR-internal > LR-neutral/LR-external. Indeed, when rotating internally the humerus, the scapulae are elevated and rotated upward [upper trapezius activity. Lastly, the clavicular head of the pectoralis major showed similar low activity in all LR variations, indicating that it plays a marginal role when the humerus is abducted laterally.LR-external, LR-neutral, and LR-internal are performed abducting the humerus, which is rotated, respectively, externally, neutrally, or internally for the entire range of motion. For LR-external, the present results showed that the deltoids sEMG RMS was ~80% in  results . Althougactivity ,28, possactivity , modificactivity , or alsoeviously . Similard upward , thus aumedial deltoid was hence lower compared to the other LR variations, where the arm was lifted almost straight on the frontal plane, increasing the length of the lever. Secondly, considering the point (ii), the forearm placed forward on the sagittal plan increased the activity of anterior deltoid as frontal humerus flexors. Indeed, greater sEMG RMS compared to LR-neutral and LR-internal was found here. Moreover, because of the forward weight that leads the humerus to rotate internally, the posterior deltoid had to counteract as an external rotator, thus stabilizing the rotation of the humerus [upper trapezius was more active during both the concentric and eccentric phase compared to LR-external and LR-neutral. It should be also noted that the flexed elbow increased the muscle length at which triceps brachii had to contract to stabilize the elbow joint [pectoralis major seemed to play a marginal role in LR-flexed, as pointed out by the low level of activation.LR-flexed, compared to the other LR variations, was performed with the flexed elbow at 90°, and the humerus rotated neutrally. However: (i) the length of the lever on the frontal plane was shorter than the other LR-variations, and (ii) the forearm was placed forward on the sagittal plane. Firstly, considering the point (i), the sEMG RMS of  humerus . Therefo humerus , so thatow joint . This miow joint . Lastly,anterior deltoid and the clavicular head of the pectoralis major. Indeed, both muscles showed the greatest activity compared with all the remaining exercises. In line, when the humerus moved towards a flexion across the sagittal plane, the sEMG RMS in anterior deltoid and the clavicular head of the pectoralis major was reported to increase [medial and posterior deltoids appeared to play a negligible role in frontal raise, in line with previous results [Upper trapezius showed relatively low activity, possibly because, when flexing the humerus, the scapulae tend to rotate internally and to be depressed [upper trapezius was similar to LR-external and LR-neutral during the eccentric phase. This might depend on the need to stabilize the scapula when the upper limb was accelerating downward, as previously reported [triceps brachii showed poor activation, possibly due to the neutral humerus rotation that per se stabilized the elbow joint on the craniocaudal axis.Frontal raise was performed on the sagittal plane, lifting up the upper limb in neutral rotation by a frontal flexion, with a straight elbow. As a whole, frontal raise appears to mainly require the activation of the increase . In cont results . Upper tepressed . Interesreported . Lastly,deltoid muscle into seven different parts [Some limitations accompany the present procedures. Firstly, the data shown are specific for the population involved and the technical requirements provided to perform each exercise. Nonetheless, although it is acknowledged that different populations may result in different findings, the present population made us confident about the goodness of the technique and muscle recruitment. Similarly, even though greater load could result in different outcomes, this may also reduce the quality of the technique execution for each exercise, and we are confident that here the load was suitable for the population. Moreover, although a dedicated authority continuously monitored the use of steroids, we could not have controlled for, and we do not know how the possible use of steroids would affect muscle activation. Lastly, some authors have divided nt parts . Howeveranterior deltoid increased its activity when the humerus was rotated externally, while posterior deltoid when the humerus was rotated internally. Medial deltoid was more active during LR with straight vs. flexed elbow. Frontal raise strongly recruited anterior deltoid and the clavicular head of pectoralis major. Triceps brachii was involved in stabilizing the elbow, and greater activity occurred when the humerus was rotated internally or the elbow was flexed. Lastly, upper trapezius showed greater activity when humerus tended to be rotated internally.In conclusion, different LR variations and frontal raise had unique muscle recruitment when performed by competitive bodybuilders. Concerning LR,


The variation in cross-sectional profile of a microgroove fabricated with focused and diverging laser irradiation is demonstrated with ray tracing. To verify the result of ray tracing, stainless-steel 304 microgrooves were manufactured utilizing the conventional lens-based and optical fiber-based laser-induced etching techniques in phosphoric acid solution. Three distinctive groove geometries, i.e., a flat surface with no groove, an intermediate stage groove, and a fully developed V-groove, were rendered for numerical analysis. For focusing mode, the first and second reflections were caused by high laser intensity and the second reflected beam could lead to variation in the groove shape such as a U-shaped groove or a V-shaped groove in accordance with etchant concentration. On the contrary, a weak laser entirely distributed at the groove sidewall could not induce a chemical reaction, leading to a V-shaped groove. The effect of process variables such as laser power (intensity) and etchant concentration on the cross-sectional profiles of a groove are closely examined through the computed simulation results. A microgroove is a key configuration of numerous microelectromechanical systems based on microthermal and microfluidic devices such as a micro heat pipe ,2,3, micAs a possible approach to realize material removal while avoiding such issues, laser-induced etching (LIE) may be employed. In LIE, the workpiece is immersed in a liquid etchant that helps the fabrication of microgrooves with almost no melting or debris due to the cooling effect. The micromachining of a workpiece in LIE is done through a thermochemical reaction between the etchant and the workpiece, while the laser beam irradiates the workpiece to locally raise the surface temperature. A relatively simple setup compared to a picosecond or sub-picosecond pulsed laser machining system is required for LIE at a substantially lower equipment or maintenance cost. Due to these advantages of LIE, the fabrication of metallic microstructures using the LIE technique has been studied for various applications ,14,15. IDespite several advantages, the typical lens-based LIE system still demands optical components such as lenses, mirrors, and a focusing lens. As a result, a complicated and routine optical alignment is frequently required, and the eye or skin of an operator could be exposed to a reflected or scattered laser beam. Furthermore, a relatively low productivity owing to the use of a single objective lens is not attractive for its application to manufacturing industries. Alternatively, an optical fiber as a low-loss light wave guide can be directly applied to the LIE system. By substituting a single optical fiber for the optical components, the whole system setup can be substantially simplified, while the safety trouble is naturally reduced. The laser beam is delivered through an optical fiber and, instead of using an objective lens, the fiber tip is directly brought to the surface of the workpiece to irradiate and micromachine the workpiece. In our previous studies, we reported that LIE utilizing an optical fiber as the laser beam guide could be effectively applied to the fabrication of metallic microgrooves , and we ® .In this study, to examine the effects of laser irradiation modes on the shape of a microgroove, ray tracing was conducted as an optical rendering. Ray tracing is a well-established technique for generating an image by tracing the path of light originating from an object. The effects of a focused laser beam from an objective lens and a diverging laser beam from an optical fiber on the cross-sectional profiles of the microgrooves are investigated using ray tracing software LightToolsXYZ stage. The workpiece was placed inside a Teflon® chamber that was installed on the XYZ stage.XYZ stage. The workpiece was placed inside a Teflon® chamber through which the etchant is continuously circulated by a peristaltic pump.In conventional LIE, the laser light from a source travels through several optical elements such as a beam expander, a polarizing beam splitter, and quarter-wave plate before being focused by a focusing lens and irradiating the workpiece as shown in 3PO4, 85%) diluted with distilled water was used as the etchant to etch the stainless-steel 304 foil. All etching processes were monitored in real time with a charge-coupled device (CCD) camera. Etching results were examined using a scanning electron microscope after an ultrasonic cleaning of the workpiece.A stainless-steel 304 foil (Fe72/Cr18/Ni10) with a thickness of 500 μm was used as the workpiece. Additionally, phosphoric acid  was used for the stainless steel 304 ,20, and To investigate the surface temperature variation of the workpiece during laser irradiation, the workpiece temperature was calculated using the thermal model in our previous research . TemperaPrior to the etching experiments, the etch threshold laser power was investigated via irradiation of the diverging continuous wave (CW) laser beam. 3PO4 (etchant) concentrations of 10% and 40% with a lens-based LIE system. The grooves fabricated at these concentrations showed a clear difference in the cross-sectional profile. Those fabricated at the low concentration in 3PO4 is about three times greater than that of the 10% (≈1.3) etchant [The etching reaction and, thus, fabrication results are highly dependent upon the etchant concentration. Since the velocity of etchant solution varies with concentration, the etchant flow over the workpiece and, more importantly, inside a microstructure under fabrication can differ significantly for low- and high-concentration cases.  etchant ,23, whicFor the optical fiber-based LIE, grooves with only a triangular cross-section were fabricated at all concentrations. The reason for this result is that the laser beam emerging from a fiber tip diverges very rapidly, where the degree of divergence depends upon the numerical aperture of an optical fiber. Due to the divergence, the average irradiance decreases rapidly with the distance from the fiber tip. Accordingly, as the etching process is completed and, thus, the groove surface retreats, the etching reaction is active only in the middle of the beam at which the irradiance remains over a threshold value. In the optical fiber-based LIE case, the aspect ratio of the groove can be adjusted by varying etchant concentration and the number of scans over the same groove. The width of microgrooves that can be fabricated with the optical fiber-based LIE depends on the diameter of optical fiber, and about minimum 50 μm at the surface can be achieved. Note that, for the conventional lens-based LIE, the groove width can be as small as 15 μm because, in this case, the laser beam spot can be focused to a much smaller size. The depth of grooves varies with laser power but it can also be adjusted by varying the number of scans. For a single scan, the groove depth reaches 100 to 200 μm depending on laser power; however, by repeatedly scanning over the same groove, the depth can be increased to 300–400 μm.P = 2 W and f = 40 mm. On the contrary, the 1/e2-radius of the laser beam emitted for the optical fiber was about 75 μm for P = 2 W and gapD = 150 μm. Since the laser intensity is inversely proportional to the laser spot area, the temperature of the irradiated surface for these two different modes of LIE may also have a large difference. Furthermore, since multiple reflections of the laser beam at the groove walls can affect the groove wall temperature, the cross-sectional shape of a groove may also be related to the intensity of the reflected beam.For experiments using the lens-based case, the radius of laser spot at the surface of workpiece was 7 μm as measured with a knife edge method for 2, while that for the diverging mode was only about 1.7 W/mm2. For these laser intensities, the maximum surface temperatures calculated using the thermal model for the focusing and diverging modes were about 1490 K and 470 K, respectively, higher than the etch threshold temperature of 360 K. Therefore, etching of the flat workpiece was expected for both irradiation modes, subsequently developing into a groove. During this initial period, the groove shapes were expected to be similar, although their sizes could be different.However, once a groove is formed, the irradiation pattern over the groove walls and the associated development of groove shape are expected to be quite different between the two modes. 2 was distributed almost over the entire sidewall of the intermediate stage groove (width = 100 μm and depth = 85 μm), as shown in On the contrary, the diverging mode showed an entirely different tendency. Unlike the focusing mode, relatively low intensity with a maximum value of about 1.3 W/mmThe intensity distributions at the bottom of intermediate-stage grooves for the focusing and diverging modes are presented in For the diverging mode, a very weak concentration of incident rays at the center of the groove bottom occurred. Moreover, the laser beam intensity at the bottom of the groove see c was almThe intensity distribution over the sidewall of the fully developed groove in 2), and the results are presented in For the diverging mode, the laser intensity over the sidewall see c and at The influence of laser irradiation modes such as focused laser beam and diverging laser beam on the cross-sectional shape of a microgroove was investigated using ray tracing for three groove geometries, i.e., an initial flat surface with no groove, a developing groove of intermediate depth, and a fully developed V-groove.Regardless of the laser irradiation modes, etching of the workpiece is expected in case of the flat surface with no groove due to the initial high laser intensity. However, once the groove is formed, an obvious difference appears between the focusing mode and the diverging mode and, thus, the probable cross-sectional profiles of a groove are noticeably distinguished. For focusing mode related to the typical lens-based LIE, the high first and second reflected laser intensities could sufficiently induce a thermochemical reaction. Above all, the second reflected laser intensity has a deeper relationship with the cross-sectional profile and, according to the etchant concentration, a U-shaped groove or a V-shaped groove can be produced. On the other hand, for diverging mode related to the optical fiber LIE, since the laser intensity distributed on the sidewall and bottom portion is too weak, no further etching occurs and the V-groove is maintained. According to this study, it was found that the ray tracing results enable prediction of the shape of the microgroove that can be manufactured through LIE.It is believed that the LIE could be a practical micromachining technique for the manufacture of microthermal and microfluidic devices such as a micro heat pipe, micro heat spreader, and micro reactor, based on metallic microgrooves.


The coupling strength, J = 32.06 ± 0.06 MHz, is measured spectroscopically with high precision. Since the coupling is weaker than the electron-nuclear hyperfine coupling A ≈ 90 MHz which detunes the two electrons, a native two-qubit controlled-rotation gate can be obtained via a simple electron spin resonance pulse. This scheme is insensitive to the precise value of J, which makes it suitable for the scale-up of donor-based quantum computers in silicon that exploit the metal-oxide-semiconductor fabrication protocols commonly used in the classical electronics industry.Silicon nanoelectronic devices can host single-qubit quantum logic operations with fidelity better than 99.9%. For the spins of an electron bound to a single-donor atom, introduced in the silicon by ion implantation, the quantum information can be stored for nearly 1 second. However, manufacturing a scalable quantum processor with this method is considered challenging, because of the exponential sensitivity of the exchange interaction that mediates the coupling between the qubits. Here we demonstrate the conditional, coherent control of an electron spin qubit in an exchange-coupled pair of  Operating donor-based quantum computers in silicon is hindered by the dependence of inter-qubit coupling on the precise donor position. Here, the authors show controlled rotation operation on exchange-coupled electron spins in the weak-exchange regime, loosening the requirements on positioning precision. For the latter, adapting the fabrication processes already in use in the classical electronics industry—silicon-based metal-oxide-semiconductor (MOS) processing6 and ion implantation9—to the construction of quantum hardware would represent a great technological head start. This was the insight that triggered the first proposal of encoding quantum information in the spin state of donor atoms in silicon10. Qubits defined by individual donor-bound electron spins have demonstrated quantum gate fidelities beyond 99.9% (ref. 11), and coherence lifetimes approaching 1 s (ref. 12). The next challenge is the demonstration of robust two-qubit logic operations, necessary for universal quantum computing. In this work, we demonstrate the key capability of performing conditional, coherent quantum operations on single-donor spin qubits in the presence of weak exchange interaction13. The weak interaction regime is crucial to ensure a mode of operation that is compatible with the inherent manufacturing tolerances of silicon MOS devices.Building useful quantum computers is a challenge on many fronts, from the development of quantum algorithmsϵ1 ≈ ϵ2, and turning on the qubit–qubit interaction J for a finite amount of time14, yielding a native SWAP gate15. The second strategy implements a controlled-Z gate by dynamical control of J. The coupling is switched on for a calibrated time period, whereby the target qubit acquires a phase shift proportional to the change in precession frequency determined by the state of the control qubit17. The third strategy implements a native controlled-rotation (CROT) gate via resonant excitation of the target qubit, whose transition frequency can be made to depend on the state of the control qubit.In their simplest form, two-qubit logic gates can be executed using three distinct strategies. The first requires the two qubits to have approximately the same energy splitting, π/2 to the target qubit. This gate requires the individual qubits’ energy splittings to differ by an amount δϵ = ∣ϵ1 − ϵ2∣ much larger than their coupling J. It was used in early nuclear magnetic resonance (NMR) experiments18, superconducting qubits19 and, more recently, was adapted to electron spin qubits in semiconductors, where the energy detuning δϵ can be provided by a difference in Landé g-factors between the two electron spins20 or by a magnetic field gradient21. For electron spin qubits, the coupling J originates from the Heisenberg exchange interaction. The main advantage of this type of gate is that it can be performed while keeping J constant—an essential feature when locally tuning J is either impossible or impractical. Moreover, the precise value of J is unimportant, as long as it is smaller than δϵ, and larger than the resonance linewidth.The CROT gate is related to the controlled-NOT operation that appears in most quantum algorithms, but imparts an additional phase of 22, the exchange interaction strength decays exponentially with distance and, when accounting for valley interference, it can even oscillate upon displacing the atom by a single lattice site23. Therefore, a two-qubit CROT gate where J can be kept constant and does not need to have a specific value (within a certain range), is highly desirable. An embodiment of such gate was proposed by Kalra et al.13, who recognized that the energy detuning δϵ between two donor electrons can be provided in a convenient and natural way by setting the donor nuclear spins in opposite states. This causes the electron spins’ energy splittings to differ by the electron-nuclear hyperfine coupling A ≈ 100 MHz.For donor electron spin qubits in silicon, two-qubit logic gates based on exchange interactions are particularly challenging. Because of the small (≈2 nm) Bohr radius of the electron wave functionJ ≫ 100 MHz (refs. 27), where the native CROT gate described above cannot be performed. A SWAP operation was recently demonstrated between strongly exchange-coupled electron spins bound to donor clusters27, albeit without coherent quantum control of the individual spins. Here, we present the experimental observation of weak exchange interaction in a pair of 31P donors, and the coherent operation of one qubit conditional on the state of the other. Achieving these results with ion-implanted donors in a MOS device  and their spins are described by the electron .The operating principle of a two-qubit CROT operation for A ≈ 100 MHz. A more general and extensive discussion of the two-donor spin Hamiltonian is given in the Supplementary Note To simplify the problem, we draw the energy levels diagrams shown in Fig. J, the electronic eigenstates become the singlet ℓ2 and ℓ5 in Fig. When the nuclei are in a parallel configuration  and is smaller than A ≈ 100 MHz. The value of J sets an approximate limit to the speed of the CROT operation13, since the spectral width of the CROT pulse  must be lower than the frequency spacing = J between resonances conditioned on opposite control qubit states20. This limitation can be circumvented to some extent by replacing the simple resonant π-pulse with more sophisticated control schemes29, as demonstrated, e.g., in quantum dot systems20. Overall, this scheme affords a wide tolerance in the physical placement of the donors.Importantly, the ability to perform a CROT gate depends only on the ability to apply a selective 31P donors, the devices include a single-electron transistor (SET) to detect the donor charge state, four electrostatic gates to control the donor potential, and a microwave antenna to deliver oscillating magnetic fields  to obtain well-isolated pairs; that is, we used the choice of acceleration energy to determine the most likely distance between donors resulting from an individual PThe ion implantation step was executed using two different strategies. We first implanted a batch of devices with a low fluence of Psee Fig. , and ada12 donors/cm2) of single P+ ions at 10 keV energy. This yields a 25-fold increase in the donor density  or the ionized D+ (N = 0) state. In particular, we can freely choose the electrochemical potential of the donors with respect to each other, i.e., which of the donors ionizes first, while the other remains neutral (see Supplementary Movie).Controlling the two pulsing gates above the donor implantation area allows us to selectively and independently control the charge state of each donor, which can be set to either the neutral N = 1) and ionized (N = 0) charge states of each donor. For example, the boundary between the  and  regions is where the second donor (target) can be read out via spin-dependent tunneling to the SET island33, while the first (control) remains ionized. This is because, when transitioning from, e.g.,  to , the lost charge is absorbed by the island of the SET, which is tunnel coupled to the donors32. At low electron temperatures (Tel ≈ 100 mK) and in the presence of a large magnetic field B0 ≈ 1.4 T, the tunneling of charge from donor to SET island becomes spin dependent, since only the 33. Therefore, the boundary  ↔  is where we can observe the spin target donor, while it behaves as an isolated system, since the control donor is ionized at all times.On the stability diagram in Fig. 31P donor35. Since we are measuring a single atom, each trace normally contains only one peak, but occasionally the nuclear spin flips direction during the scan, so a single trace can also exhibit both peaks. Since the intrinsic ESR linewidth is very narrow ), finding the resonances is a time-consuming process. To speed this up, we used adiabatic spin inversion37 with a 6 MHz frequency chirp, resulting in a large electron spin-up fraction whenever a resonance falls within the frequency sweep range. The 6 MHz width of the frequency sweeps is the cause of the artificial width and shape of the resonances shown in Fig. This expectation is confirmed by the ESR spectrum shown in Fig. c,0t) ↔  where the target donor is read out, but the control donor is in the neutral D0 charge state, with an electron bound to it. Repeatedly measuring the ESR spectrum of the target donor, now reveals four possible ESR peaks. We interpret this as evidence for the presence of an exchange interaction J between the two-donor electrons: the four ESR peaks correspond to the four possible orientations of the two donor nuclear spins, while the control donor is in the ℓ1, ℓ2, ℓ4, and ℓ5). Observing all six main ESR lines would normally require preparing the control donor electron in the ℓ5a  = 47.4 kHz, only slightly larger than the 30 kHz resolution of the measurement itself. This spectroscopic method constitutes the most accurate measurement of exchange interaction between phosphorus donor pairs obtained to date.In the numerical simulations, we vary the hyperfine coupling of target and control donors, A are far from the bulk value Abulk = 117.53 MHz and rather different between the two donors. This could be due to local variations in lattice strain and electric fields within the device, which can be substantial even on a scale  ≈10 nm. Strain, in particular, varies dramatically near the tips of the control gates40, and is well-known to cause changes in hyperfine coupling42. The possible influence of strain on the spin relaxation time T1 is discussed in ref. 43.The extracted values of J ≈ 30 MHz was measured on a second device, fabricated in the same batch and with the same high-dose P+ implantation strategy .The high-fluence devices contain many donors, as visible in the charge stability diagram Fig. . One mayc,1t) region, with the control electron in the ℓ1  or ℓ5, pink line), the Rabi oscillations have a visibility VRabi = P↑(π) − P↑(0) ≈ 0.75. In contrast, the nontrivial, conditional resonances ℓ1 and ℓ4, have a significantly lower visibility VRabi ≈ 0.5. We considered whether this could be explained by the fact that ℓ1 and ℓ4 represent transitions to the J ≈ 32.06 MHz and ℓ1 and ℓ4 is For the “trivial” resonances, where the nuclear spins are either ℓ1 or ℓ4 become inactive. Resetting the control electron to the T1, during which no excitation of the target spin would be achieved on ℓ1 or ℓ4. In this device, we measured T1 = 3.4 ± 1.3 s on the target electron spin  and ℓ1 (dependent on J), and found similar values The presence within the device of strong electric fields, which can affect the value of 40, was damaged by an electrostatic discharge before we could complete the benchmarking of the full two-qubit logic gate. Future devices will be equipped with thicker antenna to prevent this issue.The complete benchmarking of a two-qubit logic gate requires the coherent control and individual readout of both qubits, and the operation of the target qubit conditional on an arbitrary state of the control qubit. The present device, comprising a very thin ESR antenna45, that afford a greater degree of control of all tunnel couplings. Even if only one donor  happens to be readable, the control donor spin states can be read out via a quantum non-demolition (QND) method by using the target electron as ancilla qubit, as already demonstrated in exchange-coupled double quantum dot systems47. This process requires a long relaxation time T1 of the electron spins in presence of weak exchange coupling. The target electron T1 = 3.4 ± 1.3 s measured here is close to that of single, uncoupled donor electrons spins43, and indicates that an ancilla-based QND readout will be an available option for future experiments.For the readout, it is often but not always possible to read two (or more) spins sequentially using the same charge sensor. This depends simply on whether all donors electrons have a tunnel time to the reservoir that falls within a usable range . We are currently developing new device designs, inspired by the flip-flop qubit proposal31P donors implanted in 28Si. The exchange interaction J = 32.06 ± 0.06 MHz was determined by ESR spectroscopy, and falls within the range J < A where a native CROT two-qubit logic gate can be performed by applying a π-pulse to the target electron after setting the two donor nuclear spins in opposite states. These results represent the first demonstration of hyperfine-controlled CROT gate for donor electrons13—a scheme that is intrinsically robust to uncertainties in the donor location, since it only requires J to be smaller than A ≈ 100 MHz, and larger than the inhomgeneous ESR linewidth  ≈10  kHz.We have presented the experimental observation of weak exchange coupling between the electron spins of a pair of g-factors in the presence of an exchange-coupled electron, and unexplained features in the visibility of conditional qubit rotations. These effects call for detailed theoretical models of donor exchange under strain and electric fields, significantly expanding the existing theories of exchange in bulk silicon23.The present work already unveiled peculiar effects, such as the Stark shift of hyperfine coupling and 44, and studying the device yield and gate performance while subjected to realistic fabrication tolerances. The fine spectral resolution afforded by our resonant control methods will provide precious insights and experimental validation to a wide suite of theoretical models of donor physics and quantum device fabrication.Future experiments will focus on benchmarking the fidelity of a complete one- and two-qubit gate set, and studying the noise channels affecting the operations. The suitability of this exchange-based logic gate for large-scale quantum computing will be assessed by integrating deterministic, counted single-ion implantation within the fabrication process28Si with 29Si residual concentration of 730 p.p.m. (ref. 48). Heavily doped n+ regions for Ohmic contacts and lightly doped p regions for leakage prevention are defined by phosphorus and boron thermal diffusion. A field oxide (200 nm thick SiO2) is grown using a wet thermal oxidation process. The central active area is covered with a high-quality thermal oxide (8 nm thick SiO2) grown in dry conditions. Subsequently, an aperture of 90 nm × 100 nm is defined in a PMMA mask using electron-beam lithography (EBL). Through this aperture, the samples are implanted with atomic (P) or molecular (P2) phosphorus ions at an acceleration voltage of 10 keV per ion. During implantation, the samples were tilted by 7° to minimize the possibility of channeling implantation. The final P atom position in the device is determined using full cascade Monte Carlo SRIM simulations. The projected range of the implant is ~10 nm beyond the SiO2/Si interface. The size of the PMMA aperture is taken into account when determining the P–P donor spacing. Post implantation, a rapid thermal anneal (5 s at 1000 °C) is performed for donor activation and implantation damage repair. A nanoelectronic device is defined around the implantation region through two EBL steps, each followed by thermal deposition of aluminum . Between each aluminum layer, the Al2O3 is formed by immediate, post-deposition sample exposure to a pure, low pressure (100 mTorr) oxygen atmosphere. The final step is a forming gas anneal  aimed at passivating the interface traps.Silicon MOS processes are employed for the donor spin qubit device fabrication. A silicon wafer is overgrown with a 0.9 μm thick epilayer of the isotopically purified The device was placed in a copper enclosure and wire-bonded to a gold-plated printed circuit board using thin aluminum wires. The sample was mounted in a Bluefors LD400 cryogen-free dilution refrigerator with base temperature of 14 mK, and placed in the center of the magnetic field produced by the superconducting solenoid in persistent mode (≈1.4 T). The magnetic field was oriented perpendicular to the short-circuit termination of the on-chip microwave antenna and parallel to the sample surface.7 V/A gain, 50 kHz bandwidth), followed by a Stanford Instruments SIM910 JFET post-amplifier (102 V/V gain), Stanford Instruments SIM965 analog filter , and acquired via an AlazarTech ATS9440 PCI digitizer card. The instruments were synchronized by a SpinCore Pulseblaster-ESR TTL generator.DC bias voltages, sourced from Stanford Instruments SIM928 isolated voltage sources, were delivered to the SET top gate, the barrier gates and the DC donor gates through 20 Hz low-pass filters. A room-temperature resistive combiner was used to add DC voltages (Stanford Instruments SIM928) to AC signals produced by a LeCroy ArbStudio 1104. The combined signals were delivered to the pulsing SET gate and the pulsing donor gates through 80 MHz low-pass filters. Microwave pulses for ESR were generated by an Agilent E8257D 50 GHz analog source; RF pulses for NMR were produced by a Agilent N5182B 6 GHz vector source. RF and microwave signals to be delivered to the microwave antenna were combined at room temperature and delivered through a semi-rigid coaxial cable fitted with a 10 dB attenuator mounted at the 4 K plate and a 3 dB attenuation at the 14 mK stage. The SET current was measured by a Femto DLPCA-200 transimpedance amplifier at room temperature (10Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1