However, the question of whether animals with ωx desaturases have complete n–3 LC-PUFA biosynthetic pathways and hence can contribute to the production of these compounds in marine ecosystems remained unanswered. In the present study, we investigated the complete enzymatic complement involved in the n–3 LC-PUFA biosynthesis in Tigriopus californicus, an intertidal harpacticoid copepod. A total of two ωx desaturases, five front-end desaturases and six fatty acyl elongases were successfully isolated and functionally characterized. The T. californicus ωx desaturases enable the de novo biosynthesis of C18 PUFA such as linoleic and α-linolenic acids, as well as several n–3 LC-PUFA from n–6 substrates. Functions demonstrated in front-end desaturases and fatty acyl elongases unveiled various routes through which T. californicus can biosynthesize the physiologically important arachidonic and eicosapentaenoic acids. Moreover, T. californicus possess a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid via the ‘Δ4 pathway’. In conclusion, harpacticoid copepods such as T. californicus have complete n–3 LC-PUFA biosynthetic pathways and such capacity illustrates major roles of these invertebrates in the provision of essential fatty acids to upper trophic levels.The long-standing paradigm establishing that global production of Omega-3 (n–3) long-chain polyunsaturated fatty acids (LC-PUFA) derived almost exclusively from marine single-cell organisms, was recently challenged by the discovery that multiple invertebrates possess methyl-end (or  Thes14C-labelled fatty acids suggested some copepods showed the ability to bioconvert PUFA into LC-PUFA [ωx desaturases were found in several species from groups of copepods including Cyclopoida, Harpacticoida and Siphonostomatoida [Lepeophtheirus salmonis (Siphonostomatoida), none of copepod ωx desaturases has been functionally characterized yet, and therefore, it remains unknown whether the activities of the copepod ωx desaturases are conserved across the group or, on the contrary, have diversified during evolution as occurred for other fatty acyl desaturases [ωx desaturases, copepods have further enzymes such as front-end desaturases and fatty acyl elongases that, along ωx desaturases, enable complete enzymatic activities allowing the production of a variety of LC-PUFA including DHA . Total RNA was extracted from pooled (approx. 50) whole individuals using TRI Reagent  following the manufacturer's recommendations. The extracted RNA was treated with RQ1 RNase-Free DNase  to remove potential genomic DNA contamination. Subsequently, the complementary DNA (cDNA) was synthesized from 2 μg of total RNA using SuperScript III First-Strand Synthesis System for RT-PCR . The obtained cDNA was stored at −20°C until further use. All experiments were carried out in compliance with the guidelines for the care and use of laboratory animals of the Tokyo University of Marine Science and Technology.Live . 2.2ωx desaturase sequences identified by Kabeya et al. [ωx1 (NCBI acc. No. JW524768) and ωx2 (GBTC01004759), further genes encoding PUFA biosynthesizing enzymes including putative front-end desaturases and fatty acyl elongases were retrieved from the T. californicus transcriptome shotgun assembly (NCBI BioProject No. PRJNA158547 and PRJNA263967) by DELTA-BLAST. Several functionally characterized front-end desaturases and fatty acyl elongases from invertebrates including molluscs and echinoderms were used as queries [Fed1 to Fed5 and Elo1 to Elo5, respectively. After this search, a new transcriptomic dataset became available at NCBI (No. PRJNA504307). Thus, the same search against the new database was repeated and an additional elongase-like sequence was successfully obtained, which was named as Elo6. We then performed blastn search against T. californicus genome assembly  and confirmed all five front-end desaturase and six fatty acyl elongase sequences were successfully found in the T. californicus genome. Apart from the T. californicus ωx1 (JW524768) and ωx2 (GBTC01004759) found by Kabeya et al. [ωx desaturases were identified in the T. californicus genome.In addition to the two a et al. , termed  queries –43. The a et al. , no furt. 2.3ωx desaturases including the two of T. californicus was carried out by Kabeya et al. [--reorder --auto), and removal of all duplicated sequences and incomplete gappy sequences. A similar strategy was applied for elongases as follows: (i) retrieval of all metazoan aa sequences from RefSeq-specific protein-containing ‘ELO’ domain (pfam01151); (ii) and (iii) were the same as above and (iv) generation of cleaned MSA as described above and removal of potential non-PUFA elongase sequences. Each dataset was then aligned using MAFFT v. 7 [--genafpair --maxiterate 1000) and filtered through using GUIDANCE v. 2.0 [--all) [--model LG+I+G4) [https://itol.embl.de).Phylogenetic trees comparing the deduced amino acid (aa) sequences of front-end desaturases and fatty acyl elongases from a wide range of eukaryotic organisms were constructed using the maximum-likelihood (ML) method . A compra et al.  and henca et al.  with FFTFFT v. 7  with E-IE v. 2.0  with 100E v. 2.0 . The fin [--all) . The proI+G4) . The res. 2.4T. californicus ωx desaturases, front-end desaturases and fatty acyl elongases were amplified by polymerase chain reactions (PCR) using a high-fidelity DNA polymerase . All PCR runs were carried out following the manufacturer's default recommendations as follows: 10 s at 98°C for the initial denaturation, 35 cycles of 10 s at 98°C, 5 s at 55°C and 20 s at 72°C. The primer sequences containing restriction enzyme sites to enable further cloning into the yeast expression vector pYES2 (Thermo Fisher Scientific) are given in α competent E. coli . Positive transformant colonies were grown overnight in LB broth, and plasmid preparations  were sent to the DNA sequencing service  to confirm their sequences. Plasmid constructs of the corresponding T. californicus two ωx desaturases, five front-end desaturases and six fatty acyl elongases were individually transformed into INVSc1 yeast Saccharomyces cerevisiae  using the S.c. EasyComp yeast transformation kit (Life Technologies Japan). After growing the transformed yeast on S. cerevisiae minimal medium minus uracil (SCMM−ura) plates for 3 days at 30°C, one of the successful transformants from each gene was selected and used for the functional characterization assay as described below.The full-length ORF of the −ura broth and diluted to OD600 = 0.4 in one single Erlenmeyer flask for each potential substrate assayed. After the OD600 reached 1, the cultures were supplemented with 2% (w/v) galactose for the induction of transgene expression as well as with one of the potential PUFA substrates for each enzyme [ωx desaturases, the exogenously supplied PUFA were 18 : 2n–6, 18 : 3n–6, 20 : 2n–6, 20 : 3n–6, 20 : 4n–6, 22 : 4n–6 and 22 : 5n–6. For front-end desaturases, PUFA substrates included 18 : 2n–6, 20 : 2n–6, 20 : 3n–6, 22 : 4n–6, 18 : 3n–3, 20 : 3n–3, 20 : 4n–3 and 22 : 5n–3, and for elongases exogenously supplied PUFA substrates were 18 : 2n–6, 18 : 3n–6, 20 : 4n–6, 22 : 4n–6, 18 : 3n–3, 18 : 4n–3, 20 : 5n–3 and 22 : 5n–3. Since certain ωx desaturases have shown the capacity to desaturate yeast endogenous FA [T. californicus ωx desaturases were grown in triplicate Erlenmeyer flasks in the absence of exogenously added FA substrates and their FA profiles compared with those of control yeast transformed with empty pYES2 vector . Additionally, to test Δ6 desaturase activity towards 24 : 5n–3, transgenic yeast co-expressing the zebrafish elovl2 [T. californicus fed gene were grown in the presence of 22 : 5n–3 following the method established by Oboh et al. [18), 0.75 mM (C20) and 1.0 mM (C22) to compensate for the reduced uptake efficiency with the length of the carbon chain [g), washed twice in double distilled water, and lyophilized prior to preparation of fatty acid methyl ester (FAME) derivatives. FAME was prepared using Fatty Acid Methyl Ester Preparation Kit  following the manufacturer's recommendations. All fatty acids were purchased from Nu-Chek Prep, Inc. , except 18 : 4n–3 from Larodan AB  and 20 : 4n–3 from Cayman Chemicals .Each obtained transformant was individually grown in SCMMh enzyme . For ωx h elovl2  and eachh et al. . Each FAon chain . After 4. 2.5−1, 180°C to 230°C at a rate of 1°C min−1 and then 230°C for 10 min. The injection port and FID temperature were 250°C and 280°C, respectively. Helium was used as a carrier gas in constant velocity mode (30 cm sec−1). FAME was identified by the comparison of their retention times with those from commercial FAME standards. The conversion efficiency of three types of assayed enzymes towards the exogenously supplied PUFA substrates was calculated with the formula ) × 100 [FAME samples were injected on a GC-2025  gas chromatograph equipped with a capillary column  and a flame ionization detector (FID). The temperature conditions consisted of an initial ramp from 50°C to 180°C at a rate of 40°C min. 2.6n = 3) and transgenic yeast expressing the T. californicus ωx desaturases (n = 3) were carried out using Dunnett's test with p < 0.05 indicating statistical significance. The analysis was performed in R 3.4.1. (www.r-project.org).Comparisons of means of FA composition between control yeast , Caligus rogercresseyi (Siphonostomatoida) and Paracyclopina nana (Cyclopoida). A direct sister group of the copepod clade could not be clearly identified due to insufficient resolution of the tree. However, the copepod clade clustered (bootstrap = 82%) with several taxonomically unrelated species belonging to the classes Kinetoplastea , Choanoflagellata  and Haptophyta . This clade was further out-grouped with another containing sequences from Sphaeroforma arctica (Ichthyosporea), Guillardia theta (Cryptophyceae) and two Bacillariophyta diatoms, namely Phaeodactylum tricornutum and Thalassiosira pseudonana. Interestingly, front-end desaturases from other metazoan species including Vertebrata, Echinodermata and Mollusca grouped together in a clearly separated clade (bootstrap = 100%) from that containing the copepod sequences , it included several insects , a collembola (Folsomia candida), and crustaceans such as Decapoda , Branchiopoda (Daphnia pulex) and Amphipoda   containing several Arthropoda sequences including functionally characterized Elovl4-like sequences from two decapods (S. olivacea and Portunus trituberculatus) [T. californicus Elo4 was grouped in a completely distinct clade that out-grouped all other sequences  from tic tree . Within  azteca) . Interesablished . Away frculatus) ,53  and Δ15 (conversion of 18 : 2n–6 to 18 : 3n–3) desaturase activities  but not the C22 (22 : 4n–6 and 22 : 5n–6), into the corresponding n–3 products , transgenic yeast were grown in the presence of exogenously added desaturase substrates, namely n–6  and n–3 PUFA . Our results showed that, with the exception of the Fed1 and Fed3 that share Δ6 desaturase, each front-end desaturase characterized from T. californicus has a specific substrate preference. As mentioned above, Fed1 and Fed3 exhibited Δ6 desaturase activity since both enzymes were able to convert 18 : 2n–6 and 18 : 3n–3 into 18 : 3n–6 and 18 : 4n–3, respectively  grown in the presence of exogenously added PUFA substrates, namely n–6  and n–3 . All elongases except Elo6 showed activity towards all C18 and C20 substrates, but not towards C22 (18 and C20 assayed (18 PUFA (highest 2.2% towards 18 : 3n–3) and high for C20   and P. nana (Cyclopoida). These results suggest that the copepod front-end desaturase genes evolved from their common ancestor. Interestingly, the front-end desaturase sequences retrieved from T. californicus and other copepods are phylogenetically unrelated with decapod crustacean desaturases hypothesized to be Δ6 front-end desaturases [ωx desaturases [Leishmania major and P. lutheri, both characterized as Δ4 desaturases and thus playing key roles in DHA synthesis from 22 : 5n–3 [A comprehensive phylogenetic analysis of a et al.  depictedrotifers . In the aturases –60. Unliaturases –60. Indeaturases ,13). Thiaturases ,61 and oaturases  in anima2 : 5n–3 ,64. The T. californicus formed a well-supported clade with other Pancrustacea sequences. This Pancrustacea-specific clade includes the Elovl7-like elongase isolated from S. olivacea, which showed elongation capability on a range of PUFA [S. olivacea and P. trituberculatus [T. californicus, one elongase gene (Elo4) clustered within the most diverged clade, which was an out-group of all other elongase sequences. Since Elo4 showed very low activity towards all PUFA tested in the present study, it is reasonable to speculate that Elo4 does not play a prominent role in PUFA elongation in T. californicus.The ML phylogenetic analysis of elongases denoted that four out of six genes  isolated from  of PUFA . The Pan of PUFA ,66,67. M of PUFA  and cuti of PUFA . Elo6 berculatus ,53 (figu18 n–3 PUFA ALA (18 : 3n–3), a phenomenon often referred to as ‘trophic up-grading’ [T. californicus ωx desaturases studied here showed that harpacticoid copepods can efficiently produce ALA and, hence, play such a pivotal role in marine ecosystems. Indeed, the T. californicus ωx desaturases contain key enzymatic activities including Δ12 (ωx1) and Δ15 (ωx1 and ωx2) required to convert oleic acid (18 : 1n–9) into LA (18 : 2n–6) and ALA (18 : 3n–3), respectively  and EPA from the C18 PUFA LA and ALA, respectively  to DHA .  in rats , fish [5 in rats ,76,77 annstricta , and thenstricta  (figure ) to DHA . None ofcroalgae –33.ωx desaturases, five front-end desaturases and six elongases from T. californicus. The T. californicus ωx desaturases are ω3 desaturases enabling (i) the biosynthesis of LA and ALA that are precursors of LC-PUFA and (ii) the conversion of multiple n–6 PUFA into the corresponding n–3 metabolic products including LC-PUFA. Upgrading from the C18 PUFA LA and ALA to LC-PUFA is also possible in T. californicus, since the complementary action of its front-end desaturases and elongases enable multiple routes for the biosynthesis of the physiologically active compounds ARA, EPA and DHA. Collectively, the results obtained in this study demonstrate that T. californicus has a complete enzymatic complement enabling this species to produce n–3 LC-PUFA up to DHA endogenously. Therefore, harpacticoid copepods arise as primary producers of n–3 LC-PUFA in marine ecosystems and, given their widespread distribution and abundance at a global scale, it is likely that such contribution is not negligible.In conclusion, we have successfully isolated and functionally characterized two


Several central nervous system diseases are associated with disturbed cerebrospinal fluid (CSF) flow patterns and have typically been characterized in vivo by phase-contrast magnetic resonance imaging (MRI). This technique is, however, limited by its applicability in space and time. Phase-contrast MRI has yet to be compared directly with CSF tracer enhanced imaging, which can be considered gold standard for assessing long-term CSF flow dynamics within the intracranial compartment.Here, we studied patients with various CSF disorders and compared MRI biomarkers of CSF space anatomy and phase-contrast MRI at level of the aqueduct and cranio-cervical junction with dynamic intrathecal contrast-enhanced MRI using the contrast agent gadobutrol as CSF tracer. Tracer enrichment of cerebral ventricles was graded 0–4 by visual assessment. An intracranial pressure (ICP) score was used as surrogate marker of intracranial compliance.The study included 94 patients and disclosed marked variation of CSF flow measures across disease categories. The grade of supra-aqueductal reflux of tracer varied, with strong reflux (grades 3–4) in half of patients. Ventricular tracer reflux correlated with stroke volume and aqueductal CSF pressure gradient. CSF flow in the cerebral aqueduct was retrograde (from 4th to 3rd ventricle) in one third of patients, with estimated CSF net flow volume about 1.0 L/24 h. In the cranio-cervical junction, net flow was cranially directed in 78% patients, with estimated CSF net flow volume about 4.7 L/24 h.The present observations provide in vivo quantitative evidence for substantial variation in direction and magnitude of CSF flow, with re-direction of aqueductal flow in communicating hydrocephalus, and significant extra-cranial CSF production. The grading of ventricular reflux of tracer shows promise as a clinical useful method to assess CSF flow pattern disturbances in patients.The online version contains supplementary material available at 10.1186/s12987-021-00251-6. Over the last few years, the impact of cerebrospinal fluid (CSF) homeostasis for brain function has gained renewed interest, not least facilitated by the description of the glymphatic system in 2012 . These dRecently, several reviews have addressed the lack of consensus regarding magnitude and direction of CSF flow in man , 6–9. A This study examined patients with different CSF disorders with regard to how CSF tracer moves from the subarachnoid space into cerebral ventricles, and the direction and magnitude of CSF flow in the cerebral aqueduct and at the cranio-cervical junction. Two magnetic resonance imaging (MRI) modalities were used, namely dynamic intrathecal contrast-enhanced MRI with an MRI contrast agent serving as a CSF tracer for assessment of long-term flow patterns, and phase-contrast MRI, which assesses CSF flow velocity and direction through a much narrower window in time and space. The observations disclose marked variation between patient categories concerning retrograde transport of tracers from subarachnoid spaces to supra-aqueductal ventricles (referred to as ventricular reflux of tracer), and in the direction and magnitude of CSF flow in cerebral aqueduct and cranio-cervical junction. Retrograde CSF flow from 4th to 3rd ventricle was typical for communicating hydrocephalus. Moreover, the present results suggest that the MRI-derived quantitative data may be described by qualitative assessment, using a grading system for supra-aqueductal ventricular tracer reflux that is feasible in daily clinical context .This study was approved by the Regional Committee for Medical and Health Research Ethics (REK) of Health Region South-East, Norway (2015/96), the Institutional Review Board of Oslo university hospital (2015/1868) and by the National Medicines Agency (15/04932-7).The study included consecutive patients undergoing intrathecal contrast-enhanced MRI and phase-contrast MRI, as part of their neurosurgical work-up of various CSF disorders within the Department of Neurosurgery at Oslo University Hospital, Norway during the period October 2015 to June 2018. They were included after written and oral informed consent. The study was conducted according to ethical standards according to the Helsinki Declaration of 1975 (and as revised in 1983). Exclusion criteria were either a history of hypersensitivity reactions to contrast media agents, a history of severe allergy reactions in general, evidence of renal dysfunction , age < 18 or > 80 years, pregnant or breastfeeding women.The MRI protocol has previously been described , 15, andT1-weighted MRI was used for assessment of CSF space anatomy. We used axially reconstructed images (1 mm thickness) for estimation of Evans’ index  . CoronalThe protocol used for intrathecal contrast enhanced MRI has previously been described in detail , 15. Idehttp://surfer.nmr.mgh.harvard.edu/) was used for segmentation, parcellation and registration/alignment of the longitudinal data to assess the increase of T1 intensity due to CSF tracer within the cerebral ventricles; for methods description, see [ T1 signal units and adjusts for the baseline image grey scale changes between scans.With regard to image analysis, the FreeSurfer software (version 6.0) , as previously described in more detail .Volumetric flow rate The positive and negative contributions of the volumetric flow rate were calculated separately in the same manner, and the positive and negative velocities were extracted before the calculation. Thereby, net volume over one cycle was calculated by discrete integration  of Volumes over one cycle in cranial and caudal direction were calculated by integration of positive and negative volume fluxes over time. The CSF volumetric net flow rate during one cycle (ml/cycle) was determined by the sum of the positive and negative CSF flux.With regard to estimating CSF flow volume over 24 h, the daily CSF volumetric net flow rate, expressed in liter (L) over 24 h was estimated by multiplying the CSF net flow volume over one cardiac cycle with the heart rate (HR) and then multiplied with 1440 (min/day). The HR was determined over the MRI scan time that was 6 min.Aq-CCJ-ratio):Finally, we calculated the ratio between the retrograde CSF volumetric net flow rate per cardiac cycle at the cerebral aqueduct versus the CCJ cranial net flow rate per cycle, according to this formula , defined asWithin the Department of neurosurgery, overnight monitoring of pulsatile ICP is utilized as a surrogate marker of the intracranial compliance, i.e., pressure–volume reserve capacity  and the The statistical analysis was performed using the SPSS software version 26 . Continuous data are presented as mean with standard deviation, and categorical data as numbers and percentages. Difference between patient groups versus the REF cohort was determined by independent samples t-test, and differences between categorical data were determined by the Pearson Chi-square test. Pearson correlation coefficients were determined to examine associations between different variables. All observations for a specific variable were used when determining the correlation coefficients. Statistical significance was accepted at the 0.05 level (two-tailed).The study included 94 consecutive individuals who underwent a standardized MRI protocol, incorporating both intrathecal contrast enhanced MRI and phase-contrast MRI, during the period October 2015 to June 2018. Twenty-four individuals were categorized as reference (REF) patients where no apparent CSF disturbance was eventually diagnosed, and 70 patients with CSF disorders, including idiopathic normal pressure hydrocephalus , communicating hydrocephalus , spontaneous intracranial hypotension , arachnoid cyst , pineal cyst  and idiopathic intracranial hypertension , in whom a Reflux Grade of 3–4 was observed in 46/92 (50%) patients  and absolute net flow per cycle was increased with reflux grade 4 as compared with grades 0–2 . Moreover, we found a significant positive correlation between magnitudes of total aqueductal flow and enrichment of tracer in 3rd ventricle at 6 h  and 24 h , and a significant positive correlation between total aqueductal flow and enrichment of tracer in lateral ventricle at 6 h  and 24 h  and cHC patients  or the cranio-cervical junction  and 24 h , and with enrichment of lateral ventricles after 6 h  and 24 h , but not with mean ICP   is non-directional, i.e. the direction of the gradient cannot be determined from the present observations. An increasing pressure gradient (dP) was accompanied with more abnormal measures of the other independent variables of ventricular tracer transport and CSF flow. We found a positive correlation between the pressure-gradient (dP) and the degree of tracer enrichment in the 3rd ventricle Fig. a–b and lThe aqueductal pressure gradient (dP) also was associated with the MRI biomarkers of CSF space anatomy, namely Evans’ index  and callosal angle  of the cranio-cervical junction was not associated with supra-aqueductal ventricular tracer enrichment, ventricular reflux grade, ICP scores (MWA or mean ICP), or MRI biomarkers of CSF space anatomy  (data not shown).The present observations disclosed marked variation in direction and magnitude of CSF flow within and between patient groups with evidence of re-direction of CSF flow towards ventricles in individuals with communicating hydrocephalus. The phase-contrast MRI data further suggest a significant extra-cranial CSF contribution to the water component of CSF.We addressed neurological diseases characterized by different types of disturbed CSF homeostasis. Obstruction to CSF efflux is considered a main mechanism behind e.g. idiopathic normal pressure hydrocephalus (iNPH) and other communicating hydrocephalus (cHC) conditions, and is the rationale behind CSF diversion surgery (shunt surgery) . In sponAs CSF tracer, we used the MRI contrast agent gadobutrol, which is a hydrophilic molecule of about 604 Da . It shouSince MRI based quantitative estimation of tracer enrichment would require acquisition of T1 maps, and CSF flow at phase-contrast MRI requires sophisticated post-processing, not easily performed in daily clinical practice, we recently introduced the qualitative grading of ventricular tracer reflux, based on visual inspection of intrathecal contrast enhanced MRI acquisitions . VentricWe also found that higher reflux grades corresponded to higher aqueductal pressure-gradient (dP) as well as increased overnight scores of the pulsatile ICP score mean ICP wave amplitude (MWA), the latter constituting a surrogate marker of impaired intracranial compliance, i.e. impaired intracranial pressure–volume reserve capacity . This fiSince iNPH patients were older than REF patients, the ventricular reflux grades 3–4 in iNPH could potentially be attributed to age alone. On the other hand, the cohort with communicating hydrocephalus was considerably younger and comparable with the references and those with obstruction of subarachnoid CSF, indicating that high degree of reflux in communicating hydrocephalus is not merely an age-related phenomenon. Further studies are required to examine whether degree of ventricular reflux of tracer tends to increase with age.The magnitude and direction of CSF flow in the cerebral aqueduct and the cranio-cervical junction vary substantially within and between patients groups. We found antegrade net CSF flow within the cerebral aqueduct in the majority of REF patients (87%), while a higher proportion of retrograde net CSF flow was seen in some CNS diseases, such as communicating hydrocephalus. Among the 85 patients with aqueductal flow measurements, one third of patients presented with net retrograde aqueductal flow, with average net retrograde CSF flow volumes 1.039 ± 1.642 L/24 h. We previously reported retrograde aqueductal flow in 14/21 iNPH patients (estimated average net retrograde flow 1.1 ± 2.2 L/24 h) , and in 3) and the average calculated from all pixels within the region of interest. Moreover, we corrected for background velocity offset, which may be machine-dependent [A methodological strength with our approach of determining flow velocities is that CSF flow was calculated pixel-by-pixel level, i.e. flow velocity was determined for each individual pixel , mediating a significant net extra-choroidal formation of CSF water, is abundant in brain and particularly the spinal cord tissue , where pcardiac-gated phase-contrast MRI during daytime in wake individuals lying flat. The cardiac-gated phase-contrast MRI estimates net flow induced by the heartbeat. These volumes are, however, not synonymous with the total CSF production. It is well established that cardiac gated phase-contrast MRI does not incorporate CSF movements induced by respiration, which contributes significantly [2O) in humans and animals, reported free and constant exchange of water between blood, brain and CSF. He gave evidence for a CSF formation rate > 22 ml/min, i.e. > 70 times more than an estimate production rate of 0.3 ml/min.We would like to stress that the presently reported volumes of CSF flow in cerebral aqueduct and cranio-cervical junction are derived from ficantly . In a moficantly . In humaficantly , 49. In ficantly . Age andficantly . The volficantly , based oSome limitations should be noted. First, intrathecal gadobutrol is given off-label, which represents one important limitation with this method. We have, however, good experience regarding the safety of intrathecal gadobutrol in a dose of 0.5 mmol , 54. MorOne limitation with the phase-contrast MRI methodology is the accuracy of calculation of very small volumes, which is related to variability of physiological measures such as heart rate. While retrospective cardiac gating has been shown more accurate than prospective cardiac gating as it enables continuous measurements throughout the cardiac cycle , the floAnother limitation should be noted regarding our estimation of CSF flow volumes over 24 h from measurements obtained over a few minutes. Obviously, these estimates are subject to uncertainty. On the other hand, it would be expected that physiology is rather stable.The direction and magnitude of CSF flow in cerebral aqueduct and cranio-cervical junction vary considerably across disease categories, with re-direction of CSF flow towards ventricles characterizing communicating hydrocephalus. Our observations add to the increasing body of evidence that the traditional concept of CSF homeostasis, represented by the third circulation, represents an over-simplification. A significant part of the CSF water component may be produced extra-cranially. While quantification of CSF flow by using phase-contrast MRI or CSF tracer enrichment require sophisticate post-processing, a qualitative assessment of ventricular tracer reflux grade may have better utility in clinical practice.Additionalfile1: Figure S1: Examples of phase-contrast MRI of the cerebral aqueduct. For different patients, the regions of interest within the CSF of the cerebral aqueduct is shown in red and the reference regions of interest in blue . The CSF flow velocity is shown for the different pixels with mean flow velocity of all pixels indicated by a dark line, including noise level of pixels in reference tissue (colored lines) with mean noise level indicated by dark stippled line . The velocities from each pixel were assessed using MATLAB, and expressed as centimeters per second. Positive values refer to cranial CSF flow direction and negative values caudal CSF flow direction. FigureS2: Examples of phase-contrast MRI of the cranio-cervical junction. For different patients, the regions of interest within the CSF of the cranio-cervical junction is shown in red and the reference regions of interest in blue . The CSF flow velocity is shown for the different pixels with mean flow velocity of all pixels indicated by a dark line, including noise level of pixels in reference tissue (colored lines) with mean noise level indicated by dark stippled line . The velocities from each pixel were assessed using MATLAB, and expressed as centimeters per second. Positive values refer to cranial CSF flow direction and negative values caudal CSF flow direction. FigureS3: Association between the mean ICP wave amplitude (MWA) and enrichment of tracer within third and lateral ventricles. There was a significant positive correlation between the mean ICP wave amplitude (MWA) measured over-night and tracer enrichment within 3rd ventricle after 6 hours  and 24 hours , and between MWA and tracer enrichment within lateral ventricles after 6 hours  and 24 hours . Each plot shows the fit line with Pearson correlation coefficients and significance levels. FigureS4: Association between the callosal angle and enrichment of tracer within third and lateral ventricles. There was a significant negative correlation between the callosal angle and tracer enrichment within 3rd ventricle after 6 hours  and 24 hours , and between callosal angle and tracer enrichment within lateral ventricles after 6 hours  and 24 hours . Each plot shows the fit line with Pearson correlation coefficients and significance levels. FigureS5: Association between the total aqueductal flow per cycle and enrichment of tracer within third and lateral ventricles. There was a significant positive correlation between the total aqueductal flow per cycle and tracer enrichment within 3rd ventricle after 6 hours  and 24 hours , and between total aqueductal flow per cycle and tracer enrichment within lateral ventricles after 6 hours  and 24 hours . Each plot shows the fit line with Pearson correlation coefficients and significance levels. FigureS6: Association between the total flow and net flow per cycle in cerebral aqueduct and craniocervical junction. There was no correlation between the total flow and net flow per cycle in (a) the cerebral aqueduct (n=85) and (b) the cranio-cervical junction (n=32). Each plot shows the fit line with Pearson correlation coefficients and significance levels. FigureS7: Association between the CSFAq-CCJ-Ratio and enrichment of tracer within third and lateral ventricles. There was a significant positive correlation between the CSFAq-CCJ-Ratio and tracer enrichment within 3rd ventricle after 6 hours  and 24 hours , and between the CSFAq-CCJ-Ratio and tracer enrichment within lateral ventricles after 6 hours  and 24 hours . Each plot shows the fit line with Pearson correlation coefficients and significance levels. FigureS8: Association between the CSFAq-CCJ-Ratio and the mean ICP wave amplitude  but no correlation with the mean ICP . The CSFAq-CCJ-Ratio was positively correlated with the pressure gradient (dP) at the cerebral aqueduct , but not with the pressure gradient (dP) in the craniocervical junction . Each plot shows the fit line with Pearson correlation coefficients and significance levels. FigureS9: Association between the pressure gradient in the cerebral aqueduct and the biomarkers of CSF space Evan’s index and callosal angle. There was a significant positive correlation between the pressure gradient (dP) at the cerebral aqueduct and the Evan’s index , and a significant negative correlation between the pressure gradient (dP) at the cerebral aqueduct and the callosal angle . Each plot shows the fit line with Pearson correlation coefficients and significance levels.


To explore the feasibility of single-position laparoscopic radical nephrectomy (LRN) and tumor thrombectomy for left renal cell carcinoma with high-risk Mayo 0 and 1 tumor thrombus (TT).All patients with left renal cell carcinoma and venous TT (high-risk Mayo grade 0 and 1) who were performed single-position LRN and tumor thrombectomy were involved. After the renal artery was controlled by Hem-o-lok, the left renal vein was dissected through descending colon mesentery. The left renal vein was divided by EndoGIA for high-risk Mayo grade 0 TT. For Mayo grade 1 TT, part of the inferior vena cava was blocked by a bulldog clamp after milking the TT into the left renal vein and the inferior vena cava was sutured after complete excision of the TT.3 patients were involved and operations were performed successfully without conversion to open surgery. The mean operation time was 136 min and the mean estimated blood loss was 60 mL. No postoperative complications occurred.It is feasible to control left renal vein and partial inferior vena cava through descending colon mesentery in a single position during LRN and tumor thrombectomy for the treatment of high-risk Mayo grade 0 and 1 TT. Invasion of the venous system is one of the clinical features of locally advanced kidney cancer. Approximately 4–10% of locally advanced renal cell carcinomas have venous tumor thrombus (TT) . In receThe aim of the study was to explore the feasibility of single position transperitoneal laparoscopic radical nephrectomy (LRN) and high-risk Mayo 0 and 1 tumor thrombectomy and the surgical techniques were described in detail.All patients with left renal cell carcinoma and venous TT (Mayo grade 0–1) who were performed single-position LRN and tumor thrombectomy were involved. The 3 enrolled patients were males, aged 54–57 years old, with a median age of 57 years old. Urinary tract ultrasound, enhanced MRI, MRA, chest CT and bone scan were performed before surgery to determine the location, size and metastasis of the tumor. The clinicopathological data are outlined in Table Case 1 imaging examination revealed a left kidney tumor with venous TT (Mayo stage 1) and multiple lung metastases. The kidney biopsy confirmed clear renal cell carcinoma with poorly differentiated and accompanied by necrosis. Two cycles of axitinib combined with PD-1 inhibitor were provided for neoadjuvant treatment.The imaging examinations of case 2 showed a left kidney tumor with venous TT (Mayo grade 1), accompanied by left adrenal gland metastasis. The pathology from kidney biopsy demonstrated clear renal cell carcinoma and the patient received 2 cycles of axitinib for neoadjuvant treatment.The imaging examination of case 3 showed left kidney tumor with retroperitoneal lymphadenopathy, left renal vein TT (Mayo grade 0) and multiple lung metastases. The kidney biopsy showed clear renal clear cell carcinoma. The TT shrunk to high-risk Mayo grade 0 after 3 months neoadjuvant treatment of sintilimab and axitinib.After successful anesthesia, the patient was placed in a supine position obliquely 70° on the right side, fixed with a lumbar pillow, and raised the lumbar bridge. The position of the trocar was shown in Fig. The operation is performed according to the principle of treating the artery first and then treating the vein. The specific method is as follows after the left descending colon and part of the sigmoid colon lateral ligament were incised, the small intestine was pulled to the mid-abdomen. It’s worth noting that to avoid the loss of mesangial blood vessels during dissociation. A dense connective tissue can be seen in the middle of the dorsal medial side of the kidney, and the arterial pulsation can be seen under it. After open the connective tissue and the left renal vein could be seen. At the same time, the adrenal vein and reproductive vein are disconnected when separating the renal vein. The laparoscopic interventional ultrasound was used to detect the end of the TT, then the left renal vein was divided by EndoGIA for Mayo grade 0 TT and part of the inferior vena cava was blocked by a bulldog clamp after milking the thrombus into renal vein for Mayo grade 1 TT. After completely remove the TT, the inferior vena cava was sutured and finally radical nephrectomy was performed. It should be noted that when handling the inferior vena cava, we usually clamp the root of the left renal vein with a bulldog clamp. We only clamp a part of the wall of the inferior vena cava, which will not affect the return of the inferior vena cava and lumbar vein. Therefore, there is no need to deliberately isolate the lumbar vein and cut it off.All operations were successfully completed without transfer to open surgery, and no postoperative complications occurred. The mean operation time was 136 min (45–196) min and the mean estimated blood loss was 60 ml (30–100) mL. The normal diet was restored on postoperative two.No complications such as fever, intestinal obstruction, intestinal fistula, and urine leakage occurred. The wound recovered well when the patient was discharged from the hospital, and there was no wound infection. There were no complications such as bleeding and pulmonary embolism during the perioperative period.The postoperative pathology of two cases showed the tumors were clear renal cell carcinoma (postoperative pathological gross specimen was shown in Fig. Invasion of the venous system is one of the clinical features of locally advanced renal cancer, including invasion of the renal vein and inferior vena cava. The aim of the study was exploring the feasibility and safety of single-position LRN and tumor thrombectomy for left renal cell carcinoma with high-risk Mayo 0 and 1 TT. In short, the operation in our study was completed in a single position without changing the position. The surgical procedures were reasonable and the techniques could be quickly mastered, saving anesthesia and operation time. The surgical outcomes were encouraging including small trauma, quick postoperative recovery, short hospital stay, and few postoperative complications.For the left renal cell carcinoma with high-risk Mayo 0 and 1 TT, the traditional robot-assisted/laparoscopic surgical method is to remove the affected kidney, then change the position and remove the TT. Another method is performing embolization of the left renal artery before the operation and treating the inferior vena cava and TT during the operation time, then change the position to remove the left kidney. Using traditional method increases the risk of surgery such as damaging the mesangial blood vessels. At the same time, anesthesia and operation time are prolonged. Open surgery, the standard approach for treating inferior vena cava thrombus, is technically complex, has high complication rates, and requires multidisciplinary cooperation . FurtherIn 2009, our team took the lead in completing and reporting retroperitoneal LRN and tumor thrombectomy and summing up experience. In 2012, we completed the first complete LRN and grade II vena cava tumor thrombectomy in China. In view of the fact that the transperitoneal route has a greater impact on the gastrointestinal tract and the patient’s recovery is slow after surgery, in 2014, the retroperitoneal route of radical nephrectomy plus the removal of grade II inferior vena cava TT was completed for the first time in the world . Based oThe key points and techniques of this study are as follows: 1. Reasonably design the patient’s position and scientifically place the Trocar position to facilitate the operation and cooperation of the surgeon and assistant. The patient’s position and Trocar position are the key to the success or failure of a single-position operation; 2. The method of finding and processing the renal pedicles using laparoscopic procedures: For the left renal pedicle, the kidney can be pushed to the ventral side with a vascular clamp. A dense connective tissue can be seen in the middle of the dorsal medial side of the kidney, and arterial pulses can be seen below to open the connective tissue. The left renal artery can be seen . After tThe height of the TT determines the specific treatment strategy, especially the surgical strategy, so a grading system based on the height of the TT came into being. Mayo Clinic’s five-level classification of tumor thrombi has been widely used in clinical work . The 3 cIn laparoscopic renal vein/inferior vena cava TT removal surgery, because we cannot accurately determine the position of the end of the TT with the naked eye, it is very dangerous to blindly test before blocking the renal vein or inferior vena cava which increasing the risk of TT shedding. As mentioned earlier, laparoscopic ultrasonography is used in this operation to detect the location of the TT in the vein, determine the size of the TT, and detect the specific position of the end of the TT to determine how to block the vein –10. AfteThe use of neoadjuvant systemic therapy to reduce locally advanced tumors to promote surgical resection and improve survival is used in various malignant tumors . The rapIn summary, compared with traditional surgical methods, the single-position LRN and tumor thrombectomy for left renal cell carcinoma with high-risk Mayo 0 and 1 TT through abdominal cavity has the advantages like standardized methods, simple steps, less trauma, less bleeding, and quick postoperative recovery. It is a safe and effective minimally invasive technique for the treatment of renal cell carcinoma with venous TT. We initially discussed the safety and feasibility of this surgical method. However, due to the small number of clinical samples, patients and operations involved are summarized by a single center, its technology promotion, clinical efficacy and prognostic evaluation need to be further verified by multi-center, large sample size and long-term follow-up. Therefore, it is recommended to carry out a comparative study with classic laparoscopic procedures to explore the advantages of different procedures.


A greater proportion of glycolytic muscle fibers is a manifestation of skeletal muscle dysfunction in Chronic Obstructive Pulmonary Disease (COPD). Here, we propose to use the spectral analysis of the electromyographic signal as a non-invasive approach to investigate the fiber muscle composition in COPD. We recorded the electromyographic activity of Rectus Femoris (RF), Vastus Lateralis (VL), Vastus Medialis (VM) and Biceps Femoris (BF) muscles, in ten patients and ten healthy individuals, during non-fatiguing, flexion–extension leg movements. The mean (MNF) and median frequencies (MDF) were calculated, and the most common profiles of electromyographic power spectrum were characterized by using the principal component analysis. Frequency parameters showed higher values in patients with COPD than in the control group for the RF , VL  and VM  muscles during the extension movements and for the BF  muscle during flexion movements. Spectrum profiles of the COPD patients shifted towards the higher frequencies, and the changes in frequency parameters were correlated with the level of disease severity. This shift of frequencies may indicate an increase in glycolytic muscle fibers in patients with COPD. These results, along with the non-fatigable nature of the motor task and the adoption of a non-invasive method, encourage to use electromyographic spectral analysis for estimating muscle fiber composition in patients with COPD. Dysfunction of skeletal muscles is a relevant manifestation of Chronic Obstructive Pulmonary Disease (COPD) that affects motor function and limits the ability to carry out habitual activities of daily life ,2,3,4,5.While muscle strength and endurance can be assessed by non-invasive tests, such as incremental isometric contractions or maximized walking performance within a time-interval ,5, histoA typical application of sEMG is to evaluate time-domain parameters, such as amplitude or latency of electrical muscle activation. Conversely, the analysis of spectral content of the sEMG signal provides frequency-domain parameters by which the muscle fiber composition can be estimated. In fact, functional relationships between fiber type composition and response in spectral content of the sEMG have been documented by comparing changes in frequency bandwidth  with histochemical measurements of muscle fiber type composition  or analyMuscle biopsies revealed that patients with COPD show a high proportion of type II fibers ,10,11,18To date, there is a lack of literature on easy-to-administer ways of identifying and quantifying the disease-induced changes in sEMG frequency during non-fatiguing movement tasks. In the current study, the sEMG power spectrum was obtained from leg flexor and extensor muscles during simple, non-fatiguing knee flexion–extension movements. To test whether this motor task is feasible to show a shift of the sEMG power spectrum toward high frequencies, the mean frequency (MNF) and median frequency (MDF) were computed. Moreover, a characterization of the shape of spectrum profiles was performed by means of principal component analysis (PCA). Finally, we tested the sensitivity of the measured frequency-domain features with respect to the level of disease severity. p = 0.801), height (p = 0.851) and weight (p = 0.835).Ten non-hospitalized patients with moderate to severe COPD, under medical supervision at the Respiratory Medicine Unit, Policlinico Universitario di Catania , were recruited for this study. Ten healthy subjects, with anthropometric characteristics matching those of the patients, participated as a control group. In LCO) and the degree of the sensation of shortness of breath in relation to the accomplishment of common daily activities estimated by the modified Medical Research Council Questionnaire (mMRC). In The criteria of inclusion to participate to this study were the following: diagnosis of COPD according to Chronic Obstructive Lung Disease (GOLD) criteria, no disease exacerbation within the four weeks prior to the beginning of the study. The fixed exclusion criteria were presence of heart diseases, musculoskeletal traumas and/or rheumatologic diseases. The diagnosis of COPD was based on instrumental investigations such as chest X-ray and most common lung function tests. The tests measured the following parameters: forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1/FVC ratio, the diffusing capacity for carbon monoxide  to remove low-frequency artifacts produced by electrode movements. After full-wave rectification of the raw sEMG signal, the average level of sEMG background recorded 500 ms before the onset of the first extension was subtracted from the sEMG activity acquired during the leg movements. From the rectified signal, a low-pass filter   allowed MNF) and median frequencies (MDF) were the frequency-domain features computed from the PSD using the following conventional equations: jf is the frequency value of sEMG power spectrum at the frequency bin j, jP is the EMG power spectrum at the frequency bin j, and M is the length of frequency bin. Spectral analysis was performed offline on the raw sEMG data segmented in time windows corresponding to every consecutive flexion and extension leg movement. For each of these time intervals, Fast-Fourier transformation was performed using 512 points, and the power spectral density (PSD) of the sEMG signal was obtained. The mean . All the signals were sampled at 1000 Hz, with kinematic data resampled at 200 Hz for further processing.Signal processing was performed by using Matlab version R2020b .Preliminary analyses were performed to verify the presence of a normal distribution of the sample (Shapiro–Wilk test) and to verify the homogeneity of the variance between the groups when parametric statistics is applied on a small sample (Levene’s test). p greater than 0.05 for both the changes in time-domain sEMG data (sEMG area) and frequency-domain parameters (MNF and MDF). Thus, the null hypothesis that the data are normally distributed was not rejected, and we considered it appropriate to apply parametric statistics. Moreover, considering the small sample, we paid particular attention to ensure that the Levene’s test provided values of p greater than 0.05 for each parametric comparison.Appling the Shapiro–Wilk test to check the sEMG data for normality, we found values of t-test. Two-way analysis of variance (ANOVA) for repeated measures was used to detected significant differences between the two frequency-domain features (MNF and MDF); the variation between the two parameters for each muscle was the “within” repeated factor (frequency parameters) and the variation between the two groups as “between” factor (group). The interaction between the two factors was also computed. One-way ANOVA with repeated measures was performed to detect changes in sEMG amplitude and frequency parameters over the cycles of movement, in each group. In this case, the F-statistic was adjusted applying Greenhouse–Geisser correction, which produces a more conservative p-value. For all the ANOVA procedures, the magnitude of the effect was evaluated by partial eta-squared (η2p). The comparisons of sEMG area, MNF and MDF, between the two groups for each single muscle, were performed using Student’s To further characterize the sEMG power spectrum, the profiles of PSD patients and healthy participants were analyzed by means of the principal component analysis (PCA). The PCA is a technique for reducing a large number of variables describing a data set to a smaller number of independent factors or principal components (PCs). That is, the first principal component (PC1) symbolizes the most common profile across the profiles of the PSD, and the second principal component (PC2) is the next most common, after the PC1 is accounted for. To simplify the interpretation of PCs, we applied varimax rotation procedure in which the PCs axes are rotated and the correlations in the rotated component matrix are close to 1, −1 or 0. Associated to the extraction of PCs, we calculated the weighting coefficients. These indices represent the Pearson correlation coefficients between the elements of the original dataset and each of the PCs, assuming any value in the interval between −1 and 1. This procedure allows to quantify the contribution of the original PSD profiles to each PC, that is, each PC can be associated with patients or healthy individuals. r) and the related level of statistical significance (p). Disease severity was assessed by FEV1 and FEV1/FVC tests.The correlation between changes in each of the two frequency-domain features and the level of disease severity was determined by computing the Pearson correlation coefficients .Almost all participants performed the entire 10 cycles of movement. However, some participants anticipated the last cycle making the final flexion–extension movement irregular. Thus, in order to have consistent statistics across subjects, we performed the analyses over nine cycles.p = 0.026; VL: p = 0.043; VM: p = 0.047). No significant changes were observed over the four muscles for the flexion movements .During the extension movements most of the sEMG activity occurred in RF, VL and VM muscles A, with tovements B. Both gChanges in frequency-domain parameters are illustrated in t-tests, significant changes in MNF values between the groups were detected for RF , VL  and VM  muscles, but not for BF (p = 0.224). During the flexion movements (p = 0.041). The pattern of variations observed for MNF was replicated for MDF during both movement directions (p = 0.029), VL  and VM , but not for BF (p = 0.342) muscles. Changes in MDF over the flexion movements were detected only for the BF muscle .Statistical differences between groups were detected for the RF, VL and VM muscles during the extension movements and for the BF muscle during flexion movements . The values of the MDF and MNF showed no significant changes over the movement cycles for all the muscles in both groups, except for the VM in the control group, regarding the MDF measure .No interaction between group and frequency parameters was observed, except for a marginal, significant interaction observed in the RF muscle during extension movements , between each PC and each original spectral profile from the common dataset of the two groups of participants. To obtain a statistical summary of the frequency profiles observed over the dataset including all the participants, and to identify the most common patterns for each of the two groups, we performed the PCA. In r mean value ± SD: 0.58 ± 0.22) was significantly greater (p < 0.001) than the coefficients obtained for the control participants . In the case of the RF muscle , the firr mean value ± SD: 0.52 ± 0.2; COPD r mean value ± SD: 0.38 ± 0.18; p < 0.001).An opposite behavior was observed for the third PC , which had the profile shifted towards the low frequencies, and the control group showed a better correlation with respect the COPD patients (control The second PC (lilac line) explained 26% of the total variance and covered an intermediate position over the frequency distribution. In this case, no significant differences were observed between the two groups regarding the distribution of the correlation coefficients.A similar pattern was observed for the other muscles B–D. In fDetailed numerical data for all muscles and movements are reported in p < 0.05) occurred only for extension movements, with MDF and MNF exhibiting good or strong levels of correlation with respect to the disease severity for almost all the extensor muscles.In The results of the present study show that a non-fatiguing motor task is effective for identifying and quantifying the increase in frequency range of the sEMG power spectrum, along with the concomitant decrease in sEMG magnitude, in persons with COPD compared to a group of healthy individuals. Noteworthy, the changes in both MNF and MDF of the sEMG signals were correlated with disease severity , thus further supporting the use of this functional task within a clinical setting to classify the patients based on their impairment in muscle function.The decreased magnitude of the sEMG signals in persons with COPD is consistent with other studies ,25 and sTo the best of the authors’ knowledge, this is the first study reporting increased frequency of the sEMG spectrum  during a non-fatiguing movement task in patients with COPD. In previous studies, the changes in the sEMG power spectrum were used to quantify the level of fatigue associated with COPD during incremental exercise tests leading to participant exhaustion ,20,21,24In the current study, the increase in MDF and MNF in patients with COPD compared with healthy people, and the stability showed by the two frequency-domain parameters over the leg movements, indicate that the limitations in the ability of sEMG analysis to reliably detect the changes in muscle fiber distribution could be attenuated by adopting non-fatiguing leg flexion–extension movements. Furthermore, the PCA performed in the current study provided further insight into the characteristics of power profiles associated with COPD, allowing to quantify the most common profile shapes of the power frequency spectrum within each experimental group. This made it possible to evaluate any visible differences in frequency bandwidth over the power spectrum, and to spot any potential skewing in the shape of the profiles. In fact, not only were the mean and median values of the frequency spectrum higher in COPD compared to healthy participants, but also the most common spectral profiles in COPD patients were skewed to the right, indicating a peculiar change in the shape of the patient power spectrum, with a longer tail towards the higher frequencies. Another relevant result of this study regards the good correlations between each of the two frequency paraments and the level of disease severity. This point strengthens the possibility that the shift of power spectrum toward higher frequencies observed in patients with COPD is associated with switching from type I to type II fibers observed by means of invasive techniques. In fact, the MDF and MNF changes correlated with changes in the FEV1 and FEV1/FVC, showing negative relations and levels of correlation comparable with the correlation between the same marker of disease severity and the proportions of type II fibers measured by histological and molecular techniques in several studies reported in the meta-analyses performed by Gosker et al. .The results of the present study support the adoption of a non-fatiguing task as a valuable and easy-to-administer tool to better estimate muscle fiber composition by identifying and quantifying the disease-induced increase in sEMG frequency spectrum. Due to the non-fatiguing nature of this task, the patients were not asked to experience any strenuous effort that may create discomfort, especially for those patients with moderate to severe levels of disease.A non-invasive monitoring of the muscle fiber composition could be of great utility not only in the characterizing the level of disease severity, but also to monitor the effects of rehabilitation protocols addressed to restore skeletal and ventilatory muscle functionality. In fact, neuroplasticity of motor units has been well documented both in animals and humans ; for exaThe results of these studies highlight the need to employ a test to characterize muscle fiber composition and the associated adaptations after rehabilitation training. The results of the current work provide an experimental support in considering the power spectrum analysis of sEMG, obtained during a simple and affordable task for patients with COPD, as a valuable tool to assess and possibly validate the training adaptive changes in muscle fiber composition having clinical significance. It is noteworthy that in our task the magnitude of muscle activation was remarkably greater during extension than flexion phases of the movement. In fact, the knee extensor muscles  were activated to counteract the effects of gravity in the extension phase of the movement. On the contrary, the flexion phase occurred along the line of gravity, and the magnitude of muscle activation was marginal see B due to p values) and the magnitude of the experimental effect (η2p) make us confident that the data reported in the current study can also be confirmed with a larger sample. A limited sample size was used for this study, which might weaken the outcome and robustness. However, we tried to compensate for this limit by providing different statistical control tests, such as the Shapiro–Wilk test to check for normality and Levene’s test to check for homogeneity of the variance between the groups. These tests showed acceptable levels of significance. Moreover, the good level of significant differences between the two groups (The experimental design did not include histological or cellular examinations to achieve a direct determination of the muscle fiber composition. These data would have provided a greater strength to the correlations between changes in sEMG parameters and changes in fiber type distribution. The correlation of our sEMG data with the disease severity and the large use of the quadriceps muscle in many studies examining peripheral muscle biopsies of patients with COPD ,10,11,18The changes in frequency-domain features in patients with COPD and the good correlation with the disease severity, reported in the current paper, suggest that sEMG power spectrum analysis applied to a non-fatigable motor task may be suitable for estimating muscle fiber composition. Moreover, the non-invasive nature of this approach encourages to use this paradigm to monitor the progression of disease severity and the effects of physical rehabilitation programs.


The objective of this study was to characterize the effects of intramuscular botulinum toxin (BT) injections on the electromechanical delay (EMD) in spastic human biceps muscles. The EMD is calculated as the time lag between the muscle activation onset, as recorded from the surface electromyogram (sEMG), and the onset of recorded force. In a cohort of chronic stroke survivors, we compared the computed EMD derived from the spastic (injected) biceps brachii with that from the contralateral muscle. Eight participants were tested before and up to 3 months after a BT injection. At each session, participants followed an isometric trapezoidal force trajectory at 50 and 30%, respectively, of the tested maximum voluntary contraction (MVC). Joint force and sEMG signals were recorded as well. The number of zero crossings (ZC) of the sEMG during the steady-state portion of the task was also computed. The EMD post-BT was found to increase by 64 ± 10% (at 50% MVC) and 93 ± 18% (at 30% MVC) when compared to pre-BT values, while the number of sEMG-ZC, the mean MVC values, and the force-EMD slope exhibited striking reductions. These parameters, calculated on the contralateral side, remained relatively constant across sessions, with the EMD significantly lower and the MVC values much higher. We discuss potential contributing factors to an increase in EMD values on the affected side, both pre- and post-BT. The observed co-variation across sessions of the increased EMD values with the decreased ZC estimates, a surrogate of motor outflow, and, potentially, more compliant muscle fascicles suggests that the altered motor unit (MU) behavior contributes, at least in part, to the delayed force production. The process of tension generation in the neurally activated muscle fiber begins at the neuromuscular junction, followed by a cascade of electromechanical events . There iIn general, the electromechanical delay, which is partly a function of a muscle's rate of force generation, depends on two major factors: (a) the number of recruited motor units, and (b) the electro-mechanical properties of the muscle-tendon unit (MTU). The latter is dominated by intrinsic stiffness of the muscle and tendon . Neural Botulinum toxin reduces hypertonia and hyperreflexia by means of the chemodenervation at the neuromuscular junction (NMJ) . GeneralHaubruck et al. have studied the passive biomechanical properties of the muscle-tendon unit in the healthy mouse gastrocnemius muscle during stretching, and have reported a decrease of stiffness by 25% after the botulinum toxin . Howren In the last few decades, the EMD values have been quantified by several researchers in humans under several conditions. The EMD value during a voluntary contraction of the biceps brachii (abbreviated to biceps from hereon) in intact subjects was reported to be 75 ms  and 80 ±Lacourpaille et al. have studied the human biceps muscle during electrically evoked contraction and have found that 56% of the EMD is contributed by the muscle during an active force generation, while the rest is by the MTU . Mörl etThe number of turns in an sEMG signal has been used by several researchers as a rough measure of the MU activity during a voluntary contraction of the muscle as shown experimentally . During Although the clinical consequences of BT have been widely investigated , 33, theAccordingly, the objective of the current study is to characterize and to understand the effects of BT on the electromechanical delay by computing the EMD values during the isometric voluntary contractions over a period of several weeks, in the course of multiple sessions in the post-injection period. We have measured the force and the sEMG during the isometric voluntary contraction in eight stroke-affected individuals who were injected with BT, as a part of their standard clinical care in their biceps muscle. As a surrogate measure of an MU recruitment, we also computed the number of ZCs or “turns” in the respective sEMG signal over time.We report data from eight survivors of chronic stroke, who were studied before (Pre) and after (Post-W2) a BT injection, to assess the changes in the EMD subsequent to an intramuscular BT injection on the affected spastic side only. Six of the eight tested subjects were able to continue testing to the mid-segment (Post-W6), and 5 subjects were tested until the third month (until Post-W12). We have tested both the BT injected spastic biceps and the contralateral arm at different submaximal contraction levels. Details of our tested cohort are summarized in via protocols approved by the Institutional Review Board under the Office for the Protection of Human Subjects at the Northwestern University (IRB No-STU00200242).We collected the experimental and clinical data at each session from stroke survivors, across a maximum of 3 months per subject. Approximately, there were over 45 total experimental recording sessions in both stroke-affected and contralateral arm muscles. The contralateral recordings were performed in separate sessions using the same protocol. The recordings from the contralateral side were used for comparative purposes as normative (non-injected) experimental data. Details of our experimental process, data collection, and analysis are summarized in the following section. All participants gave an informed consent Two types of sEMG electrodes were used during the experiment. A single differential surface electrode (SD-sEMG)  was placed over the muscle belly in the distal region of biceps, along the fiber direction. The single differential sEMG preamplifier has a bandwidth of 20–450 Hz for surface signals, amplified by a factor of 1,000, and sampled with a band-pass filter of 20–450 Hz . The force and single differential EMG signals were digitized at a rate of 2 kHz . Force and sEMG signals were then collected and were visualized online using a software program from CED, Spike2 (version 7), and were stored on a personal computer for later analysis. The synchronized data allowed an accurate determination of the onset of sEMG in relation to the onset of torque production .In order to analyze the zero crossings of the sEMG, we utilized recordings from a novel 4 channel sensor array (SA-sEMG) electrode . The senvia a metal ring to record generated force. The other end of the load cell was firmly attached to a stable iron platform through fixture arrangements. A rubber pad was placed under the elbow to keep the forearm parallel to the ground. To restrict trunk and shoulder movement a pair of Velcro straps was attached around the subject, to the Biodex chair.Subjects were positioned in an upright sitting posture on a Biodex chair. Their upper arm joint angles were fixed at elbow flexion 120°, shoulder flexion 10°, shoulder abduction 35°, and the forearm pronation to 45° for maximum activations of biceps muscle during the elbow flexion. To further ensure isolation of the biceps, and thereby reducing contamination of force production from other muscles, the forearm was secured in a custom-made fiberglass orthopedic cast used from above the elbow up to their fingertip. A rigid 6 d-o-f force-torque sensing load-cell  was mounted on the wrist r) was calculated as the vector summation of the Fx and Fz recorded from the sensor. Real-time feedback of the force response was provided on a computer monitor. Two-dimensional visual feedback of the force trajectory in the desired quadrant plane assisted the participants to track and to maintain their force profiles as required. Details of the experimental setup and the processing are provided in Voluntary contraction force and sEMG data were recorded during a sustained isometric non-fatiguing elbow flexion task; first, in a maximum voluntary contraction (MVC) trial, then in subsequent trials of submaximal levels at 30% MVC and 50% MVC. Three good trials were performed (with an average error within 10% of the instructed trajectory) at each level with different levels performed in a randomized sequence. During each trial, all forces were simultaneously collected with an sEMG. The resultant force , then by the submaximal steady-state contraction level for a period of 10 s, and a decreasing force level at the rate identical to the rate of increment. The average FWe define the electromechanical delay as the time delay between the onset of the sEMG and the onset of force production during the increasing phase of the force trapezoid. After the initial qualitative inspection, the method was programmed to automatically detect the onset of force production. The location, where the magnitude of the sEMG trace exceeded a statistical threshold above the background noise level, was determined using a sliding window (50 samples long) as shown in To reduce the possible effects of noise in the system, 1,000 samples (500 ms) were selected from the sEMG and the force recording channel during the resting period prior to each contraction to determine the respective baseline values. The mean and the standard deviation of the selected resting period activity were calculated, the values for the affected side are summarized in The time of the initial sample of the window will be recorded, if the mean of the samples in the window exceeded the threshold. If the mean of the samples in the window did not exceed the criteria, the window was advanced one sample at a time until an onset was found. The time difference between the onset of sEMG and force was determined for both trials at each contraction level.ETH was defined as:The ZCs of the sEMG were measured during the steady-state of the force trajectory. The trials for which the EMD were detected were further analyzed for the calculation of ZC. We have only considered one incident of ZC between successive positive and negative peaks detected in sEMG. All the sEMG peaks were detected in a 5-s window (with a minimum variation of the force trace) in the sensor array sEMG signal during the steady-state period for the force trajectory. The region was selected with a minimum force variation of the resulted force trace. This sensor array recording channel, with the highest peak to baseline value, was selected for the peak analysis. The small electrode area and the high recording sampling rates of the sensor array further supported the precise detection of representing the MU firing instances. More detailed descriptions of the sensor can be found in . The sigWhere μ and δ are the mean and the standard deviation of the baseline sEMG signals before the start of the voluntary activation . The avep < 0.05.The normality of the data was analyzed by the Kolmogorov Smirnov test and the non-normality was confirmed . ThereafThe EMD values were calculated in both the stroke-affected side and the contralateral side. The EMD values were systematically higher on the affected side (79.7 ± 9 ms at 50% MVC and 127 ± 18 ms at 30% MVC contraction levels) compared to the contralateral side (25 ± 12 ms at 50%MVC and 45 ± 9 ms at 30% MVC contraction levels), as shown in p < 0.01) increase after the injection (at Post-W2) at both contraction levels compared to their respective pre-injection baseline values. In the affected side, a maximum increase of 64 ± 10 and 93 ± 18% of the EMD values were observed respectively at 50 and 30% MVC levels during Post-W2 compared to the pre-injection values as shown in 0 < 0.05, χ2 = 7.93). A decaying trend of the EMD values was observed in the following experimental sessions after Post-W2. Finally, at the end of 12 weeks (Post-W12), the EMD values decreased to 7 and 26% for the respective contraction levels as summarized in r = 0.86 ± 0.04 with p < < 0.05) was found among the time course of the EMD values at two contraction levels.Experiments were conducted longitudinally as discussed in the methods section. The EMD values were calculated across the sessions before and after the BT injection. The maximum voluntary contraction (MVC) force values, and, thus, the submaximal contraction values are varied across the post-BT recording sessions. r > 0.68) within the force and the EMD values. In order to assess the changes in the relation between the force and the EMD, the slope of the linear fit was analyzed across the experimental sessions. The slope values of the linear fit were found to systematically vary in the injected side for all the subjects as shown in The force and the EMD values were measured respectively during 30, 40, and 50% of the MVC level across the experimental sessions. After the injection, a linear regression line was fitted (min p = 0.0113) reduction of 3.5 times compared to the pre-injection values. The reduced slope value persisted until 12 weeks for the subjects (p = 0.006) compared to its pre-injection slope value. Despite the increase of the slope values compared to the post W2 were observed from Post-W6 onward, even at the end of 12 weeks (Post-W12), the values were found to be significantly lower than the pre-injection value recorded at −1.19. During this session, the slope values in the contralateral side remained high in all the experimental recording session across the time course, and the variation recorded were within 20% of all the contralateral values recorded among the subjects.During the pre-injection recording session, the slope values in the injected side (−0.49 ± 0.16) were found to be lower than the contralateral side (−0.22 ± 0.07). At Post-W2, the slope value in the affected side showed a statistically significant  than the contralateral side for all the subjects when compared to the respective contraction level. p < < 0.05) for the test cohort.The number of ZCs in the affected side was reduced after the BT injection, and the average ZCs among the subjects were found to be lower than the pre-injection baseline recording. The reduction of the number of ZCs was statistically significant (p > 0.08) for all the subjects at all contraction levels.The average reduction of ZC was 50 ± 16, 74 ± 24, and 18 ± 11% at Post-W2, Post-W6, and Post-W12, respectively, compared to the pre-injection value of the respective subjects. The number of ZCs did not return to the baseline pre-injection values when compared individually for the subjects who have studied for full 12 weeks apart from B5. Unlike the injected side, the number of ZCs across the sessions in the uninjected contralateral side remained relatively unchanged with a statistical significance . The average reduction in the slope values for the subjects at Post-W2 was 38 ± 31% compared to the Pre-session. The force-ZC slope values did not recover to the pre-injection baseline level by Post-W12. While the systematic reduction of the force-ZC was observed in the affected side, the contralateral side values did not change significantly, and no systematic difference was found.The goal of this study was to investigate the time-course of the EMD during isometric voluntary contraction in spastic biceps muscle followed by a clinically prescribed injection in the muscle of our tested cohort. Our results show that estimating EMD might provide an insight into the changes in neural control—i.e., changes in recruitment of motor units due to a BT-induced chemodenervation in spastic biceps muscle. We have measured the EMD between the sEMG activation and the resulting detectable elbow contraction force at various levels of contraction. Simultaneously, we have studied the number of ZCs in the sEMG signal based on a novel sensor array recording. Recordings from the contralateral side were used as a control reference  for the experimental analyses.The primary finding of this study was that the EMD values increase by 64 ± 10% (at 50% MVC) and 93 ± 18% (at 30% MVC), respectively, during the first recording session after the BT (Post-W2) for our test cohort, while the mean force value was also reduced by 8%. We have found maximum EMD values that occurred during the first post-injection session (Post-W2). Thereafter, the values declined gradually during the following sessions in the later stage (at Post-W6 and W12). Concurrently, the force also partially recovers back from its minimum value among four of the six subjects (except B3 and 4) who were studied for additional sessions after the initial session (Post-W2).Despite the partial recovery of the following sessions, there exists a 7 and 15% prolongation in the EMD values during session Post-W12 compared to the Pre-session baseline as shown in The highest antispastic effect of the BT induces the denervation found within 1 month of the injection affecting the contraction force and the sEMG. This has been reported earlier by several groups including us , 43.We have observed higher standard error in the EMD values at lower force levels. This is potentially due to the variation in the effect of BT in the muscle as suggested by earlier groups . A majorSince a functional alteration of the MU pool will be reflected in the sEMG, we have used the zero-crossings value of the sEMG signal as a surrogate of the recruited MU , 30. TheOur results show a dramatic reduction in the number of ZCs among our subjects that may signify a reduced number of active fibers. A reduction of active fibers after BT injection was reported earlier . We haveReduction in the force-ZC slopes provides a means of comparing the ZC despite the reduction of the contraction force during the post BT session to the respective pre-injection baseline values. Reduction of the force-ZC slope at Post-W2 indicates a disproportionate reduction of the motor outflow compared to the reduction in force in the BT-injected muscle.Disproportionately larger reductions of contraction force may have two reasons:  A changeIn order to understand the relation between EMD and the neural drive to the active MN pool, we have studied the force EMD slopes. The negative slope values for all the subjects across all the recording sessions indicate a negative correlation between the MVC levels and the EMD values  compared to their pre-injection level. This may be attributable to the architectural limitations of the shortcoming in a spastic muscle during the contraction, which also played an important role in the force generation . Moreove comparedComputation of the EMD critically depends upon the calculation method and the nature of the muscle activation task. We have defined EMD as a time delay between the detected onset of the sEMG and the respective voluntary contraction force . ExistinEMD is, in part, a measure of functional architecture and internal disruption of the muscle. This study provides a quantitative characterization of changes in the EMD and the number of zero-crossing in the sEMG signal after the intramuscular BT injections in stroke survivors, which allowed us to understand the effects of BT more clearly on voluntary contraction. The results suggest that the BT-induced reduction of spasticity results in a reduction in voluntary contraction capacity in association with the increased EMD value. The increase in EMD indicates that the BT disrupted both the fascicle architecture and the normal mechanics of force production that contributed to the clinical impairment. We have further shown the increase in EMD is at least, in part, contributed by the reduced number of firings, which was measured in terms of the zero-crossing in the sEMG signals, and does not come back to its pre-injection baseline after 12 weeks. The result of this study is the first evidence of a change in EMD affecting the force production capacity after the BT injection.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the Institutional Review Board at Northwestern University (IRB No. STU00200242). The patients/participants provided their written informed consent to participate in this study. Written informed consent was also obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SC, BA, WR, and NS participated in the study conception, design, data analysis, and interpretation. SC, BA, and NS participated in data acquisition. All authors read and approved the final manuscript.This study was funded by the National Institute on Disability, Independent Living, and Rehabilitation Research (NIDILRR-90RE5013) through Advanced Rehabilitation Research and Training (ARRT) and Rehabilitation Engineering Research Center (RERC) program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In receiver operating characteristic analysis, ALR = 1.55 had the highest sensitivity and specificity. The patients with ALR < 1.55 had a significantly poorer PFS than those with ALR ≥ 1.55. A high ALR was associated with a tumor response and good PFS in patients with advanced malignancies. The ALR based on activated cytotoxic T lymphocyte dynamics is therefore a reliable predictive marker.Immune-oncology (IO) drug therapy is effective against various types of cancer. Although several, potential, clinical predictive markers have been identified, none so far have proven reliable. Herein we evaluated changes in serum alanine aminotransferase (ALT), which is upregulated by the accumulation of activated CD8+T cells in the liver, as a potentially reliable predictive marker. We retrospectively analyzed 265 patients with advanced malignancies at three institutions between 2016 and 2019. The patients received IO drug therapy. We defined the ALT ratio (ALR) as the serum ALT value at baseline / the highest serum ALT during IO drug therapy, then determined whether the ALR correlated with the objective response rate or progression-free survival. The median follow-up was 3.1 months. We observed objective responses in 65 patients. The ALR ranged from 0.19 to 32.2 (median 1.5), and a significant ALR increase was observed in responders ( Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is also inducibly expressed on CTLs and interacts with its ligand, CD80 or CD86, to deliver an inhibitory signal to CTLs4. Both molecules inhibit antitumor immunity. Anti-PD-1 (nivolumab or pembrolizumab) and anti-CTLA-4 (ipilimumab) antibody block the inhibitory signal, thereby activating the CTLs against the cancer8. Recently, anti-PD-1 and anti-CTLA-4 antibodies were approved for use against several cancers10. These antibodies are comprehensively referred to as immune-oncology (IO) drugs. Nivolumab was effective against non-small-cell lung cancer (NSCLC)12, melanoma13, renal cell carcinoma (RCC)14, urothelial carcinoma (UC)16, squamous cell carcinoma of the head and neck (SCCHN)17, gastric cancer (GC)18, malignant mesothelioma (MM)19, Hodgkin’s lymphoma20, and hepatocellular carcinoma21. Pembrolizumab, another anti-PD-1 antibody, was also effective against NSCLC22, melanoma23, RCC24, SCCHN25, UC26, and Merkel cell carcinoma27. Ipilimumab was effective against melanoma10 and RCC28.Programmed death-1 (PD-1) is inducibly expressed by activated cytotoxic T lymphocytes (CTLs). PD-1 interacts with its ligand, B7-H1, to deliver an inhibitory signal to CTLs29, overall mutational burden30, leukocyte count33, lactate dehydrogenase34, C-reactive protein35, and adverse events38, but as of yet, no reliable predictive marker has been found39.Despite their efficacy, IO drugs benefit only a small number of patients. Therefore, reliable predictive markers allowing the identification of responders and non-responders are crucial to choosing the optimal treatment. Several studies have discussed predictive markers, such as PD-L1 expression41. Dong et al. showed a greater accumulation of CTLs in the liver of B7-H1-deficient mice than in wild-type mice42. In the present study, we evaluated changes in the serum alanine aminotransferase (ALT) level, which is a marker of liver injury, as a mechanism-based predictive marker for IO drug therapy.CTLs, which are activated by IO drugs, accumulate in the liver and undergo apoptosisIn total, 265 patients (134 with NSCLC (92 with adenocarcinoma and 42 with SCC); 38 with GC; 52 with RCC; 35 with UC; three with MM; and three with an unknown primary cancer) were enrolled . We observed a significant increase in the ALR among responders  or a trap for activated CTLs in the liver for subsequent killing (the killing field hypothesis)44. Usually, serum ALT rises immediately prior to the finding of tumor shrinkage on a radiologic modality. These findings suggested that ALR might serve as a predictive marker of IO drug therapy.The timing of the serum ALT increase varies for each IO drug. In most patients receiving pembrolizumab or a combination of nivolumab and ipilimumab, the timing of serum ALT elevation was 1–3 weeks following the initial drug infusion. However, the timing for nivolumab was different, varying from soon after the initial infusion to later in some cases. This difference may depend on the affinity of each IO drug to the PD-1 molecule. An antibody with a strong affinity might affect the immune system sooner than an antibody with a weaker affinity; pembrolizumab has a stronger affinity than nivolumab to PD-1 expressing T cellsThe present study has the limitations inherent in any retrospective analysis. The patient population varied widely. The ALR was an effective predictive marker, but presented certain problems, such as the occurrence of false negative and false positive cases. We observed no increase in the ALR in 11 (17.0%) responders while we found an increase in 67 (33.5%) non-responders.There are several possible explanations for the false negatives. First, our timing of ALT analysis was inadequate in that we failed to perform the analysis when the ALT had reached its highest value. Second, because injury to the liver was small, we did not observe elevations in the serum ALT levels. There are two reasons for the low level of injury to the liver. First, the shrunken tumor volume was too small to induce sufficient CTL activation to cause hepatopathy; in effect, there was insufficient injury to the liver to elevate the ALT level. Another reason for the minimal injury to the liver was exceptional liver function. There are also several possible explanations for the false positive results. In twelve patients, a new liver metastasis or the progression of an existing liver metastasis induced a serum ALT increase. In nine patients, other reasons, for example, autoimmune hepatitis, autoimmune cholangitis, septic shock, other drug-induced hepatopathy, etc., accounted for the elevated ALT. In ten patients, despite the attainment of SD or PD according to the RECIST criteria, a portion of the tumors showed shrinkage. We were able to identify other, clinical causes of elevated ALT as well; after excluding 21 cases of elevated ALT due to other causes from the cohort and adding ten cases showing some tumor shrinkage to the responder group, the odds ratio rose from 9.7 to 16.8. In clinical practice, the ALR may serve as a very promising predictor of the response to IO drug therapy when transitioning to the next treatment. These circumstances should be taken into consideration, and ALR should be used carefully.39. However, these factors have limitations due to cost-related concerns; these markers require additional analysis, the involvement of specialists or the use of expensive equipment. Some studies revealed that in contrast to these markers, certain blood and clinical markers, such as the leukocyte count, lactate dehydrogenase, and adverse events, can be routinely used39. ALR is also one such available blood marker. However, in this respect the ALR, based on CTL dynamics, is an effective marker of immunotherapy and has the potential to serve as a simple, reliable prognostic marker in risk stratification and provide better treatment allocation in cancer patients.The PFS duration was significantly longer in the high ALR group than in the low ALR group for four types of cancer, suggesting that the ALR can help differentiate between long and short SD among non-responders. The ALR is useful for patients receiving cancer immunotherapy because it facilitates judging whether immunotherapy should be continued or switched to an alternative treatment. Previous studies have reported several, reliable predictive markers for IO drug therapy, including PD-L1 expression and the tumor mutation burdenA high ALR was associated with the tumor response and PFS in patients with various types of cancer. The accumulation and deletion of activated CTLs in the liver might impose a burden on the liver, which induce the elevation of ALR. Thus, an increase in ALT levels during IO drug therapy is reliable because the ALR is associated with CTL dynamics.Further studies aimed at refining the use of the ALR as a predictive marker based on the mechanism of IO drug therapy are warranted.Between 2016 and 2019, we reviewed the medical records of 265 patients with advanced malignancies at Tokyo Metropolitan Tama Medical Center, Kanto Rosai Hospital, and Tomishiro-Chuo Hospital who received single-agent nivolumab, pembrolizumab or a combination nivolumab and ipilimumab treatment.The present study was approved by the ethical review board of Tama Medical Center, Kanto Rosai Hospital, and Tomishiro Central Hospital and was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice Guidelines. Comprehensive iInformed consent was obtained from all the participants.Patients received nivolumab 3 mg/kg every 2 weeks, pembrolizumab 200 mg every 3 weeks or nivolumab 240 mg every 3 weeks plus ipilimumab 3 mg per kilogram every 2 weeks in four doses, followed by nivolumab 240 mg every 2 weeks.All the patients underwent blood testing from before the commencement of IO drug therapy to the end of follow-up at intervals of 1–2 weeks. We defined ALR as the serum ALT value at baseline / the highest serum ALT during IO drug therapy.Computed tomography or magnetic resonance imaging was performed at baseline and repeated at 8- to 12-week intervals. The clinical tumor response during treatment was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 based on the patients’ medical records. We defined a clinical benefit as a complete response (CR), partial response (PR) or stable disease (SD). We defined progression-free survival (PFS) as the time from the commencement of immunotherapy to the date of progression.p < 0.05 indicating statistical significance. We compared continuous variables using the two-tailed unpaired Student’s t-test. To predict the response to, and prognosis of, IO drug therapy, we determined the optimal cutoff value of the ALR based on the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC). We constructed the distribution of the recurrence-free survival (RFS) rate using the Kaplan–Meier method.Statistical analyses were performed using the JMP® software package, with Supplementary Figure S1.