Terpenes contribute to the pharmacology, efficacy, aroma, and flavor of cannabis inflorescence, improving the experience for medical and recreational users. Terpenes are inherently volatile, resulting in the loss of terpene content as inflorescence ages. A method to establish and/or maintain a desired terpene content of cannabis inflorescence is needed. A novel packaging method was investigated for the preservation of native terpenes and the replenishment of terpenes to depleted inflorescence over various storage durations.Inflorescence samples from two different chemotypes  were obtained from a state licensed medical cannabis organization. Samples from the DJ’s Gold chemotype were depleted of terpenes whereas samples from the Cream Caramel chemotype had a terpene content representative of inflorescence available for medicinal or recreational purposes. Inflorescence samples were stored using the novel packaging approach, in airtight containers in the presence of external terpenes. Control samples were similarly stored without external terpenes. Terpene content of the inflorescence samples were quantitively determined by headspace gas chromatography mass spectrometry (HS GC-MS) after various storage durations. Main effects analysis was used to determine the impact of various parameters on the effectiveness of the system.All samples stored using the novel packaging approach had a higher terpene content than their corresponding control. 1.18% (w/w) of external terpene, relative to inflorescence weight, was the minimum amount required to maintain the initial terpene content of the inflorescence after 6 weeks of storage. Main effects analysis showed that augmentation of inflorescence terpene content was dependent upon the amount and type of external volatile utilized. The terpene profile of inflorescence samples from two separate harvests were selectively adjusted, reducing the percent difference of the two sample’s terpene profiles by 39.5%.A successful proof of concept was achieved for preservation, augmentation, and replenishment of terpenes to cannabis inflorescence over various storage durations. Inflorescence stored using the novel packaging approach is a significant step towards providing patients with cannabis inflorescence of reproducible and reliable terpene content, an important component of inflorescence efficacy. The novel approach for replenishment of terpenes to depleted inflorescence represents an exciting development for patients and manufacturers. Cannabis sativa inflorescence which had been air dried and stored for 1 week, 1 month, and 3 months, respectively, as compared to freshly harvested inflorescence  were utilized as external volatile sources. The terpene content of the inflorescence was quantitatively determined by headspace gas chromatography-mass spectrometry (HS GC-MS). Inflorescence preservation samples were analyzed for terpene content after different durations of storage and compared to initial and control. Repeatability and robustness of the approach were assessed by testing inflorescence from different cannabis chemotypes, DJ’s Gold (DjG) and Cream Caramel (Cre), as well as inflorescence from multiple harvests of the DjG chemotype. Due to age and processing conditions, the material from DjG chemotype was depleted of terpene content. The system’s ability to replenish the terpene content of depleted material was investigated. The ability of the approach to selectively adjust the terpene profile of the inflorescence was investigated by comparing the profile before and after storage in the presence of an 8-part terpene mixture. The developing method offers patients and recreational cannabis users the potential for inflorescence with a standardized terpene content, improving the reliability of the efficacy and experience.All experiments using cannabis were conducted at state licensed medical cannabis facilities in accordance with relevant state law and regulations. Cannabis samples from the DjG chemotype were obtained from Pennsylvania Medical Solutions . Experiments from Vireo Health, PA are referred to as Site One. Samples from two harvests were utilized: one harvest aged approximately 1 year (named “aged one-year”), and one harvest aged approximately 1 month (named “aged one-month”) post-harvest. The aged one-year DjG samples were ground by hand, the aged one-month DjG sample were finely ground by an industrial grinding machine. Nine samples were prepared, 4 from aged one-year and 5 from aged one-month DjG inflorescence. For the aged one-year group the samples were stored with the following external volatiles; 8-part terpene mixture, lavender EO, cinnamon bark EO, and control. The following samples were prepared for the aged one-month DjG group; 8-part terpene mixture, β-myrcene, α-pinene, argon gas, and control. 1 mL of the external volatile was added to a 1.5 mL glass vial and placed inside the jars containing the cannabis. The inflorescence samples were stirred, and 50 mg was obtained, for terpene quantitation by HS GC-MS at both 2 and 4 weeks. The profile of the 8-part terpene mixture is presented in the Supplemental Table A second set of cannabis inflorescence samples of the Cre chemotype were obtained from MaryMed . Experiments from Vireo Health, MD are referred to as Site Two. Experiments were performed on intact Cre inflorescence. The initial terpene content of inflorescence from the Cre chemotype was assessed by quantitative terpene analysis of 0.5 g. Fifteen samples were prepared, each containing approximately 1.5 g of intact inflorescence (exact weight was recorded) from the Cre chemotype in individual 4-oz airtight jars. A sponge was used as an inert matrix and added to each airtight jar. The sponge was impregnated with terpenes of the following volumes: 0 μL (control), 10 μL, 100 μL, 500 μL, and 1000 μL. A terpene blend named “Granddaddy Purps” was used as the external volatile for Site Two experiments. When adjusting for density (0.84 g/mL) these volumes represent a terpene content of approximately 0, 0.56, 5.6, 28, and 56%, weight by weight as compared to the inflorescence. The inflorescence and the inert matrix were separated by a screen so no terpenes could be transferred to the inflorescence by contact. Three samples from each group were prepared resulting in 15 total samples. To mimic patient use, samples were opened twice/week. Each sample was designated a specific storage duration  and the entire sample was ground and subjected to quantitative terpene analysis at the conclusion of the designated storage duration.All terpenes utilized in this study were derived from botanical sources. The following terpenes were sourced from True Terpenes ; β-myrcene, β-caryophyllene, linalool, α-(−)-bisabolol, D-limonene, α-humulene, α-terpineol, β-pinene, and α-pinene. Lavender and cinnamon bark EOs were sourced from Nature’s Oil . A terpene blend named “Granddaddy Purps” was sourced from Eybna Terpenes, Givat Hen, Israel. The blend is considered a β-myrcene, β-caryophyllene codominant blend by standards described by the USP Cannabis Expert Panel  were used to qualitatively and quantitatively (respectively) evaluate the impact of different variables on the novel’s system effectiveness.Analysis of variance (ANOVA), main effects analysis and other data processing was performed using Minitab  version 19.2. Main effects analysis and ANOVA  was observed for the aged one-month DjG sample which was stored with α-pinene. When compared to controls, samples stored in the presence of an 8-part terpene mixture had a total terpene content 2.6 and 2.5 times larger, for aged one-year and aged one-month DjG, respectively.The impact of terpene oxidation was investigated by displacing the air in the headspace of the container with argon. As illustrated in Table The accuracy and repeatability of modifying the inflorescence terpene profile using a formulated 8-part terpene mixture was investigated. The profile of the 8-part mixture is presented in Fig. The accuracy of establishing a specific terpene profile, of specific terpene ratios, in inflorescence samples was assessed. Inflorescence samples were stored in the presence of a formulated 8-part terpene mixture for 4 weeks. After 4 weeks of storage, each terpene’s percentage of the total terpene profile was calculated in the inflorescence samples and compared to the 8-part mixture. For example, β-myrcene represented 34.3% of the total terpene content of the 8-part mixture. After 4 weeks of storage in the presence of the mixture, the aged one-year and aged one-month DjG inflorescence had a β-myrcene content consisting of 49.0 and 46.7% of the total terpene content. The average terpene profile recovery for β-myrcene was calculated to be 139%. This recovery is graphically illustrated in Fig. The repeatability of the approach was assessed by comparing the terpene profile of the aged one-year and aged one-month DjG samples before and after storage in the presence of the 8-part mixture. Inflorescence from the two harvests of DjG had an initial percent difference in terpene profile of 55.8%, calculated by the weighted mean of the percent difference for all terpenes  for the linear fit was 0.9831.Additional validation studies were performed on inflorescence from the chemotype Cre. The initial terpene content of the Cre inflorescence was determined to be 1.49%. For preservation samples, a terpene blend named “Granddaddy Purps” was impregnated into an inert matrix and placed into an airtight jar containing the inflorescence. Varying amounts of terpenes were impregnated into the sponge, ranging from 0.5 to 57.8% of the weight of the inflorescence sample. The inert matrix for the control samples did not contain terpenes. Samples were prepared in triplicate for the 5 terpene concentrations, representing 15 preservation samples. Each sample was opened twice per week to mimic patient use. Samples were stored for 2, 4, and 6 weeks for each terpene concentration. The average loss of terpene content for the controls was 12.1%, calculated by averaging the three samples. For preservation samples using 1 mL of external volatile, a 3.5-times increase in terpene content as compared to control was observed. Figure The change in terpene profile over time a 2-week period was assessed. Terpene profiles from samples of Cre stored in the absence and presence of an external volatile were compared. Control samples after 2 and 4 weeks of storage were found to have a percent difference in terpene profiles of 41.9%  methods, chemotype genetics, as well as storage conditions and age of the inflorescence. These factors present challenges for patients and consumers seeking inflorescence of reliable terpene content.A novel method to preserve, replenish, and establish a desired terpene content of cannabis inflorescence was investigated. A total of 38 inflorescence samples were investigated in this study Table . Nine san = 27) successfully maintained a higher terpene content as compared to controls after 2 and 4 weeks (Site One) and 2, 4, and 6 weeks (Site Two) of storage, illustrating the robustness of the method. Variables that were statistically meaningful to the effectiveness of the novel system were identified by main effects analysis and ANOVA modeling. The amount and type of external terpene utilized were found to be statistically meaningful parameters towards the effectiveness of the system for terpene preservation and augmentation. Conversely, chemotype and storage time did not impact the novel’s system ability to preserve terpenes. Explicitly, all factors that were varied in the chemotype group, most notably the initial terpene content of the inflorescence, did not impact the method’s ability to preserve or augment the terpene content of the inflorescence, further illustrating the robustness of the novel system. All durations of storage were found to be effective towards terpene preservation, illustrating that the maximum storage duration has not been identified, however as results indicate it is greater than 6 weeks.The repeatability and robustness of the approach was investigated by testing the system using different chemotypes, storage times, percentages of external volatiles, and types of external volatiles. The ‘Chemotype’ group also included differences in inflorescence cultivation locations, different harvests and age, ground versus intact inflorescence, initial terpene content , and neat volatiles stored in a glass container versus volatiles impregnated into an inert matrix. Furthermore, quantitative terpene analysis for the different sites was performed by different testing labs, utilizing different analysts. Experiments at Site Two mimicked patient use by opening the storage jars twice per week. Under all conditions, all preservation samples  due to the processing and grinding methods applied to the inflorescence post-harvest. Conversely, samples from the Cre chemotype had an initial terpene content of 1.49%. This terpene content is within the expected range for inflorescence available for medicinal or recreational use  from inflorescence of the same chemotype. However, as illustrated in this work and previous research, the terpene profiles of the inflorescence may vary due to age, batch (harvest), and other factors. Results reported here illustrate the variance between inflorescence samples can be significantly reduced when the novel packaging approach is utilized.In this proof of concept study, botanically derived terpenes were utilized as the external volatile source. However, methods for extracting terpenes from cannabis are well established and terpenes extracted from cannabis could be reintroduced to the inflorescence using the novel system described here. Similar to preliminary results, utilizing the novel system with chemotype specific terpenes as the ‘external’ volatiles, is expected reduce terpene loss over time, and batch to batch variability of the terpene profile. It is worth noting that any terpenes or other volatiles utilized as an external volatile source, requires control of potential manufacturing impurities which could be introduced into the inflorescence. The reintroduction of chemotype specific terpene profiles may provide a path for pharmaceutical development via the FDA’s Botanical Drug pathway, as all ingredients would originate from the same botanical (cannabis) source, with the improved batch to batch consistency offered by the novel system.10H15) were observed to have the highest rate of terpene infusion as compared to sesquiterpenes, which contain 3 isoprene units (C15H24). Both isomers of the monoterpene pinene had the highest rate of terpene infusion for the 8 terpenes investigated . Since terpene extraction methods from both cannabis and botanical sources are well established, and can be achieved at relatively large scales, 41.3 mg of terpenes adds minimal cost to a final product. Several inert matrices of sizes ≤1 cmThe inherent volatility of terpenes often results in loss of terpene content from cannabis inflorescence over time. Unlike cannabinoids, which do not share the same volatility, and can be preserved through cultivation and processing practices, there is no readily available method for the preservation of terpenes in cannabis inflorescence. Reported here is the successful proof of concept towards the standardized control of terpene content in cannabis inflorescence, the preservation of native terpene content, selective augmentation and adjustment of inflorescence terpene profiles, and the ability to adjust the aroma of the inflorescence. Several validation parameters for the novel packaging approach were achieved in this study. Robustness was achieved by obtaining positive results for both ground and intact inflorescence, and when utilizing both neat external volatiles and external volatiles impregnated into a matrix. Intermediate precision was achieved by obtaining positive results from inflorescence at different sites, chemotypes, and analytical characterization labs. Similar results were achieved for terpene augmentations, even when utilizing different external volatiles and inflorescence from different chemotypes.Preliminary results indicate that the fundamental applications of the novel system include maintaining a desired native terpene content of the inflorescence at the time of packaging and the replenishment of terpenes to terpene-depleted inflorescence. More sophisticated applications that require further investigation include adjusting the flavors, aromas, and potentially the pharmacological effects of inflorescence by selective adjustment of the inflorescence terpene profile. The application of adjusting the aroma of cannabis inflorescence, as illustrated in this study when stored in the presence of lavender EO, may reduce the stigma of inflorescence use and increase the number of patients willing to access the medicinal benefits of whole plant products. For example, patients may be more apt to use cannabis inflorescence which possesses a lavender aroma as compared to the typical inflorescence aromas, which often have a negative association with non-cannabis users.Future directions of this research include determining the maximum storage time terpenes can be preserved, further investigating the efficiency of absorption of various terpenes, and utilizing the natural antimicrobial properties of terpenes towards inhibiting the growth of bacterial and/or fungal contaminants on inflorescence.Additional file 1.


The central nervous system (CNS) is an immune-privileged compartment that is separated from the circulating blood and the peripheral organs by the blood–brain and the blood–cerebrospinal fluid (CSF) barriers. Transmigration of lymphocyte subsets across these barriers and their activation/differentiation within the periphery and intrathecal compartments in health and autoinflammatory CNS disease are complex. Mathematical models are warranted that qualitatively and quantitatively predict the distribution and differentiation stages of lymphocyte subsets in the blood and CSF. Here, we propose a probabilistic mathematical model that (i) correctly reproduces acquired data on location and differentiation states of distinct lymphocyte subsets under homeostatic and neuroinflammatory conditions, (ii) provides a quantitative assessment of differentiation and transmigration rates under these conditions, (iii) correctly predicts the qualitative behavior of immune-modulating therapies, (iv) and enables simulation-based prediction of distribution and differentiation stages of lymphocyte subsets in the case of limited access to biomaterial. Taken together, this model might reduce future measurements in the CSF compartment and allows for the assessment of the effectiveness of different immune-modulating therapies. Under hphalitis  and Susaphalitis ) and/or phalitis ,9,10). Tphalitis ,7,11,12,phalitis ,14,15,16+ T cell subtypes in the PB and CNS compartment based on the Markov chain theory [Since disease activity and treatment responses are usually monitored using clinical and imaging parameters, repetitive lumbar punctures are difficult to justify. Therefore, CSF samples for longitudinal immune-profiling are often not available, whereas PB is more easily accessible. Thus, a model predicting treatment-related changes in the composition and differentiation state of lymphocytes in the CSF by measurements in the PB is warranted. To overcome the limitation of missing CSF data, mathematical models may allow us to get deeper insights into the kinetics of cell transmigration and distribution within the different compartments ,18. In an theory , utilizin theory . Here, o- CD4+ T-helper cells, cytotoxic HLA-DR- CD8+ T cells, CD19+ B cells, and CD56bright NK cells, as well as differentiated HLA-DR+ CD4+ T cells, HLA-DR+ CD8+ T cells, CD19lowCD138+ plasmacytoid cells, and CD56dim NK cells (1 to naïve stage cells into the CSF compartment (stage 2). Lymphocytes in the PB compartment and naïve stage (stage 1) transition within this compartment at rate β1 into the differentiated stage (stage 3). Lymphocytes in the PB compartment and differentiated stage (stage 3) transition at rate α2 into the CSF and stay differentiated (stage 4). Finally, at rate β2 naïve lymphocytes transition within the CSF compartment (stage 2) into differentiated ones (stage 4). The transition rate predicts the percentage of cell transition from one stage into another. A higher transition rate indicates that a higher proportion of cells will change their respective stage. The model implicitly assumes a sufficient large number of cells available, so that all transitions between stages can be considered independent of each other.To develop and validate a model to predict the relationship between naïve and differentiated lymphocyte subsets in the PB and CSF A, 75 patNK cells B in the NK cells . In accoNK cells C. The fo+ and CD8+ T cells in the PB were naïve, whereas in comparison the proportions of differentiated HLA-DR+ CD4+ and particular CD8+ T cells were enhanced in the CSF (+ T cells within the CSF transition with a β2 rate of 19.7744 with a 1.9-fold higher probability into differentiated ones than their respective counterparts in the PB (transition rate β1 = 10.5938). Moreover, the model predicted the distribution of naïve B-cells in the PB and CSF with high accuracy  for the PB with a high accuracy and, also, albeit to a lesser extent, in the CSF. Taken together, the proposed model correctly reproduces existing cell population distributions, whereas it is limited with regard to “non-existing” lymphocyte subsets ; the latter is expected since this scenario is currently not included in the model.First, we validated the model by comparing the predicted cell distributions in PB and CSF compartment under homeostatic conditions with the available data. Multi-parameter flow cytometry of PB and CSF from 75 patients with no sign of an inflammatory CSF as a non-inflammatory control was performed. Flow cytometry of T-lymphocyte subsets in the PB and CSF of these patients revealed that the majority of CD4 the CSF A. This s the CSF . For exaaccuracy A. Simila+ and CD8+ T cells were strongly enhanced in the CSF of RRMS patients and to a lower extent in the PB. Furthermore, we detected CD19lowCD138+ differentiated plasmacytoid cells in the CSF of RRMS patients, one of the disease hallmarks [dim NK cells in the PB (N = 216), based on PB data only, and compared them with the available CSF data  and differentiation  rates  rates C. A dire2) rates .+-mediated endotheliopathy affecting the microvessels of the brain, eye, and inner ear [+-expressing CD8+ T cells in both the PB and CSF is a hallmark of this disease [+ T-cell compartment compared to its differential diagnosis RRMS are accurately described by our model  indicating that the increased proportions of differentiated CD8+ T cells in the CSF are mainly caused by increased transition of peripheral differentiated CD8+ T cells from the PB into the CSF  reproduces the acquired PB/CSF data with high accuracy, (ii) provides quantitative factors describing the differentiation and transmigration rate, and (iii) facilitates prediction of data sets that cannot be acquired. In particular, the latter might help to reduce lumbar punctures and flow cytometry processing steps by simulation of the mathematical model.Despite offering potential treatment targets, the complex processes of transmigration of lymphocyte subsets into the CNS and activation/differentiation within the PB and CSF compartment under homeostatic and autoinflammatory conditions are still poorly understood. We have recently proposed a mathematical framework based on Markov jump processes to the distribution and migration of CD4nditions . Here, w1 into the CSF, (ii) differentiation of naïve lymphocytes with the rate β1 within the PB, (iii) transmigration of differentiated lymphocytes from the PB with the rate α2 into the CSF, and (iv) differentiation of naïve lymphocytes with the rate β2 within the CSF compartment. Due to missing markers, recirculation of lymphocytes from the CSF into the PB cannot be incorporated in the model. However, the migration rates α1/α2 are net rates including both lymphocyte migration into and back from the CSF. Nevertheless, we could demonstrate that our model does not only reproduce the acquired data of the differentiation stage and distribution of T-cell subsets and NK cells in the PB and CSF under steady state conditions, but also in distinct autoinflammatory CNS diseases including MS and SuS with high accuracy. The parameters of the model need to be determined based on existing data sets and, clearly, sufficient training data sets for each disease are a prerequisite. However, this calibration step needs to be conducted only once for each group of patients. Following calibration, the model can be used to also predict previously unknown distributions as exemplified in We developed this model, focusing on the contribution of four processes including (i) transmigration of naïve lymphocytes from the PB across the blood-CSF barrier with the rate α1,2) and differentiation rates  in addition to the qualitative prognosis as well as changes of these factors by immune-modulating therapies. Moreover, we could demonstrate that the model qualitatively coincides with the expected mechanisms of the immune-modulating therapies. For example, the transmigration blocker natalizumab resulted in a reduction of transmigration rates, whereas alemtuzumab had an impact on the differentiation rates.An advantage of the explicit mathematical model is that it provides quantitative factors describing the transmigration , and it is used for the prediction of individual patients. To eliminate statistical errors, we show a range of expected cell distributions obtained from the prediction of the model for individual patients. Due to low cell numbers and sparse CSF materials, repeated measurements in the CSF are not possible. Thus, we could not generate necessary standard deviations on the supplied patient data. The model could be made robust with respect to variations in the data and the potential impact of the measurement error could also be predicted. Although the specificity of CSF analysis could be enhanced if longitudinal data sets were available , for ethAll subjects included in this study gave their written informed consent in accordance with the Declaration of Helsinki and a protocol approved by the Ethics Committee of the University of Münster  were included in this study. All cases presented with non-specific complaints and underwent lumbar puncture during a routine diagnostic examination conducted to rule out any neurological condition. None of the healthy controls suffered from a neurological disorder, nor did they show any specific abnormalities during the neurological examination. In addition to the clinical classification, controls fulfilled the following laboratory criteria defining a non-inflammatory CSF: <5 cells/μL CSF, <20 mM lactate in the CSF, no dysfunction of the blood/CSF barrier , no oligoclonal bands (OCBs) in the CSF, and no intrathecal immunoglobulin (Ig)G, IgA, or IgM synthesis.The blood and CSF samples of control subjects without autoimmune or neuroinflammatory disorders  following treatment with VersaLyse  according to the manufacturer’s instructions. The following monoclonal antibodies were used at 1:200 dilutions: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange . Flow cytometric analysis of stained cells was performed following standard protocols. Cells were analyzed on a Navios™ flow cytometer (Beckman Coulter) using Kaluza Analysis Software  and presented using Prism 6.0 (Graph Pad). low CD14- cells. Lymphocytes were further split into T cells (CD3+CD56-), NK cells (CD3-CD56+), B cells (CD19+CD138- naïve B cells), and plasmacytoid cells . In addition, NK-cell subsets were defined by differential expression of CD56 and CD16 as CD56brightCD16dim/- (naïve) and CD56dimCD16+ (differentiated) NK cells. Finally, CD4+CD8- T-helper and CD4-CD8+ cytotoxic T-cell subsets were selected from total T cells and split into HLA-DR- naïve as well as HLA-DR+ differentiated cells, respectively. For quantification of total cell counts, FlowCount Fluorospheres (Beckman Coulter) were selected based on fluorescence characteristics.Leukocytes from the PB and CSF were gated based on established lineage markers for major cell populations . Therefo1  and two for naïve and differentiated lymphocytes, respectively, are considered. Transition between all stages will be described next. We assume linear, independent transition processes without memory. Hence, the model for transition between stages is the Markov chain model and the modelled transitions are shown in 1 to naïve stages in the compartment CSF. Lymphocytes in the compartment PB and stage naïve transition within this compartment with rate β1 into the differentiated stage. Lymphocytes in the compartment PB and differentiated stage transition with rate α2 into the CSF (and stay differentiated). Finally, with the rate β2 naïve lymphocytes transition within the CSF compartment into differentiated ones. We assume a sufficiently large number of cells available such that transition between stages can be considered independent, and we use the steady state of Markov process for prediction of the cell distribution.In more detail, we proceed as follows: The quantity we model are cell distributions. We assume that cells at different stages undergo different processes. A total of four possible cell stages X in R4 the probability to find a cell in one of the stages . Then, the number of cells Xi A0 in each stage i = 1,..,4 is obtained upon multiplication with the total number of cells A0. Since X1,.., X4 are probabilities we have X1 + X2 + X3 + X4 = 1. Furthermore, X1,.., X4, fulfills the following equations:The model can be stated in the mathematical equations as follows: Denote by X in R1, α2, β1, and β2. Provided we have k = 1,..,K, experimental measurements of cell distributions in all four stages given, i.e., Xk in R4, we use the model to estimate the rates α1, α2, β1, and β2. Since we do not have information on possible measurement error on the experimental data we consider an unweighted and unbiased least-square formulation to obtain optimal rates α =  and β = . To this end we solve the following problem:For some invertible 4 × 4 matrix A and a 4 × 1 vector b, both depending linearly on α1, α2, β1, and β2, the previous problem is nonlinear due to the occurrence of the inverse of the matrix A. It is easy to show that A is invertible for all 0 < α1, α2, β1, β2 <1. The minimization problem is solved numerically up to a tolerance of 10−16. The problem did have a numerical solution for each data set we tested.Even if A, b are linearly dependent on α


CP was determined using a time-trial (TT) protocol of three durations  interspersed by 30 min passive rest. FTP was calculated as 95% of 20-min mean power achieved during the TT. Differences between means were examined using magnitude-based inferences and a paired-samples t-test. Effect sizes are reported as Cohen’s d. Agreement between CP and FTP was assessed using the 95% limits of agreement (LoA) method and Pearson correlation coefficient. There was a 91.7% probability that CP (256 ± 50 W) was higher than FTP (249 ± 44 W). Indeed, CP was significantly higher compared to FTP (P = 0.041) which was associated with a trivial effect size (d = 0.04). The mean bias between CP and FTP was 7 ± 13 W and LoA were −19 to 33 W. Even though strong correlations exist between CP and FTP , the chance of meaningful differences in terms of performance , were greater than 90%. With relatively large ranges for LoA between variables, these values generally should not be used interchangeably. Caution should consequently be exercised when choosing between FTP and CP for the purposes of performance analysis.To investigate the agreement between critical power (CP) and functional threshold power (FTP), 17 trained cyclists and triathletes (mean ± SD: age 31 ± 9 years, body mass 80 ± 10 kg, maximal aerobic power 350 ± 56 W, peak oxygen consumption 51 ± 10 mL⋅min Sport scientists, athletes, and coaches intuitively understand that as exercise intensity increases, a point is reached where a maximal metabolic steady state occurs, beyond which perceptions of effort and physiological perturbations progress more rapidly  to be significantly correlated with FTP. Also, –1 increase above the point of a minimum ratio between blood lactate concentration and work rate). The authors concluded that despite strong correlations, the limits of agreement between the FTP estimates and IAnT were too wide to be used interchangeably. A major limitation of some of these aforementioned studies is that FTP was compared to parameters obtained from incremental exercise, which are known to be protocol dependent and may not align with indices like CP or MLSS obtained during sustained constant work rate exercise . All procedures performed were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments. Informed consent was obtained from all participants after information of the nature and any risks associated with this study were provided.Participants in this laboratory-based study were 17 moderately trained cyclists and triathletes , which enables the participant to use their own personal racing bicycle.During visit one, –1. Participants self-selected cadence throughout, and when this decreased by more than 10 rev⋅min–1 for 10 s despite strong verbal encouragement, the test was terminated. Pulmonary gas exchange was measured breath-by-breath using a Cortex MetaLyzer 3B gas analyzer , and heart rate (HR) was monitored via the ergometer. Outliers were excluded from further analysis after visual inspection by two independent researchers. MAP was calculated using the following equation:After a standardized warm-up at 150 W and for 5 min, participants completed an incremental step test until volitional exhaustion. After 3-min baseline at ∼80 W, the test commenced at an intensity of 100 W with a step-like increase of 20 W minL represents the last completed stage (W), t is time for the incomplete stage (s) and PI is the step increment (W). 2peak was taken as the highest 30-s rolling-average during the incremental test. The mean ± SD duration of the peak oxygen test was 13.5 ± 2.8 min.where PCritical power was determined using maximal self-paced TT efforts over the durations of 12, 7, and 3 min with a 30 min passive rest between efforts. The protocol started with a 5 min warm-up phase at 100 W immediately followed by a switch of the ergometer into TT mode, where resistance increases or decreases as a function of cadence and pedal force. During the TT, participants were allowed to self-pace via use of a virtual gear changer mounted to the handlebars. Feedback of elapsed time and strong encouragement was provided throughout and participants were asked to produce the highest average PO possible. Heart rate (HR) was measured continuously and rate of perceived exertion (RPE) was recorded immediately at the end of trials. HR within 10 beats of age-predicted HR maximum and RPE values above 18 were taken as an indicator for a maximal effort and accepted as a successful test.W′) were determined using two linear and one hyperbolic model:Critical power and its related maximum work above CP (P = W′/t + CP) provided the lowest combined standard error of the estimate (SEE)  and was consequently used. The linear PO vs. inverse of time model provided the lowest combined SEE for all participants (n = 17) and was therefore used for further analysis. This model was also used to calculate predicted maximal 20 min PO (p20MMP) values  + 300).The inverse time linear model (MMP) obtained during the TT . Pearson product moment correlation was used to provide an estimate of strength of association between variables, and linear regression was used to calculate the SEE associated with prediction of 20MMP and FTP from CP, and also p20MMP and pFTP, respectively. Lin’s concordance coefficient and the intraclass correlation coefficient (ICC) was used to evaluate the agreement between methods assessed by a Microsoft Excel spreadsheet. To assess differences between CP, FTP, and 20MMP a repeated-measures analysis of variance was used. Partial squared eta . The typical error of the estimate and the coefficient of variation (%)  unless stated otherwise.Data were first examined for normality using the Shapiro-Wilk test. Differences between means were assessed using magnitude based inferences  where thtion (%)  were useP > 0.05). Descriptive data is presented in W′ results from linear and hyperbolic models including combined SEE are depicted in large effect size were found between CP, FTP, and 20MMP , but not between CP and FTP , and between CP and 20MMP . CP and FTP were significantly correlated  , the ICC was r = 0.976 (95% CL: 0.915 to 0.991) and the typical error in raw unit was 13 W (95% CL: 9 to 20 W) and expressed as a coefficient of variation (%) was 5.6% (95% CL: 4.1 to 8.8%). Mean peak HR and mean RPE values for the 20 min TT, 12 min TT, 7 and 3 min TT were as follows: 184 ± 12 b min–1 and 19 ± 1; 184 ± 13 b min–1 and 19 ± 1; 181 b min–1 ± 12 b min–1 and 19 ± 1; 178 ± 14 b min–1 and 19 ± 1 (P > 0.05).All data were normally distributed (< 0.001) . There w< 0.001) . The LinMMP). The probability of a meaningful difference was >90%, however, the effect size was only of a trivial order. The results also revealed wide LoA between CP and FTP (± 24 W), a large bias (−12 W) and large intraindividual variations (± 12 W). Nevertheless, the variables were strongly correlated (r = 0.969). According to Cohen, the typical error of the estimate between CP and FTP can be interpreted as small . This, however, is arguably above the 5%, which is the commonly accepted upper limit in sport science research  and predicted 20 min PO (236 W). This might raise questions about the application rule of 5% from the 20 min FTP values as postulated by R2 = 0.939) are explained by other factors. Nevertheless, these 6.1% are suggested to notably question the interchangeable use of CP and FTP. Collectively, these findings do not support the assertion by We observed a mean difference of 7 ± 13 W between CP and FTP. Given that a 1% difference in PO (∼2.6 W in our study) is considered a “smallest worthwhile change,” the disparity between CP and FTP equates to a high probability, that a meaningful difference exists in practice . Even thtrivial effect size, between CP and 20MMP. However, previous research has shown that time-to-fatigue at CP equals ∼23 min in both untrained (MMP) is generally lower than CP.Interestingly, there were no significant differences, apart from a ntrained  and traintrained . Conversntrained  which inntrained . It is, ntrained . These dntrained . This is2 uptake  above the maximal PO estimated to be “wholly-oxidative.” Since anaerobic energy provision contributes to W′ (W′ occurs (PO at or below CP), or if expenditure of W′ occurs, then due to some other mechanism a corresponding decrease in average power must also occur. This has to be done to offset the additional energy contribution arising from expenditure of W′. In fact, it should be expected that predicted MMP over a given duration i.e.: P = W′ (1/t) + CP, where t = the duration of the task, should be close to the actual performance power. Further analysis revealed that p20MMP and 20MMP were significantly different . The discrepancy in mean values here is potentially explained by emergence of fatigue mechanisms that are not dominant during the short duration TTs used to estimate CP. For example, central fatigue has been shown to increase during a 20 and 40 km TTs compared to a 4 km TT , which suggests the domain of validity of the 2-parameter CP model should be limited to durations less than 20 min.One of the differences between CP and FTP is that the concept of CP also incorporates es to W′ , then a  4 km TT , and als 4 km TT , thus fo 4 km TT , and W′  4 km TT . Thus, iThere are certain key limitations to the present study. In the absence of pulmonary gas exchange and measurements slightly above and below both CP and FTP, we were unable to validate these estimates according to the physiological criteria which best describes the threshold phenomenon. Thus, further studies are required to establish the physiological validity of the FTP concept. Moreover, the prediction of performance PO beyond 20 min based on the CP model can be questioned. This is a limitation of the power-duration relationship modeling procedure. Furthermore, we did not conduct familiarization trials but used only experienced cyclists or triathletes. Finally, it is currently unclear if different warm-up protocols influence FTP and this should consequently be addressed in further research.MMP method. Furthermore, the LoA between CP and FTP were wide and therefore, these estimates of threshold intensity should not be used interchangeably.The “smallest worthwhile change” in PO during a sustained TT which approximates threshold intensity is approximately 1% in elite athletes . In the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University of Greenwich. The patients/participants provided their written informed consent to participate in this study.BK conceptualized the study, conducted testing, and wrote parts of manuscript. LP and AB wrote parts of the manuscripts. AK conducted testing and wrote parts of the manuscripts. NT conducted statistical analysis and wrote parts of the manuscript. CT analyzed data and wrote parts of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The application of surface electromyography (sEMG) in neurology is sometimes limited by a scientific background in the use of sEMG. Students frequently use sEMG only when developing their graduate studies. To reduce these barriers, we promoted a free Winter School on sEMG to Latin American students. The school was a 3-day event with theoretical classes and computer programming in Matlab. Lectures were delivered in Portuguese and Spanish to 50 participants. All lectures were recorded and made available on YouTube®. After the School, participants completed a written exam to receive a certificate. The written exam revealed the average effectiveness of 71 ± 20% in the comprehension of topics addressed during the school. Participants rated the School as “excellent” and considered the event as having changed their thoughts about the use of sEMG. Limited mathematical skills or background were the main barriers identified to follow the lectures and to make use of sEMG. We conclude that the Winter School had a positive impact on participant's formation, especially by showing them the importance of continuous involvement with the concepts related to sEMG to become proficient in its use. From the participant's point of view, the activity was excellent and the follow up of the school on YouTube® suggests that combining face-to-face activities followed by the online availability of lectures is a valid strategy to reinforce the learning process and to reduce barriers in the use of sEMG. Whether similar results would be achieved for a paid registration event in an economically developing region, still requires further investigation. Latin America is one of the most unequal regions in the world based on the Gini Index . TherefoFor example, in Chile, which is considered an emergent economy, social inequality remains high, like in other Latin American countries, and education is considered a consumer good. It means that even public education has a high cost, which can be higher than the cost of studying at private institutions. It results in science being transformed to privilege people with better economic conditions and credit capacity, which ends up segregating the society . In BrazIn addition to the economic barriers, there are other barriers requiring attention. These include weak, or absence of a proper educational background to move forward in concepts related to higher education. One important topic of education and research widely used in the fields of physical education, physiotherapy, and engineering is surface electromyography (sEMG). However, many barriers are observed in the use of this tool, as we will further discuss in this article.sEMG encompasses the measurement of electrical signals involved in the muscle contraction and is quantified using surface electrodes, with applications in humans  and othehttp:www.robertomerletti.it and www.seniam.org, where a wide number of sources for historical and updated academic material are available to help elementary and advanced sEMG studies.For example, to promote better use of the sEMG in clinics, hospitals, and gymnasiums, knowledge of several basic concepts from anatomy, neurophysiology, and physics, among others, will be required. The user needs to properly locate the electrodes and be aware of particularities depending for example on the muscle evaluated , 18. SimThe real condition is that most health professionals from Latin America do not receive a background in mathematics sufficient enough to understand and use sEMG in their professions. Many students seek training in health courses just to get away from mathematical work. National scientific journals related to human movement sciences are available, and most of the papers published in these journals, addressing sEMG, are the result of laboratory research developed during master's and doctoral programs. These papers are accessible to students, but the use of scientific papers during the study of these topics needs to be strongly promoted. This establishes an important barrier to sEMG applications that have been considered when promoting regular congresses in the field, like the Brazilian Congress of Biomechanics, and the Congress of the Chilean Association for Human Movement Sciences. But this limitation is not observed only in economically developing countries (EDC).The evaluation of the knowledge from participants working in developed countries also suggested that neurology residents show a knowledge gap regarding the use of sEMG, resulting in up to a quarter of residents expressing a lack of confidence when using techniques like sEMG . FurtherThe knowledge about sEMG may also impact on aspects related to patient management. When considering the use of sEMG in a clinic or hospital, a patient's education about sEMG is also important. We consider that in some cases, not informing the patient or participant about what the examination is, and how it works, may increase stress and possibly change participants' behavior when realizing that many electrodes are going to be attached to their own body, and this misunderstanding may lead to limited clinical results. Among middle-aged patients undergoing sEMG examination, 52.1% of the patients either received no information about what sEMG is, or, the information provided was very poor or incorrect . On the Considering all these aspects, we promote educational activities that reduce the knowledge gap and barriers faced when using sEMG, observed among health professionals who want to or already do use of sEMG without proper training in basic concepts related to its use. In 2018 we organized the first Winter School on sEMG Digital Signal Processing for Latin American students. Here we describe this educational activity, and the impact it had as perceived by the participants. We also share our experience in the selection of the topics for lectures and the methodology for the development of the course. The barriers faced when using sEMG are also discussed.The Winter School lasted for 3 days during September 2018 and was held at the Universidade Federal do Pampa, a Brazilian public university established in a remote region of the Rio Grande do Sul state in Brazil. Registration was free of charge and we received 50 applications for registration. The event was advertised on social media and through email lists of different institutions. There was no limitation in the background level required or the purpose of the participants registering to the school, and the program was available to all participants before completing the free registration. We did not control registration according to the level of knowledge of the participants because it has been suggested that merging students with different levels of expertise may bring education advantages especially for those who are less experienced . Upon ren = 24, 49%) or graduate students , and the other 25% were young investigators with a master's or Ph.D. degree in human movement sciences or physiology, one post-doctoral fellow, and one high school student. We did not collect data regarding personal information like the age of the participants, but informally we can report that most of the participants were young with age ranging between 17 and 30 years. A higher number of female participants was noted, which may in part relate to the fact that most of the participants were from the physiotherapy field, which in Brazil usually has a higher percentage of female graduates.The 50 participants had different backgrounds  and came from cities from the south of Brazil (border with Argentina and Uruguay) to participate in the school that was conducted based on a group-learning methodology and thinking-based learning activities. The school was advertised to other countries from Latin America and we consider that the main limitation for attendance of participants from other countries was the high cost to travel and stay abroad. All the participants had a connection with academia being either undergraduate  to 10 , only 10.2% of the registered participants indicated a score higher than 5. The school's program was designed to include theoretical and practical activities in a friendly environment. We organized a single room meeting, with coffee and finger food; lectures were delivered using media projectors, whiteboards, and questions were allowed at any time. The organizers worked to have the entire class actively participate, and when the participants showed signals of tiredness or if they missed the concepts , a short break was proposed.The main goal of the school was to promote a solid basic background, and therefore, concepts related to muscle contraction, joint angles, and ground reaction forces were briefly addressed to ensure that all participants were familiarized with the origins of biological signals that were used for the examples. In this regard, differences between kinetic, kinematic, and sEMG signals were discussed, and sEMG generation and interpretation were often mentioned, commented on, and explained during the course. Topics were organized to promote a progressive level of complexity. The entire program was covered in 3 days within a total of approximately 20 h of activities. Topics were organized in three blocks:10%: the importance of the proper knowledge related to signal processing, its application in sEMG analysis, and where to find relevant material to study;10%: important aspects of data acquisition, mostly related to hardware characteristics;5%: examples of signal processing methods that are common among different techniques (like kinematics and kinetics) in human movements sciences;75%: data processing concepts: limits, derivation, integration, sums, complex numbers, functions, Euler identity, definitions and classification of signals, discrete acquisition, Nyquist theorem, aliasing, time domain and time-series, frequency domain, Newton Prism experiment and frequency decomposition, Fourier series, harmonics, Fourier transform, spectrum, inverse Fourier, and signal filtering. We also focused on the interpretation of sEMG alterations and distortions, cross-talks, amplitude analysis, windowing functions, cancelation of signals, onset-offset analysis, frequency analysis, co-activation, synergy, and linear decomposition into basis functions. We also included some concepts of the Teager energy operator and prosthesis control, but these last topics were only briefly mentioned.The approach to these topics was always based on evidence from the literature. Basic topics related to the mathematics involved in sEMG processing and analyses were discussed based on scientific papers, and the explanation of concepts for signal processing and analysis was always followed up by a discussion of topics related to data processing and the interpretation of results from scientific papers, i.e., topics for non-engineers began with basic math and sinusoidal wave analysis. When a practical situation was needed to illustrate concepts, as well as to discuss how a decision on data analysis or processing affects the results of sEMG analysis, scientific articles were used, i.e., filter coefficients, signal decomposition, or alias signal . Papers considered for the examples were always related to the general study area of the participants such as those addressing gait biomechanics, jump landing, and for some cases, papers reporting the use of EMG associated with prosthesis control and biofeedback, especially to give a contextualized health scenario for non-engineers.The voluntary tutors to the school were one professor with a Ph.D. in human movement sciences, two Ph.D. students in biological sciences, and one MSc. in engineering, and a MSc. in kinesiology and clinical biomechanics. In addition to the handout and the introductory references already mentioned, the teaching activities were mostly based on the textbooks “Digital Signal Treatment” , “Discrehttp://youtube.com/neuromechTV). Slides used to explain the mathematical concepts and examples of data analyses were also uploaded to Researchgate as “Escola de Inverno: Fundamentos de processamento de sinais discretos em biomecânica.”An important concern when planning the school was the need for a follow up on the educational activities, and the importance of revisiting the concepts discussed during the lectures. This is why all the lectures were video recorded and uploaded with free access on YouTube®. Lectures are available for free in the non-monetized official channel of the Applied Neuromechanics Group, the NeuromechTV  to 10 . Participants graded the advertisement given to the school when registration was open as 9.83. The handout and reference list provided upon registration were considered pertinent, and about 50% of the participants said they read the material before the school started. The strategies employed to discuss the concepts on signal processing were graded as 9.33, and the performance of the tutors was graded as 8.83. The overall grade for the activities was 9.85, and the free registration was considered to be of 60% importance on a scale from 0 to 100, in which we asked whether participant registration was conditioned by the free registration.The written exam completed by participants after the school showed average effectiveness of 71 ± 20% (ranging from 40 to 100%) in properly answering the 15 questions related to the topics studied. We observed that better answers were found for questions addressing those topics in which more time was spent on during the lectures and for topics we highlighted as important during the lectures .The school had a significant number of undergraduate students from physical education and physiotherapy courses who participated, as we detailed in the previous section. As observed, when experiments and data processing and analysis using electrocorticography techniques were developed with undergraduate students, the enrollment in educational activities similar to that developed in our school may help increase their interest in following a postgraduate science program . FurtherWe do not believe that the different backgrounds of the participants limited the development of the activities in the school. Nevertheless, in schools aimed at more complex topics, or those prepared to solve particular problems, the heterogeneity of the participants may have different effects on the activity outcomes. We did not strictly control the methods of each activity, but in general, we conducted a team-based learning approach, which is also known to benefit learning in the laboratory environment . We alsoConsidering the topics addressed in the school, a good amount of time was spent on explaining filtering, especially because filtering is a fundamental step in the sEMG data analysis because of its susceptibility to low-frequency noise, baseline noise, and movement artifact noise , 27. FilThe activities developed in the school also considered concepts related to data acquisition. We aimed to provide basic concepts and straightforward recommendations on how a sEMG examination should be conducted. When it comes to professor basic knowledge of sEMG procedures is required, improving teaching practices as well, and we consider that a specific school for teachers would be a valuable activity for the future. The results from sEMG examination can be a good platform to promote learning sessions and tutorials on how muscles are controlled, to discuss concepts of supraspinal control, reflexes, movement production, and regulation . AlthougWe used a strategy of continuous online education by making all the lectures of the school permanently available online after the school was completed. Webinars and online classes are not a novelty these days, but most of the crash courses available are segmented for a very specialized audience, and sometimes assume that the viewers already have a good background. We believe that the recordings available online could promote additional learning when participants watch the classes again, also resulting in revalidation of the concepts, similar to what has been discussed when records of surgical procedures are integrated into patient care . FurtherFinally, the main barriers identified by the tutors and participants were mathematical skills or background, i.e., algebra, calculus, and mathematical logical thinking. Furthermore, in many cases, fear of the numbers and programming a computer, and the insecurities and anxiety about colleagues from developed countries—who might be better prepared—were other barriers identified among the participants in Latin America. However, important social barriers also exist in regions where investment in research is not aligned with the economical aims of institutions, thus negatively impacting science education. We therefore recommend the organization of basic science schools for health careers in Latin-America, and to frequently develop critical lectures to fill educational gaps, using theoretical frameworks similar to those of engineering and other sciences, but adding innovative learning strategies from modern pedagogy in health sciences, that considers the biological profile of the students and professionals of health and life sciences.The Winter School had a good impact on participant's formation and may have motivated other groups from Brazil to promote similar educational activities during 2019 as we observed schools in topics related to biomechanics and muscle function being promoted. From the participant's point of view, the activity was excellent, and the follow up of the school on the YouTube® channel, considering the social media engagement, suggests that combining face to face activities followed by online availability of lectures can be a valid strategy to sustain the interest of students on these topics.We recommended frequent organizations of basic science Schools in health careers in Latin-America at all levels i.e., bachelor, Master, PhD, and post-doc, as an initiative to fill educational gaps using theoretical frameworks similar to those that engineering and other sciences adopt, as innovative learning strategies for modern pedagogy in health sciences is advisable. Furthermore, we believe that providing schools that are conducted 100% online could significantly benefit a larger number of students and professionals.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants.CD, AM, and MK: conceived the project; delivered lectures; analyzed data; wrote and approved the final version of the manuscript. FC: conceived the project; approved funding; delivered lectures; analyzed data; wrote and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Testosterone has been postulated to be involved in ALS causation.CSF levels of free testosterone and dihydrotestosterone were measured in 13 ALS patients  and 22 controls .CSF free testosterone levels did not show any significant differences but CSF dihydrotestosterone levels were significantly decreased in all male and female ALS patients.DHT is probably integral to survival of motor neurons. In patients predisposed to develop ALS, there is possibly a sort of “testosterone resistance” at level of blood–brain barrier [BBB] existing right from birth and is likely the result of dysfunctional transport protein involved in testosterone transfer across the BBB. In these patients, lesser amount of testosterone is able to breach the BBB and enter the central neural axis. Lesser amount of testosterone is available to 5 α reductase in the anterior pituitary to be converted to DHT and lesser amount of DHT is generated. There is inadequate negative feedback suppression of LH at the level of anterior pituitary by DHT. As a result of higher LH levels, testosterone levels rise in the peripheral testosterone fraction [the fraction outside the BBB] and this explains the various physical attributes of ALS patients like lower Ratio of the index and ring finger lengths (2D:4D ratio), increased incidence of early onset alopecia etc. This deficiency of DHT leads to motor neuron death causing ALS. CSF levels of dihydrotestosterone were measured in 13 ALS patients [7 males and 6 females] and in 22 controls [12 males and 10 females], and we found that CSF dihydrotestosterone levels were significantly decreased in all the male and female ALS patients dihydrotestosterone probably plays a key role in ALS pathogenesis. All CSF samples were collected in the morning between 9:00 a.m. and 11:00 a.m. as some studies  Patients fulfilling the diagnosis of clinically definite ALS and clinically probable ALS as per the El Escorial Criteria (EEC)  Controls were enrolled from surgery, gynecology and obstetrics and from orthopedics wards. CSF was obtained from male and female controls undergoing lumbar puncture for spinal anesthesia for surgery.Exclusion criteria used for both patient and control selection:(a) Subjects not giving consent for study. (b) Subjects suffering from cryptorchidism, testicular malignancy or any other testicular pathology. (c) Subjects taking anabolic hormones. (d) Subjects suffering from pituitary or adrenal disease. (e) Female subjects with history of PCOD or history of hirsuitism. (f) Subjects with family history of ALS or any form of motor neuron disease or frontotemporal dementia [FTD].CSF concentrations of free testosterone and dihydrotestosterone were measured using solid phase enzyme‐linked immunosorbent assay (ELISA) based kits sourced from IBL International.3Clinical and demographic data and CSF concentrations of free testosterone and dihydrotestosterone in ALS patients and controls are summarized in Tables t test to evaluate whether there was any significant difference between the 2 groups.The data sets were found to have a normal distribution using Kolmogorov–Smirnov [K‐S] goodness‐of‐fit test. Thus, we applied students Results are summarized asp = .344].CSF testosterone values in Male ALS patients and controls ‐No significant difference [p = .000009]. CSF testosterone values in female ALS patients and controls‐No significant difference [p = .917]. CSF dihydrotestosterone values in female ALS patients and controls—significant difference was found with values significantly less in ALS patients [p = .000783].CSF dihydrotestosterone values in Male ALS patients and controls—Significant difference was found with values significantly less in ALS patients. (Marynick, Havens, Ebert, & Loriaux, s Denef,  provideds Denef,  done on s Denef,  have fous Denef,  suggest s Denef,  also supInterestingly, the anterior pituitary and the hypothalamic/preoptic area possess 5 α reductase type 2 isoenzyme whereas the glial cells and the neurons predominantly express the 5 α reductase type 1 isoenzyme. It was also found that in the rat pituitary, 5 α reductase type 2 isoenzyme is located mainly in the gonadotropes. Various authors have concluded that type 1 5 α reductase is constitutively expressed in the mammalian brain and appears to have a neuroprotective role and mutations in type 1 5 α reductase lead to death of the organism in gestation itself (Mahendroo, Cala, Landrum, & Russell, . For the same reasons, receptor dysfunction is unlikely to be the culprit. In addition, a study (Bruson et al., Based on data from the above studies, we postulate the following hypothesis‐DHT or one of its metabolites is probably integral to survival of motor neurons and in ALS, it is lack of DHT in the motor neurons which leads to their death. CSF levels of DHT are probably the final arbiter of LH release at the level of anterior pituitary. Entire DHT fraction in the central neural axis is derived from the testosterone fraction which penetrates the BBB since DHT itself cannot penetrate BBB. Dubey et al.  had founConclusions—Our study implicates dihydrotestosterone deficiency in the neural axis as an important component of ALS pathogenesis. More studies evaluating these parameters and also concurrent evaluation of serum testosterone, serum dihydrotestosterone and LH levels would shed more light on ALS pathogenesis. Long‐term serial studies on asymptomatic familial ALS gene carriers would be invaluable in delineating the pathogenesis of this dreaded disease.None.Dr Nishit Sawal: Conceived the entire idea , recruited all patients , wrote the manuscript and uploaded it. Dr Jasbinder Kaur: Did the CSF ELISA testosterone and dihydrotestosterone evaluation of all patients and controls and co‐authored the manuscript. Kamaljeet Kaur: Did the CSF ELISA testosterone and dihydrotestosterone evaluation of all patients and controls and co‐authored the methods portion of the manuscript. Dr Satinder Gombar: Recruited Controls for the study. There is no conflict of interest for any of the authors. The authors take full responsibility for the data, the analyses and interpretation, and the conduct of the research; full access to all of the data; and the right to publish any and all data.Appendix S1Click here for additional data file.


Sortilin C-terminal fragments (shortened as “sorfra”) can deposit as senile plaque-like lesions within AD brains. The course and pattern of sorfra plaque formation relative to Aβ and pTau pathogenesis remain unknown. In the present study, cerebral and subcortical sections in postmortem human brains (n = 46) from aged and AD subjects were stained using multiple markers . The course and pattern of sorfra plaque formation relative to Thal Aβ and Braak NFT pathogenic stages were determined. Sorfra plaques occurred in the temporal, inferior frontal and occipital neocortices in cases with Thal 1 and Braak III stages. They were also found additionally in the hippocampal formation, amygdala, and associative neocortex in cases with Thal 2–4 and Braak IV–V. Lastly, they were also found in the primary motor, somatosensory, and visual cortices in cases with Thal 4–5 and Braak VI. Unlike Aβ and pTau pathologies, sorfra plaques did not occur in subcortical structures in cases with Aβ/pTau lesions in Thal 3–5/Braak IV–VI stages. We establish here that sorfra plaques are essentially a cerebral proteopathy. We believe that the development of sorfra plaques in both cortical and hippocampal regions proceeds in a typical spatiotemporal pattern, and the stages of cerebral sorfra plaque formation partially overlap with that of Aβ and pTau pathologies.Amyloid plaques and neurofibrillary tangles (NFTs) are hallmark lesions of Alzheimer’s disease (AD) related to β-amyloid (Aβ) deposition and intraneuronal phosphorylated tau (pTau) accumulation.  Senile plaque deposition and neurofibrillary tangle (NFT) formation are considered as the two primary neuropathological features in Alzheimer’s disease (AD); both were initially identified in human brains using classic histological preparations  . The β-aThal staging divides Aβ pathogenesis into five phases: (1) occurring in isocortex; (2) spreading into allocortex ; (3) spreading into subcortical structures  and white matter; (4) involving brainstem ; and (5) involving pons  and cerebellum . The abosortilin fragments as “sorfra(s).” Our initial characterization suggests that this extracellular proteopathy is associated with neuritic-like Aβ plaques, but not with diffuse plaques or subpial and vascular amyloidosis. Notably, sorfra plaques are not detectable in some commonly used transgenic mouse models of AD  or in aged non-human primates , whereas the other hemibrain was fixed in formalin for 2 weeks. Blocks (∼0.5 cm thick) from the fixed hemibrain were then prepared from the temporal lobe at the level of midhippocampus, the frontal lobe at the level of the anterior horn of the lateral ventricle (LV) and including the precentral gyrus, and the occipital lobe at the level of the posterior horn of LV. Blocks were also taken from subcortical regions including the striatum, the diencephalon with insular cortex, the amygdala complex and basal forebrain nucleus, the midbrain at the level of the substantia nigra, the pons at its middle level, the medulla at the middle level of the olive, and finally a cerebellar slice containing cortex and dentate nucleus. These blocks were then transferred into 30% sucrose in 0.1 M phosphate buffer, embedded, and then sectioned at 40 μm using a cryostat. Sections from each block were collected and placed into the wells of culture plates filled with phosphate-buffered saline . Each well contained four sections at an equal distance . The sections were rinsed with PBS to remove the embedding medium and then stored in a cryoprotectant at −20°C before staining.All sections to be stained were removed from the culture plates stored in the −20°C freezer and rinsed with PBS to remove the cryoprotective chemicals. Relevant sections were immunolabeled with the following antibodies. A monoclonal mouse anti-Aβ 6E10 antibody , a rabbit anti-pTau antibody , a rabbit antisortilin antibody targeting the intracellular C-terminal domain  , and a r2O2 in PBS for 30 min and 5% normal horse serum in PBS with 0.3% Triton X-100 for 1 h. Sections were then incubated with the primary antibodies according to the above dilutions at 4°C overnight on a laboratory rotator. The next day, sections were incubated with a pan-specific biotinylated horse anti-mouse, rabbit, and goat immunoglobulin G at 1:400 for 1 h. All sections were then rinsed in PBS and then incubated with the tertiary ABC reagent at 1:400  for 1 h. The final immunoreactive product was visualized in 0.003% H2O2 and 0.05% 3,3’-diaminobenzidine. An additional set of sections from all the cases was stained with the modified Bielschowsky silver stain to visualize both neuritic plaques and NFTs  for the initial assessment of immunolabeling pattern. All sections were then imaged using the 20 × objective on a Motic-Olympus microscope equipped with an automated stage and imaging system , which could yield a final autofocused, montaged, and magnification-adjustable image covering the entire area of a glass slide. Motic scanned images from adjacent sections with different labelings were examined comparatively from low to high magnification. Specifically, the presence, distribution, and burden of amyloid plaque pathology were cross-validated in 6E10- and BACE1-stained sections, with the latter displaying dystrophic presynaptic terminals typically arranged as rosette-like clusters in neuritic plaques . Similarp < 0.05 . The stages of sorfra plaque formation were also compared with the Thal Aβ and Braak NFT stages assessed microscopically for individual and groups of brains.We carried out quantitative analysis on the amount of sorfra plaques in selected cerebral regions to assess the spatiotemporal progression of sorfra plaques relative to Thal Aβ and Braak NFT stages. This quantification was carried out in representative limbic regions, as well as associative and primary cortical areas, including the hippocampal CA1, subiculum (Sub), inferior temporal gyrus (ITG), superior temporal gyrus (STG) in the temporal lobe; the lateral orbit gyrus [OrG(l)] and superior frontal gyrus (SFG) in the frontal lobe; and the lateral occipitotemporal gyrus [OTG(l)], area 18 and area 17 in the occipital lobe. Images from the above areas were extracted from the original Motic files at 4 × magnification and saved as TIFF documents. In each extracted microscopic field, the area occupied (%) by sorfra plaques and the total regional area (gray matter) were obtained by using the OptiQuant software , as described previously . QuantitFigures were prepared from representative cases using the Motic-scanned images from various brain regions. Thus, a global view image at 2 × (plaque immunolabeling) or 4 × (pTau immunolabeling and silver stain) magnification was prepared for a given brain region, with high-power images from representative subregions provided as additional panels to illustrate the details of labeled profiles. Figures shown as main documents are summarizations of labelings in representative sections covering multiple neuroanatomical structures, emphasizing the differences between, and the evolution of, the neuropathologies. Supplemental figures are also provided to illustrate the global view (using an atlas format), as well as enlarged views of labelings in selected regions. Schematic figures were prepared to illustrate the spatiotemporal progression of sorfra, Aβ, and NFT lesions, based on the observational and morphometric data obtained from the current study, and in reference to the original documentation of Thal Aβ and Braak NFT staging .Primary age-related tauopathy (PART) refers to the occurrence of tauopathy in the absence of Aβ deposition in the human brain, which has been well documented in literature . In thisOverall, sorfra plaques were not found in aged human brains with PART in this present study. To assess the early development of sorfra plaques and the pattern of distribution, we examined sections from aged brains with early stages of Aβ and pTau pathology. Overall, less sorfra plaques were found relative to Aβ deposition in selected cerebral cortical areas in the cases with Thal phase 1 pathology, in which the NFT pathology ranged from Braak stage I–III; that is, involving the entorhinal to limbic regions as assessed with pTau immunolabeling and silver stain. The regional pattern and extent of pathology in the temporal, frontal, and occipital lobe structures from a representative case are described below with detailed illustrations –4 and suThe C-terminal sortilin antibody visualized a small amount of extracellular lesion in the temporal neocortical areas . Sorfra In comparison, Aβ labeling was present across the neocortical regions from the STG, MTG, and ITG to FG . β-AmyloSorfra plaques were observed in the basal or inferior frontal neocortical gyri, with a higher density in the anterior [OrG(a)] than in lateral [OrG(l)] orbit gyri . No plaqIn comparison, Aβ plaques occurred with an overall low density across the basal as well as the lateral and superior frontal gyri, appearing largely as compact plaques localized to the middle layers of the cortex . HoweverSorfra plaques occurred with an overall low density across the entire occipital cortical subregions, but localized primarily to the basal or temporal cortical regions . Thus, sIn comparison, Aβ labeling appearing predominantly as compact plaques was present in low density over the basally located occipitotemporal gyri, as well as medially and superiorly located visual cortical areas . CAA andTo explore the regional progression of sorfra plaques beyond the above early stage, cerebral and subcortical sections from brains with Thal Aβ (2–4) and Braak NFT (III–V) stages were examined. The distribution and amount of sorfra, Aβ, and pTau pathologies were compared using the Motic scanned images from adjacent sections of the temporal, frontal, and occipital lobes. Again, for the purpose of cross-validation, BACE1 and silver stain in neighboring sections were examined. The results described below were obtained from the brain of a demented patient, with Aβ and pTau pathologies at Thal phase 4 and Braak stage IV, respectively.A large number of sorfra plaques existed across the temporal neocortical regions and also over the entorhinal cortical areas and all subregions of the hippocampal formation . Thus, tLarge amounts of Aβ labeling were also present across the neocortical and entorhinal cortical areas, and the hippocampal formation . The oveSorfra plaques were present across the laterally located SFG, MFG, and IFG and the basally located OrG(l) and OrG(a) . HoweverIn contrast to sorfra plaques, Aβ labeling occurred in the lateral and basal gyri of the frontal lobe with comparable density, whereas the overall amount of deposition was dramatically increased relative to the aforementioned early stage of lesion in the frontal lobe . MorpholSorfra plaques were present in a substantial amount in area 18, whereas much fewer lesions occurred in area 17 . The plaAccording to the Braak NFT staging, tauopathy in the primary motor and sensory neocortical functional areas indicates that the pathogenesis has reached stage VI. In the NIH “ABC” scoring system, the occurrence of Aβ plaques, silver-stained neuritic plaques and NFTs in areas 17 and 18 mark stage C, or the end stage of AD neuropathology . In the In both the lateral  and medial  parts of area 4, sorfra plaques occurred in a substantial amount over the cortical gray matter . The plaRelative to the distribution described in the preceding result section, sorfra plaques occurred apparently in area 17 in addition to area 18 . NotablyFollowing examination of sections from multiple cases at various Thal Aβ and Braak NFT stages, we did not find a presence of sorfra plaques in subcortical structures. Results detailed below were obtained from a brain with Thal Aβ 5 and Braak V stage and showed a lack of sorfra plaques in the striatum and diencephalon with extensive Aβ and pTau pathologies –12. OtheIn the frontal cerebral sections at the level of the mammary bodies, immunolabeled profiles over multiple telecephalic and diencephalic structures were visualized . Sorfra β-Amyloid deposition was broadly present in the cerebral and subcortical structures . In the Briefly summarizing the findings from the midbrain, pons, and cerebellar sections , Aβ labeBased on cross-region comparison in the same brain and between cases as described above, sorfra plaque formation was recognized to progress in cerebral subregions in a stage-dependent manner. To depict the spatiotemporal progression quantitatively, the fractional areas occupied by sorfra plaques were measured in selected neuroanatomical regions, with the areas of interest (AOIs) including representative basally/inferiorly and superiorly located cerebral regions. Thus, the AOIs were the gyral portions of the OrG(l) and SFG, respectively, in the frontal lobe; the middle portions of hippocampal CA1 and the subiculum, and the gyral portions of the ITG and STG, respectively, in the temporal lobe; and the gyral portion of the OTG(l), the middle segment  of area 17 (A17), and the gyral portion of area 18 (A18) in the cuneate gyrus, respectively, in the occipital lobe .n = 4, P < 0.0001, F = 7.74, df = 8,27). Specifically, differences  existed between the basal and superior cortical areas in the frontal  and occipital  lobe cortices, according to Bonferroni multigroup comparison (post hoc) test. Differences also existed for CA1/Sub relative to ITG/STG respectively, but neither between CA1 and Sub, nor between ITG and STG.In four brains with sorfra plaques localized primarily to the temporal, basal frontal, and basal occipital neocortical regions , the aren = 4, P < 0.0001, F = 6.95, df = 8,36). Post hoc test indicated a significant difference between the SFG and OrG(l) in the frontal lobe. Between the temporal subregions, only the difference between CA1 and Sub reached statistical significance. The mean of A17 was significantly lower than that of the OrG(l), Sub, ITG, STG, OTG(l), and A18, respectively.In five brains with sorfra plaques occurring in the hippocampal formation and occasionally in area 17 , the aren = 4, P < 0.004, F = 3.84, df = 8,27). Intergroup comparisons reported that statistically significant differences existed only for A17 relative to Sub and ITG, respectively.In four brains with sorfra plaques occurring in the primary visual cortex , the fraWhile β-amyloidosis and tauopathy occur prominently in the cerebrum, both lesions can develop in subcortical regions. Thus, Aβ deposition and tauopathy are commonly found in subcortical structures in aged or AD brains exhibiting relatively advanced Thal Aβ and Braak NFT stages of neuropathology . AccordiWe show here that, in contrast to Aβ and pTau pathologies, sorfra plaques do not develop in subcortical structures even in the cases with end-stage Thal Aβ and Braak NFT lesions. As shown in the frontal cerebral sections at the midthalamic levels, sorfra plaques are essentially absent in the striatum, basal forebrain nucleus, thalamus, and hypothalamus wherein substantial amount of β-amyloidosis and tauopathy is present. On the other hand, sorfra plaques are clearly present in the insular cortex and amygdala in the same sections. As illustrated in the In line with the existing literature , we findOn the basis of the microscopic characterization and quantitative analysis, we propose that the development of sorfra plaques in human cerebrum can be divided into three phases or stages . Thus, sThe clinical course of AD is characterized by progressive memory loss and decline of executive cognitive functions, with neurological dysfunctions occurring at the end disease stage involving failures of sensory, motor, and autonomic functions . RegionaThe present study indicates that the extent and spatiotemporal progression of sorfra plaque pathology show some similarity to but do not fully parallel those of cerebral β-amyloidosis or tauopathy . Sorfra 2, respectively (Silver stains (including the Bielschowsky method) are routinely used for postmortem pathological diagnosis of AD, partially because microscopic differentiation of amyloid plaque types based on Aβ immunohistochemistry is not reliable . More imectively . AccordiOur current multisection and multipathology comparative analyses show that silver-stained neuritic plaques can be constituently variable to a great extent because Aβ, sorfra, and pTau pathologies onset and progress differentially between cerebral structures and cortical subregions. Such a constitutional heterogeneity of neuritic plaques can be clearly recognized in the visual cortex. Thus, before sorfra plaque pathology reaches stage C and NFT pathology reaches stage VI, silver-stained neuritic plaques in area 17 essentially consist of Aβ and BACE1-enriched dystrophic neurites  . PatholoBased on the comparative microscopic examination of brain sections labeled for multiple pathological markers and quantitative analysis, we conclude that (1) sorfra plaques are essentially a cerebral proteopathy; (2) the development of sorfra plaques in cortical and hippocampal subregions proceeds with a stereotypic spatiotemporal order; (3) stages of cerebral sorfra plaque formation partially overlap with that of Aβ and pTau pathologies; (4) silver-stained neuritic plaques are constituently heterogeneous in context of molecular pathology.The datasets generated for this study are available on request to the corresponding author.Use of postmodern human brains was approved by the Ethics Committee for Research and Education at Xiangya School of Medicine, in compliance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).X-XY acquired funding, conceptualized the study, and wrote the manuscript. TT, JJ, Q-LZ, Y-NL, and AP were responsible for study methodology. TT, JJ, LW, and Y-NL curated data. JM reviewed and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.