TM assay 1.3K platform. A total of 1,125 proteins were analyzed. There were 86 downregulated proteins and 52 upregulated proteins in ASD (FDR < 0.05). Combining three different algorithms, we found a panel of 9 proteins that identified ASD with an area under the curve (AUC) = 0.8599±0.0640, with specificity and sensitivity of 0.8217±0.1178 and 0.835±0.1176, respectively. All 9 proteins were significantly different in ASD compared with TD boys, and were significantly correlated with ASD severity as measured by ADOS total scores. Using machine learning methods, a panel of serum proteins was identified that may be useful as a blood biomarker for ASD in boys. Further verification of the protein biomarker panel with independent test sets is warranted.Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and social interaction and restricted, repetitive patterns of behavior, interests, or activities. Given the lack of specific pharmacological therapy for ASD and the clinical heterogeneity of the disorder, current biomarker research efforts are geared mainly toward identifying markers for determining ASD risk or for assisting with a diagnosis. A wide range of putative biological markers for ASD is currently being investigated. Proteomic analyses indicate that the levels of many proteins in plasma/serum are altered in ASD, suggesting that a panel of proteins may provide a blood biomarker for ASD. Serum samples from 76 boys with ASD and 78 typically developing (TD) boys, 18 months-8 years of age, were analyzed to identify possible early biological markers for ASD. Proteomic analysis of serum was performed using SomaLogic’s SOMAScan Autism spectrum disorder (ASD), a heterogeneous neurodevelopmental disorder, is characterized by deficits in social communication and social interaction, with restricted, repetitive patterns of behavior, interests, or activities . ASD impASD is currently diagnosed based on behavioral criteria because its underlying disease mechanisms and associated medical, neurological, and psychiatric comorbidities are poorly understood –7. HowevBased on our current understanding of the etiology of ASD, many blood-based biomarker candidates have been investigated , particuTM platform, incorporating machine learning of the associated demographic and clinical data, for biomarker discovery.The objective of the present study was to conduct a proteomic analysis of serum from boys with and without ASD using the SomaLogic SOMAScanThe study protocol and subsequent amendments were submitted by The Johnson Center for Child Health and Development  and approved either by the Austin Multi-Institutional Review Board (for samples collected before October 2017) or IntegReview Institutional Review Board (for samples collected from October 2017 onwards). The study was carried out in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all participants and/or their legal guardians before study participation. Subjects with a genetic, metabolic, or other concurrent physical, mental, or neurological disorder were excluded.A total of 154 male pediatric subjects were enrolled. The ASD group was comprised of 76 subjects with a mean age of 5.6 years (SD 1.7 years). The TD group was comprised of 78 subjects with a mean age of 5.7 years (SD 2.0 years). The ethnic breakdown was as follows: 73 White/Caucasian, 32 Hispanic/Latino, 17 African American/Black, 5 Asian or Pacific Islander, 23 multiple ethnicities or other, and 4 not reported . Co-morbFor the ASD group, all subjects were assessed by a clinical psychologist with research-reliability training using both the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview–Revised (ADI-R). A clinical diagnosis was made based on these data and overall clinical impression using DSM-5 criteria. In addition, ADOS diagnostic algorithms consisting of two behavioral domains: Social Affect (SA) and Restricted and Repetitive Behaviors (RRB) were used to determine an ADOS total score, which provides a continuous measure of overall ASD symptom severity. These scores can be used to compare ASD symptom severity across individuals of different developmental levels ,26 and wFor the TD group, all subjects underwent a developmental screening using the Adaptive Behavior Assessment System—Second Edition (ABAS-II) to rule out developmental concerns. TD subjects were excluded if they had any first- or second-degree relatives diagnosed with ASD.All subjects were healthy, defined as being fever-free for 24 hours, and presenting with no clinical symptoms. A fasting blood draw was performed on ASD and TD subjects between the hours of 8–10 am in a 3.5 ml Serum Separation Tube using standard venipuncture technique. The blood was gently mixed by 5 inversions and then stored upright for clotting at room temperature for 10–15 min. Blood was centrifuged immediately after the clotting time in a swing bucket rotor for 15 min at 1,100–1,300 g. After centrifugation was completed and the turbidity and hemolysis of the serum had been recorded, 250μl aliquots of serum were transferred to 1.0ml coded cryovials and then stored at -80°C. Serum was shipped on dry ice to SomaLogic  for analysis.TM platform 1.3k was used for analysis, and assays were run by SomaLogic. SOMAmer aptamer reagents consist of short single-stranded DNA sequences with ‘protein-like’ appendages that allow tight and specific binding to protein targets.The SOMAScanTM analytes. In this study, 192 proteins failed to pass quality control (QC). After removing these proteins, 1,125 proteins were analyzed. The protein abundance data were normalized by taking log10 transform and then z-transformation. To deal with outliers, any z-transformed values less than -3 and greater than 3 were clipped to -3 and 3, respectively. To discover proteins for ASD prediction, three different methods were deployed: random forest (RF), t-test, and correlation-based methods.The assay measured 1,317 proteins in 150μl serum in 154 samples to identify an optimal subset of proteins to be used as a panel for ASD prediction. An additional 14 samples (7 ASD and 7 TD) were included as blinded duplicates to assess the variability of SOMAScanRF, a well-known decision tree-based ensemble learning method, produces consistent results even without hyper-parameter tuning. At the same time, it measures feature importance by observing how random re-shuffling of each predictor influences its model performance. To train RF models and calculate feature importance, an R package, ‘randomForest’, was used. In this study, we chose MeanDecreaseGini (mean decrease in Gini Index), a weighted measure of the average reduction in node impurity within a random forest, as the surrogate representing a protein’s importance in predicting ASD versus TD. With the normalized data, we trained an RF model 1,000 times. Each protein’s importance value was then averaged over the 1,000 runs. The 10 proteins with the highest averaged importance values were chosen for the RF-based prediction model.A t-test, which determines if there is a significant difference between the means of two groups, is a widely used approach to discover biomarkers in biological data. In this study, the 10 proteins with the most highly significant t-test values were selected for the prediction model.A correlation approach, which measures the statistical relationship between two variables, was used to calculate each protein’s correlation with ADOS total scores (SA + RRB), as a measure of ASD severity. Based upon the absolute values of each protein’s correlation coefficient, the 10 most highly correlated proteins were selected as the correlation-based predictive proteins.After identifying the top-10 predictive proteins from each of the 3 methods , we found 5 proteins that were common to each method used. These were considered ‘core’ proteins, leaving 13 additional proteins that were not part of the core. A prediction model trained with the 5 core proteins was taken as a baseline model. Next, we investigated whether the addition of one or more of the 13 proteins provided any additive predictive power.A logistic regression model was used with datasets based upon the RF model, the t-test model and the correlation model, taking the subjects’ assigned group (ASD or TD) as output variables. We randomly assigned 80% of subjects to a training dataset and the remaining 20% of subjects to a test dataset. We then calculated the trained model’s area under the curve (AUC) for the test dataset as an evaluation metric. This process was repeated 1,000 times so as to obtain a rigorous evaluation while suppressing any bias which might be caused by favorable data splits.limma::goana function in R. From its gene ontology results, the Top-20 biological process pathways were extracted and reported.A pathway enrichment analysis was performed for the optimal proteins. Entrez Gene IDs corresponding to the optimal proteins were fed to a Finally, to evaluate possible confounding factors, we examined the impact of ethnicity, co-morbid conditions/clinical diagnoses, age, and medication use  on the 9TM platform. Three computational methods were combined to search for a panel of proteins with high predictive power for ASD. The top-10 proteins were sought using RF analysis, t-test analysis between ASD and TD groups, and a correlation analysis with ASD severity . The 9 proteins selected for the optimal ASD biomarker panel exhibited an average of 6 to 13% variability between the duplicate assays.To determine the accuracy of the SomaScanFinally, ethnicity, allergies, age, and medication use were analyzed as independent variables using t-tests or Spearman’s rank correlation, as appropriate. Neither ethnicity nor a diagnosis of allergies had an effect on the mean protein counts of the optimal proteins . For ageTM platform. Nine proteins were identified based upon a novel combination of machine learning methods with random forest analysis, t-test analysis, and correlation analysis with ADOS total scores that produced an accurate identification of ASD in boys. Five of the proteins, IgD, suPAR, MAPK14, EPHB2, and DERM, were present in all three analyses and were considered core proteins in the panel. Four proteins providing additive power were combined with the 5 core proteins, and together, the 9 proteins resulted in an AUC of 86% . These proteins have pathway significance related to a number of processes, including negative regulation of CD8-positive, alpha-beta T cell proliferation, immune response, neuron projection retraction, MAPK14 activity, and glutamate receptor signaling, all of which have previously been associated with ASD [The goal of the present study was to identify a blood biomarker profile for ASD from >1,200 proteins using the SOMAScanwith ASD . EthniciImmune system aberrations have been reported in ASD for some time . AbnormaA third core protein, mitogen-activated protein kinase 14 (MAPK14), was significantly lower in ASD versus TD serum. MAPK14 is activated in response to stress and inflammation . In two Another core protein, EPHB2, is linked to NMDA glutamate receptor activity. Interestingly, several lines of evidence suggest an imbalance between excitatory (glutamate-mediated) and inhibitory (GABA-mediated) neurotransmission, which may be a common pathophysiological mechanism and treatment target for ASD –55.All but one (eIF-4H) of the 9 optimal proteins overlapped with the top-10 highly correlated proteins indicating that the biomarker panel was associated with ASD severity, as measured by ADOS total scores. IgD levels were negatively correlated with ADOS total scores. Previous studies have reported similar trends. For example, IgG levels, and to a lesser extent IgM levels, were found to be significantly negatively correlated with total scores measured on the Aberrant Behavior Checklist . suPAR lIn a previous study, we investigated 110 proteins using the MesoScale Discovery platform, and two proteins were found to be most important: IL-8 and TSH 8 –60. SimiTM plates containing >1,200 protein markers/well, well-to-well differences may add variability to the data. To address this, we ran duplicate samples on a subset of ASD and TD samples to determine the variability of the measurements, and for the 9 optimal proteins, the variability in measurement for each protein was <14%. The complex phenotypic heterogeneity of ASD also presents some limitations when performing biomarker studies. To address this, we used standardized diagnostic criteria to classify individuals with ASD, as well as incorporating an analysis of ethnicity, co-morbid conditions/clinical diagnoses, and medication use. Total ADOS scores were correlated with the optimal biomarker proteins strengthening the association between the biomarker panel and ASD-associated behaviors. Co-morbid conditions, such as anxiety, epilepsy, ADHD, and gastrointestinal disorders, occurred at very low frequencies in our cohorts and could not be analyzed The most commonly reported clinical diagnosis, seasonal allergies, and medication use had no effect on the core proteins. Finally, dietary intervention and use of nutritional supplements were difficult to assess accurately due to the limited information collected and our inability to verify the data provided, especially since some subjects were not under the care of a nutritionist or dietitian at the time of study participation.There are some limitations to the present study. Although the sample size is acceptable for a discovery study, the data presented here are preliminary, and a larger validation study is needed to be certain of the value of the biomarker panel. Due to the increased prevalence of ASD in boys, this study only enrolled boys, which does not allow for an investigation of gender-specific differences. Furthermore, although there was no effect of age on the panel of proteins selected, a prospective study is planned that would shed light on the stability of these proteins over time. When making aptamer measurements on SomaScanTM platform. A panel of 9 proteins was identified using three computational methods: RF, t-testing, and correlation analysis with ASD severity scores. The 9 proteins were significantly different in ASD compared with TD boys, they were significantly correlated with ASD severity, and several of these proteins have been mechanistically  and genetically  linked to ASD. The panel of proteins exhibited an AUC of 86% . This novel set of proteins has the potential to be an efficacious blood-based biomarker for the early identification of ASD in boys, particularly since behavioral and developmental assessments are not easily administered in very young children. While the use of machine learning for ASD diagnosis is still in its infancy, identifying key proteomic biomarkers may also lead to targeted intervention strategies as we further elucidate the functional processes associated with ASD and the mechanistic interplay between brain structure and behavior.The present study used serum samples from ASD and TD boys to search for a panel of proteins with diagnostic accuracy for the identification of ASD. Over 1,100 proteins were examined on the SomaScanS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.


Among the tree parts, trunks, roots, leaves and branches accounted for 50%, 22%, 16% and 12% of the total tree biomass, respectively. The average carbon content ratio was 0.49 (range: 0.41–0.52). The average carbon density of forest ecosystems was 205.72 t·hm−2, and the carbon storage of the forest ecosystems was 131.35 Tg (standard deviation: 31.01) inside study area. Soil had the highest carbon storage (65.98%), followed by tree (32.81%), herb (0.78%) and litter (0.43%) layers. Forest age has significant effect on biomass, carbon content ratio, carbon density and carbon storage. The carbon density of forest ecosystems in study area was spatially distributed higher in the south and lower in north, which is influenced by climate, topography, soil types and dominant tree species.The natural mountain forests in northwest China are recognized as a substantial carbon pool and play an important role in local fragile ecosystems. This study used inventory data and detailed field measurements covering different forest age groups , structure of forest  and trees  to estimate biomass, carbon content ratio, carbon density and carbon storage in Altai forest ecosystems. The results showed that the average biomass of the Altai Mountains forest ecosystems was 126.67 t·hm Terreste effect ,4. Forese effect . EvaluatAlthough carbon storage studies of forest ecosystems have been carried out all over the world, the estimated carbon density values vary due to the wide diversity of vegetation types, forest age, tree structure, climate, regional characteristics, research scales, data sources and applied methodologies ,7,8,9,10To further understand forest carbon storage, the more precise carbon sequestration studies focusing on various forest ecosystems in different regions are needed. Northwest China is located in the hinterland of the Eurasian continent. It has a typical inland desert environment and desert irrigation-based economy, which causes simple and vulnerable ecosystem structure with low biomass in natural ecosystems. Local forests play an essential role as ecological barriers for improving the environment, maintaining ecological balance, and ensuring the sustainable development of oasis . The nat(1)0), or they are highly related to age groups (H1) of the natural mountain forest inside study area.The biomass, carbon content ratio, carbon density and carbon storage in different parts of forest ecosystems, such as trunk, branch, leaf, herb, etc., have no obvious difference in age groups (H(2)0), or there is a relation (H1) in the natural mountain forest of arid central Asia.The variation of tree species (composition and DBH pattern) is not related to forest age groups and soil carbon , where the forest has developed. Within forest zone, the annual mean temperature is −4.0–2.0 °C, and the annual precipitation is 400–600 mm, which increases with the altitude rises, and decreases from northwest to southeast .Larix sibirica, which distributed on the shady and semi-shady slopes [Abies sibirca at the humid northern slopes, and with a few Picea obovate in river valleys at the lower altitude. The deciduous forest is mainly dominated by Betula pendula, Populus tremula, etc. There were only a few disturbances inside the forest of the study area: at the lower limit of the forest, a few loggings and small tree planting in 1950s [The local mountain coniferous forest is the south end of the Siberian taiga, which represents the boreal coniferous forest wedging into the prairies . The domy slopes . It alsoThe brown coniferous forest soil and gray forest soil are the main soil types beneath the forest . Soil paThe data used in this study were collected by field measurements and from dataset of Xinjiang forest resources survey. The survey of Xinjiang forest resources was carried out in 2006, and part of the Chinese 7th National Forest Resources Inventory [2, 28.3 m × 28.3 m) was placed . If the soil depth of some locations was less than 100 cm, the soil samples were taken until the soil parent materials was reached. The soil samples were air-dried, crushed and passed through a sieve, and then the soil organic matter (OM) was determined by potassium dichromate external heating method.Trees: diameter at breast height (DBH) (at 1.3 m above ground) and tree height were measured for all the trees which DBH was larger than or equal to five centimeters (DBH ≥ 5 cm) . TherefoInside each sampling plot, tree, herb, litter and soil layer were investigated with different methods:Within these 35 plots, nine plots were selected covering different forest age groups. Twenty-seven trees were randomly selected in these nine plots (three trees in each plot), parts of leaves, trunks, branches and roots of these 27 trees were collected as samples. These samples’ fresh weights were measured at field, and then brought back to laboratory for dry weight and other analysis. Considering there are only a few dead trees in the sampling plots, tree parts of dead trees were collected and measured if they were found in the field. These samples were included in the tree layers in this study.2). Inside 105 quadrats, all the above-ground part and underground roots of grass, and all the litters were collected. The fresh weight was measured at field, and dry weight was measured in the lab.Herb and litter layers: three small quadrats (1 m × 1 m) were randomly selected in each sampling plot , herb and litter samples were air-dried, crushed and passed through a 0.15-mm sieve, and their organic carbon was determined by using potassium dichromate oxidation methods.W, D and H represent the biomass per tree, DBH and tree height, respectively; a and b are parameters.Biomass of herb and litter layers were directly calculated from field sampling and measurements in laboratory. Tree biomass was estimated by an allometric equation :W = a, biomass was calculated separately according to tree species  Table 3Table 3. Carbon density (CD) of tree, herb and litter layers were calculated by multiplying biomass and carbon content ratio (CCR), and then adding them  together.iS) of each layer was calculated as [iC represents the CCR, iD is soil bulk density, iE is soil thickness, iG is the volume percentage of the gravels with the diameter of more than 2 mm.The soil CD (0–100 cm) was the sum of each layer’s CD. The soil organic CD  and least significant difference (LSD) test.The average CCR of the Altai Mountains forest ecosystems was 0.49 (range: 0.41–0.52), which was significantly influenced by forest age . CCR of −2 (p < 0.05), and the difference between young and mature/old-growth forest was significant. Biomass of the entire vegetation layer increased with forest age: biomass of per unit area in mature and old-growth forest was the highest, while young forest was the lowest. Biomass of tree and litter were positively related to forest age, while herb biomass peaked in young forest   . The avep < 0.05): for trunk, the difference between young and mature/old-growth forest was significant; for root, differences between young and mature/old-growth forest, and the differences between near-mature forest and old-growth forest were significant. The CD of trunk and root were increased with increasing forest age, while CD of leaf and branch were peaked in young and middle-age forest  at the range of approximately 20%, some are similar. The variation may be caused by different methods and vegetation types.The values of average forest CD vary among studies. the continuous biomass expansion factor method was used to build linear and hyperbolic models of biomass-stand volume and calculated the average CD of vegetation as 41.00 t·hm2 t·hm−2 ,31. ChenThe carbon density distribution of forest ecosystems is higher in south and lower in north inside study area. This spatial pattern is determined by many factors, such as climate, topography, vegetation, soil types, etc. ,35,36.The Altai Mountains lie in a northwest–southeast direction, forming a horn shape with the high and wide end to the northwest, and a narrow and lower end to the southeast merging into the Gobi Desert. Along the Irtysh River valley at the Altai Mountains piedmont, the cold and wet airflows of Atlantic and Arctic oceans from the northwest are the only moist airflow sources for the regions. The mountains in the northwest intercept most of the airflows, and therefore the climate is humid and warm comparing to other parts of the study area. The local forest coverage is relatively low, but with high soil organic carbon. In the middle region of the Altai Mountains, forest is widely distributed in Aletay, Fuhai and Fuyun, where is cooler and less humid than the northwest. At the southeast, due to the decreasing altitude and the effects of Siberia Mongolia high pressure dry anticyclone, the climate is cold and dry. Although the forest area decreases in Qinhe region at southeast, the local carbon density of forest ecosystems is relatively high because of the high coverage ratio of mature and old-growth forest .Larix sibirica [Inside study area, the brown coniferous forest soil and gray forest soil are main soil types; both of them have high soil organic carbon. Gray forest soil is the most widely distributed forest soil in Altai mountains, which is suitable for growing sibirica . The forLarix sibirica and Picea obovate, increase from 72.1% to 100.0% . The number of Larix sibirica increases greatly, while the trend of Picea obovate is minor  and fires [Larix sibirica and Picea obovata. In addition, the forest type is mature and old-growth forest, which leads to the highest carbon density inside study area.nd fires . The forThe studies on carbon storage and forest age of Altai Mountains forest ecosystems showed that carbon storage was significantly correlated with forest age, meaning that the carbon storage increases with forest age increases, which is consistent with the common pattern in China . The AltLarix sibirica increase from northwest to southeast (Larix sibirica reaches peak (31–42%) around 10 cm −2. Trees have the highest biomass (120.84 t·hm−2), followed by herb (4.22 t·hm−2) and litter layer (1.61 t·hm−2). Among the tree parts, trunks, roots, leaves and branches accounted for 50%, 22%, 16% and 12% of the total tree biomass, respectively.The average biomass of the Altai Mountains natural forest ecosystems is 126.67 t·hm(2)−2, and the carbon storage is 131.35 Tg in Altai Mountains forest ecosystems. Soil and tree layers are the main local carbon pools.The average carbon content ratio is 0.49, the average carbon density is 205.72 t·hm(3)Forest age has significant effect on biomass, carbon content ratio, carbon density and carbon storage. For parts of tree and forest ecosystems, forest age effect for carbon content ratio was the most obvious, while the biomass of trunk and carbon density of trunk and root were significantly correlated with forest age.(4)The change of dominant species coincides with the spatial pattern of vegetation carbon density inside study area, which implies a potential increase of local carbon storage in the future.Mountain forests in arid northwest China function as a substantial carbon pool, and have great significance for forest resource conservation and in maintaining the balance of the local “mountain–oasis–desert” ecosystem. In this study, we used detailed field measurements covering different forest age groups, structure of forest  and trees  to calculate biomass, carbon density and carbon storage in the Altai forest ecosystems. The major conclusions can be summarized as follows:


Targeted canonical variate analysis (CVA) explained 85–90% of the variance ratio across the two vintages, indicating persistent and stable elemental fingerprints of wines at a sub-regional level. A sixth, newly founded neighborhood was correctly grouped separately from the others using a Soft Independent Modeling of Class Analogy (SIMCA), indicating the potential of elemental fingerprints for wine authenticity.The compositional authentication of wine is of great interest, as the geographic origin of the grapes is often associated with quality, uniqueness, and authenticity. Previous elemental fingerprinting studies mainly discriminated wines from different countries or regions within a country. Here, we report the use of element profiles to distinguish commercial Pinot noir wines from five sub-regional appellations or neighborhoods within one American viticultural area (AVA). Fifty-three single cultivar wines were collected over two harvests and analyzed using microwave plasma-atomic emission spectroscopy (MP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS). Of 62 monitored elements that were quantified with fully validated methods, 24 and 32 elements differed significantly across the neighborhoods and vintages, respectively ( History has long-attributed wine sensory properties to the vineyard sites on which the grapes were grown. The concept of appellations and defined districts based on geography is also central to authenticity and truthful labeling and can be traced back to the establishment of the demarcated Douro Region of Portugal in 1756 .Today, there are 246 AVAs in the United States, of which 139 are located in California, with some of these AVAs overlapping or existing as smaller AVAs within larger AVAs )As seen in Last, Coetzee et al. similarly found that variations in the concentration levels of B, Ba, Cs, Cu, Mg, Rb, Sr, Tl, and Zn were good indicators to distinguish different cultivars of red and white wines from different estates within a single wine district in South Africa . The stuThe uptake of minerals by grapevine roots is known to involve several transport systems. One of these, an ATPase proton-monovalent cation exchanger, is responsible for the uptake of group 1 elements . While potassium is the major element in this group taken up in many soils, different rootstocks and a lower availability of potassium would be expected to give rise to different concentrations in the others. This was born out in several previous studies ,22,23,36Among the 2017 samples, wines from a new neighborhood, Eastern Hills (EH), were included in the analysis. Elemental differences in Al, Mn, Ni, Zr, Mg, Si, Yb, Er, Cr, and S were found to be discriminating EH wines from the other neighborhoods; except for S, all listed elements were the highest in concentration in the EH samples. Using this new neighborhood, we were able to further validate the distinctiveness and uniqueness of the neighborhood elemental fingerprints. We first included the EH data into the 2017 data for visual statistical interpretation using CVA and then Soft Independent Modeling of Independent Classes (SIMCA) as a multiclass classification method that is able to classify unknown samples as being either within a class or outside. Samples from the new neighborhood EH should be classified by the SIMCA algorithm as originating from none of the five original neighborhoods, if this new neighborhood is indeed characterized by a unique elemental fingerprint. First, half of the 2017 dataset with the original five neighborhoods  was used to establish and calibrate the model . Then, the second half of the dataset, with the addition of the Eastern Hills (EH) samples, was then used to test the developed model .A visual presentation of the prediction models for each wine sample is shown in The cumulative explained variance for the first three components of the SIMCA model for each class  was very high, ranging from 99.97% for GV and SH to 99.99% for LR, MR, and SP. This indicates that each class is separated from the others . The senDespite these limitations, interestingly, none of the EH wine samples were placed into any of the existing classes . This men = 53), the obtained results are of high importance to the wine industry and food authenticity testing at large. The strengths of this study are: (i) the collection of wines from different sub-regions within a single AVA, (ii) the analyses of wines of two different harvests, (iii) the use of commercial wines while maintaining some control over the winemaking process known to affect the elemental profile, and (iv) the application of a SIMCA classification algorithm that has not been previously applied to wines.Although the overall sample size is small  could be distinguished from other wines produced in Hawke’s Bay . SIMCA mAs indicated previously, the major limitation of this initial survey is the low number of wine samples from each neighborhood collected from the different wineries. Although wineries are known to change the elemental compositions , the efIt is unknown whether and, if so, how much these existing elemental differences contribute to any perceivable flavor differences in the finished wines. Finally, a better understanding of the variability within the wine production process is needed to test whether the elemental fingerprints of finished wines, i.e., wines that have been fined, blended, bottled, and stored, would similarly be grouped by neighborhood. Similarly, elemental analyses of the vineyard soil and water used in the vineyard and winery, as well as any other viticultural and/or enological materials, could provide insights into the underlying causes for the observed, distinct elemental patterns by neighborhood.Since this study was done over the course of 4 years, there were slight changes to the methods used to analyze the wine samples from the two vintages, e.g., different analysts and slight method modifications and improvements based on the advancements of the lab. Data validity was checked and ensured through the use of certified reference materials and spiking experiments for all employed analytical methods.https://russianrivervalley.org/discover/neighborhoods). In 2017, another three wines were obtained from Eastern Hills (EH), which has been added as an additional neighborhood after the beginning of this study.A total of 53 commercial Pinot noir wines from single vineyard lots located within the Russian River Valley AVA in Bordeaux region, Northern CA, USA, were used in this study. Samples were aged between 4–5 months in neutral oak barrels and unfiltered, unfined, and not treated for physical instabilities. Samples were collected from each Russian River Valley neighborhood based on the local initiative aiming to understand the Pinot noir diversity in the AVA ; in eachAll sampled wines were transferred to the Food Safety & Measurement Facility at the University of California, Davis  where they were stored at 4 °C until analysis. In 2015, samples were transferred in sterile 50-mL plastic centrifuge tubes  taken directly from fermentation vessels, while the 2017 harvest wines were stored in glass bottles.3, 67–69%) and hydrochloric acid  were purchased from Fisher Science . Due to the lack of a wine-certified reference material (CRM), NIST 1643 (e/f)  and CRM-TMDW-B and CRM-TMDW (High Purity Standards) were used and adjusted to account for the ethanol sample matrix. Element-free centrifuge tubes (15 and 50 mL) were purchased from VWR .Multielement standards  and single element standards (P and Mn) were obtained from SPEX CertiPrep . Single element standards for B and S were obtained from High Purity Standards , while Si, Rb, and Sr were purchased from Crescent Chemical Company, Inc. . Items purchased from Agilent Technologies  included ionization buffer solution ; two internal standard solutions  and ICP internal standard ); Environmental Spike Mix; Calibration Mix Majors; and single element standards Mg and K. All samples, quality controls, and calibration standards were prepared with ultrapure water . Ethanol (200 proof) was obtained from Koptec , and optima-grade nitric acid (HNO3 (w/w) and mixed with ICP internal standard solution and ionization buffer solution (diluted to 2000 mg/L with 1% HNO3 before use) in a mixing tee prior to entering a micromist nebulizer and a double-pass cyclic spray chamber. Matrix-matched calibration curves prepared in 4% ethanol and 5% HNO3 were established between 0.01 and 50 mg/kg ; 1 and 200 mg/kg (Mg); 0.01 and 2 mg/kg (Mn); and 1 and 700 mg/kg (K and P). Drinking water reference standard CRM-TMDW-B was diluted 3-fold in 4% ethanol and 5% HNO3, and sample spikes at two concentrations (low: 10 mg/kg and high: 100 mg/kg) were analyzed throughout the sample sequence to monitor the instrumental drift. Sample blanks of 4% ethanol and 5% HNO3 were used for the continuing calibration blank (CCB) and 1-mg/kg (10 mg/kg for Mg and 50 mg/kg for K/P) standards served as the continuing calibration verification (CCV) samples. Each wine was prepared in triplicate, and each sample was analyzed in triplicate, whereby each element was monitored with two or three different emission wavelengths. Wavelengths used for quantitation were free of interferences and chosen based on previous reports , microwave plasma-atomic emission spectroscopy  was used for the 2015 samples. Samples were diluted 5-fold with 5% HNO reports  31]. Li. Li3 . All wines were diluted 3-fold in 5% HNO3 (v/v) prior to analysis and mixed in a mixing tee with the Internal Standard Mix (diluted 10-fold in 1% HNO3 to 1 mg/kg) before entering the micromist nebulizer , with the quartz glass spray chamber held at 4 °C. Elements were monitored in no gas, helium (flow rate = 5mL/min (year 1) and 4.3 mL/min (year 2)), high-energy helium (flow rate = 10 mL/min), or oxygen mode (flow rate = 30%)  . In the 3) 6-point calibration curves for each element were established between 0.1 and 500 µg/kg using multielement standards. Two different drinking water CRMs were diluted 10-fold in 4% ethanol and 5% HNO3 (CRM-TMDW and NIST 1643e) and analyzed throughout the sequence for quality control. In addition, samples were spiked with Environmental Spike Mix at two concentrations (low: 8.3 µg/kg and high: 83.3 µg/kg) and analyzed throughout the sample sequence. Sample blanks and 1-µg/kg calibration points served as CCBs and CCVs.Matrix-matched . A specific gas mode and isotope were selected for each element based on the limits of detection (LOD) and reported the background equivalent concentrations (BEC) .3 and 1% HCl in metal-free centrifuge tubes. Furthermore, all calibration curves were prepared with at least 6 calibration points for each element, which began at 0.05 µg/kg. All other instrument parameters were the same as listed in the section above. In order to analyze the elements with high concentrations , wines were further diluted 250-fold and analyzed. Each wine was prepared and analyzed in triplicate at the two different dilution levels. The diluted samples were stored in 4 °C and centrifuged prior to analysis.For the 2017 analyses, the method of analysis was slightly changed. Trace and microelement profiling were still completed with ICP-MS, as well as quantification of the major elements after a larger sample dilution. Furthermore, hydrochloric acid was introduced to all diluents to help stabilize some elements in the sample solution and to mitigate memory effects. For trace and microelement profiling, samples were first diluted 5-fold with 3% HNO3, 1% HCl, and 3% ethanol when analyzing the 5-fold diluted wine samples. Since the percentage of ethanol in the higher diluted samples was negligible, ethanol was not included in the calibration standards, blanks, and CRMs solutions while analyzing the higher diluted samples. In all cases, 6-point calibration curves were created for all quantified elements  in the two harvest years. Analysis of variance (ANOVA) for each element (p < 0.05) was then performed considering neighborhoods within the Russian River Valley AVA as factors. All analyses were done with R  using RStudio , with the additional packages car, [emmeans, [multcomp [candisc package) [For statistical analysis, elements that were not detected in any of the wine samples were removed from the analysis. Elemental concentrations below the LOD were set to LOD/100 for the multivariate data analyses. Multivariate analysis of variance (MANOVA) was conducted to test for significant differences among the neighborhoods  .mdatools package [Further testing was completed using the Soft Independent Modeling of Class Analogy (SIMCA), which was performed using the  package . SIMCA i


However, a strong correlation was observed between self-reported pain and perceived level of stress during experimentally-induced muscle pain . Furthermore, there was a moderate correlation between the levels of sAA at baseline and during experimental pain . In conclusion, this study could not show any association between the levels of sAA and perceived pain and or/stress. However, since a significant strong correlation could be observed between perceived stress and pain intensity, this study indicates that experimentally-induced muscle pain could be used as a stress model.Salivary alpha-amylase (sAA) is a marker of psychological stress and might also be a potential marker for pain-associated stress due its non-invasive, cost-effective, and stress-free collection. The current study aimed to investigate whether the levels of sAA are influenced by experimentally induced muscle pain. In this study, 26 healthy, pain-free and age-matched participants (23.8 ± 2.6 years) were included, 13 women and 13 men. Prior to the experiment, questionnaires assessing health and anxiety were completed. Muscle pain was then induced through intramuscular injection of 0.4 mL hypertonic saline (56.5 mg/mL) into the masseter muscle and unstimulated whole saliva samples were collected at baseline before injection, 2 min, and 15 min after injection. A commercially available colorimetric assay was used to analyze the sAA. Perceived pain and stress were assessed using a 0–100 Numeric Rating Scale for each sample. There were no significant differences in sAA levels prior and after injection of hypertonic saline ( Saliva is produced by three major glands and several minor accessory glands. Human saliva has several important functions, such as moistening and lubricating the oral tissues and functioning as a taste solvent and a swallowing agent. Salivary alpha amylase (sAA) is one of the most essential enzymes in saliva and is mostly synthesized by the parotid gland ,2,3,4.Secretion of alpha amylase from the salivary glands is controlled by autonomic nervous system (ANS) and substantial literature reveals that secretion of sAA seems to correlate to sympathetic activity under conditions of stress ,6,7,8,9.Self-reported pain is important for adequate therapy, but in some cases, where patients are unable to subjectively assess pain, an objective method would be preferable. Saliva sampling has a lot of advantages: it is easy to collect, non-invasive, cost-effective, and stress-free . SalivarThe aim of this study was to investigate if the experimentally-induced muscle pain influences the level of sAA, and if the levels of sAA correlates with the pain intensity. The hypothesis was that experimentally-induced muscle pain will affect the ANS and consequently result in increased secretion of sAA.Twenty-six healthy participants, 13 men and 13 age-matched women, were included in the study, with a mean ± SD age of 23.8 ± 2.6 years. The participants were recruited through an advertisement on social media and among undergraduate dental students at Karolinska Institutet, Huddinge, Sweden.2. Participants also had to maintain exceptional oral hygiene on the day of collection.Inclusion criteria were: (a) good general health; (b) age ≥ 18 years; and (c) body mass index <30 kg/mExclusion criteria were: (1) any current pain; (2) a pain-related diagnosis according to the diagnostic criteria for temporomandibular disorders (DC/TMD) ; (3) a dAll participants received information regarding the objectives and procedures of the study and gave their informed written consent before the start of the experiment. The study protocol was approved by the Swedish Ethical Review Authority, Sweden  and followed Good Clinical Practice and the guidelines according to the Helsinki declaration.All participants who fulfilled the inclusion criteria underwent a clinical examination according to the Swedish version of the DC/TMD axis I . The eviPrior to saliva collection, participants were instructed to fill in validated questionnaires to assess the degree of depression, somatic symptoms, and current and general anxiety. The perceived health questionnaires (PHQ) were used to assess anxiety (PHQ-9) and somatization (PHQ-15). PHQ-9 consisting of nine questions regarding depression, asking how often over the past two weeks the participants have been bothered by various feelings related to depressive symptoms, with four possible answers 0 (not bothered), 1 , 2 , and 3 . Scores ranged between 0 and 27, and scores of 5, 10, 15, and 20 were considered cut-off values for mild, moderate, moderately severe, and severe depression, respectively. Studies of the reliability and validity in adults shows that the PHQ-9 has a 61% sensitivity and 94% specificity ,31. The The experiment began by randomizing the side for intramuscular injection of hypertonic saline. During the study, unstimulated whole saliva was collected at three time points: at baseline before induced pain, during experimentally-induced muscle pain, and 15 min after the induced pain for measurement of the sAA activity. Before each saliva sampling, the perceived pain and stress were assessed using a Numeric Rating Scale (NRS). The scale range was from 0–100, where 0 was no stress or pain experienced and 100 was maximal stress or worst pain experienced.After the first saliva sample was collected, muscle pain was induced by an intramuscular injection of hypertonic saline 0.4 mL (56.5 mg/mL) into the prominent part of the masseter muscle during approximately 30 s. One minute after the injection the participant assessed perceived pain and stress on a 0–100 NRS. A second saliva sample was collected for another 3 min and the participants were asked again to assess the perceived pain and stress on the NRS. The third and last saliva sample was collected 15 min after the injection and the participants were asked to once more assess the perceived pain and stress on the NRS. They were also asked to assess how stressed they felt during the whole experiment using the Rated Perceived Exertion (RPE) graded from 0 (no perceived stress) to 10 (maximum perceived stress). The Rated Perceived Exertion scale was used to measure the experienced exertion .The participants were instructed not to eat, drink, or brush their teeth a minimum of one hour prior to sample collection. To avoid variation of sAA secretion due to the diurnal changes during the day, most of the samples were collected in early afternoon.Unstimulated whole saliva was collected while the participants were seated comfortably with eyes open and head slightly tilted forward, as previously illustrated by Jasim et al. . Particig for 15 min in order to remove mucins and debris. All samples were diluted and assayed in duplicate in order to reduce the margin of error. sAA activity were measured using a commercially available enzymatic assay kit according the instructions from the manufacturer .Saliva samples were thawed, vortexed, and centrifuged at 1500× p < 0.05, a power of 80%, an estimated mean difference of 35%, and a standard deviation of 60% (based on a previous study not yet published).The sample size was based on a power calculation showing that 25 participants were needed to be able to reject the null hypothesis, given a significance level of U-test since most variables did not show normal distribution. Analysis of variance (Friedman’s ANOVA) was used for repeated measurements to analyze the variation of sAA, perceived stress, and pain over time. When significant, post-hoc analysis with Wilcoxon matched pair-test was applied and adjusted for multiple comparisons according to Bonferroni.The Shapiro-Wilks test was used to study the distribution of the data. Differences in descriptive variables between males and females in the study were tested with the Mann-Whitney p < 0.05. The statistical analyses were performed using Statistica version 13 .The Spearman correlation test was used to test for significant correlations between variables with additional Bonferroni adjustments. Descriptive data are presented as mean ± SD or median and interquartile range (IQR). For all analyses, the significance level was set at n = 26) included in the experiment are presented in p > 0.05), which is why data further on are presented on a group level.Descriptive data of the participants (p = 0.04).sAA levels showed no significant differences between sexes at baseline or after injection. However, the sAA level 15 min after injection was significantly higher in males compared to females .Although the levels of sAA were higher before (87 ± 46.5 U/mL) than during (80.5 ± 37 U/mL) and after (81.7 ± 63.4 U/mL) experimentally-induced muscle pain, see 2 = 29.7; p < 0.001) and level of pain  between the time points prior, directly after, and 15 min after experimentally-induced muscle pain. The post-hoc analysis with Bonferroni correction adjusted for multiple comparisons (p < 0.0017) showed a significant increase in perceived stress directly after pain was induced , which then significantly decreased 15 min after injection  and was at that time point (15 min after injection) normalized, showing no significant difference compared to baseline .There was a significant change in perceived stress (Xp = 0.0000) and showed a decreasing pattern, but did not return to baseline levels 15 min after injection   B.s = 0.687; p < 0.0001). Furthermore, there were also a strong correlation between the perceived stress and level of pain during experimentally-induced muscle pain There were no significant correlations between sAA and pain and/or stress after adjustment for multiple comparisons. The sAA levels assessed prior to injection showed a moderately strong correlation to sAA levels directly after experimentally-induced muscle pain (rThe main finding of this study was that experimentally-induced pain, i.e., acute pain, did not affect the levels of sAA, which seems to be in contrast to previous studies ,21,22,23sAA in the current study design, although non-significant, showed a tendency towards declining when acute pain was induced. The non-significant alternation in the sAA level during conditions of pain contrast previous studies investigating sAA in acute and chronic pan settings ,21,22,23Participants reported at baseline a low level of stress, but the levels increased significantly when pain was present and returned to similar levels as baseline when pain disappeared A.. NocicHypertonic saline injections have been used extensively to induce muscle pain, since the quality of the pain mimics acute clinical muscle pain ,37. The The relationship between subjective pain and sAA are inconclusive. Some studies report a significant association, while others report no differences. Common in all studies is that they use perceived pain intensity as a tool to assess pain ,20,22,23Several studies have reported higher pain intensity and a lower pain threshold and pain tolerance in women compared to men ,58, whicA strength in our study design was the healthy homogeneous group and the controlled study environment. It was ensured via the anamnesis and the oral examination that the participants were healthy, and they were matched for age and gender to eliminate the influence of these factors on enzyme activity or reported stress levels. The pain model applied are well-known, and numerous studies have shown good reliability ,37,38. TNevertheless, our findings should be interpreted within the context of certain limitations. For instance, the pain induced had a short duration, which does not accurately mirror pain under normal pathological circumstances, and the participants were aware of the experimental pain, which may have influenced the stress response. However, the experimental protocol was very rigorous, which should remove such a confounding factor. Furthermore, one factor that may have affected the sAA levels is the human salivary amylase gene, of which the expression is affected by the amount of starch in the diet . When anTo conclude, this study could not show any association between the levels of sAA and perceived pain and or/stress, which agrees with the variety of different results regarding the levels of sAA. This indicates that it might not be pain itself that influences the levels of sAA, but instead the duration of the perceived discomfort/unpleasantness induced by pain. Furthermore, this study showed that even experimentally-induced pain induces stress, based on the significant strong correlation observed between perceived stress and pain intensity. Hence, this study indicates that experimentally-induced muscle pain could be used as a stress model. However, further studies need to confirm this outcome.


Radiofrequency of the Gasserian ganglion can be used for ophthalmic herpetic neuralgia (OHN), but it is associated with complications. This study aimed to use the supraorbital nerve for computed tomography- (CT-) guided radiofrequency thermocoagulation to treat refractory OHN.  This was a retrospective case series study of patients with simple or combined OHN treated at our hospital between 06/2012 and 06/2018. The numerical rating score (NRS), spontaneous pain, allodynia, gabapentin dosage, paracetamol/oxycodone dosage, patient global impression of change (PGIC) score, Barrow numbness score, postoperative 360-day recurrence rate, and complications were recorded before the operation and at 1, 30, 90, 180, and 360 days after the operation. P < 0.001). Compared with 1 day after the operation, numbness was decreased at 30, 90, 180, and 360 days after the operation (P < 0.001). Compared with baseline, the number of patients with allodynia at each time point after the operation was decreased (P < 0.001), but without a difference for spontaneous pain (P=0.407). No subjects showed drooping eyelid, corneal ulcers, eyeball damage, decreased vision, and other severe complications.  Compared with baseline, the NRS was decreased, and PGIC was increased at postoperative 1, 30, 90, 180, and 360 days, and the gabapentin and paracetamol oxycodone doses at postoperative 30, 90, 180, and 360 days were decreased (all  CT-guided supraorbital nerve radiofrequency thermocoagulation for the treatment of OHN can effectively relieve pain and reduce the dose of analgesics, without any serious complication. This study suggests that this technique is feasible and applicable to clinical practice. Herpes zoster (HZ) is one of the clinical syndromes associated with the reactivation of latent varicella-zoster virus (VZV). HZ typically occurs years after primary VZV infection (varicella or chickenpox) –4. The aCumulative trigeminal nerves  can cause head and facial HZ, of which herpes zoster ophthalmicus (HZO) is the most common, which can account for 10%–20% of patients with HZ , 8. HZO Radiofrequency (RF) is a reliable method for the treatment of neuropathic pain in patients who are refractory to conservative treatments. RF is divided into pulsed radiofrequency (neuromodulation) and radiofrequency thermocoagulation (neurolytic impairment). Pulsed radiofrequency has an unclear analgesia mechanism. Clinically, for the treatment of refractory herpetic neuralgia, a single-pulse radiofrequency has a short effective duration, and repeated operations are often necessary to prolong the curative effect, which will increase the patients' burden . MeanwhiThe aim of this retrospective study was to examine whether the use of another target, i.e., the supraorbital nerve, showed benefits for computed tomography- (CT-) guided radiofrequency thermocoagulation for the management of refractory ophthalmic herpetic neuralgia. The results could provide another option for the management of refractory ophthalmic herpetic neuralgia.This was a retrospective case series study of patients with simple or combined HZO treated at the pain department of our hospital between June 2012 and June 2018. The procedure was approved by the hospital in 2011 (#2011–037). The data were from a prospectively maintained database using Haitai 3.0 software. The data themselves were extracted from the charts after 2018. The study was approved by the ethics committee of our hospital. The need for individual consent was waived by the committee.The inclusion criteria were as follows: (1) diagnosed with HZO , and theAll operations were performed by the same surgeon, who was a chief physician who had been engaged in the diagnosis and treatment of pain for 20 years. The patient was placed supine on the operating table in the CT treatment room, comfortably fixed, with eyes closed. ECG was monitored routinely, and oxygen inhalation was given. The supraorbital foramen (notch) was identified using CT. When a complete supraorbital foramen (notch) could be seen, the CT layer was selected as the puncture layer, the puncture path was designed, and the puncture point was marked. After routine disinfection and draping, a radiofrequency puncture needle (length of 50 mm and inner diameter of 0.7 mm), exposed end of 2 mm , was punctured vertically along the puncture path to the supraorbital orifice or the notch bottom  under thThe patients continued to take oral gabapentin . The dose for patients with NRS 1–3 points was halved, while it could be doubled in patients with NRS 4–6 points; each increase or decrease lasted for more than 1 week. Patients with NRS >6 points were rescued with paracetamol oxycodone tablets , no more than four tablets/day.Before the operation and at 1, 30, 90, 180, and 360 days after the operation, the following data were recorded routinely: NRS, occurrence of spontaneous pain, occurrence of allodynia, gabapentin dosage, paracetamol/oxycodone dosage, the patient global impression of change (PGIC) score , the Bart-test. Data with a skewed distribution were expressed as medians ; comparison between pretreatment and posttreatment data was conducted using the Friedman rank-sum test, and multiple comparisons were made using the Wilcoxon rank-sum test. Categorical data were analyzed using the Pearson chi-square test. Two-sided P values <0.05 were considered statistically significant.Statistical analyses were performed using SPSS 16.0 . Continuous data were first tested with the Kolmogorov–Smirnov normality test. Data in line with a normal distribution were expressed as means ± standard deviations, and comparison between pre- and posttreatment data was conducted using repeated-measures ANOVA and the post hoc paired Thirty-six patients among eighty patients with HZO completed the radiofrequency thermocoagulation intervention. Five patients were lost to follow-up, and 31 patients were finally analyzed in the study. Their characteristics are listed in χ2=80.37, χ2=55.53, χ2=38.00, and χ2=47.32; all P < 0.001). The differences in gabapentin and paracetamol oxycodone doses at postoperative 1 day were not statistically significant . Compared with 1 day after the operation, numbness was decreased at 30, 90, 180, and 360 days after the operation .The last follow-up was on September 1, 2019. The NRS, gabapentin dosage, paracetamol/oxycodone dosage, PGIC before and after the operation, and postoperative numbness are shown in χ2=46.25, P < 0.001), while the number of patients with spontaneous pain at each time point after the operation did not show a significant difference . In addition, there were no significant differences between the number of patients with allodynia and the number of patients with spontaneous pain before operation , but the number of patients with spontaneous pain at each time point after the operation was higher than the number of patients with allodynia .The number of patients with allodynia and spontaneous pain is shown in At 360 days after the operation, four patients (13%) had recurrences, but they were treated at other hospitals, and the details are unknown. Twenty-nine (94%) patients had varying degrees of subcutaneous swelling at the supraorbital area within 1 week of surgery, but no patients showed drooping eyelid, corneal ulcers, eyeball damage, decreased vision, and other severe complications. After 1 week without special treatment, they returned to normal on their own.Radiofrequency of the Gasserian ganglion can be used for ophthalmic herpetic neuralgia, but it is associated with risks and complications –17. ThisThe Gasserian ganglion, which is a concentration site of primary sensory neuron bodies in the head and face, is the ideal target for radiofrequency thermocoagulation in the treatment of refractory ophthalmic herpetic neuralgia –17. RadiThe ophthalmic nerve erupts from the skull via the supraorbital fissure and is immediately branched into the lacrimal, nasociliary, and frontal nerves . The nasβ fibers sprout from the deep lamellar to lamellar I and lamellar II in the spinal dorsal horn after nerve injury and occupies the upward pain pathway of the Aβ and C fibers [Among the 31 patients, the pain score decreased significantly after radiofrequency thermocoagulation, and the relief of allodynia was significantly better than that of spontaneous pain. Spontaneous pain is considered to be caused by central sensitization . This tyC fibers , which bC fibers . Since tWith the relief of pain after radiofrequency, the doses of analgesics such as gabapentin and paracetamol oxycodone after the operation could be decreased accordingly. In the present study, there was no significant change in the doses at postoperative 1 day, which may be related to pain and discomfort due to subepithelial swelling in the supraorbital area after thermocoagulation. Meanwhile, PGIC, as an indicator that effectively reflects the therapeutic effect, was improved postoperatively compared with baseline (after nerve block), indicating that the treatment is effective.Numbness is an unpleasant sensory experience and is also a cause for the low acceptance of nerve impairment methods . In the This retrospective study has some limitations. The sample size was small. There was no control group. In addition, the patients combined with HZ in the supratrochlear nerve were excluded, but some cases might have been missed since there is a somatic overlap between both nerves. Therefore, further research and observations are needed.In conclusion, radiofrequency thermocoagulation of the supraorbital nerve is a feasible method for treating ophthalmic herpetic neuralgia, without serious complications. This could be a suitable method for the management of ophthalmic herpetic neuralgia.


Research on rare diseases has specific problems, such as low or small patient groups, limited public awareness, and limited financial support. By definition, a rare disease affects not more than 50 per 100,000 individuals, but with over 6000 unique, rare diseases, more than 300 million people are affected worldwide. Especially, genetic screens are difficult to perform for rare diseases. Due to selective inbreeding in dogs, often these rare diseases present at high frequency in specific dog breeds. This paper in the special issue on “(epi) genetic disorders in companion animals” describes an example of how a novel gene was found that regulates copper accumulation in the liver in a specific dog breed, the Bedlington terriers, and describes an example of how gene products titrate each other’s function on the liver copper accumulation in Labrador retrievers. These two examples clearly show the power in dog genetics for both veterinary and human medicine. Although inbreeding is under great societal scrutiny due to its consequential large number of inherited diseases, dog genetics will directly positively influence animal welfare, in addition to basic knowledge of biochemical regulation systems, and lastly, it will be beneficial for people suffering from rare diseases.Wilson’s Disease is a rare autosomal recessive disorder in humans, often presenting with hepatic copper overload. Finding the genetic cause of a rare disease, especially if it is related to food constituents like the trace element copper, is a Herculean task. This review describes examples of how the unique population structure of in-bred dog strains led to the discovery of a novel gene and two modifier genes involved in inherited copper toxicosis. COMMD1, after the discovery in 2002, was shown to be a highly promiscuous protein involved in copper transport, protein trafficking/degradation, regulation of virus replication, and inflammation. Mutations in the ATP7A and ATP7B proteins in Labrador retrievers and Dobermann dogs resulted in a wide variation in hepatic copper levels in these breeds. To our knowledge, numerous dog breeds with inherited copper toxicosis of unknown genetic origin exist. Therefore, the possibility that men’s best friend will provide new leads in rare copper storage diseases seems realistic.  Copper storage disorders are considered rare diseases. Although the European Union (EU) and the United States have different definitions of rare diseases , it is clear that for each individual rare disease, no large patient cohort exists. However, taking into account that around 6000 unique rare diseases are described, the total number of people affected by rare diseases is estimated to be well over 300 million [Wilson disease (WD) and Menkes disease (MD) are rare genetic diseases associated with disturbed copper fluxes in the body. Copper accumulates in the liver in WD patients due to a mutation in the ATP7B gene, which is involved in intracellular copper excretion. In contrast, MD patients suffer from reduced copper levels in various organs, e.g., the brain, due to a mutation in the ATP7A gene, which is responsible for the distribution of copper within the body. The burden on the patient’s quality-of-life is further dependent on their variable clinical presentation. This review paper focuses on the utilization of canine genetics to discover causative and modifier genes of hepatic copper storage diseases in dogs. First, a brief overview is presented on WD and MD, as well as three other very rare copper toxicosis disorders. Second, the unique population structure of dogs will be described to show its feasibility in studying copper-related disorders. Third, examples of a simple Mendelian inherited copper-storage disease  and one with a more complex mode of inheritance in specific dog breeds will be presented. Finally, a perspective on how to implement canine patients with a rare disease in (advanced) pre-clinical research to the benefit of a patient with rare copper storage diseases is discussed, although the clinical presentation can be similar but not necessarily identical when comparing these two species.+ and oxidized as Cu++). Because of these strongly opposing situations, copper’s intracellular free concentrations need to be kept within very narrow limits [The trace element copper is like the ancient Roman mythological god of the beginnings, duality, and transitions Janus (or more modern Dr. Jekyll and Mr. Hyde). On the one hand, a good person, and on the other hand, an evil one . Indeed,w limits . Regulatw limits . Copper w limits ,4. Furthw limits . Once exwww.rare-liver.eu (accessed on 30 October 2020)) states Wilson disease as a rare disease, with an estimated clinical prevalence of 1 per 30,000 in the general European population. This is half the prevalence compared to China and several Asian countries [In 1912, Dr. Wilson described 12 patients with neurological presentations caused by an “unknown toxic” compound. It took another 17 years before this “unknown toxic” turned out to be hepatic copper overload, historical review in [ountries ,9. For mountries . Recent ountries , an obseIn 1993, the causative gene for WD was discovered, being the copper transporter ATPase2 (ATP7B) ,13. The ATP7B gene. These rodent models owing to their mutations in the ATP7B gene, do not fully recapitulate the variable clinical presentation of human WD patients [ATP7B mutations ) [ATOX1, XIAP (X-linked inhibitor of apoptosis), MTHFR , and COMMD1  [ATP7B gene and/or by the activity of modifier genes and/or environmental factors.The ATP7B protein is responsible for about 95% of the excretion of hepatic copper into the bile. The ATP7B protein consists of eight transmembrane regions forming a membrane-channel, whereas the copper-binding domains are in the N-terminal part within the cytosol. Several established model animals, based on ATP7B mutations, are described to facilitate research. The most frequently studied rodent models include the “Toxic milk” mouse, the TX-J mouse with different mutations in the ATP7B gene, and the ATP7B knockout (ATP7B-/-) mouse . The Lonpatients . This vae 2020)) ,20,21,22erriers) ,25,26,27www.orphanet.net (accessed on 2 December 2020)). This rare X-linked copper deficiency disorder is caused by mutations in the ATP7A gene [In contrast to WD, which is associated with copper accumulation in the liver, Menkes disease (MD) is a disease caused by reduced copper levels in various organs. Beginning in the 60′s, the first description of a family with a progressive neurological collapse very early in life was published . This tiP7A gene ,31,32. TP7A gene . As for P7A gene ,36,37,38ATP7A, ATP7B, or COMMD1 (COpper Metabolism Murr1 domain-containing protein 1) genes seem not to be involved [Indian Childhood Cirrhosis (ICC), Endemic Tyrolean Infantile Cirrhosis (ETIC), or Idiopathic copper toxicosis (ICT) are very rare copper overload diseases related to copper homeostasis ,40,41. Iinvolved ,43,44. Ainvolved .Generally speaking, copper-related disorders are rare to very rare, which in itself comes with specific research limitations, as described in the introduction. The failed wool production in sheep and the inherited copper toxicosis in Bedlington terriers are examples of the benefits to carefully observe diseases in animals. Below, we will move away from human diseases and focus on non-genetically modified animals, more specifically on dogs, as genetic powerhouses to dissect simple and complex genetic diseases.www.omia.org (accessed on 2 December 2020)) lists almost 800 traits or disorders described in dogs, compared to around 400 for cats. The initiatives for more concerted investigations of the dog genome were first described in a “briefings” in Science in 1990 entitled Canine Genome Project (doi:10.1126/science.248.4960.1184). In 1993, the Dog Genome Project, founded by Rine and Ostrander, appreciated the value of canine genetics for cancer research [www.dog10Kgenomes.org (accessed on 5 January 2021)) aims to describe the enormous phenotypical variation between dog breeds in molecular terms [At present, the OMIA database , life became a lot easier for researchers in canine genetics and molecular cell biology. In the next paragraphs, inherited copper toxicosis is used to exemplify the potential of canine genetics for human biomedicine.Canis lupus familiaris) as a genetic magnifying glass. What makes dogs, especially for inherited copper toxicosis, so well-suited [Some of the drawbacks related to rare diseases can be tackled by using dogs  and in BDE-cells (canine liver cells) resulted in elevated intracellular copper levels, even in short-term cultures [The first description of copper toxicosis in Bedlington terriers was reported in 1979 . In 1999oxicosis . The preoxicosis . The curoxicosis ,72,73,74cultures ,72. Livecultures ,76. In lcultures ,78,79.Longitudinal studies on COMMD1-deficient dogs revealed a progressive development of hepatitis similar to the development of chronic hepatitis in men ,81, yet While investigating its mechanism of action, several un-anticipated functions of COMMD1 were described. This review only briefly addresses the plethora of non-copper transport functions of COMMD1, ranging from protein trafficking/degradation, regulation of virus replication, inflammation, and possible roles in oncology. For a more detailed description of the numerous actions of COMMD1, the readers are referred elsewhere ,87,88,89ATP7B mutation in Dutch and USA Dobermanns increased hepatic copper levels. A mutation in ATP7A was also found. However, there were too few cases to draw conclusions for the US cohort [ATP7A is a modifier gene for ATP7B is astonishing, given their high level of homology. In this breed, the high prevalence of mutations in the ATP7A gene showed the power of canine genetics to find modifier genes. The fact that these mutations are in a gene that is causative for MD, further highlights the beauty of canine research. It is unlikely that this would have been discovered in men where MD is affecting 1 in 300,000 people. It turned out that COMMD1 mutations were involved in neither Labrador nor Dobermann copper toxicosis [RETN (coding for protein RESISTIN) was discovered as a novel modifier gene in copper toxicosis in Labrador retrievers [RETN mutations with hepatic copper accumulation.Labrador retrievers are among the most popular dog breeds worldwide. To emphasize the power of canine genetics to dissect genetic causes in complex genetic diseases, as little as 235 dogs were enrolled in a canine-specific genome-wide association study (GWAS). This study revealed that an Arg1453Gln substitution in the ATP7B protein was related to increased hepatic copper levels, whereas a Thr327Ile mutation in the ATP7A protein partially rescued the ATP7B phenotype . SimilarS cohort . Beauty oxicosis . Recentltrievers . RESISTIFelis catus), copper levels in other felidae were measured, including bobcats, tigers, cougars, coyotes, and lions [ATP7B gene [Although less stringent, inbreeding occurs in the feline pet population. Much less is known about the genetic background of hepatic copper accumulation in cats. The first description was in 1995 in Siamese cats  ) and the Dutch government provided us with a grant to exploit the downside of genetic inbreeding in dogs to the mutual benefit of men and their best friend. In addition to the likelihood that novel (modifier) genes involved in rare diseases, such as copper accumulation, will be found, this work will also create a genetic base for more realistic large animal models [Copper-related diseases in humans are often rare diseases but account for about one-third of the chronic hepatitis cases in dogs . This rel models ,82,124.Current deep sequencing strategies that are exploited in men will find numerous genetic variants. Whether this will lead to a causality of the novel variants with the phenotype will remain difficult in view of the limited sample size in the case of (very) rare diseases. In addition, taking into account that canine inbreeding is tremendously higher than in humans, much fewer variants will be detected in dogs. The causal association between a novel variant and a specific phenotype will, therefore, be much more easily established. This strategy and the implementation of state-of-art sequencing technology in men and dogs has been nicely described .Dogs are ideal for testing nutritional interventions, as, opposed to people, we can keep their dietary intake the same for prolonged periods of time. As already mentioned, copper transporters also transport zinc. By increasing dietary zinc, the absorption of copper will, therefore, be reduced. In dogs, a commercially available diet with low levels of copper  and high levels of zinc  was able to reduce hepatic copper levels in Labrador retrievers with a history of copper storage disease and kept them clinically healthy up till 43 months after starting dietary treatment . As an a


Saccharomyces cerevisiae and comparable with Homo sapiens. Indeed, comparison of plant MCs with those from these better characterized species has been highly informative. Here, we review the most recent comprehensive studies of plant MCFs, incorporating the torrent of genomic data emanating from next-generation sequencing techniques. As such we present a more current prediction of the substrate specificities of these carriers as well as review the continuing quest to biochemically characterize this feature of the carriers. Taken together, these data provide an important resource to guide direct genetic studies aimed at addressing the relevance of these vital carrier proteins.Mitochondrial carriers (MC) are a large family (MCF) of inner membrane transporters displaying diverse, yet often redundant, substrate specificities, as well as differing spatio-temporal patterns of expression; there are even increasing examples of non-mitochondrial subcellular localization. The number of these six trans-membrane domain proteins in sequenced plant genomes ranges from 39 to 141, rendering the size of plant families larger than that found in  Furthermore, it was demonstrated that the AtSFC1 carrier catalyzes a fast counter-exchange transport and low uniport of substrates, as well as exhibiting a higher transport affinity for tricarboxylates than dicarboxylates [In Arabidopsis, one of the MCF members (At5g01340), named as SFC1 carrier, exhibits 35% similarity with the ACR1 transporter from yeast . The yealutarate . The SFCexchange . The Aran source . Despiteiposomes . Surprisoxylates . IntriguAtMPT1 , AtMPT3  and AtMPT2 , and all of them are related to mitochondrial Pi carrier (PiC) from human and yeast [AtAAC1–3; 53,065 protein copies/mitochondria) and PiC1–2  are the most abundant proteins in the IMM [+ symport (or Pi/OH− antiport) and thus supplying phosphate required for the ATP synthesis [Apart from ADP, the transport of phosphate (Pi) through the IMM is essential for the oxidative phosphorylation of ADP to ATP. In Arabidopsis, three genes encodes mitochondrial Pi carriers, namely nd yeast ,105. Biond yeast ,107, thund yeast ,135 rema the IMM . In agreynthesis ,137. Recynthesis ,139. NotT. brucei, drosophila, mouse and humans [S. cerevisiae it was demonstrated that MPC is a hetero-dimer in its functional state providing the basis for the structure elucidation of the functional complex [AtMPC1 interacts with NRGA1 and plays a role in the regulation of stomatal movement and pyruvate cellular content [AtMPC protein complexes is required for cadmium (Cd) tolerance and also prevention of Cd accumulation in Arabidopsis [AtMPC complexes are composed of two elements, the AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). Interrupting the formation of AtMPCs by silencing AtMPC1 element, the synthesis of acetyl-coenzyme A was supplemented by glutamate and thus sustaining the activity of TCA cycle reactions and glutathione synthesis following exposure to Cd stress [Pyruvate, the final product of glycolysis in the cytosol, must be transported into mitochondria to supply the carbon skeletons for oxidative metabolism through the TCA cycle reactions. The transport of pyruvate through the IMM must be performed by specific carriers. While candidates for mitochondrial pyruvate carriers (MPCs) have not been identified in the classic MCF yet, the identity and functionality of a series of MPCs, non-MCF members, have been reported in yeast, d humans ,141,142.d humans ,145,146  complex . In plan complex . Besides complex . This fa complex ; that sa complex . Further complex . Interes content . In addi content . Recentlbidopsis . In the d stress . ClearlyAtmIT1 (At2g30160) and AtmIT2 (At1g07030) [AtmIT1 and AtmIT2 proteins exhibit the classical MCF characteristic feature and were predicted to localize to the mitochondria by proteomic study [AtmIT1 or AtmIT2 can rescue the phenotype of the yeast mutant defective in mitochondrial Fe transport , from Oreochromis niloticus, catalyzes the transport free Fe and not a chelated Fe complex. In addition, it was shown that it is selective for alkali divalent ions [AtmIT1 and AtmIT2 transporters seem to be important for mitochondrial Fe uptake and also for the correct mitochondrial function, and consequently, they are necessary for the proper growth and development of the plant [Initially, mitochondrial iron (Fe) transporters, namely Mitoferrins (mIT), were identified and characterized in drosophila, zebrafish and humans ,153,154.1g07030) . These p1g07030) . In addiic study  which haic study . The ricic study . Similar mutant; ). In mam+ uptake . Moreoveent ions . In the ent ions . Of notehe plant ,155.Research into the metabolic roles of plant MCFs has made impressive advances since the last comprehensive reviews were published some eight to nine years ago ,135. Thi