The leaves, inflorescences, and fruits of hawthorn have long been known for their therapeutic properties. A wide range of hawthorn products, including liqueurs, are manufactured, due to the technological potential of the raw material as well as the richness of its volatile compounds. This study aimed to determine the effect of the liqueur production method and various methods of fruit preservation on the quantitative and qualitative composition of volatile compounds in the liqueurs produced. Hawthorn fruits saturated with sucrose and non-saturated with sucrose, fresh or preserved through one of three methods: freezing, air-drying, and freeze-drying, were used for liqueur preparation. The samples were analyzed using a gas chromatograph–mass spectrometer. They were found to contain 54 volatile compounds classified into 12 groups of chemicals. All 54 identified volatile compounds were detected in the liqueur made from hawthorn fruits non-saturated with sucrose and preserved by freeze-drying. In this liqueur type, 12 of the identified volatile compounds occurred in the highest concentration when compared to the other treatments. Among all volatiles, the following compounds were present in the analyzed liqueurs in the highest concentrations: dodecanoic acid ethyl ester (11.782 g/100 g), lactones (6.954 g/100 g), five monoterpenes (3.18 g/100 g), two aromatic hydrocarbons (1.293 g/100 g), isobensofuran (0.67 g/100 g), alcohol—2-methyl-2-propanol (0.059 g/100 g), and malonic ester (0.055 g/100 g). Among all analyzed liqueurs, the one made from the fruits non-saturated with sucrose and frozen was characterized by the smallest diversity of volatiles, which were present in the lowest concentrations in that liqueur. Crataegus) belongs to the Rosaceae plant family. It is most common in Asia, North America, and Europe [C. monogyna, C. laevigata, and C. mexiana [Hawthorn -2-hexenyl benzoate, -α-farnesene, and (Z)-3-hexenyl, and docasane. They also observed differences in volatile compound composition, which were affected by species examined and preservation method of the experimental material. They identified, i.a., (E)-β-damascenone only in the dried material, whereas δ-cadinene, germacrene D, α-selinene, β-selinene, δ-cadinol, selina-3,7(11)-diene, spathulenol, and valencene only in the fresh material from C. azarolus. Özderin et al. [A wide range of hawthorn products is manufactured due to the technological potential of the raw material, as well as the richness of its volatile compounds. The latter has been confirmed by Agiel et al. , who iden et al.  detectedRaw plant materials are usually seasonal and susceptible to fast decay. They need to be preserved to make them available all year round while their composition changes as little as possible . For yeanalewki), are one of the types of alcoholic beverages produced in Poland from wild-growing raw materials [The beverage market, including alcoholic drinks, represents a dynamically developing industry branch. In order to succeed on this market, producers develop novel types of beverages and search for exceptional aromatic raw materials. Liqueurs, also called tinctures  and various methods of fruit preservation (involving low temperatures or water activity decrease through its evaporation using different techniques) on the changes in the quantitative and qualitative composition of volatile compounds in the liqueurs produced.The analyzed liqueurs  containep-xylene and ethyl hexanoate, whereas trans-p-menth-8-en-2-one was not detected. In turn, the concentrations of six volatiles, including all alkenes, were statistically the highest, and concentrations of hexanol, cis-p-menth-8-en-2-one, and trans-p-menth-8-en-2-one were below the detection level in the liqueur made from fresh fruits saturated with sucrose (FS).Compared to the other analyzed liqueurs, the one produced from fresh hawthorn fruits non-saturated with sucrose (FNS) had statistically significantly higher concentrations of three out of four aldehydes identified in different treatments. In the liqueur produced from FnNS, eleven compounds present in other treatments were not detected at all, whereas concentrations of four compounds determined in these liqueurs were statistically significantly the lowest compared with the other liqueurs . The liqp-menth-8-en-2-one, trans-p-menth-8-en-2-one, p-menth-6,8-dien-2-one, and methyl eugenol. The liqueur produced from HS of hawthorn fruits had statistically the highest concentrations of ethyl acetate, 9,12,15-octadecatrienoic acid, ethyl ester, and ethyl stearate but did not contain 1-butanol, p-menth-1-en-4-ol, cis-p-menth-8-en-2-one, and trans-p-menth-8-en-2-one. The saturation of accessory hawthorn fruits with a sucrose solution before preservation probably contributed to a greater distinctness of volatile compounds, because the concentration of only one compound  was statistically the lowest in liqueurs produced from these fruits compared with the other analyzed drinks. However, the liqueurs made from fruits non-saturated with sucrose had a significantly higher number of volatile compounds occurring in higher concentrations compared with other liqueur types.On the other hand, the liqueur made from hawthorn fruits saturated with sucrose and freeze-dried (DS) had statistically significantly the highest concentration of 2-methylbutanal but none of the volatiles identified in the other liqueurs, namely 1-butanol, 2-ethylhexanol, cis-The pre-treatment (saturation with a sucrose solution) and preservation methods of hawthorn fruits contributed to changes in the concentrations of volatile compounds in particular groups of compounds . The metThe liqueurs made from fresh fruits non-saturated with sucrose  had the In the case of liqueurs made from fruits saturated with sucrose , the ratp-cymene). In turn, jams produced from Crataegus azarolus L. fruits were found to contain 56 volatile compounds [p-cymene in concentrations similar to those found in the liqueurs produced from FnNS and DS hawthorn fruits. In the studied jams, the aforementioned authors also identified limonene. Its concentration was 50% higher than that determined in the liqueurs produced in our study from FnNS and DNS hawthorn fruits. Both the liqueurs made from hawthorn fruits and the hawthorn fruits analyzed by Agiel et al. [p-cymene, p-menth-1-en-4-ol, and p-menth-6,8-dien-2-one (carvone). Both in the present study and in the research by Agiel et al. [Gasiński et al. , who anaompounds , includil et al.  identifio-cymene, α- and β-ionone, eugenol, 2-decanone, and geranic oxide. In a study conducted by Vukoja et al. , the addp- (o-) cymene, diethyl methylsuccinate, ethyl decanoate, ethyl tetradecanoate, dill ether, 5,6-dihydro-2H-pyran-2-one, limonene, and p-menth-6,8-dien-2-one. These compounds, detected in the highest concentrations in liqueurs, significantly affected their flavor bouquets, by imparting the following flavor notes  hawthorn fruits had higher concentrations of 10 out of the 54 identified volatile compounds . The following flavor notes (defined from literature data) were perceptible in the analyzed liqueurs: fusel oil, sweet, balsam, whiskey, citrus, floral, chocolate, fruity wine, waxy, sweet, apricot, banana, brandy, pear, and cedar camphoraceous . The useor notes : ethereaor notes  contribuompounds  contribuompounds , which iCrataegus azarolus L. and Crataegus pallasii L., noticed the effect of the fruit-preservation method on the concentration of volatile compounds in the analyzed samples. They detected (E)-β-damascenone only in the dried samples of Crataegus azarolus L., whereas α-selinene, β-selinene, δ-cadinene, germacrene D, selina-3,7(11)-diene, δ-cadinol, spathulenol, and valencene were only detected in the fresh samples of Crataegus azarolus L., and -farnesol only in the dried samples, regardless of the hawthorn species.In another study, Sun et al.  identifiThe aim of the PCA was the additional verification of the obtained results. Multidimensional analysis provides a simple visual representation of the complex analysis of the collected data. This allows us to attempt to answer various questions, e.g., if there are relationships between the applied preservation method and the volatile compounds present in the sample or which preservation techniques provide similar results concerning the concentrations of volatile compounds. As a result of the information about the degree of correlation between the preservation method and the type and concentration of the volatile compounds, it is possible to select the best method of fruit preparation to achieve the final product, i.e., a liqueur with a rich, full-bodied, and pleasant flavor.PC1 explains 51.84% of data variance for 1-butanol, hexanol, 2-ethylhexanol, benzyl alcohol, and 2-hexyloctanol, whereas PC2 explains 20.53% of data variance for 2-methyl-2-propanol and hexanol. The biplot analysis shows a high correlation between PC1 and 1-butanol, 2-ethylhexanol, and 2-hexyloctanol. The arrangement of the vectors of these variables indicates a positive correlation between them. What is more, a moderate correlation was noticed between hexanol and PC1 and a slightly weaker one between hexanol and PC2. In addition, positive correlations were noted among 1-butanol, 2-ethylhexanol, and benzyl alcohol, as well as between benzyl alcohol and 2-hexyloctanol. PC2 strongly correlated with 2-methyl-2-propanol. In the case of this compound, correlations with other variables were either weak or none . Moreover, a negative correlation was noticed between 2-hexyloctanol and hexanol.The analysis of the distribution of cases relative to the variables allowed us to distinguish a few clusters of points that represented fruit-processing variants. The liqueurs made from the raw materials from the group A, including DNS, HNS, FnS, and DS, were characterized by a high concentration of 2-methyl-2-propanol . Those pThe analysis of the distribution of cases relative to the variables allowed us to distinguish a few clusters of points that represented fruit-processing variants. The liqueurs made from the FS, DS, and FnS fruits (group C) had a higher concentration of 2-methylbutanal. Liqueurs made from FNS had high concentrations of benzylaldehyde, 3-methylbutanal, and 3-(2- or 4-) methylbenzaldehyde. The liqueurs produced from the HNS, DNS, and HS fruits (group D) had low concentrations of all analyzed compounds, whereas liqueurs made from FnNS fruits had moderate concentrations of the analyzed compounds equal to the arithmetic means of all eight fruit-processing variants.o-xylene, whereas PC2 explains 28.63% of the data variance for ethylbenzene and p- (o-) cymene. The biplot analysis shows a relatively strong correlation between toluene and o-xylene and PC1, as well as a relatively strong correlation between PC2 and ethylbenzene and p- (o-) cymene. Positive correlations were demonstrated among toluene, o-xylene, and styrene, and among p- (o-) cymene, ethylbenzene, and p-xylene. A lack of or a very weak correlation was observed between styrene and p- (o-) cymene and between styrene and ethylbenzene. p- (o-) cymene and ethylbenzene, whereas those made from DNS fruits had very high concentrations of ethylbenzene and p- (o-) cymene compared with the liqueurs produced in the other processing variants. The liqueurs produced from the HNS and FnNS fruits (group F) had high concentrations of styrene, o-xylene, and toluene; however, the first also contained p-xylene, while the other contained significantly higher concentrations of toluene, o-xylene, and styrene. The analysis of the distribution of cases relative to the variables allowed us to distinguish a few clusters of points that represented fruit-processing variants. The liqueurs from a group E, produced from the FNS, FS, FnS, and HS fruits, had low concentrations of the analyzed compounds. The liqueurs produced from DS fruits had slightly higher concentrations of The analysis of the distribution of cases relative to the variables allowed us to distinguish a few clusters of points that represented fruit-processing variants. Liqueurs from the group G, i.e., those made from the DNS and HNS fruits and especially those produced from DS fruits, had a high concentration of diethyl methylsuccinate. The liqueurs prepared from the FNS, FS and HS fruits (group H) had high concentrations of ethyl-9-hexadecenoate; 9,12-octadecadienoic acid, ethyl ester; 9,12,15-octadecatrienoic acid; ethyl ester; and butanedioic acid diethyl ester; however, the contents of these compounds were higher in liqueurs made from FNS and HS fruits than in those made from FS fruits. The liqueurs produced from FnS fruits had rather low concentrations of all analyzed compounds, whereas those produced from FnNS fruits had the highest concentration of diethyl malonate.p-menth-1-en-4-ol, cis-p-menth-8-en-2-one, trans-p-menth-8-en-2-one, p-menth-6,8-dien-2-one, and limonene, and PC2 explaining 18.33% of the data variance for 3-pinanone. The biplot analysis demonstrates a very strong correlation between PC1 and p-menth-1-en-4-ol, cis-p-menth-8-en-2-one, trans-p-menth-8-en-2-one, and p-menth-6,8-dien-2-one, and a relatively strong correlation between PC1 and limonene. The arrangement of the vectors of these variables points to a positive correlation between them. In turn, PC2 correlated strongly with 3-pinanone, while there was no correlation or a very weak correlation between this compound and the other analyzed compounds.cis-p-menth-8-en-2-one, trans-p-menth-8-en-2-one, p-menth-6,8-dien-2-one, and limonene.The analysis of the distribution of cases relative to the variables allowed us to distinguish a few clusters of points that represented fruit-processing variants. Liqueurs made from the FS, DS, FnS, FnNS, HNS, and HS fruits (group I) had low concentrations of all analyzed compounds. The liqueurs produced from the FNS fruits had a high 3-pinanone concentration, whereas those made from the DNS fruits had especially high contents of The analysis of the distribution of cases relative to the variables allowed us to distinguish a few clusters of points that represented fruit-processing variants. The liqueurs from group J, i.e., those produced from the DNS and HNS fruits, had high concentrations of dill ether  and 5,6-dihydro-2H-pyran-2-one. The liqueurs from group K, namely those made from the DS, HS, FS, FNS, and FnS fruits, had relatively low concentrations of the examined compounds. In turn, the liqueurs produced from FnNS fruits had high concentrations of acetophenone, acetic acid, and methyl eugenol.Crataegus laevigata (Poir.) DC.) collected (15 October 2015) at the consumption maturity stage on sunny day from a natural habitat located near a forest and far from asphalt roads and industrial buildings, in Jędrzejowo, Poland (50°38′22″ N 20°18′15″ E). The hawthorn grew on loamy, sandy–silty soil with a moderate humus content (1.6–1.75%) and a pH of 4.6–5.0. The soil had average contents of phosphorus—below 10 mg P2O5·100 g−1; potassium—15–20 mg K2O·100 g−1; magnesium—7–10 mg Mg·100 g−1; and mineral nitrogen—10–20 mg·kg−1 [The experimental material included fruits (3500 g) of two-necked hawthorn  to the final product . The fruFruits pre-treated and preserved as described above were used to prepare liqueurs according to the scheme presented in The samples were analyzed by HS-SPME-GC/MS using a GCMS-QP 2010 Plus gas chromatograph–mass spectrometer  and a 50/30 μm DVB/CAR/PDMS fiber . Two GC columns , one non-polar  and the other polar , were used. Columns of different polarity were used separately .m/z; the temperature of the ion source was 250 °C. The quadrupole electron ionization (70 eV) mass spectrometer was operated in full scan mode in the range of 35–450 The parameters of the SPME autosampler Combi Pal System, AOC-5000  were set at: equilibration time: 30 min; temperature: 50 °C; exposition time: 15 min; temperature: 50 °C; and desorption in the splitless port: 240 °C for 2 min.−1, oven temperature: 37 °C (10 min), then increased to 132 °C (4 °C/min) and 240 °C (8 °C/min). The total time of the analysis was 60 min. Chromatographic conditions were set at: flow : 1 mL·minn-paraffins—with values calculated as Linear Retention Indices (LRI).The substances were identified by the mass spectra libraries NIST08, NIST08s, and FF NSC1.3, as well as by RI: retention index. RI data were retrieved from a reference database of the National Institute of Standards and Technology and compared—based on analyses of Results were expressed as the profiles of volatile compounds, i.e., percentage of the selected peak area in the sum of the peak areas of all the analyzed compounds.n = 3). The results obtained were subjected to statistical analysis using STATISTICA 13.3 software  and expressed as mean ± standard error of the mean. Either a one- or two-way analysis of variance was performed. The significance of the differences between the means was estimated based on Tukey’s post-hoc test at p < 0.05.All analyses were performed in triplicate . The PCA was used for those groups of volatiles whose individual components were most highly correlated. It was performed using STATISTICA 13.1 software .The saturation of hawthorn fruits with a sucrose solution followed by their preservation using various methods caused changes in the qualitative and quantitative compositions of volatile compounds. In total, 54 volatile compounds were identified in the produced liqueurs. The liqueurs produced from fruits non-saturated with sucrose contained all of the identified volatiles.cis-p-menth-8-en-2-one and trans-p-menth-8-en-2-one.The liqueurs produced from fruits preserved at low temperatures were characterized by the smallest diversities and the lowest concentrations of volatile compounds. Fruit preservation involving water-activity reduction (upon freeze-drying and air-drying) contributed to a more distinct flavor bouquet of the liqueurs (the greatest number and diversity of identified volatile compounds). Only liqueurs made from these fruits contained Freeze-drying turned out to be the best method of hawthorn-fruit preservation applied to obtain liqueurs with the richest profile of volatile compounds. However, the traditional air-drying technique also provided good results in terms of the aromatic bouquet of the final products. On the other hand, the saturation of fruits with sucrose solution before their preservation did not produce a satisfying effect as regards the profile of flavor compounds. In the present work, the authors studied the effects of different types of fruit treatments on the profile of aroma compounds in hawthorn liqueurs. However, raw material was collected once from the restricted area, and liqueurs were produced in one series. Therefore, further research is needed to estimate the influence of different botanical origins of the raw materials on the selected characteristics of the final products.


To evaluate the effects of long-term tumor necrosis factor (TNF) inhibition on the risk and age at onset of Parkinson disease (PD), we performed a 2-sample Mendelian randomization study using genome-wide association studies (GWAS) summary statistics.TNFRSF1A, the gene encoding TNF receptor 1 (TNFR1), were identified as predictive of pharmacologic blockade of TNFR1 signaling by anti-TNF therapy, based on genetic associations with lower circulating C-reactive protein . The effects of TNF-TNFR1 inhibition were estimated for PD risk  and age at PD onset  using GWAS data from the International Parkinson's Disease Genomics Consortium and 23andMe, Inc. To validate variants as proxies of long-term anti-TNF treatment, we also assessed whether variant associations reflected anticipated effects of TNFR1 inhibition on Crohn disease, ulcerative colitis, and multiple sclerosis risk .Genetic variants in the vicinity of TNF-TNFR1 signaling inhibition was not estimated to affect PD risk  or age at onset . In contrast, genetically indexed TNF-TNFR1 signaling blockade predicted reduced risk of Crohn disease  and ulcerative colitis  and increased multiple sclerosis risk . Findings were consistent across models using different genetic instruments and Mendelian randomization estimators.Our findings do not imply that TNF-TNFR1 signaling inhibition will prevent or delay PD onset.This study provides Class II evidence that TNF-TNFR1 signaling inhibition is not associated with the risk or age at onset of PD. Moreover, results obtained from a sample of patients with IBD may not be generalizable to individuals without IBD.Inflammation in the periphery is hypothesized to contribute to the pathogenesis of Parkinson disease (PD), particularly when present in the gastrointestinal tract.5 In Mendelian randomization studies relating to pharmacologic exposures, genetic variants that perturb the expression or function of a drug's target (often a specific protein) are used to anticipate the effects of modulating this target with therapeutic use.6 Due to Mendelian randomization principles, associations of this genetic variation with diseases are not expected to have arisen from confounding or reverse causation, thus aiding causal inference.7 In this study, we used a Mendelian randomization design to further evaluate the therapeutic potential of targeting TNF signaling for PD prevention or treatment, leveraging genetic variation in or near the gene encoding TNF receptor 1 (TNFR1) that is indicative of the blockade of proinflammatory TNF signaling via this receptor.2Mendelian randomization is a valuable approach to help answer questions in pharmacoepidemiology.8 We focused on single nucleotide polymorphisms (SNPs) in the vicinity of the gene TNFRSF1A, which encodes TNFR1, the principal effector of proinflammatory signaling following TNF agonism.2 We determined the extent to which genetic variation in this gene region is indicative of long-term TNF signaling blockade, based on associations of the regional SNPs with circulating markers of systemic inflammation reported by large genome-wide association studies (GWAS) of C-reactive protein (CRP) and cell count measures.10 We then combined data on selected variants with corresponding association statistics from GWAS of PD traits to test the effects of TNF-TNFR1 signaling inhibition on (1) the risk of PD, measured as self-reported or clinically ascertained disease status; and (2) age at PD onset, measured as self-reported age at motor symptoms manifestation, or age at PD diagnosis when the former was unavailable.12 This study provides Class II evidence that TNF-TNFR1 signaling inhibition is not associated with PD risk or age at onset, according to criteria of the American Academy of Neurology.This study used a 2-sample Mendelian randomization design .8 We foc14 Therefore, researchers have investigated the development of novel therapeutics to selectively antagonize TNFR1 without inhibiting TNFR2-mediated signaling.16 An effective response to anti-TNF treatment leads to lower systemic inflammation, reflected by reductions in circulating inflammatory biomarkers, such as CRP.17 Thus, to validate whether SNPs in the vicinity of TNFRSF1A index the modulation of proinflammatory TNF-TNFR1 signaling, we extracted data on associations of these variants with circulating CRP from a large GWAS meta-analysis  and mean platelet volume (MPV)—from independent GWAS data , whereas TNFR2 signaling contributes to tissue repair and neuron survival.WAS data .10,18TNFRSF1A is located close to genes that encode other proteins with known immune-related roles, such as lymphotoxin β receptor gene LTBR .In total, 23 SNPs were selected solely from within the gene's genomic coordinates and a narrow flank in either direction . The choice of a narrow flanking region was adopted to minimize the possibility that the selected variants might associate with PD traits via pathways other than TNF-TNFR1 signaling, given that 11 This yielded a sample of 37,688 cases and 981,372 controls. Genetic association data for age at PD onset were based on a GWAS comprising 28,568 PD cases from a subset of the cases sampled by the IPDGC and 23andMe, in which the mean age at onset was 61.7 years (range 20–97).12 These samples are described further in Genetic association data for PD risk were derived from a meta-analysis of 16 case–control samples from the International Parkinson's Disease Genomics Consortium (IPDGC) and 23andMe, using the same protocol as adopted in a recent GWAS.19 However, sample overlap is likely to be nominal in this study because the exposure and outcome GWAS were conducted with largely independent samples: samples for CRP and cell count GWAS were derived primarily from population-based cohorts assembled by the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium,9 and PD GWAS samples were derived mainly from independent case–control studies assembled by the IPDGC and the 23andMe user base  to the set of SNPs in the region with CRP association p values under 0.05, to select independent SNPs with the strongest evidence for association with systemic inflammation. Mendelian randomization results based on this selection criterion were then obtained using Wald estimation, given that a single SNP (rs767455) was retained for analyses. Mendelian randomization estimates can be biased when the genetic variants used in analysis are weak instruments for the exposure being indexed.29 Thus, to indicate the strength of this SNP as an instrumental variable, the F statistic for its association with CRP was estimated from the F distribution based on the p value and sample size of SNP–CRP association, with 1 degree of freedom.We conducted Mendelian randomization models based on 3 approaches. In the primary analysis, we applied conservative linkage disequilibrium (LD) clumping (r2 < 0.8). The clumped SNPs were then filtered by requiring false discovery rate–corrected CRP associations with p values under 0.05, and further associations of the variants with both WBC and MPV (using an unadjusted p threshold <0.05), ensuring consistent and robust associations of variants with inflammatory signaling were retained. Next, Mendelian randomization estimates based on the set of retained SNPs were calculated using an extension to the inverse variance weighting (IVW) method with principal components to account for the residual correlations between selected SNPs.30 This method relies on reference data to adjust for estimated correlations between the SNPs, for which we used data on 502 individuals of European ancestry of the 1000 Genomes project, phase 3.31 Principal components explaining over 99% of variance in the weighted correlation matrix were used in the IVW estimator.For secondary analyses, we aimed to retain more variants—which may explain more variance in CRP and therefore potentially improve the power of Mendelian randomization models—by including all regional SNPs after liberal LD clumping .Third, after examining the functional annotation of SNPs in the gene region, we also assessed specific associations of a single SNP (rs1800693) with traits, since this SNP is suggested to affect alternative splicing of the 33 In plots, outputs from logistic regression models were presented as log-odds to scale appropriately alongside βs from linear regression models. Corresponding odds ratios (ORs) and 95% confidence intervals (CIs) for binary outcomes are reported in the Results. Given that we had strong priors for non-null results from positive control analyses, we did not adjust these results for multiple testing correction.All Mendelian randomization outputs were scaled to be expressed per 10% reduction in circulating CRP, indicative of the direction of effect anticipated from TNF inhibition.TwoSampleMR and mendelianRandomization packages.34All analyses were performed in R version 3.6.1 (2019-07-05) using For each GWAS study included in the present work, written informed consent was obtained from all participants and the studies were approved by relevant ethics review boards. Because our study was based on existing summary-level genetic results—i.e., without accessing individual-level genetic data—no additional ethical approval was required.9 Summary statistics from GWAS of WBC and MPV are publicly available at bloodcellgenetics.org/.35 Full PD GWAS data can be obtained via research project applications to 23andMe, Inc. and the IPDGC.The majority of genetic association statistics used in this study are presented in TNFRSF1A were present in the CRP data. One SNP in the region—rs767455—was retained following conservative LD clumping (p = 7.8 × 10−8). It is in high LD with the presumed functional variant (rs1800693) in this region (r2 = 0.84)32 and the variant is also associated with circulating WBC (p = 4.2 × 10−3) and MPV (p = 3.7 × 10−6), with consistent directions for all 3 inflammatory markers. Using this variant in Mendelian randomization models, no effects of TNF-TNFR1 inhibition on PD risk  or age at onset  were predicted .A total of 23 SNPs in the vicinity of clumping . This exredicted . In contp < 0.05. Among these 4 SNPs, one was not associated with either WBC (p = 0.7) or MPV (p = 0.95) and was therefore removed, leaving 3 variants that were associated with all 3 inflammatory markers for secondary Mendelian randomization models . Although we showed no support for the potential of blocking TNF-TNFR1 signaling to lower PD risk, the results should be interpreted carefully in comparison to the observational study for several reasons. First, the 4 TNF inhibitors investigated by Peter et al.4—adalimumab, certolizumab, golimumab, and infliximab—all directly neutralize soluble or transmembrane TNF and disrupt both the TNFR1- and TNFR2-mediated pathways as well as other potential biological effects induced by TNF by any other pathways,36 whereas our results are specific to the selective inhibition of TNF-TNFR1 signaling transduction. However, we note that TNF-TNFR1 signaling is proposed as the major TNF-related neurotoxic pathway to intervene on for PD therapy.14–37 Second, given that IBD is rare, our findings are more relevant for the general population. The models test whether TNF-TNFR1 signaling blockade affects PD onset regardless of whether or not individuals have a background of severe systemic inflammation.38 Consequently, we do not exclude therapeutic benefits in individuals with certain underlying conditions, such as IBD. Third, Mendelian randomization estimates relate to an averaged effect of long-term exposure to the TNF-TNFR1 inhibitors across the life course; therefore, a null result may reflect either a true lack of an overall effect on PD risk at any stage of life or a sum of opposing, period-specific effects that balance out in our models. This scenario might be possible if, for instance, TNF-TNFR1 signaling inhibition is beneficial for PD in older age due to anti-inflammatory effects, but detrimental earlier in life for other reasons . Mendelian randomization estimates are also averaged across any tissue-specific effects, whereas therapeutic administration of TNF inhibitors may be limited to effects induced in the periphery. Fourth, because the 4 TNF inhibitors examined by Peter et al.4 are large molecules that do not cross the blood–brain barrier (when intact), they are assumed to mitigate peripheral inflammation principally, unless IBD has affected the integrity of the blood–brain barrier to allow TNF inhibitors to act in the cerebral parenchyma. However, the possibilities of opposing period- or tissue-specific effects of TNF inhibition on PD risk are speculative.By comparing patients with IBD with and without anti-TNF treatment in a cohort study, Peter et al.In addition to the risk of PD, we also considered whether TNF-TNFR1 signaling inhibition may affect the age at PD onset for its relevance to the course of disease progression in lieu of large GWAS of longitudinally measured PD progression, which are not currently available. For a progressive disease such as PD, an exposure affecting the age at onset could do so by either delaying the pathogenesis of PD  or modifying the rate of PD progression once the disease process has commenced. In the present study, the long-term blockade of TNF-TNFR1 signaling did not affect the age at PD onset, implying limited potential for TNF inhibitors to affect PD progression (while keeping the aforementioned caveats about interpreting genetic vs pharmacologic results in mind).The strengths of our study include the use of large-scale genetic data in a Mendelian randomization design to facilitate causal inference, robust metrics for the Mendelian randomization analyses , and positive control analyses that indicate the validity of using the chosen genetic variants to predict therapeutic effects. This study's main limitations are that we could not examine whether there is an interaction between inflammatory status and TNF inhibition on PD risk or index the effects of TNF inhibition on direct measures of PD progression.39 Hence, our findings should be considered as part of discourse on the repurposing potential of TNF inhibitors for PD prevention and treatment, particularly if selective inhibitors of TNF-TNFR1 signaling become available. Future Mendelian randomization studies could use large-scale GWAS of PD progression to help evaluate the disease-modifying potential of selective TNFR1 inhibitors for PD treatment.In conclusion, our findings do not support the notion that long-term blockade of TNF-TNFR1 signaling will prevent or delay PD onset. Human genetics provides valuable guidance for therapeutic programs because the chances of successful drug development are substantially improved when genetic evidence links a drug's target to an indication for therapeutic use.


Among the tools useful for the management of neuropathic pain, ultrasound presents several advantages, shown by the literature. We assessed the scientific production about neuropathic pain and ultrasound from different points of view: general topics, journal categories, geographical origin and lexical analysis. We searched papers on PubMed using the Medical Subject Headings “neuropathic pain” AND “ultrasound”. We collected data about the journals where the papers were published, the country of the affiliation of the first author. For the lexical analysis, we evaluated the presence of selected words in the papers, and we built a graph representing the connections among words and papers. The papers were focused on the use of ultrasound as a diagnostic tool and guide for the therapy, assessing its application in different diseases such as Morton’s neuroma and piriformis syndrome. The most represented journal category was anesthesia while the most common country the United States of America. The lexical analysis confirmed the importance of ultrasound for diagnosis of specific disease and treatment of pain. The described approaches provide a multiperspective evaluation of the literature and may support the interpretation of the information contained by the papers. Neuropathic pain (NeP) is a relatively common condition that affects the daily life of patients . It can The first challenge in the management of the NeP is related to the differentiation between NeP and the other types of pain, in particular nociceptive . Proper The second challenge in NeP management is related to the treatment choice, because the therapeutic options may have various efficacy and numerous side effects . The posSince the NeP is associated with many diseases, its impacts are not only related to the general quality of life of the patients, but also to the management itself. Indeed, the symptom may be extremely severe and influence the psychological status causing a lowering of the patient’s adherence to the treatment. Furthermore, the rehabilitation itself has shown an important reduction of its effectiveness when the NeP is present ,9.For these reasons, one of the most important targets in the management of the NeP concerns the identification of the primary reason for the symptoms. The number of diseases or clinical situations causing the NeP is very large and includes traumas, acquired of genetic neuropathies, drug-induced nervous diseases, radiculopathies, central nervous system diseases . If the Considering the diagnostic tools, imaging techniques represent essential completion. These instruments can characterize from a morphological point of view, body parts. This can reveal the possible alteration in specific organs and can help in the identification of the conditions causing the NeP. Among these techniques, ultrasound (US) presents peculiar features, which make it the natural evolution of physicians’ hands and eyes . US can The aim of our paper was the assessment of the literature about the NeP and the US from multiple points of view. In particular, we considered: (1) the overall information about the literature concerning NeP and US; (2) the information about the journal categories and the geographical origins of the papers; (3) the lexical data to identify the relationships between the words and the published papers. With these approaches, we intended to summarize the current state of the art of the literature about NeP and US.We searched on PubMed the papers about NeP and US using the following Medical Subject Headings (MeSH): “neuropathic pain” and “ultrasonography”. The two MeSH terms were linked by the Boolean operator “AND”, to collect the papers that included both terms. We just used the following filters: date (papers published in the last 10 years) and language (English). The search was performed on 24 February 2021. The titles and the abstracts of the found papers were exported as a text file using the specific PubMed function. After this initial collection, we excluded the non-pertinent papers based on the titles and the abstracts. The papers were considered non-pertinent when they concerned about non-human subjects or when NeP or US were not indicated as subjects of the study in the abstracts.For each paper, using the titles and the abstracts, we evaluated: the main topic of each paper (diagnosis or treatment), the year of publication, the main disease of the study and the relative outcome measures.After the exclusion of non-pertinent papers, we have considered the journal in which each paper was published. We associated with this journal its main category as visible in Journal of Citation Reports. For each category, we calculated the total number of papers included and then we considered the percentage of recurrence of the category with respect to the total number of the papers.For the assessment of the geographical origin of each paper, we considered the affiliation of the first author. In the case of multiple affiliations, just the first one was considered. For each country, the percentages with respect to the total number of papers were calculated and represented as a world map .The lexical network based on graph-theory (LENGTH) is an approach previously published that visualizes and analyzes the relationships of the words contained in the assessed papers. For the LEGTH, we imported the text file obtained from PubMed, after the exclusion of the non-pertinent papers, in the freeware software TXM 0.8.0  . This alIn our case, the nodes were the papers and the words, while the edges were the connections between them. The software can calculate specific parameters indicating the quantity and the quality of the connections. In particular, the degree indicates the number of the connections, which represents, for the words, their frequency. The modularity class shows the aggregation of nodes and, in our case, which words are “close” to others. The closeness centrality and the betweenness centrality indicate how well the nodes are connected with the others  Figure .We found 158 papers on PubMed using the MeSH terms and the filters. Nine of these papers were excluded because not considered relevant for the aim of the study. Hence, the final number of the selected papers was 149. Among these, we found 57.05% of papers mainly focused on treatment, while 42.28% were focused on diagnosis. The topic of one paper was equally related to diagnosis and treatment. The literature about NeP and US showed a continuous growth with the major recent increases in 2016 and 2019. Concerning the assessed diseases, Morton’s neuroma was the most frequent, present in 15.57% of the studies, followed by piriformis syndrome in 14.75% and herpes/postherpetic neuralgia in 11.48% . The mosUS showed a high level of effectiveness in studying the body structures with high capability to depict alterations useful for the diagnosis . In the The journal categories of the selected papers covered a wide range of medical fields . With 32The geographical origin revealed the publications about NeP and US interest each continent . United The LENGTH approach revealed PAIN was the most frequent word in the titles and abstracts . It was The software can automatically locate the nodes to better visualize the associations and the modularity calculation supports the interpretation of node aggregation. Hence, we found seven node groups representing the major subtopics of the literature analysis. In particular, ULTRASOUND was associated with the terms related to the guiding procedures, while PAIN was associated with BLOCK and the terms defining the symptoms. The piriformis syndrome revealed an association with the injection, while Morton’s neuroma (and relative words) was especially connected with the diagnosis. Finally, PATIENT was related to the severity characterization of the symptoms and NERVE showed interactions with the peripheral nervous system and the nerve stimulation. SCIATIC appeared isolated from the other terms.The centrality measures provided additional information about the words. Although ULTRASOUND was the fourth term for its frequency, it presented the highest level of connections in the graph, together with PAIN. The word DIAGNOSIS, while showed a relatively low degree, had higher centrality values compared to the words indicating the diseases, but lower than the words indicating the treatments. Similar behavior was visible for IMAGING. Considering the different syndromes, we found relatively high degrees, but the specific terms (MORTON and PIRIFORMIS) showed a low level of closeness centrality.The worldwide literature confirms the important contribution of US in management of the NeP. Many studies underline its positive impact in guiding the treatment, especially for the nerve block or the intramuscular injection ,26,30. AIn the management of the disease, US-guided injections appear more effective than blind procedures because, seeing the impaired organ to treat and the surrounding tissues, they limit the side effects, often related to the diffusion of the drugs in wrong sites ,26.In piriformis syndrome, Wu and colleagues assessed patients with clinical suspicion of piriformis syndrome with a low-frequency convex probe able to evaluate muscle and the sciatic nerve and with a high-frequency linear probe to study the nerve. They found piriformis muscle and sciatic nerve were enlarged and hypoechoic in the impairment side of the patients. Finally, the authors found a diagnostic sensitivity of around 70% for the muscle and nerve ad a very high specificity for the sciatic nerve . In our Interestingly, the term SCIATIC is apparently isolated, despite its relative common frequency in the selected papers. Its closeness centrality is relatively low. These data should indicate the specificity of most of the studies about sciatic impairment. This specificity is likely considered for the terms related to Morton’s neuroma. They appear relatively far from the other words and present a very low level of closeness centrality. This parameter is a measure of the distance of a node from the other ones. Hence, these speculations about the goodness of the connections of the node may be made. Obviously, they should be associated with the other parameters, in particular the word frequency.The five most common terms in our analysis, which present the highest centrality values, include PAIN and ULTRASOUND. This is due to the MeSH terms used for the research. However, even if the MeSH term “neuropathic pain” was used, the word NEUROPATHIC just appears in the sixteenth position, considering its frequency, about 1:6 in comparison with PAIN. This is likely due to the omission of the adjective neuropathic in most of the times in which the pain is cited. However, among the five common words, PATIENT, NERVE and GUID* are present. This is interesting because the papers likely focus on clinical perspective and many of them consider the nerve impairment related to the neuropathy . FinallyThe geographical origin of the papers shows stimulating data. First, the number of papers, as expected, is not directly correlated with the population. The United States of America, as often visible, is a very prolific country, while China and especially India, the two most populated countries in the world, do not show a significant amount of published paper. This should be related to their relatively recent approach in the worldwide scientific literature. In our analysis, Turkey confirms its increasing impact, already visible in other scientific productions related to US. Interestingly, two small countries, Cyprus and Luxembourg, and a very large one, Brazil, showed one single paper. The results underline the dissociation between the population and the literature production, and they should be further studied considering, for each country, different points of view: culture, language, history, society, income, healthy policies, education systems and, specifically, relations with the virus diffusion .In general, the LENGTH approach reveals the connections among words and papers, considering the lexical point of view of the papers. This method may reveal information enclosed in the texts and allow speculations about the way to convey specific messages by the words. The calculation of the parameters may be relevant because of the quantification of the position and the impact of a word in the network. Finally, the considerations should be always related to the “usual” revision process to support and translate them into useful information. The approach can be used for the visualization of the state of the art of literature, can support the consideration of the information contained in the papers and can help in identifying the topics to widely assess.The approach presents some limitations. First, paper exclusion depends on the abstracts and the examiner’s experience. Second, the word selection itself depends on experience, because, despite the calculation of the frequencies, some words are not considered pertinent. Additionally, the words are analyzed in the titles and abstracts and not in the entire document, but this allows a simplification of the analysis without the restriction due to the unavailability of some papers.In conclusion, the management of the NeP includes US application both for the diagnosis and for the treatment. For the diagnosis, specific diseases such as Morton’s neuroma and piriformis syndrome can be evaluated. For the therapy, US is a fundamental help in guiding the injection and in performing nerve blocks. The published papers mainly belong to journals of anesthesiology and radiology and cover countries of all continents. Finally, the lexical analysis confirms the US impact for diagnosis and treatment and reveals specific relationships with words indicating organs, diseases, techniques, and clinical components.


Long-term potentiation (LTP) is regulated in part by metaplasticity, the activity-dependent alterations in neural state that coordinate the direction, amplitude, and persistence of future synaptic plasticity. Previously, we documented a heterodendritic metaplasticity effect whereby high-frequency priming stimulation in stratum oriens (SO) of hippocampal CA1 suppressed subsequent LTP in the stratum radiatum (SR). The cytokine tumor necrosis factor (TNF) mediated this heterodendritic metaplasticity in wild-type rodents and in a mouse model of Alzheimer’s disease. Here, we investigated whether LTP at other afferent synapses to CA1 pyramidal cells were similarly affected by priming stimulation. We found that priming stimulation in SO inhibited LTP only in SR and not in a second independent pathway in SO, nor in stratum lacunosum moleculare (SLM). Synapses in SR were also more sensitive than SO or SLM to the LTP-inhibiting effects of pharmacological TNF priming. Neither form of priming was sex-specific, while the metaplasticity effects were absent in TNFR1 knock-out mice. Our findings demonstrate an unexpected pathway specificity for the heterodendritic metaplasticity in CA1. That Schaffer collateral/commissural synapses in SR are particularly susceptible to such metaplasticity may reflect an important control of information processing in this pathway in addition to its sensitivity to neuroinflammation under disease conditions. In a unique form of metaplasticity in area CA1 of the rodent hippocampus, prior electrical activity (priming activity) in either stratum oriens (SO) or stratum radiatum (SR) inhibits subsequent LTP and promotes LTD at a separate set of synapses in SR. This heterosynaptic effect is independent of postsynaptic neuronal firing and activation of N-methyl-D-aspartate receptors (NMDARs), metabotropic glutamate receptors (mGluRs) and gamma-aminobutyric acid (GABA) GABA-A and –B receptors. Instead, the priming effect appears to involve activation of non-neural cells, such as astrocytes, across the hippocampal network4 Accordingly, we have hypothesized that gliotransmitters are released onto nearby synapses to cause the metaplastic effect.Long-term potentiation (LTP) and long-term depression (LTD) are widely accepted candidates as neural mechanisms underlying memory formation. Moreover, past neuronal state can regulate LTP and LTD via metaplasticity, the activity-dependent changes in neuronal state that regulate future synaptic plasticity, and that may participate in the maintenance of synaptic thresholds within normal limits7. Recently, we reported that TNF but not IL-1β mediates the long-range metaplastic inhibition of LTP in CA1 of rat hippocampus, while pharmacological priming by TNF but not IL-1β protein causes a similar inhibition of LTP8. Further, we reported that both the electrical and pharmacological TNF-mediated metaplastic effects involve activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK)8. However, the receptor subtype that TNF binds to in order to activate these kinases is unknown. TNF is a pleiotropic pro-inflammatory cytokine that binds to two receptor subtypes, TNF receptor I  and TNF receptor II . TNFR1 activation is associated with an enhanced glutamate receptor responsiveness10, neuropathic pain11, and the progression of AD12. Moreover, TNFR1 is required for the amyloid-beta (Aβ) mediated inhibition of LTP in the dentate gyrus15 and CA116. Therefore, we hypothesised that TNF binding to TNFR1 is essential for the metaplastic inhibition of LTP in CA1.Several cytokines, notably tumour necrosis factor (TNF) and interleukin-1 beta (IL-1β), suppress LTP in the CA1 region of the hippocampus18. This raises the question whether SR synapses are also more sensitive to TNF-mediated metaplasticity effects than SO synapses. Equally, it is unknown whether synapses in the third main dendritic layer of CA1, the stratum-lacunosum moleculare (SLM) that contains the pyramidal cell apical tuft dendrites, is more like SO or SR in its sensitivity to TNF-mediated inhibition of LTP. To address these questions, we compared the relative sensitivity of SO, SR and SLM synapses to electrical and TNF priming. Further, since all our prior studies used male animals, we compared the priming effects between males and females.Interestingly, Aβ causes inhibition of LTP in SR but not in SOWe found that the metaplasticity effect occurred differentially across strata, as electrical priming in SO only inhibited LTP in SR but not in SO or SLM. Similarly, SR synapses were more sensitive to TNF priming than SO or SLM synapses. Electrical and TNF priming effects in SR occurred in both females and males and required TNFR1 activation.19. In this paradigm, following a 45 baseline recording period, priming stimulation (6 trains of high-frequency stimulation (HFS) are given to the SO afferents and 30 min later, LTP is 30 induced in the Schaffer collateral afferents in SR through two trains of theta-burst stimulation . In confirmation of our previous results, priming in SO  = 4.750, p = 0.025). Here, post-hoc tests revealed no differences between experiments except for a significantly greater SO LTP in the SLM experiment relative to that in the SO experiment (p = 0.02), likely because of the inclusion of GABA receptor antagonists throughout the SLM experiment (see Methods) to ensure adequate LTP induction in SLM under control conditions20.To assess the pathway specificity of the priming effect, we first sought to replicate the previously reported finding that priming inhibits subsequent LTP in SR, using male Sprague–Dawley ratsSO Figs. a,g cause6 and as a priming stimulus8. Here, a 10 min bath application of 1.18 nM TNF protein caused significant inhibition of LTP induced 30 min later in SR compared to ACSF controls  exhibited a similar pathway specificity. TNF at a low concentration (nM range) has been found to be effective in inhibiting LTP at SR synapses both when given concurrentlyp = 0.0004; Fig. p = 0.001; Fig. Although 1.18 nM TNF had no effect on LTP at the SO and SLM synapses, we asked whether this was a relative or absolute difference in TNF sensitivity. Accordingly, we tested the efficacy of a higher concentration of TNF in inhibiting subsequent LTP at these synapses. We found that 5 nM TNF was sufficient to inhibit LTP at SO synapses  could also generate the priming effect. Similar to HFS priming, two trains of TBS priming in SO not only produced homosynaptic LTP in SO .TNF plays a critical role in mediating the heterodendritic metaplasticity effect in area CA1 of the rat hippocampus+/+ mice  mice. Similar to TNFR1−/− mice, priming stimulation in TNFR1+/− mice produced a significant homosynaptic LTP in SO, but in this case a partial priming-mediated inhibition of LTP in SR . However, the finding in SO is consistent with our previous report that priming stimulation in SR, which also inhibits SR LTP, failed to inhibit LTP in SO19.LTP is vital for learning and memory and is regulated in part by metaplasticity. In one particularly interesting form of metaplasticity, priming stimulation in SO, the basal dendritic zone of CA1 pyramidal cells, inhibits LTP induction 30 min later at Schaffer collateral/commissural synapses located a few hundred microns away in the apical dendritic zone in SR8. Moreover, delivery of recombinant TNF to slices mimicked the priming effect elicited by SO stimulation8, supporting previous studies using similar paradigms25. Here, we confirmed the role of TNF in priming-mediated inhibition of LTP and showed that it acts via the TNFR1 receptor, as both the electrical and TNF priming effects were absent in TNFR1−/− mice. Notably, this was true for both male and female mice, demonstrating a lack of sex differences in the priming effect generally and the TNF mechanism in particular. The present experiments also showed the TNF concentration that is sufficient to inhibit SR LTP failed to affect either SO or SLM LTP, corresponding well with the sensitivity of SR synapses to the electrical priming effect. On the other hand, TNF delivered at the higher concentration of 5 nM could inhibit SO and SLM LTP. Thus, these strata have a higher threshold for TNF-mediated LTP inhibition compared to SR. This is consistent with the inhibition by amyloid-β of SR but not SO LTP, through a TNFR1-dependent mechanism13.Recently we reported that TNF release mediates the long-range metaplasticity effect between the SO and SR dendritic compartments27. Moreover, CA2 pyramidal cells also project mainly to SO28. Postsynaptically, the basal and apical dendrites of CA1 pyramidal cells differ in size, shape and geometry, electrical properties, and responsiveness to neurotrophic factors31. In addition, dendrites in SO exhibit more mushroom spines and a higher magnitude of LTP than SR dendrites32. It is also possible that the pyramidal cell dendrites in SO and SLM with their bushy dendritic arbours may more readily generate synaptically evoked NMDAR spikes and plateau potentials34, which may make LTP induction there particularly resistant to counteracting influences. Mechanistically, it is notable that participation of nitric oxide (NO) is necessary for SR LTP but not for SO LTP, indicating a differential molecular mechanism of LTP expression in apical versus basal dendrites in CA135. This may be due to the absence of endothelial NO synthase in SO, which is the primary source of NO in SR37. On the other hand, the enhancement of LTP in SO relative to SR is dependent on the expression of Type II cadherins32.What could explain the sensitivity of SR synapses to the metaplasticity effect, especially relative to SO synapses that nominally have the same glutamatergic afferent inputs? Some clues may come from the anatomical, physiological and molecular differences between these pathways. For example, although there are indeed Schaffer collateral/commissural inputs to both SR and SO, the cells of origin are not entirely overlapping as SR receives its projections preferentially from proximal CA3 ipsilaterally, while SO receives its projections more from distal CA3 and contralateral CA338. In addition, the temporoammonic pathway displays distinct plasticity properties in response to the same stimulation strength  compared to Schaffer collateral/commissural synapses in vivo39. Conceivably, there could also be a differential regulation of TNF activity in SLM and SO compared to SR due to variations in TNFR1 distribution or activation threshold. However, we have not been able to detect differences in TNFR1 distribution across the CA1 strata, nor in the expression of downstream enzymes such as p38 MAP kinase or JUN kinase, using immunofluorescence imaging techniques .SLM synapses also display synaptic properties different from other hippocampal synapses. For example, in the developing hippocampus, the electrical stimulation that produces substantial LTD at SR synapses is less effective at SLM synapses19, which might otherwise explain why the suppression effect becomes evident so early post-tetanisation. Moreover, the priming stimulation in SO does not affect baseline transmission in SR except for a transient depression on occasion, indicating that a presynaptic change in transmitter release is unlikely to explain the effect. The inhibitory mechanism does rely on activation of p38 MAP kinase, JUN kinase and extracellular signal-related kinase8. However, the subcellular localisation of these kinases and their actions remains to be determined, especially given that astrocytes appear to also play a role in mediating this metaplasticity effect4.The mechanisms by which electrical priming and TNF act to inhibit SR LTP have yet to be fully characterised. Our previous experiments showed that this is not due to altered GABAergic inhibition, nor to altered NMDAR currents in SR40, and plays a prominent but paradoxical role in the brain because of its differential regulatory actions in healthy conditions versus during neurological disease or injury. TNF treatment on primary hippocampal neurons prevents hypoxia and NO-induced neuronal death41, ameliorates hippocampal damage in acute brain insults42 and stimulates synthesis and secretion of nerve growth factors43. In addition to a role in neuroprotection, as mentioned above, TNF has emerged as a regulator of synaptic function in the hippocampus, such as the trafficking of GABA-A44 and AMPA receptors45. Additionally, TNF acting on TNFR1 is critical for low-frequency stimulation-induced LTD in CA1 SR46 and metabotropic glutamate receptor-mediated LTD in the dentate gyrus13. Here we have confirmed an additional role for TNF, namely the pathway-specific metaplasticity evoked by both HFS and the more physiologically relevant TBS protocol.TNF is produced in the CNS by neurons, astrocytes, and microglia47. The output connections of CA1 with subicular and cortical regions are important for the replay of this information through the hippocampal and cortical circuitry and retrieval during memory retention trials as triggered by output from CA348. Since the metaplasticity effect decays over a 1.5–2.5 h period, it is conceivable that the initial period of memory storage and consolidation is accompanied by a subsequent transient period of reduced memory storage functionality, i.e. LTP induction, to prevent interference from later information coming via CA3 while still allowing potential updating based on current information arising via the direct entorhinal inputs to SLM. The affected information could be particularly relevant to non-spatial recognition memory, mediated preferentially by the proximal CA3 input that projects to distal CA1 SR49. Note that the duration of the metaplasticity effect at physiological temperatures in vivo could be rather shorter than that noted by Holland and Wagner in vitro50.What could be the behavioural relevance of the sensitivity of SR synapses to prior activity in either SR or SO afferents? SR inputs arise mainly although not exclusively from proximal CA3, where auto-associative connections mediate pattern completion and memory storage that is passed to area CA1 to consolidate activity there, such as place cell activity51, mediates synaptic scaling in cortical synapses52, and participates in experience-dependent synaptic plasticity in the developing mouse visual cortex53. On the other hand, at higher concentrations, TNF inhibits LTP in both CA1 and the dentate gyrus of the hippocampus54. High levels of TNF have been detected in multiple sclerosis55, Alzheimer's disease (AD)56, and Parkinson's disease57. Moreover, animal and human studies indicate a correlation between increased TNF levels with cognitive and memory impairments59. Consistent with this, we have reported that the elevated TNF level in an APP/PS1 mouse model of AD generates an aberrant engagement of the heterodendritic metaplasticity mechanism responsible for the LTP impairment in these mice8, consistent with the efficacy of a scavenger biologic (XPro1585) in rescuing LTP in a 5xFAD mouse model60. Moreover, genetic and pharmacological block of TNFR1 prevents inflammation and cognitive impairment in APP/PS1 mice61 and less susceptibility to hippocampal epileptic seizure9. Together these data indicate a major role for TNF acting on TNFR1 in mediating cognitive impairment in neurodegenerative disease and suggest TNF as a therapeutic target.TNF may deviate from its functions in healthy conditions at constitutive levels versus its level in a diseased condition. At physiologically relevant concentrations, TNF plays a role in controlling the strength of glutamatergic synapses in the hippocampal neuronsIn summary, CA1 pyramidal cells have a finely tuned sensitivity of LTP to prior activity in the network that differs between afferent pathways and dendritic compartments. This differential sensitivity may play an important role in the efficiency and consolidation of memory storage. Endogenous TNF acting on TNFR1 plays a fundamental role in mediating these pathway-specific heterodendritic metaplasticity effects. Maintaining the TNF concentration at an optimum level may be essential for synaptic homeostasis in the brain, while activity-dependent and transient elevations may play a critical role in regulating CA1’s learning, memory and retrieval functions.62  and their wild-type (WT) C57BL/6 littermates were used in this study. All of the animals were bred in specific-pathogen-free colonies at the University of Otago Breeding Station. Once delivered to the laboratory, the rats were kept in standard open-top cages , while in a different room, the mice were group-housed  in individually ventilated cages after weaning. All animals were given PuraWool  as nesting material and acclimatized to the vivarium for at least one week prior to use. They were monitored daily with health status recorded on approved monitoring sheets. The light/dark cycle and ambient temperature were kept at 23 ± 2 °C, and 12:12 h, respectively, with the lights turned on at 6 a.m. The University of Otago Animal Ethics Committee approved the study objectives and design, as well as all animal handling and manipulation techniques, which were carried out in accordance with New Zealand animal welfare legislation. The study was designed in accordance with ARRIVE guidelines.Adult Sprague–Dawley (SD) male rats (6–8 weeks), and male and female mice (12–35 weeks) with a functional knock-out of the tumor necrosis factor 1 receptor+/− mice (heterozygous), male TNFR1-KO mice obtained from The Jackson Laboratory were bred with female C57BL/6 mice present in our facility. TNFR1+/− mice were crossed with TNFR1+/− mice to produce TNFR1+/+ (wild type), TNFR1+/−, and TNFR1−/− mice. To check the genotype of the animals, the DNA was isolated from mouse tail samples using the lysis buffer . The DNA isolated was subjected to polymerase chain reaction (PCR) , using the primers for TNFR1+/+ , TNFR1+/−  and TNFR1−/−  purchased from Invitrogen. To analyse and visualise the amplicons resulting from PCR, 1% agarose gel was prepared along with the addition of ethidium bromide (EtBr) at a final concentration of 0.5 μg/ml, the samples were loaded on to the precast wells followed by separation at 100 V for 1 h. The gel was exposed to UV light and a picture was taken using the gel documentation system . The band appearing at 470 bp was identified as TNFR1+/+, 300 bp as TNFR1−/−, while bands appearing at both 470 bp and 300 bp as TNFR1+/−. The genotyping was done in-house for the first three generations and for subsequent generations the mouse tail tips were outsourced to Transnetyx   continuously gassed with carbogen (95% O2–5% CO2). Hippocampi were dissected free, and the ventral portions removed. CA3 was removed manually with a knife cut to separate CA1 connectivity from the rest of the hippocampus. Using a Leica VT1000 S vibroslicer, 400 μm thick slices (3–6 slices from each hemisphere) were prepared. Slices were placed in a humidified incubation chamber with artificial cerebrospinal fluid (ACSF) and incubated at 32 °C  and gassed with carbogen. Before beginning the recordings, the slices were left at the interface between ACSF and humidified air for 30 min at 32 °C and then at room temperature for 90 min to equilibrate. Following incubation, up to two slices at a time were immersed in a humidified recording chamber at a temperature of 32.5 °C ± 0.5 °C and perfused with ACSF at 2–2.5 ml/min.Male Sprague–Dawley rats (n = 79) were deeply anaesthetized with ketamine . Rats were decapitated, and brains were quickly removed and immersed in ice-cold sucrose cutting solution , TNFR1−/− (n = 52), and TNFR1+/− (n = 23) mice were given open-drop isoflurane anaesthesia (1 ml) in a closed desiccation jar before decapitation via guillotine. Horizontal brain sections of 400 μm thickness containing the hippocampus were prepared from each hemisphere. Mouse slices were then treated as per the rat slices, except that area CA3 was left intact. The experimenters were blind to genotype during data collection and analysis using a coding sheet held by a third party, with sex and genotype being quasi-randomly interspersed.Male and female TNFR1To activate synaptic inputs and generate field excitatory postsynaptic potentials (fEPSPs), 50 μm Teflon-insulated tungsten monopolar stimulating electrodes were used, connected to custom-built programmable constant current stimulators. Stimulating electrodes were placed centrally in CA1 stratum radiatum (SR), stratum oriens (SO), and/or stratum lacunosum moleculare (SLM) to elicit fEPSPs . Similarly, the temporoammonic (TA) pathway was activated to elicit at least 2 mV field responses at a stimulation strength of 0.1 mA in SLM. Baseline stimulation was then set at 40% of the maximum fEPSP slope elicited at a stimulation strength of 0.2 mA. Baseline stimulation was delivered every 30 s, alternating between the priming stimulating electrode placed in SO and the test stimulating electrode in either SR, SO or SLM. For the TNF priming experiments, baseline stimulation was delivered every 30 s to the pathway of interest.Control experiments included 75–80 min of baseline stimulation followed by LTP induction and 60 min post-tetanization recording. As an a priori criterion, slices which exhibited changes in baseline responses > 10% were excluded from the study. SO LTP was induced by a single train of theta-burst stimulation (TBS), each train consisting of 10 bursts of 5 pulses at 100 Hz, 200 ms between bursts, at baseline stimulation amplitude; in SR, the same stimulation was repeated twice with 30 s between the trains. The LTP stimulation protocol for SLM required four trains of TBS (with 10 pulses in each burst and pulse duration of 200 µs) with 30 s between trains. Because of the strength of synaptic inhibition in SLM, LTP induction in SLM also necessitated the bath application of a mixture of a GABA-A receptor antagonist (0.2 µM gabazine) and a GABA-B receptor antagonist (2 µM CGP 55845 hydrochloride). Thus, the LTP induction protocols were adjusted to deliver approximately the same degree of control LTP in each pathway.8 or 2 trains of theta-burst stimulation , 30 min before LTP induction in the test pathway. TNF protein priming entailed bath application of exogenous TNF for 10 min, with a washout period of 30 min before LTP induction8. Recordings from control and primed slices generated from the same animal were quasi-randomly interspersed throughout each experimental day. For the priming experiments, TNF protein  was reconstituted at 10 μg/mL in filter-sterilized PBS containing 0.1% bovine serum albumin as a vehicle. In this study, we used rats for the pathway specificity and the TBS priming experiments whereas mice were used for the investigation of TNF receptor subtype involved.We used two different electrical priming protocols in SO: either 2 sets of 3 trains of high-frequency stimulation 8. A Grubb’s test was used to discard slices with outlier LTP values. The graphs were plotted, and statistical analyses  were performed using GraphPad Prism software . Tukey’s post-hoc analyses were utilized to find the difference between groups (significance set to p ≤ 0.05). All data are expressed as mean ± SEM unless otherwise stated.FEPSPs were recorded in real-time and analyzed offline using custom-built software based on the LabVIEW library. The initial slopes of fEPSPs were measured, and the baseline value was calculated as the average fEPSP slope for the 10 min preceding LTP induction. All synaptic responses throughout the experiment were then expressed as a percentage of baseline. LTP was calculated by averaging the responses generated in the final 10 min of the experiment. The data for each group were pooled across animals with individual slices being the experimental unit of analysis (n’s). Group sizes were n = 5–9, as based on our previous studies of this metaplasticity effectAll methods and procedures were approved by the University of Otago Animal Ethics Committee under animal ethics number AUP-19-42 and AUP-18-51. All the experiments were performed by adhering to the ethically approved protocols and in accordance with the New Zealand Animal Welfare and Biosecurity Legislation.Supplementary Information.


The structure adjustment and layout optimization of water pollution-intensive industries (WPIIs) are crucial to the health and sustainable development of the watershed life community. Based on micro-detailed data of Chinese industrial enterprises from 2003 to 2013, we analyzed and revealed the spatial differentiation characteristics and influencing factors of WPIIs in the Yellow River Basin (YRB) from 2003 to 2013 by constructing a water pollution-intensive index and integrating kernel density estimation and geographically weighted regression models from a watershed perspective. The results show that: (1) the scale of WPIIs in the YRB showed a growth trend from 2003 to 2013, and the output value increased from 442.5 billion yuan in 2003 to 6192.4 billion yuan in 2013, an increase of 13 times. (2) WPIIs are generally distributed in an east-west direction, and their spatial distribution is river-side, with intensive distribution in the downstream areas and important tributaries such as Fen River and Wei River. (3) WPIIs are generally clustered in high density downstream, but the spatial clustering characteristics of different industries varied significantly. The chemical industries, paper industries, etc. were mainly concentrated in downstream areas. Processing of food from agricultural products was distributed in the upper, middle and downstream areas. Resource-intensive industries such as coal and oil were concentrated in energy-rich midstream areas. (4) Natural resource endowment was the main factor affecting the distribution of WPIIs in the midstream and upstream areas of the basin, and technological innovation played a significant role in the distribution of downstream industries. The level of economic development and industrial historical foundation promoted the geographical concentration of industries. The scale of wastewater discharge and the proximity of rivers influenced the concentration of industries in the midstream and downstream. Large river basins are the cradle of the development of human civilization, and river systems and their organically connected river basin life systems continue to provide natural resources, inland navigation, ecological environment and many other functions and services for human survival and development . With ChThe Yellow River Basin (YRB) is an important energy, chemical, raw material, and basic industrial base in China, it has undertaken a large number of high energy-consuming and high-polluting industries and become an important agglomeration of PIIs ,5. In reDomestic and foreign research on industrial development and its environmental pollution mainly focuses on the pollutant emission pattern of industry or on the manufacturing industry and its environmental effects ,9,10, thRelevant scholars focused on analyzing the influence mechanism of PIIs. People have proposed a series of theories to explain the motives and patterns of the industrial layout. The factor endowment theory and law of comparative advantage emphasize that labor, natural resource endowments, capital and technology have important impacts on industrial location, which determines the direction of national and regional specialization ,21. The 3-N) and the scale-intensity method, we can quantitatively identify WPIIs. (2) Taking the YRB as an example, the spatial differentiation characteristics of WPIIs in the YRB from 2003 to 2013 were analyzed and revealed by fully exploring and correlating the industrial characteristics and basin features from a basin perspective. (3) We revealed how different factors can have positive or negative effects on WPIIs in different regions of the basin. Our results can provide a reference for quantitatively identifying WPIIs and exploring the heterogeneity of their influencing factors.The above studies provide us with a good theoretical basis and empirical examples, but there are still several shortcomings: At present, studies on WPIIs are still rare, and the industrial point source pollution, which is difficult to depict in detail, cannot provide strong support for water pollution prevention and control in areas with prominent water problems. In addition, the watershed is an organic life system formed by a series of rivers, the upstream is connected to the downstream, the left bank influences the right bank, and the tributaries act as the main stream. But the existing studies seldom consider WPIIs into the watershed system, which makes it difficult to reveal the linkage between the development and protection of the industry watershed connected by the river system. Therefore, this study mainly discusses the following: (1) by constructing water pollution-intensive index through industrial wastewater discharged (IWW), chemical oxygen demand discharged (COD) and ammonia nitrogen discharged , and the sample scope was all state-owned and non-state-owned enterprises above designated size. First of all, according to the comprehensive measurement results of the WPPI, the industries with a comprehensive index greater than 0.2, according to the Industrial Classification for National Economic Activities (GB/T 4754-2011), the large industry category codes and names of 22 (manufacture of paper and paper products), 26 , 17 (manufacture of textile), 13 , 28 , 15 , 06 , 25 , 14 (manufacture of foods) were selected as the research category of WPIIs in this paper. Note that, except for 06  belonging to the “category B” mining industry, the rest are all “category C” manufacturing industries. Secondly, the above data were pre-processed through various methods, such as filtering, deduplication, repair, and elimination. Finally, geocoding was performed based on the detailed address information of WPIIs, the latitude and longitude information of WPIIs from 2003 to 2013 was obtained and spatially processed, and the WGS_84 geographic coordinate system was converted to the WGS_84_Albers projection coordinate system. The indicator data of the influencing factors came from the China City Statistical Yearbook, while some missing data came from the statistical yearbooks of various provinces and cities, and the statistical bulletin of national economic and social development.3-N) were selected to participate in the calculation. The above data were obtained from China Environment Statistical Yearbook (2014). The formula used were as follows:I is the water pollution-intensive index; iE is the scale of the i pollutant discharge of each industry, i.e., “the i pollutant discharge of a certain industry/the i pollutant discharge of all industries”; iP is the i pollutant discharge intensity of each industry, i.e., “the i pollutant discharge of a certain industry/the industrial sales value of that industry”; iE and iP are normalized. Through the geometric averaging treatment of the scale and intensity of the three pollutants discharge, and then averaging, the water pollution intensive index of each industry can be obtained. The results are shown in PIIs are industries that produce large amounts of pollutants directly or indirectly during the production process without adequate treatment. The current academic research on the definition of PIIs mainly includes three methods: the pollutant emission scale, pollutant emission intensity, and pollution control cost methods ,32. WPIIThe standard deviation ellipse is a common method for analyzing the spatial distribution characteristics of point-like geographical elements, which can accurately reveal the spatial distribution patterns of WPIIs in terms of orientation characteristics, dispersion degree and distribution range. Among the basic parameters, the center point represents the central position of industrial layout, the azimuth angle represents the main trend direction of industrial distribution, and the long and short semi-axes represent the direction of industrial distribution and its spatial distribution range, respectively .n is the number of enterprise points in the neighborhood, h is the bandwidth, k  is the kernel function, and (x − ix) is the distance from the estimated value x to the enterprise point ix. After several trials, the final output cell was 2.5 km × 2.5 km, and the search radius was 15 km.Kernel density estimation is a non-parametric estimation method. It is used to calculate the spatial smoothing estimation value of the feature point and its density in the neighborhood. Through the visual form to characterize the spatial distribution pattern of geographic elements in the study area, it can effectively measure the spatial concentration of WPIIs. The estimated value of the kernel density reflects the density of enterprises, and the larger the value, the denser the distribution of enterprises. The kernel density distribution function was as follows :(4)f(x)=iy is the dependent variable at point i; ikx is the value of the kth independent variable at the i-th point; i-th sample; kth variable at point i; and The traditional regression model is mainly based on the least square method to estimate the parameters average or global. The geographically weighted regression model (GWR) extends the traditional linear regression model. Its regression coefficient is no longer a global unified single value, but the spatial location is included in the regression parameters, which can reflect the influence of factors on the distribution of WPIIs that vary with the spatial location, and it is used to explore the spatial non-stationarity and spatial difference of regression coefficients . The essThe scale of WPIIs in the YRB showed a growth trend from 2003 to 2013, with the output value increasing from 442.5 billion yuan in 2003 to 6192.4 billion yuan in 2013, an increase of 13 times . Within The statistics of the water pollution-intensive index, output value and number of WPIIs in 2013 are shown in All WPIIs in the YRB show a significant east-west spatial distribution pattern from 2003 to 2013 . The disWithin the basin, the distribution directions of WPIIs in the upper, middle and lower reaches of the basin from 2003 to 2013 were similar, with an overall northeast-southwest direction (in the direction of the main tributaries of the Yellow River) and a symmetrical distribution along the river. In the upstream, the central axis of the industry was in the line of Lanzhou-Yinchuan-Hohhot, and the distribution center shifted from the junction of Yinchuan and Ordos to the northeast to the territory of Ordos. The coverage area moved to the northeast and the WPIIs were clustered along the northeast-southwest direction. In the midstream, the industrial axis was in the line of Taiyuan-Linfen-Xi’an, along the Fen River, and the distribution center was always located in Linfen. The WPIIs were clustered in the northeast-southwest direction. In the downstream, the WPIIs were symmetrically distributed in the northeast-southwest direction and along the main stream of the Yellow River, with the center of distribution located at the junction of Liaocheng and Jinan.To further analyze the spatial relationship between WPIIs and rivers in the YRB, the number of enterprises within 10 km of the main stream of the Yellow River and its main tributaries were counted for WPIIs in 2013. The results are shown in The kernel density analysis of 9 WPIIs in 2013 was carried out. The spatial distribution of all WPIIs in the YRB had significant watershed differentiation characteristics, with the intensity of downstream > midstream > upstream a. In theThere were industry differences in the spatial clustering of WPIIs b–j. Manu3-N and COD, forming a number of high-density cores mainly in Dezhou and Liaocheng, with Lvliang and Xi’an as the cores; the enterprises formed two tandem clusters along Fen River and Wei River, and also formed a tandem cluster along the main stream of Yellow River from Yinchuan to Bayannur. Manufacture of foods formed a high-density core in Dezhou and Zhengzhou, with a small concentration near Xi’an. Manufacture of wine, drinks and refined tea formed a high-density core in Zhengzhou, Jiaozuo and Jiyuan, in addition to a small cluster near Xi’an.Processing of food from agricultural products, manufacture of foods, manufacture of wine, drinks and refined tea mainly rely on the convenience of raw materials, and were distributed in the upstream, midstream and downstream, but the most dense distribution occurred in the downstream areas. Processing of food from agricultural products was the main contributor of NHMining and washing of coal, processing of petroleum, coking, processing of nuclear fuel, as resource-intensive industries, are highly dependent on energy, so were mainly distributed in energy-rich regions. Shanxi, Shaanxi, and Inner Mongolia are the main supply bases of China’s energy resources, and mining and washing of coal was highly concentrated here. A high-density core was formed at the junction of Lvliang and Jinzhong, a cluster belt formed along the core to the periphery, and a small cluster belt is formed between Yulin and Ordos. Processing of petroleum, coking, processing of nuclear fuel were concentrated in the midstream and downstream, forming a high-density core in Dongying, an important oil base, and a small high-density core in Lvliang and Yulin, and it is connected to the periphery to form a dense belt.Drawing on relevant references, the spatial distribution characteristics of WPIIs, and taking into account the availability of data, this paper analyzes the factors influencing WPIIs from four aspects: resource endowment, socio-economic, pollution discharge, externality and transportation:Resource endowment affects WPIIs mainly through natural resources, labor, and technology level ,37. AmonSocio-economic factors are mainly characterized by the level of economic development and industrial structure. A better foundation of economic development, a more complete infrastructure and a large market capacity have an important impact on the industrial layout, represented by per capita GDP . The regPollution emission factors are characterized by the scale of industrial wastewater discharge and the distance from the WPIIs to the river. The scale of industrial wastewater discharge and the proximity of rivers have a direct impact on the discharge and treatment of wastewater, which are characterized by the industrial wastewater discharge and the nearest distance from the WPIIs to the river, respectively .Externality and transportation factors are characterized by foreign investment and transportation. Foreign investment can provide various resource required by enterprises, thus affecting the spatial distribution pattern of WPIIs, which is represented by utilization of foreign capital . The impSince Ordinary Least Squares (OLS) is highly diagnostic in terms of factor covariance, etc., it is first analyzed by OLS, and the results are shown in The influencing factors and the degree of role of WPIIs were usually different in different regions .Resource endowment. The impact of natural resource endowments on the distribution of WPIIs gradually decreased from west to east, negatively a. The poSocio-economic factors. The effect of economic development level on WPIIs was significantly positive, and the intensity of the effect decreased from east to west c. The stPollution discharge factors. The impact of industrial wastewater discharge scale on WPIIs was significantly positive, and the degree of impact decreased from east to west, indicating that the scale of pollution discharge is an important factor affecting the distribution of WPIIs in the middle and downstream e. Due to3-N) to quantitatively identify WPIIs. Comparing the first national pollution source census Bulletin (2010) [This paper focuses on industrial point source pollution that has a serious impact on the water environment and water pollution events. The water pollution-intensive index was constructed by combining the scale-intensity method with industrial wastewater discharged (IWW), chemical oxygen demand discharged (COD), and ammonia nitrogen discharged (NHn (2010) , the resWPIIs such as petrochemical industry, textile industry and agro-food processing industry in the YRB are mostly traditional industries which are facing the problems of low resource utilization, high consumption and high pollution. The concentration of WPIIs along the river has caused huge pressure on the water environment, resulting in some sections of the YRB approaching the upper limit of environmental capacity. The layout of WPIIs in the YRB is influenced by factors such as economic development, industrial structure and pollution discharge. These results verified the analysis of Zhou et al. . Zhou etTherefore, it is of great value and significance to fully understand the location, scope and degree of agglomeration of WPIIs and their sub-industry in terms of spatial distribution and their influence mechanisms, to promote the transformation and upgrading of traditional industries, to promote the transfer of WPIIs, and to connect the ecological protection and high-quality development of the YRB, especially the protection of water resources, water environment and water ecology.The scale of WPIIs in the YRB showed a growth trend from 2003 to 2013, with the output value increased from 442.5 billion yuan in 2003 to 6192.3 billion yuan in 2013, an increase of 13 times. Within the basin, the spatial distribution of industries is extremely uneven, showing a significant geographical differentiation of downstream > midstream > upstream. The WPIIs in the YRB showed east-west distribution characteristics, and the distribution center was basically located at the junction of the middle and lower reaches. The distribution of WPIIs in the upper, middle and lower reaches of the river basin was similar all along the northeast-southwest direction  and basically showed symmetrical distribution along the river. The spatial distribution of WPIIs was riverine and densely distributed in the downstream areas and along the important tributaries of the Fen and Wei rivers.The WPIIs were clustered in high density in the downstream areas, and formed a cluster area centered on Zibo and Dezhou, as well as dense belts along the Fen River and Wei River. There were industrial differences in the spatial clustering of WPIIs. The chemical and textile industries were mainly concentrated in downstream areas. Processing of food from agricultural products was distributed in the upper, middle and lower reaches. Resource-intensive industries such as coal and oil were concentrated in the energy-rich midstream areas.The spatial differences in the effects of each factor on the distribution of WPIIs were significant. Natural resource endowment was the main factor promoting the concentration of WPIIs in the middle and upstream of the basin, while the downstream had overcome resource constraints. The level of technological innovation had a significant impact on the eastern and downstream areas of the basin. The level of economic development and the historical foundation of industry promoted the geographical concentration of industry and formed a certain path dependence. The scale of wastewater discharge and the proximity of rivers also had a strong role in promoting the clustering of industries in the middle and downstream areas.We offer some policy recommendations in this paper. (1) The local governments need to control the total amount of pollutants discharged according to the water environment capacity of the region. This can be implemented similarly to carbon emission trading, combined with the regional water environment capacity, implementation of the emission trading scheme, and reasonably laying out the transferred industries. (2) The implementation of environmental regulations should be adapted to the regional development stage and factor endowment. Environmental controls at river junctions should be appropriately strengthened to avoid transboundary pollution. (3) Industrial parks are an important means of industrial layout and development. Increasing the admission rate of WPIIs to the parks helps to centralize the layout of pollutant treatment facilities. At the same time, it promotes technological innovation and the transformation and upgrade of low-end industries, and encourages the development of high value-added and technology-intensive industries.


The Intergovernmental Agency on Development defines the Karamoja Cluster Fig.  as a croThe Karamoja region in northeast Uganda has long been characterized as under-developed and insecure, with repeated episodes of violent livestock raiding within Karamoja itself, and affecting neighbouring areas in Kenya, South Sudan and Uganda. Since the late 1970s, ‘modern’ forms of raiding evolved using automatic weapons, causing considerable loss of human life  and the wider Cluster, the KRSU organized Karamoja’s first international research conference Pathways to Resilience in the Karamoja Cluster, in Moroto in May 2019. The call for conference papers was broad in scope and allowed for submissions related to any sector but with a clear and direct link to livelihoods and resilience. Papers could be original research, reviews or project evaluations, especially evaluations showing livelihoods’ impacts. The conference called for papers from the ‘Karamoja Cluster’ and within this regional framing was the notion of towns such as Moroto in Karamoja (Uganda) and Lodwar in Turkana (Kenya) being regional hubs and connected, cross-border centres of economic activity and trade. This framing drew heavily on the international conference on “The Future of Pastoralism in Africa” in Addis Ababa in 2011 ; peace and conflict (four papers); mobility, land and water (four papers); resilience, risk and change (three papers); diversified livelihoods (three papers); human nutrition (five papers); education and health (three papers); emergency and extractives (two papers); and programming experiences (three papers). The papers were presented over 3 days and were preceded by three keynote presentations.This Special Issue of Pastoralism presents selected papers from the conference, preceded by a commentary that was drawn from the opening keynote presentation . As expected, livestock and issues related to the role of livestock in livelihoods and livestock management were featured heavily in the conference. In a second keynote presentation, Frank Muhereza provided an overview of recent livestock and pastoralism policies in Ethiopia, Kenya, South Sudan and Uganda and noted the mixed policy narratives around pastoralism but a constant thread of commercialization ambitions by national governments in contexts of continuing conflict and contested access to land Muhereza . Where lThe third keynote presentation focused on gender and livelihoods in Karamoja and how changes in livestock ownership led to corresponding changes in the roles of women and in particular, an increasing domestic burden in terms of household incomes and diets Stites . The genTwo papers in the Special Issue describe the changing livelihoods of pastoralists and their engagement with the state and development actors. In Ethiopia’s lower Omo valley, Fana Gebresenbet (this issue) associates a decline in the food security and livelihoods of Bodi agro-pastoralists to a large-scale government irrigated sugar plantation, developed near the Gibe III dam from the early 2010s. In this case, increasing market engagement by the Bodi was mainly in the form of distress sales, which contrasted with the local government views of this engagement as a positive development. In Turkana County in northwest Kenya, Gregory Akall (this issue) focuses on the development impacts and issues around small-scale irrigation from the mid-1960s. He describes an increasing array of pressures on secure access to land by pastoralists and, as livelihoods declined, shifts towards negative forms of diversification, especially for women and girls.Against the myriad of aid organizations, and development and humanitarian programmes in the region—especially in Karamoja and Turkana—Cory Rodgers (this issue) examines the quality of the interaction between outsiders and pastoralists, and the specific issue of customary land tenure in a refugee resettlement scheme in Turkana. He argues that despite community participation being widely accepted in a rights-based approach to development, meaningful participation was limited and the aid organization in question relied on dialogue with urban professionals and politicians. The key finding that this organization had limited understanding of local context or limited capacity to engage directly with pastoralists resonates loudly across other areas of the Karamoja Cluster.The Pathways to Resilience conference in Karamoja took place in a period of relative peace and stability, and a general sense of optimism that the region could emerge finally from decades of armed violence, and political and economic isolation. At the same time, the conference papers clearly described the challenges ahead for resilient livelihoods in the Karamoja Cluster, especially for households living on the edges of pastoralism or no longer owning livestock. In common with other pastoralist areas of East Africa, the Cluster is characterized by substantial numbers of households who are livestock-poor and cash-poor and who make ends meet through mixes of local social support, negative diversification activities and, in some areas, social protection programmes. Where innovation occurs, it is often among those with sufficient wealth, e.g. the division of herds into a ‘herd for the market’ and a ‘herd for milk and savings’. The pathways to resilience for women and girls are especially uncertain, and education might be seen as a critical element for providing access to better paid and safer employment, beyond activities such as charcoal production, brewing or causal labour. Yet notably, we received no papers that described educational approaches or models in the Cluster that demonstrated improved education for girls or boys, that considered the need to adapt conventional schooling to the pastoralist context, or which assessed the affordability of education for poorer households. In 2016, in Karamoja (Uganda) alone, there were 20 aid organizations implementing or supporting 29 education projects (Karamoja Resilience Support Unit In addition to more research on delivering effective and affordable education, other research opportunities included growing trade and social ties across borders, such as between Karamoja and Turkana, and the flows of goods, services and people between towns such as Moroto and Lodwar. Whereas issues of land governance and tenure have been well-documented and openly discussed for Turkana, the issue of land access in Karamoja remains under-researched and few, if any, aid programmes work directly on the policy and institutional arrangements that are needed to secure access to productive rangeland. There are many concerns in the Karamoja case, with for example, nearly 25% of land in Karamoja being subject to government-issued exclusive mineral exploration and location licences, that were reportedly shrouded in anonymity (Rugadya et al. Since the 2019 conference and during the preparation of this Special Issue, the pastoralist areas covered by the conference were affected by COVID-19 restrictions, and in the Karamoja case, people faced an unprecedented mix of market closures, movement restrictions, price rises and hindrances to livestock and crop production, with serious impacts on food security and livelihoods (Arasio et al.