Montanera  are subjected could be overcome by pre-cure freezing. Three sets of Montanera Iberian presas  (n = 15) were established to assess the impact of frozen storage -0, or non-frozen, 3 and 6 months—previous to the technological process of curing—on the quality traits of the dry-cured product Montanera Iberian dry-cured lomito. Similar seasoning and curing processing conditions were applied to all sets. Lower productive performance due to higher weight loss during curing, and lower colour intensity were observed in pre-frozen dry-cured lomitos. The fatty acid profile was more saturated, and the oxidative status increased as a result of pre-cure freezing. On the matter of texture, all parameters were modified, highlighting the higher values of hardness and shear force of pre-frozen dry-cured lomitos. The time that raw material was frozen exerted a slight, thus helping manufacturers to better address the gap between industry and consumer demand with minimal effect on quality traits.The seasonality to which dry-cured products from Iberian breed pigs finished in  Montanera system (free-range rearing in which feed is based exclusively on ad libitum consumption of acorns mainly from Quercus ilex and grass) attaining the highest organoleptic [Montanera Iberian dry-cured products are subjected to seasonality due to the seasonal production of the natural resources of dehesa (rangelands with evergreen oaks and pastures found in the southwest of the Iberian Peninsula used for finishing Montanera Iberian pigs); acorns and grass (November to March). Thus, given that the highest consumption of Iberian dry-cured products occurs from November to December (owing to the Christmas season) there is a time gap between the commercial outlet of Montanera Iberian products and their consumers’ demand.Dry-cured products from Iberian breed pigs are valuable products, with those from pigs finished in noleptic  and nutrnoleptic . HoweverMontanera Iberian dry-cured products, and may help to overcome its implicit seasonality. Despite the technological and economic advantages it may have for the Iberian industry, the high commercial value and quality attained by Montanera Iberian dry-cured products [The freezing of the primal cuts prior to the technological process of curing would balance the production and demand of products  make it Montanera Iberian ones with a short technological process (60–70 days) which means that they would launch to the market in months with low commercial demand. In this line, only Ortiz, Tejerina, Contador, de Andrés, Petrón, Cáceres-Nevado, and García-Torres [Montanera Iberian dry-cured loins as affected by the pre-cure freezing practice. There are some specific designations of origin which do not contemplate such practice for products marketed under this designation. On the other hand, the European regulations for the freezing of animal products and the Spanish Iberian Quality Standard (IQS) do not mention this practice, which has led to a situation of uncertainty and lack of regulation and control of pre-cure freezing at industrial level. There is little scientific literature assessing the impact that pre-cure freezing practice has on quality characteristics of Iberian dry-cured products, which has been carried out on Iberian products from animals reared indoors and fed on commercial fodder (intensive system) ,4,5,6 soocess 60–0 days whpresa  is one of the most consumed primal cuts from Iberian pigs [presa can undergo a curing process, of a similar length to that followed by the loin, giving rise to Iberian dry-cured lomito, the commercial name given to the Iberian dry-cured presa, highly appreciated by consumers owing to its sensory characteristics. However, to the best of our knowledge, there is no literature dealing with Iberian dry-cured lomito.The Iberian ian pigs . In addiMontanera Iberian dry-cured lomito could contribute to the generation of knowledge of the factors that can condition the homogeneity of the Iberian dry-cured products and the standardisation of quality in general. Furthermore, it would specifically assess its effect on a product with short technological process, but with different physico-chemical characteristics to Iberian dry-cured loin [Montanera Iberian dry-cured lomito could be affected.Thus, the study of the pre-cure freezing practice in red loin  in an atMontanera Iberian presa)) on the quality traits of Montanera Iberian dry-cured lomito.Within this framework, the aim of the current research study was to assess the impact of pre-cure freezing practise  were used in the present study. Muscles proceed from Iberian Retinto breed animals crossed with Duroc pigs (50:50) finished in Montanera system. A total of 45 muscles Montanera, i.e., growing phase, animals were reared in open-air corrals and subjected to a feeding based on commercial feedstuffs. Thus, the average daily gain (ADG) during growing phase was 256 ± 28.3  g/day, whilst the average daily feed intake (ADFI) was 1.29 ± 0.08 kg/d. After that, animals started the finishing phase in Montanera system  [Montanera phase were as follows.Previous to finishing phase in Montanera was carried out in the dehesa of Valdesequera farm, . The length of Montanera was 67 days, comprised between November 2018 and January 2019. During this phase, animals were fed ad libitum on fallen acorns from Quercus ilex and grass. The animals had free access to water. The stock rate was 0.60 pigs per hectare, which was established with margins in order to guarantee that acorns would not run out before the fattening was completed [Montanera phase, the animals were weighted in order to calculate their ADG; 673.1 ± 34.9 g/d, attaining an average live weight of 148.4 ± 6.15 kg after finishing Montanera, with 14 months old. Subsequently, animals were transported to a local slaughterhouse , Spain). During transport animals were not supplied with feed or water [ompleted . At the or water . During or water . The conor water , the Royn = 45). Afterwards, intermuscular fat and connective tissue were removed, and counterparts were individually packaged without vacuum in nylon/polyethylene bags , and cooled for 24 h (4 °C). Later, Iberian presa were allocated into three sets before curing: non-frozen (T0) (718.34 ± 88.16 g) (n = 15) and pre-frozen dry-cured lomitos for 3 (T3) (721.9 ± 69.95 g) (n = 15) (ii) and (iii) 6 (T6) (712.6 ± 116.37 g) months (n = 15).After carcass quartering, which was carried out 4 h after slaughtering , SV muscpresa belonging to non-frozen (T0) set were immediately cured. Iberian presa from T3 and T6 sets were subjected to freezing and freezing storage during 3 and 6 months, respectively. Freezing was performed in a freezing chamber by means of cold air (−20 ± 2 °C) flowing at 20 km/h. Thus, the rate of freezing ranged from 1 to 5 cm/h. Frozen storage times and the freezing method was selected to be in line with the usual practices in the Iberian manufacturing industry. The Iberian presa in a cold chamber at refrigerated temperature (4 ± 1 °C). Thereafter, the Iberian presa were subjected to curing process. The average weight of the Iberian presa after freezing storage for 3 or 6 months and subsequent thawing was 692.3 ± 67.81 g and 672.8 ± 113.81 g, respectively.Thawing was conducted by placing the Iberian presa were seasoned with a mixture of salt (2.5%), paprika (0.6%) and 0.9% additives; E-250, E-252, and E-331. Subsequently, Iberian presas were stored at 4 °C for 48 h in darkness to allow the seasoning mixture to spread into the meat. Afterwards, Iberian presa were stuffed into 10 cm-diameter collagen casings and subjected to the technological process of curing. For the next 30 days they were kept at refrigerated temperature (4 °C) and high relative humidity (RH) (above 75%). During the rest of the time , the temperature increased from 10 °C to 16 °C while the RH decreased to 60–65%. The average dry-cured lomitos’ weights according to pre-cure freezing sets were 439.1 ± 60.09, 395. 9 ± 47.02, and 373.4 ± 66.32 g for non-frozen (T0), pre-frozen dry-cured lomitos T3, and T6, respectively.Seasoning, processing, and curing length were similar in the three loin sets and conducted according to regular practices of the Iberian manufacturing industry. Thus, all Iberian The dry matter (DM) was conducted in accordance with standard method of the Association of Official Analytical Chemists .v/v) [Intramuscular fat (IMF) was extracted and quantified gravimetrically by means of chloroform/methanol  . The chloride content (NaCl) was analysed by means of Volhard method .0 is the weigh at the beginning and Wf the weigh after curing, respectively. The weight loss during curing was calculated as follows:L*), redness (a*) and yellowness (b*) coordinates were calculated using a Minolta CR-400 colorimeter  with illuminant D65, a 0° standard observer and a 2.5 cm port/viewing area. The saturation index or chroma (C*), defined as (C = (a2 + b2)0.5), and hue angle (H°) as arctangent (b*/a*) were calculated. The measurements were taken at five randomly selected sites on the steak of each sample (Iberian dry-cured lomito) and averaged.Lightness (2O 55:45 [α- and γ-tocopherol were extracted by means of a solution of KOH 11.5% in EtOH/H2O 55:45 . SubsequSeparation of main fatty acid methyl esters (FAMEs) was carried out from IMF previous extracted  by meansThe oxidation of lipids was carried out by the 2-thiobarbituric acid (TBA) method .For the purpose of protein oxidation, the carbonyl groups formed during incubation with 2,4-dinitrophenylhydrazine (DNPH) in 2N HCl was measured . Two texture analyses were performed; Texture Profile Analysis (TPA) and Warner-Bratzler test (WBSF) by means of a texturometer TA XT-2i Texture Analyser .3 cubes of dry-cured lomito, which were axially compressed to 50% of it an original height with a 20 mm diameter (P/20) flat plunger using a 25-kN load cell applied at a crosshead speed of 2 mm/s through a 2-cycle sequence. Thus, hardness (N/cm2), springiness (cm), cohesiveness (dimensionless), gumminess (N cm s2), chewiness (N cm s2), and resilience (dimensionless) were determined from force–deformation curves [TPA was determined at 50% deformation ,23 on unn curves .2) was measured to cut samples.For the WBSF test, samples—10 × 30 × 10 mm slices (width × length × thickness)—were cut with a Warner–Bratzler blade (HDP/BS) in a direction perpendicular to the muscle fibres. The maximum shear force  and ei(j) is the residual error.The data were analysed using the statistical SPSS package SPSS.PC+ v. 20.0. The model included the pre-cure freezing time  as factor for the one-way ANOVA analysis of variance of variables related to the proximate composition, weight losses, instrumental colour, antioxidant and fatty acid profile, oxidative status and instrumental texture of the p ≤ 0.05.The data were presented as mean ± standard deviation (SD). Tukey’s HSD test was applied to compare the mean values of each group. Statistical significance was set at ® Trondheim, Norway) to check the overall effect of the pre-cure freezing practice on Montanera Iberian dry-cured lomito samples distribution and to explore the multivariate relationships among variables.A principal component analysis (PCA) was performed by means of Unscrambler X software (CAMOp ≤ 0.05) due to pre-cure freezing. Thus, DM was higher in Montanera Iberian pre-frozen dry-cured lomitos compared to those manufactured from non-frozen raw material. No effect was observed owing to freezing storage time of the Montanera Iberian presas (p > 0.05).With regard to proximate composition , differeIn refer to IMF content, values were comprised between 26.54 ± 2.23 and 27.83 ± 1.80 g/100 g of moisture-free dry-cured meat. The salt content value ranged from 2.74 ± 0.23 to 2.94 ± 0.50. Pre-cure freezing did not exert any significant differences in either of the two parameters.Montanera Iberian pre-frozen dry-cured lomitos—irrespective of the previous freezing time of the raw material (T3 and T6)—with respect to Montanera Iberian dry-cured (T0) ones (p ≤ 0.05). The higher WL due to water losses during the technological process of curing was observed for a*) and yellowness (b*) decreased due to the freezing of Montanera Iberian presas before curing irrespective of the freezing time (p ≤ 0.05). In contrast, no differences in lightness were observed due to pre-cure freezing (p > 0.05). Chroma and Hue differences might be explained by the modifications of the abovementioned coordinates, especially redness and yellowness. Specifically, Chroma was higher in dry-cured lomitos manufactured from non-frozen (T0) Iberian presas, whilst the pattern followed by Hue was not clear, with the highest value yielded by Montanera Iberian pre-frozen dry-cured lomitos (T3) (p ≤ 0.001).Referring to the instrumental colour, redness (p > 0.05), with values ranging from 18.84 ± 0.84 to 20.14 ± 1.84 and from 2.48 ± 0.29 to 2.66 ± 0.30 for α- and γ-tocopherol, respectively.Results for the antioxidant composition as affected by pre-cure freezing practice are shown in p ≤ 0.001). More specifically, Montanera Iberian pre-frozen dry-cured lomitos yielded lower values of both C18:2 n-6 and C18:3 n-3. Additionally, for the former, the time that raw material—Iberian presas—was frozen and stored influenced the ultimate value of this fatty acid (p ≤ 0.001). On the other hand, Montanera Iberian pre-frozen dry-cured lomitos revealed higher values of C16:0, with T6 ones reaching the highest value (p ≤ 0.001). The n-6/n-3 ratio was comprised between 8.80 ± 1.27 and 13.36 ± 2.37, revealing significant differences on account on the pre-cure freezing practice (p ≤ 0.001). Thus, an increase in the ratio was observed in Montanera Iberian pre-frozen dry-cured lomitos, irrespective of the time raw material was frozen before the technological curing process.The oleic acid (C18:1 n-9) was the most abundant fatty acid, yielding values from 51.57 ± 2.09 to 52.41 ± 1.66 followed by the palmitic acid (C16:0) 24.01 ± 1.00 to 25.51 ± 1.14 . The preMontanera Iberian dry-cured lomitos manufactured from Iberian presas frozen/thawed yielded higher values of lipid and protein oxidation (p ≤ 0.001). In the matter of oxidative status , MontaneMontanera Iberian dry-cured lomitos.p ≤ 0.001). Thus, Montanera Iberian pre-frozen dry-cured lomitos yielded higher values for hardness and chewiness and lower values for springiness, cohesiveness, and resilience. Nevertheless, the time that raw material—Montanera Iberian presas—were stored frozen (3 or 6 months) did not derive in any differences for the above-mentioned parameters.The pre-cure freezing practice provided differences on all textural parameters evaluated in the TPA test (p ≤ 0.001), regardless of freezing storage time of the Montanera Iberian presas.With respect to WBSF test, the pre-cure freezing practice resulted in higher values of shear force  1, as it was responsible for more than 50% of the variance. Thus, samples from pre-frozen dry-cured lomitos placed in the left of the plot, mainly in the upper quadrant, whilst dry-cured lomitos samples from frozen/thawed Iberian presas tended to have positive scores in the PC 1 as well as in PC 2 axis, the latter explaining the 13% of the variance.The Montanera Iberian pre-frozen dry-cured lomitos with respect to dry-cured ones (T0) may be explained by their higher WL throughout the technological process of curing. These results were not unexpected, since proof has been shown of the higher amount of water released during curing process in Montanera Iberian pre-frozen dry-cured loins [Montanera Iberian presas) [presas) to avoid the decrease in production yield.The higher DM content observed in ed loins  and thesed loins . The WL  presas) . The difMontanera Iberian dry-cured lomito were slight higher than those observed for Montanera Iberian dry-cured loins, which could be associated to the higher IMF content of the former [lomito neither on the evaluation of the influence that factors related to production or technological processing have on their quality characteristics to compare our results.The results obtained for DM, in the current research for e former ,27. To oMontanera Iberian pre-frozen dry-cured lomitos may be attributed to the oxidation of red pigments. Thus, Iberian presas might have yielded a lower myoglobin due to freezing storage [lomitos would expect to have lower a* than those elaborated from non-frozen raw material. Variations in redness coordinate is an aspect to consider because of the importance consumers assign to it in the case of cured products [Montanera Iberian pre-frozen dry-cured lomitos, the lack of correlation in the current study with the lipid oxidation [Montanera Iberian pre-frozen dry-cured loin. In refer to redness as affected by pre-cure freezing practice, the lower values observed for  storage  so the rproducts , so may xidation , which ixidation  and thatxidation  for MontMontanera dry-cured lomitos were higher than those reported for Montanera Iberian dry-cured loins, whilst yellowness was slight lower [In general terms, values of redness observed for ht lower .Montanera Iberian dry-cured loins. In this line, Martín-Mateos [presas. Therefore, its stability after the curing process was expected.The lack of differences provided for α-tocopherol due to pre-cure freezing practice suggest the great stability of these antioxidants after pre-cure freezing, which is consistent with findings obtained by Ortiz, Tejerina, Contador, de Andrés, Petrón, Cáceres-Nevado, and García-Torres  in Montan-Mateos  previousMontanera Iberian dry-cured lomitos were higher than those identified in Montanera Iberian dry-cured loins [longissimus thoracis et lumborum and Serratus ventralis muscles, respectively [Serratus ventralis could be due to its oxidative nature as opposed to the glycolytic one of the longissimus thoracis et lumborum [On the other hand, in general terms tocopherols content values observed for ed loins . This miectively . More inlumborum .Montanera Iberian lomitos agreed in general terms with those previously reported for Montanera Iberian dry-cured loin, whilst C16:1 was lower and C18:2 n-6 and C18:3 n-3 higher [The values observed for C16:0, C18:0 and C18:1 n-9 fatty acids in 3 higher ,27. Montanera Iberian pre-frozen dry-cured lomitos, lower PUFA values and, in consequence, higher percentage of SFA would be expected. These results agree with those found by Ortiz, Tejerina, Contador, de Andrés, Petrón, Cáceres-Nevado, and García-Torres [Montanera Iberian dry-cured loin, although in the latter study the increase in SFA after pre-cure freezing did not reach a significant level. The differences observed for the n-6/n-3 ratio resulted from the variations of both C18:2 n-6 and C18:3 n-3, and in any case, the mean values attained were lower than those observed for the case of Montanera Iberian dry-cured loin [The decrease in the unsaturation with pre-cure freezing was not unexpected. Thus, the decrease in PUFA group as well as the individual fatty acids that comprise it might be ascribed to lipolysis and lipid oxidation processes, since the oxidation susceptibility is dependent on the level of unsaturation of fatty acids ,34. Thusa-Torres  for the red loin .Montanera Iberian dry-cured lomitos in comparison with those described for Montanera Iberian dry-cured loins [Serratus ventralis is an oxidative muscle, it may be more susceptible to oxidative processes as a result of factors related to technological processing than Longissimus thoracis et lumborum one [lomito vs. dry-cured loin to the pre-cure freezing practice. Additionally, the higher lipid oxidation values observed in Montanera Iberian pre-frozen dry-cured lomitos might be explained by the probably higher initial values of the raw material (Iberian presas) because of freezing storage [lomitos with respect to dry-cured ones (T0). The higher extent of both lipid and protein oxidation of Montanera Iberian dry-cured lomitos as result of pre-cure freezing are consistent with results reported by Ortiz, Tejerina, Contador, de Andrés, Petrón, Cáceres-Nevado, and García-Torres [Montanera Iberian dry-cured loins. Lorido, Ventanas, Akcan, and Estévez [On the matter of oxidative status, in general terms, higher rates of lipid oxidation were observed for ed loins . This coorum one , from wh storage ,28. Like storage , might ea-Torres  for Mont Estévez  also fouMontanera Iberian pre-frozen dry-cured lomitos. Additionally, the higher protein oxidation observed for Montanera Iberian pre-frozen dry-cured lomitos might have enhanced the formation of cross-links [Montanera [Concerning instrumental texture properties, the higher hardness after pre-cure freezing (T3 and T6) might be related to the higher weight loss observed for ss-links  and protss-links . The patontanera  and indoontanera . Among tontanera . So, in Serratus ventralis and Longissimus thoracis et lumborum [Mean values of hardness were in general lower than those reported for Iberian dry-cured loins . Discreplumborum , the typlumborum .presas after the freezing storage, although this latter author did not study the texture properties after curing process. Ortiz, Tejerina, Contador, de Andrés, Petrón, Cáceres-Nevado, and García-Torres [Montanera Iberian pre-frozen dry-cured loins compared to those manufactured from non-frozen raw material. Discrepancies among studies might derive from the differences in the myofibrillar structure and physic-chemical characteristics between muscles from both dry-cured lomitos and loins are obtained; Serratus ventralis and Longissimus thoracis et lumborum, respectively [The increase in shear force in the WBSF test with pre-cure freezing practice disagreed with previous scientific literature. Thus, Martín-Mateos  reporteda-Torres  found loectively . Montanera dry-cured lomitos observed for WBSF were in general similar to those described in Montanera dry-cured loins [lomito, nor how its textural parameters are affected by technological or productive related factors to compare our results.The mean values of ed loins . To our ed loins ,7,25. HoMontanera Iberian dry-cured lomitos samples manufactured from non-frozen Iberian presas might be due to the negative loadings of redness (a*), polyunsaturated fatty acids such as C18:2 n-6 and C18:3 n-3 and some textural parameters such as springiness, cohesiveness, and resilience, which yielded higher values in these samples in comparison with pre-frozen dry-cured lomitos. In contrast, the positive loadings of lipid, protein oxidation, WL, DM, saturated fatty acids, and some textural parameters such as hardness and shear force could have led to the pre-frozen dry-cured lomitos samples to positive scores along the PC1, confirming the higher values of these parameters after pre-cure freezing. Nevertheless, when the pre-cure freezing time was considered, the separation was not clear. The slight differences exerted by the freezing storage time of raw material (Iberian presas) previous to technological process of curing on dry-cured lomito could explain the inability of the model to graphically discriminate between dry-cured lomito samples belonging to T3 and T6. Indeed, only hue coordinate, C16:0 and C18:2 n-6 were affected by pre-cure freezing time (3 vs. 6 months).The variations in the quality parameters above-mentioned as a result of pre-cure freezing allowed the separation of the samples in the new space defined by the principal component (PC) 1 and 2 according to the pre-cure freezing effect. More in detail, the negative scores of Montanera Iberian dry-cured products, providing insight into the Iberian meat industry in order to overcome the seasonality to which these products are subjected.The findings of the current research study contributed to the generation of knowledge regarding the influence of the latest manufacturing-related factors on the quality of lomitos may solve the gap between the industry and consumer demand, but could impact the productive performance of these, given the higher weight loss during technological process of curing and dry matter of the pre-frozen dry-cured lomitos. Therefore, pre-cure freezing should be considered by manufacturers to adjust the technological process conditions and length to the technological aptitude of the raw counterparts. Additionally, the pre-cure freezing may exert an effect on some quality traits such as instrumental colour, fatty acid profile, oxidative status, and textural properties.Our findings suggest that the use of frozen/thawed counterparts to obtain dry-cured Further studies should study its impact on sensory attributes to explore whether this practice may affect the consumer’s acceptability. The effect of pre-cure freezing practice on shelf life should be addressed.


Reactive oxygen species, including RNS, contribute to the control of multiple immune cell functions within the tumor microenvironment (TME). Tumor-infiltrating myeloid cells (TIMs) represent the archetype of tolerogenic cells that actively contribute to dismantle effective immunity against cancer. TIMs inhibit T cell functions and promote tumor progression by several mechanisms including the amplification of the oxidative/nitrosative stress within the TME. In tumors, TIM expansion and differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced by cancer and immune cells. Nevertheless, the role of GM-CSF in tumors has not yet been fully elucidated. In this study, we show that GM-CSF activity is significantly affected by RNS-triggered post-translational modifications. The nitration of a single tryptophan residue in the sequence of GM-CSF nourishes the expansion of highly immunosuppressive myeloid subsets in tumor-bearing hosts. Importantly, tumors from colorectal cancer patients express higher levels of nitrated tryptophan compared to non-neoplastic tissues. Collectively, our data identify a novel and selective target that can be exploited to remodel the TME and foster protective immunity against cancer. NonetheC) model , 14. ReaC) model  or most C) model .in vivo significantly affects the immune landscape at the tumor site. Importantly, GM-CSF nitration in the Tryptophan residue can be also found in human colorectal cancer specimens, where we observed higher levels of both GM-CSF and nitrated-Tryptophan in comparison to the surrounding non-transformed ones.Notably, the release and overload of inflammatory reactive species are prominent features of cancer. In particular, high levels of nitrated proteins have been detected in several human tumors, including prostate, colon, liver, breast, and ovarian neoplastic tissues . Our groCollectively, our data identify the nitrated-GM-CSF as a novel player of tumor-related immunosuppression, which can be pharmacologically targeted in novel immune-based strategies against cancer.C57BL/6 and OT-I (C57BL/6- Tg (Tcra Tcrb) 1000Mjb/J mice were purchased from Charles River Laboratories. All experiments were performed with mice of 8 weeks maintained in the animal facilities of the Venetian Institute of Molecular Medicine (VIMM). Experiments were performed according to the guidelines approved by the local ethics committee.The encoding sequence for GM-CSF were taken from pubmed and obtained by gene synthesis from GenScript. This sequence codifies the full length of GM-CSF plus the 6XHis tag introduced at the C-terminus after AgeI restriction site. To allow further cloning, two cloning sites were introduced at 5’ and 3’. The plasmid obtained from GeneScript were cloned HINDIII/NHE into pcDNA3.1-hygro (Invitrogen). To generate the mutant version of GM-CSF, a site-specific mutagenesis approach was used with the following oligos:mut W>L for: 5’-ccatcactgtcacccggcctCTgaagcatgtagaggccatc-3’mut W>L rev:5’-gatggcctctacatgcttcAGaggccgggtgacagtgatgg-3’.MCA-203 fibrosarcoma cell line was maintained in DMEM (Lonza) supplemented with 10% heat-inactivated FBS (Gibco), 2 mM L-glutamine (Gibco), 10 mM HEPES (Lonza), 55 uM 2-mercaptoethanol (Gibco), 150 U/ml streptomycin, 200 U/ml penicillin obtained from the American Type Culture Collection (ATCC). Two additional cell lines were generated by the stable transfection with either electroporation or the usage of Lipofectamine 2000 (Invitrogen) of MCA cell line with the plasmid encoding either GM-CSF or its mutant version GM-CSF W30L. Transfected cells were selected and thus culture in the media used for MCA-203 supplemented with 0,3 ug/ml Hygromicin (Invitrogen).6 MCA203-GM cells or MCA203-W30L cells. Tumors were measured every 2 days by a digital caliper. At day 21 post-tumor challenging, mice were euthanized, and their tumors explanted and processed for flow cytometry or immunofluorescence analysisC57BL/6 mice were injected on the right flank with 10Explanted tumors were cut in small pieces with a scissor; pieces were covered with a digestive solution composed of collagenase IV (1 mg/ml) hyaluronidase  and DNase  and incubated at 37°C; every 10 minutes tumors were mechanically processed using a 5 ml pipette. After 1 hour, cells were collected and washed twice in complete medium to remove all digestive solution.in vitro assays at 20 ng/ml.Recombinant mouse GM-CSF was purchased from Miltenyi Biotech (130-095-742). Cytokine nitration was performed by adding 1mM peroxynitrite (Millipore) to the recombinant protein at 37°C for 15 min in a final volume of 100ul PBS (Lonza). After incubation, the samples were extensively dialyzed in PBS for 5 hours using the Slide-A-Lyzer Dialysis cassette kit, 3,500 MWCO (Thermo Fisher Scientific). Finally, cytokines were collected and used for 18 material  using a Ultimate 3000 HPLC system (Dionex – Thermo Fisher Sicentific) and separated using a linear gradient of acetonitrile/0.1% formic acid from 3% to 40% in 15 min at a flow rate of 250 nl/min. Eluted peptides were analyzed using a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled online with the LC system through a nano-spray ion source (Thermo Fisher Scientific). The MS system operated in a data dependent mode, by acquiring a full MS scan at high resolution in the Orbitrap analyzer, followed by the MS/MS scans in the linear Ion Trap of the 10 most intense ions. Data were searched using Mascot Search Engine (Matrix Science) against the SwissProt database . Precursor and fragment ion tolerance were set to 10 ppm, and 0.6 Da respectively. An error tolerant search was done to highlight possible modifications and nitration of Tryptophan 30 (W30) was identified as one of the main post-translational modifications, which was present at high concentration only in the sample treated with peroxynitrite. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  either untreated or treated with peroxynitrite or with degraded RNS were digested using sequencing-grade modified trypsin (Promega) with a protein/enzyme ratio of 50:1, as described in . Peptide6 cells were plated into 6-well plate in medium supplemented with 20 ng/ml of GM-CSF (Miltenyi) native or nitrated by peroxynitrite (Millipore) in combination with 20 ng/ml of IL-6 and cultured for 5 days at 37°C in 5%CO2.C57BL/6 mice were sacrificed, tibias and femurs were removed to flush the bone-marrow under sterile conditions. Red cells were lysed with ACK Lysing Buffer (Lonza). To obtain MDSCs 2x10in vitro bone marrow differentiation or cells isolated from tumor-bearing mice, were blocked with FcR binding sites and stained with the following antibodies: anti-CD11b (clone M1/70), anti-Ly6G (clone 1A8), anti-F480 (clone CI:A3-1), anti-Ly6C (clone HK1.4), anti-pStat3, anti-p-Stat5, anti-p-Erk, anti-CD45.1 (cloneA20). Aqua Live/Dead®dye (Invitrogen) was used to analyze cell viability. Intracellular staining was performed using BD Cytofix/Cytoperm Kit in association with BD Phosflow Perm Buffer III. Flow data were acquired with FACSAriaII and analysed with FlowJo software .Cells derived from Mouse tumors were collected and fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose and frozen in OCT. The samples were cut with cryostat (7μm), incubated with blocking solution  2 hours at room temperature. The following primary antibodies were used: anti-nitrotyrosine (Millipore), anti-nitrotryptophan (JaICA), anti-F480 (Abcam), anti-CD31 (R&D Systems). The appropriate secondary antibodies were used. For quantitative analysis, 10 different and non-contiguous regions of interest (ROIs) were randomly selected, and percentage of positive areas was obtained for each marker. Images were acquired with a Leica TCS SP5 confocal microscope. Images and colocalization indexes were analyzed by ImageJ Software.Human colorectal specimens (n=6) were obtained from the Padua Hospital in accordance with the ethical standards. This study was examined and approved by the Ethics Committee of the local Health and Social Services  in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Specimens were frozen in OCT, cut with cryostat (7µm) and analysed with doublestain IHC Kit (Abcam) according to manufacturer’s instructions. For quantitative analysis 10 different and non-contiguous regions of interest (ROIs) were randomly selected, and percentage of positive areas was obtained for each marker. Images were acquired by Olympus IX81and analyzed by ImageJ Software.Gapdh). The values obtained were converted by the formula 2(−ΔΔCt) to be expressed as fold changes in regulation compared with the reference sample.To confirm the presence of GM-CSF encoding sequences, RNA extraction was performed using RNeasy Mini Kit (QIAGEN). cDNA was obtained with SuperScript II reverse transcriptase and poly dT primers (Invitrogen). PCR was performed in a volume reaction of 10ul containing TaqMan Universal PCR Master Mix (Applied Biosystem) and 20 ng cDNA with the following couple of primers (5’ – 3’ etc). We used Applied Biosystems 7900HT-Fast Real-time for PCR reaction and fluorescence detection. All measures were performed in duplicates and were analyzed by the ΔΔCt relative quantification method: arithmetic means of the cycle threshold (Ct) values were calculated and target gene mean Ct values were normalized to the respective endogenous control . Where required, MDSCs (derived from immunomagnetic sorting) were added at decreasing percentages (12% and 6%). T Cell proliferation was evaluated by FACS and data analyzed by FlowJo software  using the FlowJo’s Proliferation Tool by tracking cell generation according to software indications.MLPC cultures were prepared by mixing γ-irradiated C57BL/6 splenocytes with OT-I/CD45.1 splenocytes in order to obtain 1% OVA-specific T lymphocytes in the final culture. OVA-specific lymphocytes were previously CFSE-labeled  according to manufacturer’s instructions for the proliferation assay. A total of 0.6x10The GM-CSF concentrations in cell culture supernatants and in serum from mice were evaluated using mouse GM-CSF Quantikine ELISA kit  according with manufacturer’s instructions.Total protein extract was obtained with RIPA buffer. Protein extracts were separated by 4-12% Bold NuPage (ThermoScientific) and transferred onto PVDF membranes (BioRad). After blocking with 3% albumin (Sigma-Aldrich) and primary antibody incubation phospho-STAT5, phospho-Erk1/2, Erk1/2 -Cell Signaling- and STAT5 -Santa Cruz- 1:1000), the membranes were incubated with an anti-rabbit peroxidase-conjugated secondary antibody . Chemiluminescence was obtained by the ICL Substrate , and images were obtained with an imaging ImageQuant LAS 500 .Statistical comparison between two groups was carried out using the Student’s T test or Mann Whitney test. For multiple comparisons, One-way Anova test, followed by a Tukey post-hoc test was performed. Results with a p-value ≤ 0.05 were considered as significant. In figures, asterisks were used as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Analyses were performed using Graph Pad Prism (version 7).-); the presence of reproducible modifications as nitration/nitrosylation and/or oxydation was evaluated by tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS). Our results indicated that a single Tryptophan residue, in the SPITVTRPW*K peptide was stably nitrated, as demonstrated by a +45Da shift in the tryptophan residue  upon peroxynitrite treatment (in vitro fully competent MDSCs from bone marrow precursors (BM-MDSCs) by adding GM-CSF and IL-6 cytokines as previously published  or GM-CSF treated with degraded ONOO- (control) alone, or in combination with IL-6. After 5 days of culture, we phenotypically profiled harvested culture cells by multiparametric flow-cytometry (FACS). Interestingly, FACS analysis revealed a higher expansion of the PMN-MDSC subset in cells differentiated in presence of N-GM-CSF, alone or in combination with IL-6, in comparison to cells differentiated with the unmodified cytokine. No significant differences were observed for the Mo-MDSC subset. Moreover, N-GM-CSF exposure induced an increment of macrophages (Ly6Clow/- Ly6G- F4/80+) and a concomitant reduction of Ly6Clow/-Ly6G- cells  or a full-mutant sequence (named W30L), where we introduced a point mutation at the site of protein nitration. In more detail, we substituted the RNS-targeted amino acid residue  with a no-targetable one, by a site-specific mutagenesis approach. We changed the codon encoding W in position 30 of murine GM-CSF with the one for leucine (L). This substitution was driven by a bioinformatic analysis where different homologues and orthologues protein sequences where aligned. Interestingly, our analysis indicated that this position is well conserved, with very few cases where other amino acids are encoded. Moreover, the codon for leucine has the highest substitution score according to two independent substitution matrix (PAM250 and Blosum) and is not known to be sensitive to nitration/nitrosylation such as other aromatic residues or polar residues  and that were able to secrete comparable amount of GM -CSF  obtained from BM cell cultures upon the stimulation with either MCA203-GM or MCA203-W30L secretome. However as expected, cell supernatants were less effective than the recombinant cytokines in differentiating myeloid cell populations  but we observed a significant reduction in the percentage of PMN-MDSCs (Ly6G+) and macrophages (Ly6C-/Ly6G-/F4/80+) in mice MCA203-W30L-bearing mice compared to the MCA203-GM controls  and macrophages in the spleens of W30L tumor bearing mice as compared to the control  model  model .in vitro peroxynitrite-treated protein we singled-out a specific aromatic residue, a tryptophan (W) which can be stably and consistently nitrated by RNS. The propensity of such residues to be modified by RNS was strengthened by a bioinformatic approach (homology modeling of the human GM-CSF) that clearly confirmed the accessibility of such residues for the interaction with radical species. Importantly, we found that RNS-modified GM-CSF can act as an amplifier of cancer-related immunosuppression by enhancing the generation and activity of myeloid suppressive cell subsets.Taken together, all these findings indicated that the biology of the GM-CSF in tumors needs to be further expanded in order to reconcile the dual nature of this cytokine in cancer. Our study shed light on GM-CSF activity in tumors by indicating that this cytokine acts as a pliable element in cancer hosts. Indeed, we found that GM-CSF represents a new target for RNS in tumors. Furthermore, we investigated the functional significance of RNS-induced PTM for the biology of this cytokine. Specifically, by performing MS/MS analysis on A key process occurring during tumor progression is the pathologic expansion and activation of distinct myeloid cell subsets that suppress adaptive immunity , collectFor this reason, MDSCs are now recognized as a worthy diagnostic marker and importantly as a key target for cancer immunotherapy. Our data showed that N-GM CSF promotes the expansion of PMN-MDSCs and macrophages, which represent highly immunosuppressive subsets.in vivo data confirmed that the nitration of GM-CSF represents a founding element for MDCS expansion and recruitment in tumor tissues. To deal with this issue, we engineered a fibrosarcoma cell line by introducing a single point-mutation in GM-CSF sequence, generating a tumor model that stably secretes a GM-CSF mutant which is insensitive to RNS-induced PTM. Indeed, the substitution of the site of RNS attack –a tryptophan- redraws the immune landscape at the tumor site. Tumors secreting the genetically-modified GM-CSF insensitive to RNS action (the W30L mutant) display a significant decrease in the number of both PMN-MDSC and macrophages (TAM) at the primary neoplastic foci. This occurs with comparable serum levels of the “native” versus mutated GM-CSF. For ethical reason we ceased tumor observation at day 21. We found no differences in the overall growth of tumor masses between tumor cell lines secreting either WT or W30L GM-CSF at this time-point. Nonetheless, the volume of W30L tumors was slightly decreasing from day 15, reasonably suggesting that the decreased recruitment of immunosuppressive subsets at the primary site could affect tumor growth on a longer timeframe. Consistently, the rate of nitrosivative stress was significantly decreased in W30L tumors compared to control as indicated by the reduced expression of both nitro-tyrosine and nitro-tryptophan markers.The induction of MDSC phenotype and activity relies on multiple signaling pathways downstream cytokine stimulation, including GM-CSF. The canonical pathway triggered by the GM-CSF binding to its receptor involves primarily MAP kinases and STAT family members. In particular, STAT5 phosphorylation sustains the maturation of dendritic cells and it is commonly associated with pro-inflammatory responses. The duality in GM-CSF molecular signature is consistent with the complexity of cytokine-driven responses and it could be explained on the basis of GM-CSF dosage, signaling amplitude, and timing. Remarkably, we found that N-GM-CSF significantly amplifies the molecular signaling leading to MDSC expansion and activity compared to the native, unmodified GM-CSF. Our data indicated that N-GM-CSF can boost GM-CSF signaling through the STAT5 pathway. In tumor tissues where RNS are present at a very high extent  the post2-, and H2O2  in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the institutional protocols for animal care and approved by “Ministero della Salute”. All experiments followed committee guidelines CEASA 15/3/2012.Conceptualization, BM and IM. Investigation BC, AA, CC, RS-R, AP, GIT, GA, FZ, CM, and LA. Writing- original draft, BM and IM. Critical data discussion, VB and AV. Funding acquisition, BM. All authors contributed to the article and approved the submitted version.Grant from the Italian Ministry of Health, BANDO GIOVANI RICERCATORI, No. 2009-GR-2009-1558698; Agnellini R.H. A. was granted by Cariparo Fundation Fellowship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E. coli BL21 (DE3) as the vector to express the RBP4 recombinant protein and prepared anti‐RBP4 mAb using hybridoma technology. We performed immunohistochemical analysis on hepatocellular carcinoma (HCC) and adjacent tissues by using this anti‐RBP4 mAb. In addition to the high‐purity RBP4 recombinant protein, we successfully developed the anti‐RBP4 mAb with high affinity and specificity; it binds to natural RBP4 protein and is suitable for immunohistochemical analysis.The expression level of retinol‐binding protein 4 (RBP4) protein is closely related to liver damage and plays an important role in the diagnosis and prognosis of cancer. However, the preparation of anti‐RBP4 mAb or exploration on the application of anti‐RBP4 mAb has not been reported thus far. In the present study, we constructed a pET30a‐RBP4 recombinant vector, used  The expression level of RBP4 protein is closely related to liver damage and plays an important role in the diagnosis and prognosis of tumours. In this study, we successfully developed an anti‐RBP4 mAb with high affinity and specificity; it is bound to natural RBP4 protein and is suitable for immunohistochemical analysis. DABdiaminobenzidineDE3E. coli BL21ECLenhanced chemiluminescenceHCChepatocellular carcinomaiELISAindirect enzyme‐linked immunosorbent sandwich assayIHCimmunohistochemistryIODintegrated optical densityIPTGisopropyl‐β‐d‐thiogalactosideKEGGkyoto encyclopedia of genes and genomesNEK2‐Hisnever in mitosis gene‐A‐related expressed kinase 2‐HisRBP4retinol‐binding protein 4SAA4‐Hisserum amyloid A‐4‐HisSDS/PAGEsodium dodecyl sulphate/polyacrylamide gel electrophoresisSHBG‐Hissex hormone‐binding globulin‐HisTCGAthe cancer genome atlasRetinol‐binding protein 4 (RBP4) is a member of the lipocalin super family and is mainly secreted by the liver. RBP4 is secreted into the blood to combine with retinol, playing a crucial role in the transport of retinol and vitamin A . In 2005We expressed the RBP4 recombinant protein using the prokaryotic expression system. After purification, the recombinant protein was immunised to BALB/c mice to prepare anti‐RBP4 mAb. Anti‐RBP4 mAb could be used to detect the RBP4 expression level by immunohistochemistry (IHC) on the basis of the expression level of RBP4 in HCC tissues found on The Cancer Genome Atlas (TCGA) database. Therefore, the proposed mAb has advantages of high affinity and specificity for RBP4 protein, thereby providing insights in advancing auxiliary examination of related diseases.E. coli BL21 (DE3) were purchased from Beijing Quanshijin Company. The plasmid pET‐30a was donated by the School of Life Science and Technology of Guangxi University. SPF‐grade 4‐week‐old female BALB/c mice, weighing 14 g‐17 g, were sourced from Shanghai Slack Laboratory Animal Co., Ltd. [SCXK (Shanghai) 2017‐0005], with certificate number: 20170005000441. All mice were housed in specific pathogen‐free facilities and cared for in Laboratory Animal Center of Guangxi Medical University [SCXK (Gui) 2020‐003]. Sterile surgery was conducted in the Experimental Animal Centre of Guangxi Medical University. All animal experiments had been approved by the Laboratory Animal Welfare and Ethics Committee of Guangxi Medical University (NO. 201911011) and performed in accordance with national ethics regulations. In the experimental design, we had achieved the minimum necessary amount by optimising the experimental scheme. In the process of the experiment, we used a relatively mild injection technique and appropriate amount of adjuvant to reduce the serious side effects of animals and reduce the pain of the animals. Other materials purchased include the following: total RNA extraction and transcription kit, 10× T4 DNA ligase , RBP4 gene primers , plasmid extraction kit , Hind III and BamH I enzymes , 2× Fast Pfu PCR enzyme , Ni‐Agarose Resin , SDS/PAGE protein gel reagents and BCA kits , Freund’s complete adjuvant, Freund’s incomplete adjuvant, HRP‐labelled goat anti‐mouse secondary antibody , Bovine Serum Albumin (BSA) , gradient RT‐PCR instrument , gel image analyser , ultrasonic cell disruptor , western blot electrophoresis imaging system , EVOS FL automatic cell imager  and HCC tissue sample chip .Normal human liver cell line HL‐7702, HCC cell hep3B, HCC cell huh7 and SP2/0 mouse myeloma cells were purchased from the Shanghai Academy of Biological Sciences and Cell Collection and Chinese Academy of Sciences. Strain DH5α competent cells and HL‐7702 and Huh7 were cultured in DMEM containing 10% FBS, and Hep3B was cultured in MEM containing 10% FBS. Mouse myeloma cell line SP2/0 was cultured in RPMI‐1640 medium containing 10% FBS, hybridoma cell lines were cultured in RPMI‐1640 medium containing 20% FBS and 20% HAT, and monoclonal hybridoma cell lines were cultured in RPMI‐1640 medium containing 20% FBS and 20% HT. All the cells were incubated at 37 °C in 5% CO2.RBP4 primer sequences were AAAGGATCCGAGCGC GACTGCCGAGTGAG (forward) and GGGAAGCTTCTACAAAAGGTTTCTTTCTG (reverse). Total RNA of a normal human liver cell line HL‐7702 was extracted as raw material, and RBP4 gene was obtained by RT‐PCR amplification. The reaction conditions are as follows: 95 °C 3 min, 95 °C 30 s, 61 °C 30 s, 72 °C 90 s, and 35 cycles; 4 °C infinite cycle. A small amount of pET‐30a plasmid was extracted with the kit, and the pET‐30a plasmid and RBP4 gene were digested with BamH I enzyme and Hind III enzyme, respectively. The ligated vector was transformed into competent DH5α and plated onto LB kanamycin plates. The next day, a single colony was picked from the plate and cultured in LB, and the recombinant plasmid was identified by restriction enzyme digestion with BamH I and Hind III.The amino acid sequence of RBP4 protein was obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the position of its signal peptide was analysed by SignalP5.0. The antigenic index of RBP4 protein was predicted by DNAstart software, and the appropriate sequence was selected for expression. The RBP4 plasmid and pET30a empty plasmid were divided into two groups, namely the IPTG‐induced group and the uninduced group. The IPTG‐induced group was added with isopropyl‐β‐d‐thiogalactoside (IPTG) to a final concentration of 1.0 mmol·L−1, and the expression was induced at 37 °C for 4 h. The bacterial pellet was broken by ultrasonic disruption, and the bacterial supernatants and the precipitates were collected separately. The expression form of the recombinant protein was identified by 12% sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE). According to these protein expression steps, different induction temperatures  and different induction IPTG concentrations  were considered to determine the optimal expression conditions. The supernatant protein solution was added to a chromatography column containing 2 mL Ni agarose gel packing and incubated overnight at 4 °C on a shaker to purify the recombinant protein. Proteins were eluted with 150 mm imidazole elution concentration, which was increased from 50 mm to 200 mm to determine the optimal elution condition, and the elution peaks containing a large amount of RBP4 protein were collected. This solution was purified by 250 mmol·L−1 KCl staining. Finally, the RBP4 recombinant protein containing His tag was confirmed by western blot using anti‐His‐tag antibody.The pET30a‐−1, and the BALB/c female mice were immunised with multiple subcutaneous injections at an interval of 14 days, using recombinant RBP4 protein at a concentration of 1 μg·μL−1 as the immunogen. For the second immunisation, the dose of 80 μg·mouse−1 was mixed with the same volume of Freund’s incomplete adjuvant, and the interval was 2 weeks. The dose of the third and fourth immunisations, identical to the second, was given at 2‐week interval. Seven days after the fourth immunisation, the iELISA was used to detect the serum titre of mouse tail vein blood. In summary, the 96‐well microplate was coated with 100 μL RBP4 recombinant protein (2 μg·mL−1) at 4 °C overnight. The positive serum and negative serum samples were diluted in a threefold ratio, from 1 : 1000 to 1 : 729,000, and the 0.1% BSA solution was used as a blank control. The HRP‐IgG goat anti‐mouse  was added and incubated at 37 °C for 30 min. Under dark conditions, the substrate colour solution was added with 100 μL·well−1. Finally, the reaction was stopped by 2 m sulphuric acid, and the OD450nm value was measured. The serum dilution corresponding to positive serum (P)/negative serum (N) value ≥ 2.1 was used as the serum titre of immunised mice. In addition, immunised mice with serum titre greater than 1 : 10,000 were selected for the next cell fusion.The first dose was mixed with the same volume of complete Freund’s adjuvant at a dose of 150 μg·mouseMice with serum titre greater than 1 : 10,000 were selected for cell fusion. Three days after booster immunisation of mice, spleen cells and SP2/0 cells are fused at a ratio of 2 : 10 under the concentration of 50% PEG. After fusion, the hybridoma cells were cultured in 1/3 HAT selection medium. After 9 days, the hybridoma cell supernatants were determined by iELISA. The positive hybridoma cell lines were selected and subjected to three subclonal screening by the limiting dilution method.7 monoclonal hybridoma cells were injected into the mice abdominal cavity to prepare ascites. The monoclonal antibodies were purified by the rProtein G affinity chromatography column. The mouse mAb isotyping  detection kit was used to detect mAb isotype. The iELISA method was used to determine the antibody affinity constant. According to the checkerboard square method, the RBP4 recombinant protein was coated on the 96‐well microplate at the concentration of 4, 2, 1 and 0.5 μg·mL−1, and the selected mAb was serially diluted with a double dilution ratio . The calculation method of affinity constant Ka was the same as that reported in the previous study [−1, and the sample diluent was used as a negative control. According to the iELISA method, the anti‐RBP4 mAb was diluted at 1 : 20,000 and then tested. HCC cell line hep3B, HCC cell huh7 total protein, SHBG‐His recombinant protein, RBP4‐His recombinant protein and RBP4 protein standard (R&D) were electrophoresed in 12% SDS/PAGE gel and transferred into the PVDF membrane. The membrane was incubated with purified anti‐RBP4 mAb  overnight at 4 °C. HRP‐IgG goat anti‐mouse  was added and incubated for 60 min at room temperature, the membrane was washed with TBST buffer for three times and then developed with enhanced chemiluminescence (ECL).BALB/c mice were sensitised intraperitoneally with 500 μL of paraffin. After 7 days, the 1 × 10us study . Recombin = 1), II (n = 4), II ~ III (n = 2) and III (n = 3). A summary of the pathological characteristics and the patient characteristics is presented in Table image‐pro Plus 6.0 software was used for semi‐quantification of the staining. In summary, three areas of each sample were captured randomly at 40× the magnification of EVOS FL automatic cell imager, and the measurement parameter was the integrated optical density (IOD). The parameter setting referred to the method of Li Gang et al. [The tissue chip contains tissue samples from 10 hepatocellular carcinoma (HCC) patients and the corresponding adjacent tissues. In accordance with the improved nuclear grading scheme of the Edmondson and Steiner system, all tissue samples were pathologically diagnosed as hepatocellular carcinoma, and the histological grade was categorised as grade I ~ II  was a web‐based tool, which allowed researchers to perform analyses of TCGA gene expression data [P value was calculated and displayed on the web page.The mRNA expression levels of ion data . In addit‐test. One‐way ANOVA was used to analyse the differences amongst multiple groups. P < 0.05 was considered statistically significant. Origin 9.1 was used to calculate the antibody concentration corresponding to the EC50 of the mAb.ImageJ image software was used to evaluate the grey value of western blot bands and Coomassie brilliant blue staining band. IBM SPSS Statistics 22.0  was used for statistical analysis. The differential expression of RBP4 between HCC and adjacent non‐tumorous liver tissues was determined using the two‐tailed paired Student’s RBP4 gene was cloned into pET30a. Specific primers were used to amplify the RBP4 gene fragment with a size of ~ 600 bp, and the extracted cDNA of human normal liver cell line HL‐7702 was used as a template    were evident in lanes 1 and 2, indicating that the ein Fig. . The pro00) Fig. . Further00) Fig. . Under o mm Fig. . The RBP mm Fig. . The pur mm Fig. .After immunising the mice with purified RBP4 recombinant protein as the immunogen, the serum titre was determined by iELISA. The polyclonal antibody serum titre of three mice could reach 1 : 729,000 Fig. . The ave−1 RBP4 recombinant protein was 0.197, 0.154, 0.166 and 0.122 µg·mL−1, respectively    Fig. . This fi01) Fig. . At the 17) Fig. .−1, and the urine concentration is 0.7 mg·L−1. Serum RBP4 transports retinoid proteins to target organs and reabsorbs them back into the blood through renal tubules. Therefore, RBP4 is often used as a clinical indicator for early diagnosis and efficacy evaluation of renal diseases [RBP4 protein, as a secreted protein of the retinol family, is widely distributed in blood, urine and other body fluids. The normal concentration of human serum RBP4 protein is 25–70 mg·Ldiseases . Liver idiseases . Some stdiseases . The detdiseases . Researcdiseases . The negdiseases , 23, whiE. coli was selected as a protein expression tool for expressing RBP4 recombinant protein, considering its characteristics of prokaryotic expression system with clear genetic background, strong operability, mature and stable technology, large yield and low cost [m was beneficial to the efficient recovery of the target protein, but still a small amount of other protein was found, probably affecting the effect of RBP4 protein as an immunogen. After the initial purification by the Ni affinity chromatography column, RBP4 recombinant protein was further purified by KCl silver‐stained gum recovery to obtain a higher purity RBP4 recombinant protein. During the gum recovery process, the imidazole, urea and salt ions in the RBP4 recombinant protein solution were also removed, reducing the loss of the recombinant protein in the final dialysis process, which was conducive to obtaining a large amount of high‐purity recombinant protein. The RBP4 recombinant protein was successfully identified by western blot, indicating that the recombinant protein expressed in the form of inclusion bodies maintained the integrity of the linear epitope and had compelling antigenicity after renaturation.In the clinical testing, antibodies are the commonly used tools. At present, reports on the preparation of monoclonal antibodies against RBP4 are few. The RBP4 protein was analysed by bioinformatics analysis, and the N‐terminal amino acid sequence (1‐18aa) of the RBP4 protein was found to be the secretion signal peptide sequence. Thus, the amino acid sequence with favourable epitope specificity (19‐201aa) was selected for the protein expression. low cost , 25. Aftlow cost , 27. TheThe results of serum titre showed that the serum antibody titres of mice could reach more than 1 : 729,000, which was much higher than the currently reported literature , 29. Thi8 L·mol−1. The affinity constant of mAb was referred to as the specific binding strength of the antigenic determinant and the antibody. Higher affinity was associated with stronger specificity. When the affinity constant was between 107 and 1012 L·mol−1, it belonged to the high‐affinity antibody [Low‐dose and long‐term immunisation programme was conducted, and the antibodies secreted by the five monoclonal hybridoma cell lines were demonstrated to be IgG subtypes, which could bind to antigens stably and effectively. They are often adopted as stable and widely used monoclonal hybridioma subtypes. Our research results also showed that the average affinity constant of RBP4 mAb was 3.98 × 10antibody , and theThe TCGA database contains comprehensive cancer genome data, including mutations, copy number variations, mRNA expression, miRNA expression and methylation data . TCGA isThe liver is the main secretion site of RBP4 protein. When it is damaged, it affects the synthesis of RBP4, resulting in the decrease in serum RBP4 concentration, accompanied by the aggravation of liver damage. With the deepening of studies, researchers found that the expression level of RBP4 was closely related to cancers. In head and neck cancer , breast However, the current study also had several shortcomings in the process of preparing monoclonal antibodies. For example, only two types of antibodies secreted by the newly prepared five monoclonal hybridoma cell lines could be used for detection by western blot and immunohistochemistry simultaneously, and the three remaining strains could only be used for immunohistochemical detection. This finding might be because the anti‐RBP4 monoclonal antibodies secreted by the three strains specifically recognised the internal conformational epitopes of natural RBP4 protein, rather than the linear epitopes on the protein surface. As for the application of anti‐RBP4 mAb in immunohistochemical detection, we will consider to expand the sample size to detect HCC patients at different pathological grades in the future. In addition, the application value of anti‐RBP4 mAb in the diagnosis of HCC will be evaluated.In conclusion, the recombinant RBP4 protein with high concentration, high purity (98%) and outstanding antigenic activity was successfully expressed and obtained. The expression and purification method had important reference value for the development of a large‐scale standardised production process of RBP4 recombinant protein. The anti‐RBP4 mAb was prepared; it had high affinity and strong specificity and could specifically bind to the natural RBP4 protein and suitable for immunohistochemical analysis.The authors declare no conflict of interest.All data in our study are available from the corresponding author on reasonable request.HL performed the experiment, analysed data and wrote the original draft. WS, LCY, QLC, SPH performed the animal experiment. XH, YSL, FJW analysed the data and revised the manuscript. MH designed the study, supervised experiments, MH and XH supported the study. All authors reviewed the final manuscript.


Hepatitis E virus (HEV) infection can lead to a variety of neurological disorders. While HEV RNA is known to be present in the central nervous system, HEV quasispecies in serum and cerebrospinal fluid (CSF) have rarely been explored. We studied the virus’ quasispecies in the blood and the CSF of five patients at the onset of their neurological symptoms. The samples of three patients suffering from meningitis, neuralgic amyotrophy and acute inflammatory polyradiculoneuropathy were taken at the acute phase of the HEV infection. The samples from the other two patients were taken during the chronic phase (5 years after HEV diagnosis) when they presented with clinical signs of encephalitis. We sequenced at least 20 randomly polyproline regions of the selected virus clones. Phylogenetic analysis of the virus variants in the blood and the CSF revealed no virus compartmentalization for the three acute-phase patients but there was clear evidence of HEV quasispecies compartmentalization in the CSF of the two patients during chronic infection. In conclusion, prolonged infection in the immunocompromised condition can lead to independent virus replication in the liver and the tissues, producing viruses in CSF. HEV is the most common cause of hepatitis worldwide. It was originally thought to be a disease of developing countries, where genotypes 1 and 2 predominate and where it is transmitted by ingestion of fecally contaminated water ,4. In viMost patients with an acute hepatitis E infection have no symptoms, while the symptoms of an acute infection are indistinguishable from those of the hepatitis caused by other acute viral infections . HEV repWe aimed to investigate virus diversity in the serum and the CSF in five patients with neurological manifestations observed during the acute or the chronic phase of HEV infection.3) due to HIV. The clinical manifestations of two patients were described previously [The five HEV-infected patients with neurological manifestations all had HEV RNA in their blood and CSF samples . They weeviously ,15. The Virus RNA was isolated from the blood and CSF samples using a QIAamp Viral RNA kit  and a fragment covering the PPR was amplified by reverse transcription PCR (RT-PCR). A nested reverse transcriptase PCR (RT-PCR) was used to amplify a 325 nt fragment of the HEV genome. RT-PCR was performed with a SuperScript III One-Step RT-PCR system  under the following conditions: 30 min at 50 °C, 2 min at 94 °C, and 50 cycles of 30 s at 98 °C, 30 s at 55 °C, and 35 s at 72 °C. The sense primer was 1710-S (GAGTGCCGYACKGTGCTYGGGAATAA) and the antisense primer was 3050-AS (ACATCRACATCCCCCTGYTGTATRGA). Then, a nested PCR was performed with an Expand High Fidelity PCR system  under the following conditions: 30 s at 98 °C and 30 cycles of 30 s at 98 °C, 30 s at 55 °C, and 30 s at 72 °C. The sense primer was IS-G3 (ACCYTGTAYACTCGNACCTGGTC) and the antisense primer was IA-G3 (ACCTTRGCRCCGTCAGGRTAGGT).Escherichia coli competent cells  according to the manufacturer’s protocol and the resulting transformants were grown on ampicillin plates. At least 20 clones were randomly selected for sequencing. PCR products were sequenced on both strands using the dideoxy chain termination method  on an ABI 3130XL analyzer .The purified PCR products were quantified by means of spectrophotometry. We then directly ligated 10 ng cDNA into 10 ng of TOPO XL-2 . Recombinant plasmids were used to transform We used the MAFFT version 7.45 sequence alignment tool followed by IQTREE version 1.6.12 to construct a bootstrapped tree (1000 replicates) to obtain the maximum likelihood model. Finally, Interactive Tree Of Life (iTOL) v3 was used to visualize the tree . If seveWe studied the virus quasispecies of three patients at the acute phase of HEV infection presenting with different neurological manifestations: meningitis (patient 1), neuralgic amyotrophy (patient 2), and acute inflammatory polyradiculoneuropathy (patient 3) . For theWe also investigated the virus quasispecies in the blood and the CSF of the two chronically infected (5 years) HEV patients who presented with clinical signs of encephalitis (patients 4 and 5) . PhylogeThe phylogenetic tree for patient 4 quasispecies showed distinct clusters : one incThe phylogenetic tree for patient 5 also revealed distinct populations of virus variants in the blood and the CSF . One cluSeveral neurological manifestations associated with HEV infection have been described and in vitro studies suggest that HEV can replicate in the brain. Our comparison of the HEV quasispecies in the blood and the CSF of the patients with neurological manifestations indicates that there is no compartmentalization of HEV quasispecies in the CSF at the acute phase of infection and that it arises only during a chronic infection.Close to half (42.9%) of patients with neurological manifestations can have HEV RNA in their CSF , and sevViruses enter the brain by a variety of means, primarily via neuronal transport or by crossing one of the several barriers to the central nervous system, including the blood–brain barrier and the blood–cerebrospinal fluid barrier (choroid plexus) . An in vOur investigation of the HEV quasispecies in the blood and the CSF of the five patients with acute or chronic HEV infections and neurological manifestations found distinct HEV variants, indicating compartmentalization only in chronic-phase patients. This agrees well with a previous study that found virus compartmentalization in the CSF of a chronically HEV-infected kidney transplant recipient suffering from peripheral demyelinating polyradiculoneuropathy after 33 months of HEV infection . A virusThe duplication of the virus’ PPR region in patients with clear compartmentalization of the HEV quasispecies in the CSF was detected only in the samples collected when the neurological symptoms occurred. The sequence encoding this region can vary in both composition and length, depending on the HEV subtype ,8,24,25.In conclusion, compartmentalization of the virus in the central nervous system seems to require a prolonged infection. The cells that support HEV replication in the human central nervous system have yet to be identified.


The present results revealed not only detailed information regarding the CFL morphology, but also inter- and intra-individual laterality regarding the CFL traveling angle. It is likely that the differences in the quality and quantity of mechanical stress against each leg may have caused this morphologic laterality of the CFL.The lateral ligament complex of the ankle is involved in a large proportion of ankle sprains. The calcaneofibular ligament (CFL) is often involved in severe injuries. The purpose of this study was to evaluate the anatomical variation and laterality of the CFL to improve our understanding of the mechanisms of CFL-related injuries. This study utilized 110 paired ankles from 55 formalin-fixed Japanese cadavers . The length and width of the CFL and the angle created by the CFL and long axis of the fibula (CF angle) were measured after exposing the CFL by careful dissection from the surrounding tissues. The results revealed that each parameter exhibited a wide range of values and showed unique patterns of frequency distribution, among which only the length was normally distributed. Among the parameters, only the CF angle showed no significant correlation with the other parameters. Analysis of laterality revealed that the mean left CF angle was significantly greater than the value on the opposite side ( The lateral ligament complex of the ankle is composed of the anterior talofibular ligament (ATFL), posterior talofibular ligament (PTFL), and calcaneofibular ligament (CFL) Fig. . InjurieAs reviewed by Matsui et al. , severalOnly Wiersma and Griffioen  have refThe purpose of this study was to evaluate variations of the CFL using an in-depth approach and in view of laterality to further the understanding of the mechanisms of CFL injury.We assessed 110 paired ankles from 55 formalin-fixed Japanese cadavers (33 men and 22 women) used in a gross anatomy course conducted in 2014 and 2015 in the Faculty of Medicine, Saga University, Japan. In all cadavers, no traces of prior injury or surgical treatments were observed in the ankle region. The ages at the time of death ranged from 48 to 100 years [mean ± standard deviation (SD) 78 ± 12 years]. This study was approved by the local ethics committee of Saga University.During the gross anatomy course, the bilateral lower legs of the cadavers were partly dissected. During this process, the skin was removed and the subcutaneous tissue, deep fascia, and muscles of the lower leg were exposed while the lateral ligaments of the ankle were left intact. First, we removed the peroneus longus and brevis tendons. Then, after observing the fiber directions, the CFL was dissected carefully from the surrounding tissues, including the peroneal tendon sheath, lateral talocalcaneal ligament (LTCL), and arch-forming fibers that unite the CFL and ATFL. The CFL was then assessed as follows.The free length of the CFL was measured between the fibular origin and the most proximal insertion on the calcaneus (Milner and Soames All measurements were performed by a single researcher (H.Y.) by setting both the talocrural and subtalar joints in the 0° position and using a caliper  or goniometer  on each CFL. A minimum unit of records was defined as 0.5 mm for distance or 5° for angle. The accuracy of the measurement technique was determined by repeating all measurements in technical triplicates. The findings in all specimens were photographed using a NEX-5R digital camera  for future reference.p value of 0.05. The Shapiro-Wilk test was used to evaluate normality. Laterality between grouped data from each side  was examined by a paired t test or Wilcoxon signed-rank test. Pearson’s correlation coefficient or Spearman’s rank correlation coefficient was calculated to investigate laterality at the individual levels  and the relationships among parameters.All statistical analyses were performed using IBM SPSS Statistics version 22, and the level of statistical significance was set at a During sample preparation, we noticed a cadaver  in which the left CFL was deficient and excluded it from the study. Therefore, data from 108 paired ankles from 54 cadavers  were included in the subsequent analysis.Table p = 0.08).The frequency distributions of the CFL dimensions are shown in Fig. To examine the relationships among parameters, correlation coefficients were calculated Table . Among pp < 0.05).The collected data from each side were compared to examine inter-individual laterality Table . Among tTo visualize and examine intra-individual laterality for each parameter, we prepared scatter plot diagrams and calculated correlation coefficients between the two sides Fig. . There wp < 0.05) (Table t test (p = 0.06)  Table , and the5) Table . These r5) Table . In addi5) Table . Thus, i5) Table . Conside6) Table . As the The present results revealed a wide range and unique pattern of frequency distribution for each parameter Fig. , among wIt has been previously documented that the CFL crosses over the deep side of the peroneus longus and brevis tendons. In this study, we confirmed this structural relationship in all 108 samples. Considering this positional proximity, Shinohara et al.  speculatBurks and Morgan  describeIn the present study, as information on handedness of the cadavers was unavailable, we could not analyze the laterality of the CFL by separating them into right- and left-handed individuals. This is a limitation of this study.In conclusion, the present results revealed not only detailed information regarding CFL morphology, but also inter- and intra-individual laterality regarding the CFL traveling angle. It is likely that the differences in the quality and quantity of mechanical stress against each leg may have caused this morphologic laterality of the CFL.


There is an urgent need for objective markers of Alzheimer’s disease (AD)-related cognitive impairment in people with Down syndrome (DS) to improve diagnosis, monitor disease progression, and assess response to disease-modifying therapies. Previously, GluA4 and neuronal pentraxin 2 (NPTX2) showed limited potential as cerebrospinal fluid (CSF) markers of cognitive impairment in adults with DS. Here, we compare the CSF profile of a panel of synaptic proteins  to that of NPTX2 and GluA4 in a large cohort of subjects with DS across the preclinical and clinical AD continuum and explore their correlation with cognitive impairment.42:40 ratio, CSF Aβ1-42, CSF p-tau, and CSF NFL. P values were adjusted for multiple testing.We quantified the synaptic panel proteins by selected reaction monitoring in CSF from 20 non-trisomic cognitively normal controls (mean age 44) and 80 adults with DS grouped according to clinical AD diagnosis . We used regression analyses to determine CSF changes across the AD continuum and explored correlations with age, global cognitive performance (CAMCOG), episodic memory  and CSF biomarkers, CSF Aβadj.p = .04) and age (adj.p = .0008) and was the best correlate of CSF Aβ42:40 (adj.p = .0001), p-tau (adj.p < .0001), and NFL (adj.p < .0001). Compared to controls, mean VAMP-2 levels were lower in asymptomatic adults with DS only (adj.p = .02). CSF levels of Neurexin-3A, Thy-1, Neurexin-2A, Calysntenin-1, Neuroligin-2, GluA4, and Syntaxin-1B all strongly correlated with NPTX2 (p < .0001), which was the only synaptic protein to show reduced CSF levels in DS at all AD stages compared to controls (adj.p < .002).In adults with DS, VAMP-2 was the only synaptic protein to correlate with episodic memory (delayed recall These data show proof-of-concept for CSF VAMP-2 as a potential marker of synapse degeneration that correlates with CSF AD and axonal degeneration markers and cognitive performance. However, neuropsychological assessment can be confounded by substantial inter-individual variation in intellectual disability (ID). Therefore, there is an urgent need for objective markers of AD-related cognitive impairment in people with DS to improve diagnosis, monitor disease progression, and assess response to disease-modifying therapies.Alzheimer’s disease (AD) is the leading cause of death in adults with Down syndrome (DS), with a cumulative incidence that exceeds 90% in the seventh decade –4. CurreSynapse loss is an early event in AD  and one Neuronal pentraxin-2 (NPTX2), a protein involved in inhibitory circuit dysfunction , is a prThe aim of this study was to evaluate a comprehensive panel of alternative synaptic proteins  as surrogate markers of early AD-related cognitive decline in non-trisomic cognitively normal controls (n = 20) and a large cohort of adults with DS (n = 80) from across the preclinical and clinical AD continuum, exploring their relationship to cognitive performance. The panel comprises 8 proteins that were shown to be specifically expressed at the synapse in human frontal cortex postmortem tissue and show CSF alterations that precede clinical symptoms and markers of neurodegeneration in sporadic AD . We alsoThe primary objective of this study was to evaluate a comprehensive panel of synaptic proteins as surrogate markers of early AD-related cognitive decline in adults with DS from across the preclinical and clinical AD continuum, specifically exploring their relationship to cognitive performance and AD biomarkers.1-42, Aβ42/40 ratio, t-tau, p-tau) concentrations using our validated cut-offs for sporadic AD [This is a single-center, cross-sectional study of CSF levels of synaptic markers in adults with DS, sporadic AD patients and cognitively normal controls. The study (IIBSP-BMS-2018-103) was approved by the local ethics committee  and was conducted in accordance with the Declaration of Helsinki. All participants gave their written informed consent to participate in the study. Non-trisomic controls were selected from the Sant Pau Initiative in Neurodegeneration (SPIN) cohort, a prospective longitudinal cohort at Hospital Sant Pau, Barcelona, Spain . Adults radic AD . For aduradic AD  and undeThe level of ID in adults with DS was categorized according to the Diagnostic and Statistical Manual of Mental Disorders (DSM), Fifth Edition, as mild, moderate, severe, or profound ID, based on caregivers’ reports of the individuals’ best-ever level of functioning and the Kaufmann Brief Intelligence Test (KBIT) . As prev1-42, Aβ1-40, NFL, total tau, and p-tau at threonine residue 181  [CSF samples were collected following international consensus recommendations  as previMA, USA) .We monitored a set of 22 proteotypic peptides corresponding to 10 proteins  using the previously described selected reaction monitoring (SRM) method . BrieflyAll statistical analyses were performed in R version 3.4.3 . We exclp = .20) and dDS  but lower in aDS . The male to female proportion was comparable across clinical groups (p = .45). The level of ID in the adults with DS was classified as either mild/moderate (78% of cases) or severe/profound (22% of cases), a proportion that was comparable across clinical groups (p = .37). Cognitive tests were restricted to individuals with mild or moderate intellectual disability. As would be expected, cognitive scores were sequentially lower in pDS  and dDS  compared to aDS. As previously reported [42:40 ratio  was lower in all DS groups compared to controls, while mean CSF p-tau and t-tau levels were higher in pDS and dDS compared to controls . CSF NFL levels were available for DS only and were elevated in pDS and dDS compared to aDS (p < .00001).Table r = .70 to .96, n = 78-80, p < .0001). They also all correlated with NPTX2 . VAMP-2 showed the weakest correlation with all other proteins . In controls, all proteins showed weaker pair-wise correlations than in the DS group, although NPTX2, Neurexin-3A, Thy-1, Neurexin-2A, Calysntenin-1, Neuroligin-2, GluA4, and Syntaxin-1B were moderately correlated in at least one pair-wise combination . VAMP-2 did not correlate with any other protein in controls . We took VAMP-2 forward for further analyses due to its relative independence from NPTX2.The synaptic panel analyzed here includes 7 synaptic proteins previously unpublished in this cohort  and their comparison to 2 synaptic proteins, NPTX2 and GluA4, previously reported in the same cohort . We firsp = .04). Mean CSF VAMP-2 intensities were lower in the aDS group compared to controls  and compared to the symptomatic group . CSF VAMP-2 intensities were comparable to controls in pDS  and dDS . This relative increase in CSF VAMP-2 at late AD stages in adults with DS is supported by Fig. r = .43, n = 79, p < .0001). This association was also observed in a linear regression analyses adjusting for degree of ID . Conversely, CSF VAMP-2 inversely correlated with age in controls . The control and DS regression lines for VAMP-2 were non-overlapping at the earliest age included in the study (22 years old) and did not intercept until the age of 42. CSF VAMP-2 was not associated with AD diagnosis when controlling for age p = .61).Figure 42:40 ratio  and directly correlated with p-tau,  and NFL . To determine whether low CSF VAMP-2 is related to AD biomarker positivity in asymptomatic DS, we compared CSF VAMP-2 SRM intensities in the aDS group stratified by positivity for CSF Aβ1-42 using our validated in-house cut-offs for sporadic AD. Compared to controls, CSF VAMP-2 SRM intensities were lower in individuals positive for CSF Aβ1-42  but not in individuals negative for CSF Aβ1-42 . Thus, low CSF VAMP-2 is related to AD biomarker positivity and changes over the course of AD and with age in adults with DS.Figure p = .31), pDS (p = .71), and dDS (p = .73). To determine whether neurodevelopmental factors may influence CSF VAMP-2 concentrations, we compared VAMP-2 across ID groups stratified by < 35 or 35+  and older (p = .87) age groups. Furthermore, CSF VAMP-2 SRM intensities were not associated with K-bit score  or when controlling for age . While Fig. r = − .32, p = .02) and delayed  recall in the mCRT test but not with CAMCOG scores . However, since ID had a greater impact on CAMCOG score than on mCRT in our previous study [t = − 4.04, p = .0002) and VAMP-2  were associated with CAMCOG score , while VAMP-2  but not ID  was associated with immediate recall in the mCRT test . We observed a similar association with delayed recall . Therefore, VAMP-2 was associated with both CAMCOG and mCRT score in adults with DS even when controlling for ID. VAMP-2 was not associated with any of the cognitive measures when age was included as a covariate .We next explored the relationship between CSF VAMP-2 and measures of intellectual and cognitive impairment in adults with DS. Mean CSF VAMP-2 SRM intensities were comparable across individuals with, mild, moderate, and severe intellectual disability Fig. a in aDS us study , we perfp = .03), Neuroligin-2 (p = .02), Neurexin-2A (p = .02), Neurexin-3A (p = .02), and Thy-1 (p = .03) were associated with ID in individuals aged < 35, suggesting some influence of neurodevelopmental factors on CSF concentrations of these proteins, albeit that the associations did not survive adjustment for multiple testing . The correlation of VAMP-2 with immediate recall (adj.p = .17) and association with CAMCOG (adj.p = .41) did not survive adjustment for multiple testing. Variables associated with at least one synaptic protein (adj.p < .05) in DS are shown in Fig. adj.p = .04) and age (adj.p = .0008) and was the best correlate of CSF Aβ42:40 (adj.p = .0001), CSF p-tau (adj.p < .0001), and CSF NFL (adj.p < .0001). On the other hand, NPTX2 was the best correlate of CSF Aβ1-42 , showed the greatest fold-change across all AD stages , and was the only synaptic protein to show changes in pDS . NFL and p-tau remain the best correlates of cognitive performance in this population and were not altered in the aDS group compared to controls .Finally, we compared these findings for VAMP-2 to the other synaptic panel proteins and to NPTX2 and applied a strict adjustment of p values to account for multiple testing. Calsyntenin-1  and did not converge until the age of 42. This finding suggests a distinct CSF profile of VAMP-2, and potentially a different mechanism of synaptic pruning, between healthy aging and the presence of AD pathology and/or triplication of chromosome 21. It is possible that the relatively lower CSF VAMP-2 levels in younger adults with DS compared to controls is a result of reduced VAMP-2 expression from birth due to neurodevelopmental factors. However, several lines of evidence suggest that CSF VAMP-2 levels change as a function of AD as opposed to ID: (a) the association between CSF VAMP-2 and age was independent of ID, (b) CSF VAMP-2 was comparable to controls in adults with DS negative for the CSF amyloid marker, (c) the findings reported here for this genetically determined form of AD replicate the nonlinear CSF profile of the 8 synaptic panel proteins previously reported across disease stages in sporadic AD , and (d)We have previously evaluated NPTX2 as a synaptic marker in the same cohort reported here . SimilarA previous study reported that CSF NPTX2 correlates well CSF levels of two other synaptic proteins, SNAP-25 and neurogranin in sporadic AD CSF . In thisThe novelty of VAMP-2 is that it was the only synaptic protein evaluated here to correlate with age (a surrogate measure of disease progression in DS) and mCRT in the DS population. Similar to our previous study, we found that ID had a greater impact on CAMCOG score than on mCRT  such thaWhile DS and autosomal dominant cohorts with available CSF are scarce, further replication of the findings reported here in independent genetic AD and DS cohorts would be valuable. A limitation of this study is the cross-sectional design, particularly in the analysis of cognitive decline. Longitudinal studies are needed to fully establish the prognostic value of VAMP-2 in DS cohorts.NFL remains the best CSF correlate of cognition in this population and this work opens the door to future studies exploring the prognostic capacity of CSF VAMP-2 in adults with DS and sporadic AD. The data reported in this manuscript show proof-of-concept for CSF VAMP-2 as a potential marker of synapse degeneration that correlates with CSF AD and axonal degeneration markers and cognitive performance. Whether VAMP-2 could be a useful addition to NFL to specifically monitor synapse engagement and therapeutic response, particularly in AD clinical trials would be an interesting avenue worth pursuing; as anti-tau and anti-Aβ are common therapies, there is a need for an alternative surrogate measure of cognitive performance not directly affected by the drug. An ELISA-based immunoassay to facilitate the quantification of VAMP-2 in patient CSF is in development. Moreover, as VAMP-2 is also detectable in blood , whether


Handwashing with soap has received considerable attention due to its importance in the prevention and interruption of the transmission of diseases. Regardless of the positive effects of handwashing with soap, developing countries still have a low rate of handwashing.The study aimed to determine the individual, household and community-level factors associated with handwashing behavior among households in EswatiniUsing the Eswatini Multiple Indicator Cluster Survey conducted in 2014, a secondary analysis was done of the households surveyed. A total of 1,520 households nested in communities with complete data on handwashing practices were included in the analysis. Univariate, bivariate analysis and multivariate multilevel logistic regression were used to establish the factors that were associated with handwashing behavior.The prevalence of handwashing among households was 56% in 2014. Households whose heads were aged 35–54 and 55 years and older were more likely to practice handwashing ; and  compared to those aged 15–34 years. Households with a pit latrine or no toilet facility at all, were less likely to practice handwashing ;  respectively compared to those with a flush toilet. Region of residence was a community-level variable associated with lower odds of handwashing, with those from the Hhohho  and Manzini region  compared to Lubombo region. Households from communities where access to mass media was high were more likely to practice handwashing  compared to those from communities where access to mass media was lowHouseholds headed by young adults, with pit latrine or no toilet facility at all and lived in the Hhohho and Manzini regions and with low access to mass media, should be targeted for interventions aimed at improving handwashing practices. Globally, approximately 6.3 million children died, and over 500 thousand deaths were due to diarrhea in 2013Handwashing with soap (HWWS) has received considerable attention due to its importance in the prevention and interruption of transmission of viruses and bacteria causing diarrhea, common flue, ARIs, pneumonia, and (COVID-19), hence reducing the magnitude of diseasesThe practice of HWWS is founded on the global agenda of Sustainable Development Goals (SDGs), especially target 6.2 of goal 6, which aims to achieve access to adequate and equitable sanitation and hygiene for all and end open defecation, paying special attention to the needs of women and girls and those in vulnerable situations by 2030Even though several studies have been conducted in other countries to investigate the factors associated with handwashing behavior, they applied a single-level analysisRegardless of the importance of HWWS as a public health intervention, few studies have been conducted in Sub-Saharan Africa (SSA) that investigated the correlates of handwashing behavior let alone the application of multilevel approaches. In Eswatini, only descriptive reports The study adopted a secondary cross-sectional analysis using the 2014 Eswatini Multiple Indicator Cluster Survey (EMICS). The MICS is an international initiative that is nationally representative of the population and is utilized to assist countries in monitoring and tracking indicators on children, women, and men's health and development. MICS uses standardized structured survey questionnaires administered through face to face interviews at the household levelThe 2014 EMICS adopted a two-staged sampling strategy that involved systematic sampling of enumeration areas (EAs) and randomly selecting 15 households from each EAIn the survey, there were 5,211 households selected; however, 4,865 (93.4%) completed the interview. Out of the households that were interviewed, only 1,809 had a place for handwashing observed during the household interview; 1,533 had water available in the household, and 272 did not have water in the household. The total number of households included in the analysis were 1,520.Outcome variable: The study outcome variable is handwashing behavior. The household was deemed to be practicing handwashing if it had a place for handwashing, and water was available with soap/ detergent at the specific place coded as 1. In contrast, those that had a place for washing hands but without water and soap or detergent in the particular place were coded 0. These were assessed through rapid observation during the household interview and is recommended to be the most efficient methods in community surveys Explanatory variables: To understand the determinants of handwashing behavior, several individual, households, and community-level factors were utilizedStata 15 was used for the analysis. To account for the complex MICS design, weights were applied through the svy command. Frequencies and percentages were used to describe the study sample. Bivariate analysis with chi-square test statistics was performed to test the independence of the distribution between the independent variables and handwashing behavior. The vector inflation factor (VIF) was used to test for strongly correlated explanatory factors, and no factors were strongly correlated to each other (see Table S1). The command melogit was used to fit the multilevel logistic regression to identify the factors that were significantly associated with handwashing behavior. Multilevel analysis was considered appropriate to account for the hierarchical nature of the MICS and to be able to estimate community-level effects on the outcome variableIn the final model , at the At the household level, the toilet facility was associated with handwashing behavior. Consistent with model 3, in the final model, households with a pit latrine and no toilet facility at all were less likely to practice handwashing, ;  respectively compared to households with a flush toilet, holding other factors constant in the model.At the community level, even after controlling for the individual and household level factors, households from Hhohho and Manzini regions were less likely to practice handwashing, , and  respectively compared to households located in the Lubombo region. In the final model, households from communities where access to mass media was high were more likely to practice handwashing,  compared to those from communities where access to mass media was low.The random-effects results were also reported . There wThe study found that overall the practice of handwashing was 56% in Eswatini and that several factors were associated with handwashing behavior. The findings showed a 9 points increase in the prevalence of handwashing with soap in 2014 when compared to 47% in 2010 Evidence from the literature suggests that the older the household head, the more likely the practice of handwashing in the householdEvidence in the literature reported a positive relationship between education and handwashing behaviorSeveral studies found a significant association between the type of toilet facility in the household and handwashing behaviorThis study also found that households from the Hhohho and Manzini regions were less likely to practice handwashing when compared to the Lubombo region. The higher odds of handwashing behavior in the Lubombo region are surprising when compared to the other regions, given that it is the least developed, followed by ShiselweniExposure to mass media has been found to play a significant role in the adoption of a positive attitude towards handwashing behavior. In a study done in Kenya, media exposure was associated with the formation of hygiene behaviors, including handwashingThere are some limitations associated with the study. First: social desirability bias could affect the findings since the data was self-reported This study confirms the relationship between age of the household head, type of toilet facility, region of residence, the proportion of households with access to mass media in the community, and handwashing practices. To achieve remarkable progress on handwashing practices by 2030, intervention should target households headed by young adults, with pit latrines or no toilet facility at all, and live in the Hhohho and Manzini region. This study highlights that individual, household, and community-level factors have a significant role in determining handwashing behavior among households. The study results suggest that handwashing behavior could be improved if interventions consider the individual, household, and community factors that influence handwashing. Therefore, interventions should be integrative of the individual, household and community-based approaches especially in disadvantaged communities