Partially cystic thyroid nodules (PCTNs) are a kind of thyroid nodule with both solid and cystic components, and are usually misdiagnosed as benign nodules. The objective of this study was to determine the ultrasound (US) characterizations with a TIRADS Grade-4a or higher partially cystic thyroid nodules (PCTNs) which are associated with being malignant or benign. In this study, 133 PCTNs with a TIRADS Grade-4a or higher were enrolled; 83 were malignant and 50 were benign. TI-RADS classification can detect malignant PCTNs, and its sensitivity, specificity, positive predictive value, negative predictive value, and accuracy are 39.8%, 96.0%, 94.3%, 49.0%, and 60.9%, respectively. Univariate analyses revealed that nodule shape, margin, and structure were related to PCTNs’ being benign and malignant, among which nodules taller-than-wide, with an irregular shape, non-smooth margin, eccentric sharp angle, or edge sharp angle were significantly associated with malignancy while ovoid to round nodules, smooth margins, multiple separation, and eccentric obtuse angle structures were significantly associated with a benign nature. For the solid part of PCTNs, its free margin, echo, and calcification are related to benign and malignant PCTNs. Among them, the free margin of the solid part is non-smooth, hypoechoic, and microcalcification, which are related to malignant PCTNs, while the free margin of the solid part is smooth, isoechoic, macrocalcification, non-calcification and are related to benign PCTNs. Calcification of solid part and free margin are important factors for predicting malignant PCTNs. In addition, nodules’ composition, blood flow signal, and other factors had nothing to do with PCTNs’ being benign or malignant. In the multivariate Logistic regression analysis, solid part calcification  and free margin  were revealed to be the strongest independent predictors for malignancy (P<0.05). Our study indicated that understanding the ultrasound characteristics of malignant PCTNs, to avoid misdiagnosed PCTNs patients, is important to make a precise diagnosis and prognosis of PCTNs. Partially cystic thyroid nodules (PCTNs) are a kind of thyroid nodule with both solid and cystic components, accounting for approximately 20.9% of thyroid nodules . Since mFine-needle aspiration biopsy (FNAB) is a reliable and cost-efficient method to evaluate thyroid nodules, with an overall accuracy of 98% and a false-negative rate below 5%. Current guidelines in China only consider the size (1.0-1.5 cm) as a criterion for FNAB, regardless of the cystic portion . SimilarThyroid ultrasonography(US) is an essential diagnostic technique for assessing the malignancy risk of thyroid nodules , 7, but The study was approved by the Ethical Committee of the First Affiliated Hospital of Zhengzhou University in China and was performed in accordance with the Declaration of Helsinki for human studies. The requirement of informed consent from human subjects is sometimes waived by institutional review boards (IRBs) for protocols that include a retrospective review of images acquired for clinical diagnostic purposes. From January 2016 to January 2020, 133 PCTNs patients who underwent thyroidectomy surgery were retrospectively enrolled in this case-control study. The inclusion criteria were: 1) patients with mixed echoic thyroid nodules that were confirmed by our central ultrasound examination before surgery; 2) no invasive procedure such as thyroid surgery or FNA was previously performed; and 3) all PCTNs were suspicious for malignancy (preoperative TIRADS grade 4a and above) and underwent surgical resection. In addition, the exclusion criteria were as follows: 1) without PCTNs pathologic examinations after surgery and 2) the color Doppler report was not checked by the chief physician. Finally, 133 PCTNs patients were included in this study; there were 59 males and 74 females, ranging in age from 9 to 71 years old, with an average age of 46.32 ± 12.27 years. The longest diameter of the nodules is 9 to 48mm. According to Chinese Thyroid Imaging Reporting and Data System( C-TIRADS)  connected to an 8-14 MHz linear transducer (PLT 1202S probe). Patients took the supine position to fully expose the neck. Ultrasonography was performed by two attending physicians in the same medical group with more than 8 years of experience in thyroid ultrasonography (>2000 cases/year), and the results were checked by one chief physician who had 24 years of experience in thyroid ultrasonography (>4000 cases/year) to prevent any artificial bias and ensure optimized image quality. The depth, gain, and focus of scanning were adjusted according to physical status after locating the thyroid nodules, and then clear 2D images were displayed and stored. Then, color Doppler ultrasound was applied to inspect the thyroid nodules and surrounding tissues, so as to determine the blood flow inside the foci and the surrounding tissues. The range was set to 9.8 cm/s, the frame rate was 8 frames/s, the color gain was 50 dB, and the filter was 6.Images were acquired using a Toshiba AplioUS images in our study were retrospectively evaluated by two experienced physicians (each with more than 8 years of experience in thyroid ultrasonography) who were blinded to the other imaging results and clinical as well as histopathological data. Two physicians performed the following US finding for each entire nodule: size (the longest diameter), internal content (predominantly solid vs. predominant cystic), shape , margin (smooth vs. non-smooth), and structure . The internal solid portion inspection includes: free margin(smooth vs. non-smooth), echo , calcification , and blood flow signal level 0, 1, 2, 3). The ultrasound results report uses the 2020 Chinese thyroid nodule ultrasound malignant tumor risk stratification guideline C-TIRADS recommended term . The ultFigure 1All analyses were conducted using SPSS version 24.0 . Normally distributed enumeration data were expressed as cases and percentages (%), measurement data were expressed as mean ± standard deviation  Table 2.Multivariate Logistic regression analysis showed Table 3 TI-RADS is thyroid imaging reporting and data system for ultrasonography, mainly to classify the benign and malignant degree of thyroid nodules. Among them, the malignancy of TI-RADS 4a may be about 2-10%, TI-RADS 4b is up to 10-50%, TI-RADS 4c is 50-90% and TI-RADS 5 is >90% . PCTNs aAccording to the analysis of the results of ultrasound images in this study, the solid component in some cystic nodules with eccentric sharp angle, edge sharp angle, non-smooth free edge, nodule hypoechoic, or microcalcification was associated with malignant PCTNs (P<0.01). A related report found that, compared with benign nodular goiters, PCTNs more frequently presented with calcification, hypoechogenicity of the solid part, hypoenhancement, heterogeneous enhancement, centrifugal perfusion, peak intensity index <1, time to peak index ≥1, and area under the curve index <1 on preoperative US and CEUS . A recenA domestic study in 2014 showed that the free margin of the solid part of PCTNs was divided into two groups: smooth and non-smooth. In this study, our findings are consistent with the above conclusions. We can explain this phenomenon by the fact that the tumor growth is heterogeneous, invasive, migratory, and has no complete capsule formation.In most TI-RADS grading systems, the proportion of solid parts in the nodule and the blood flow signal are used as independent predictors in the risk assessment of thyroid nodules . HoweverMultivariate Logistic regression analysis showed that PCTNs solid part calcification and free margin were important factors for predicting malignant PCTNs, of which the non-smooth free margin and microcalcification in the solid part were poor factors for predicting malignant PCTNs, showing the key to predicting benign and malignant PCTNs. It is the observation of the general shape of the nodule(free margin) and the calcification of the solid part.A suspicious FNAB is the most common indication for thyroidectomy. However, its reliability in large nodules is not clear. Osman described a lower sensitivity 55.5-71.4%) and higher false-negative rate (6.7-9.7%) in nodules larger than 4 cm, and only 17.3% patients were detected as having cancer , 14; mea5.5-71.4%Not all surgeons are equally experienced in performing thyroid surgery. Studies have shown that surgeons who perform a large number of thyroidectomies each year have fewer complications than those who perform fewer operations. Such data do not mean that low-volume surgeons should not perform these operations. However, before undertaking a difficult operation, the surgeon should consider whether that patient might be better served by referral to a specialized institution for care. Meanwhile, in solitary small low-risk PTCNs, hemithyroidectomy, thanks to its lower complication rates, is still the safest standard of care, avoiding unnecessary morbidity .This study has some limitations. First, the retrospective assessment of static ultrasound images of PCTNs in this study was less accurate than prospective dynamic ultrasound images. Secondly, the number of benign cases in this study is small, as patients are usually recommended surgical treatment when the TI-RADS grade is 4 or above in the ultrasound diagnosis. For nodules below grade 4, ultrasound follow-up or ultrasound-guided fine-needle aspiration is often recommended. Since this study did not use all PCTNs as candidates and only selected patients with surgical and pathological results, selection bias is inevitable for patients with only ultrasound results or puncture results. In addition, no hyperechoic nodules were found in this study, which may be a characteristic feature of benign PCTNs nodules.TI-RADS can detect malignant PCTNs, and its sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate are 39.8%, 96.0%, 94.3%, 49.0%, and 60.9%, respectively. For PCTNs, nodule shape, margin, and structure were associated with benign and malignant PCTNs (P < 0.01). Among them, nodules taller-than-wide, with an irregular shape, non-smooth margin, eccentric sharp angle, and edge sharp angle were associated with malignant PCTNs, while ovoid to round nodules, smooth margins, multiple separation, and eccentric obtuse angle structures were associated with benign PCTNs. For the solid part of PCTNs, its free margin, echo, and calcification were related to benign and malignant PCTNs. Among them, the free margin of the solid part being non-smooth, hypoechoic, and microcalcification are related to malignant PCTNs, while the free margin of the solid part being smooth, isoechoic, macrocalcification, and non-calcification are related to benign PCTNs. The calcification of solid part and free margin are important factors for predicting malignant PCTNs. In addition, nodules with predominant cystic components and low blood flow signal levels cannot be ignored in clinical diagnosis and treatment. These findings are helpful for clinicians to more accurately identify benign and malignant PCTNs, avoid missing diagnosis and misdiagnosis of PCTNs patients, and are crucial for the clinical treatment and prognosis of PCTNs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Review Committee of First Affiliated Hospital of Zhengzhou University. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.CW, YY, SW and KJ designed the study. CW, DZ and CL collected the clinical data, ultrasonography images and histopathological/cytopathology results. YL and MZ performed the statistical analysis. CW, ZY and ZW drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Nature Science Foundation of China (No. 81871962), National Scientific Center Project (No. 62088101) and the Industry-University-Research Innovation Fund in Ministry of Education of the People’s Republic of China (No.2018A01013).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this study, the effect of several agronomical practices on the chemical composition of hemp inflorescences, a potential novel food that needs to be further studied, was observed. Here, the case study of inflorescences from Ferimon cultivars is discussed and submitted to different agronomical practices (irrigation and fertilizers) in different years, and the inflorescences harvested in different periods were analyzed by a multimethodological approach. Targeted and untargeted methodologies allowed cannabinoids, total phenolic content, metabolite profile and antioxidant activity to be determined. The biomass and inflorescence yields were also reported. The whole data set was submitted to ANOVA-simultaneous component analysis. The statistic results allowed us to observe that irrigation was responsible for the (–)-Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) increment. THC, cannabichromene (CBC), cannabigerol (CBG), succinate, and fructose resulted as higher in full female flowering than in the period of seed maturity. On the other hand, nitrogen supplementation led to an increase of iso-leucine, valine, and threonine. The obtained results underlined both the potential food application of hemp inflorescences, due to the rich chemical profile, and the strong effect of agronomical practices, mainly irrigation and harvesting, on the qualitative and quantitative characteristics of its metabolite profile. Cannabis sativa L.) is an herbaceous, annual plant of eastern Asian origin, whose cultivation was forbidden in many countries for a long time due to the presence of tetrahydrocannabinol (THC), a psychoactive secondary metabolite contained in different parts of the plant. Recently, the European Union reintroduced the cultivation of some genotypes of Cannabis sativa L., also called industrial hemp, with a THC content lower than 0.2% w/w for fiber, seed, and inflorescence production [Hemp  and phosphorus (P) fertilizers play an important role in crop growth and development . The knoectively . Moreoveectively ,26 has bIn the Mediterranean environment, another important agronomic factor for industrial hemp production is water availability, since during the cropping period, the climate is generally characterized by drought and high temperatures. Therefore, for the crop in a semi-arid Mediterranean environment, watering is often needed. Cosentino et al.  have sugThe harvesting period also plays a pivotal role in the chemical profile of inflorescences. Ingallina et al.  have fouTherefore, up to now, the research has been focused on the study of individual agronomical practices and their effect on cannabinoid content. A more complete chemical profile of hemp inflorescences has been reported in a few studies where only the harvesting period effect has been investigated ,13.In this paper, the combined effect of nitrogen and phosphorus fertilizer, irrigation, harvesting time and cultivation year on inflorescence chemical profile was investigated considering as a case study the monoecious Ferimon cultivar. A multimethodological approach based on both targeted  and untargeted (NMR) methodologies and chemometric analysis was applied.2O) 99.97 atom% of deuterium and 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) were purchased from Euriso-Top . Magnesium oxide (MgO), potassium phosphate monobasic (KH2PO4), and potassium phosphate dibasic (K2HPO4) 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu reagent, gallic acid (GA), and sodium carbonate (Na2CO3) were purchased from Merck Life Science .Gradient grade water, acetonitrile, chloroform, methanol, and formic acid were purchased from Merck Life Science . Cannabinoid reference standards dissolved in methanol, namely, cannabidivarin , cannabigerol , cannabidiol , cannabidiolic acid , cannabinol , (–)-Δ9-tetrahydrocannabinol ((–)-Δ9-THC, 1 mg/mL), Δ9-tetrahydrocannabinolic acid ((–)-Δ9-THCA, 1 mg/mL), (–)-Δ8-tetrahydrocannabinol ((–)-Δ8-THC, 1 mg/mL), and cannabichromene  with purity ≥ 99%, were purchased from Merck Life Science . Deuterated water (D−1 of dry soil (Kjeldahl method), available P2O5 41 mg kg−1, and exchangeable K2O 534 mg kg−1. The climate of the experimental area is typically Mediterranean, characterized by a dry and hot summer and humid and mild winter. Weather data  were recorded daily from an automatic meteorological station located 100 m from the experimental fields. Meteorological data are reported at 10-day intervals for the period in which the experiment was conducted . The soil is volcanic with the following main characteristics at 0–30 cm depth: 11.2% clay, 14.7% silt, 74.1% sand, pH 6.7 , organic matter 1.2% (Lotti method), total N 0.93 g kgonducted . As expeonducted . As a co2O5 ha−1 as triple superphosphate. After applying the fertilizer, the soil was disked twice for seedbed preparation. The hemp variety used in this study was Ferimon, a French monoecious cultivar listed in the EU database of registered hemp varieties and commonly used for seed production, that also showed good performance for inflorescence yield in the Mediterranean environment of Central Italy [−1. In both years, in order to avoid emergence failure, the field was irrigated just after sowing by sprinkler irrigation. Ten days after emergence, the hemp seedlings were thinned manually in order to reach the density of 80 plants m−2, in accordance with the density used in a similar environment in recent research on hemp cultivation [−1 as ammonium nitrate, split into three doses applied at 15, 30 and 45 days after crop emergence . Following each N fertilization, a manual hoe was applied to incorporate the N fertilizer and to control inter-row weeds. Potassium fertilizer was not applied due to the high levels present in the soil. Irrigation water was supplied in the irrigated plots by means of drip irrigation placing on the soil surface one pipeline per row immediately after hemp seedling thinning. The amount of water input was determined by evapotranspiration estimated by class A pan evaporimeter and converted by crop coefficients, returning 90% of the evapotranspirated water from the crop. The hemp inflorescences were harvested twice: at full female flowering stage (A), and at seed maturity stage (B). In 2017, the following experimental treatments were applied in factorial combination: (i) one irrigation regime (irrigated crop); (ii) two doses of phosphorus (0 and 90 kg P2O5 ha−1); (iii) two doses of N fertilization (0 and 150 kg of N ha−1). In 2018, further variables were considered, by applying the following treatments: (i) two irrigation regimes (irrigated and not-irrigated crop); (ii) three doses of phosphorus ; and (iii) three doses of N fertilization , One week before hemp sowing, where foreseen, the plots were fertilized with phosphorus (P) at a dose of 45 or 90 kg Pal Italy . Industrtivation . Where f2 (9 m × 15 m), the subplot size was 67.5 m2 (4.5 m × 15 m), and the sub-sub-plot size was 22.5 m2 (4.5 m × 5 m). In both years, the main plots in the field trial were separated by 8 m wide alleys for equipment operation.In 2017, the experimental design of the field was a split-plot, where the main plots were represented by the phosphorus fertilization, and the sub-plots were the N fertilization. In 2018, the experimental design of the field was a split-split-plot, where the main plots were represented by the irrigation regimes, the sub-plots were the phosphorus fertilization, and the sub-sub-plots were the N fertilization. The main plot size was 135 m2 area, were manually cut just above the soil surface. The aboveground biomass was split into two fractions: stems and inflorescence by cutting the upper part of the plant stem, and the stem ramifications when present, at the insertion level of the first basal flower of the inflorescence. Both flowers and floral leaves were included in the inflorescence for determining the chemical profile, whereas the seeds present at the second inflorescence harvest were manually separated. The fresh material was immediately weighed, and a sub-sample was oven dried at 70 °C until constant weight to determine the dry matter content of stems, inflorescences, and seeds when present. The remaining part of the inflorescences were pulverized with liquid nitrogen by using mortar and pestle and then was preserved at a temperature of −80 °C until subsequent laboratory analyses.Immediately after hemp seedling thinning, a 2.0 m central row was randomly selected to carry out phenological observations, that were performed daily when the flowering stage was approaching. Two inflorescence harvests were carried out during the hemp reproductive period: the first one was carried out at full female flowering (50% of the bracts formed), encoded as phase 2302, whereas the second harvest occurred at seed maturity (50% of seeds hard), encoded as phase 2306 . At eachIn order to have information relevant for the economic sustainability, the biomass and inflorescences yield, inflorescences/biomass ratio (inf/bio) have been measured and reported.Fresh inflorescences were dried at 70 °C for 48 h before extraction. After drying, 100 mg of inflorescences were extracted with 4 mL of methanol in an ultrasonic bath for 30 min. The residual pellet was then extracted other two times. For each extract, an aliquot of 1 mL was filtered with a 0.45 μm PTFE membrane and finally analyzed.Analyses were carried out by using an Ultimate 3000 ultra-high-performance liquid chromatography (UHPLC) , with a binary gradient system, an automatic injector, a thermostatic column compartment, and a diode array detector. The system was controlled by Chromeleon Chromatography Data System software . All separations were performed by using a Titan C 18 column ) with a mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The used elution conditions were the same previously described .w/w (g for 100 g of dried inflorescence) ± SD (standard deviation). Three replications were made for each sample.Cannabinoid concentrations were expressed as % 2O, containing a 1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) as an internal standard, and then transferred into a 5 mm NMR tube. NMR analyses were carried out on a Bruker AVANCE 600 spectrometer operating at the proton frequency of 600.13 MHz and equipped with a Bruker multinuclear z-gradient 5 mm probe head . 1H spectra were acquired at 28 °C using the following parameters: 256 transients, suppression of residual water signal (HDO) using a pre-saturation, 5 s recycle delay, 45° pulse of 7.5 μs, and 32 K data points. Metabolites identification, see For the untargeted NMR analysis, Bligh–Dyer extraction procedure was applied, following the previously described conditions . The hyd2O 7:3 v/v solution. Samples were then shaken for 1 min, sonicated in an ultrasonic bath for 5 min, and finally centrifuged for 10 min at 2500× g. After supernatant removal, the residual pellet was extracted again using the same conditions. Both obtained supernatants were united to a final volume of 5 mL.The extraction of phenolic compounds was carried out as follows: 0.5 g of fresh hemp inflorescences were added to 2.5 mL of MeOH/H−1: a calibration curve was created, with a concentration range from 15 to 500 mg/L (R2 = 0.9925). The measurements were carried out in triplicate for each sample and reported as mean value ± SD.Total phenolics content (TPC) was determined using the Folin–Ciocâlteu method, optimized in a previous work for hemp inflorescences . TPC was50 value ± SD; EC50, corresponded to the extracts’ concentration providing 50% of radicals scavenging activity. Gallic acid dissolved in methanol, ranged between 1 and 100 mg/L, was used as reference compound.The antioxidant activity of hemp hydroalcoholic extracts was determined by applying DPPH and TEAC assays. The free radical scavenging activity of fresh hemp inflorescence extracts was evaluated by means of DPPH assay, according to a previously reported method . The sca2 = 0.9963).The Trolox equivalent antioxidant capacity (TEAC) of hemp inflorescence extracts was estimated by the ABTS radical scavenging assay, according to Omono et al. , with soThe assays were carried out in triplicate for each sample and reported as mean value ± SD.To evaluate the effect of the different cultivation conditions on the total defined chemical profile of hemp inflorescences, ANOVA-simultaneous component analysis (ASCA) was used. ASCA  is a mulIf a term is found to be statistically significant, interpretation of the observed effect in terms of the multivariate profile recorded is made by SCA of the corresponding effect matrix. As explained above, under the constraints of ANOVA, the use of SCA is equivalent to a standard PCA on the matrix.Since the effect matrices are only made of the mean profiles of the different factor/interaction levels, for the sake of a better interpretation, it is common practice, after the SCA model is calculated, to also project onto the resulting component the residuals, so as to have an idea of the expected within-level variability .The obtained results, combined with the application of chemometric analysis, allowed us to observe the effect of the considered agronomical variables  on the quali-quantitative features of the Ferimon metabolite profile.The chemical profile of Ferimon inflorescences was investigated by applying both targeted  analyses focused on cannabinoids and total phenols determination and NMR untargeted methodology to obtain the metabolite profile. The results will be discussed separately.Cannabis sativa L. methanolic extracts allowed us to quantify Δ9-THC, CBD, Δ9-THCA, CBDA, CBG, and CBC. The concentrations, expressed as % w/w of dry matter ± SD, are reported in Cannabinoids. The UHPLC-DAD analysis of −1 of N) have shown a different cannabinoids profile with the presence of CBDV and CBN not detected here. The cannabinoid profile of the Ferimon hemp cultivar grown in Slovenia in 2017  [All the analyzed samples were characterized by a THC content below the legal limit, whereas CBD turned out to be the most abundant cannabinoid. In a previous study , the infeported)  has showNMR metabolite profile. The NMR metabolomic profile of hydroalcoholic Bligh–Dyer inflorescence extracts was obtained using literature data  relativeTotal phenolic content (TPC) and antioxidant activity. The TPC and the antioxidant activity of Ferimon inflorescence were determined by means of DPPH and ABTS assays, see To take into account the whole set of data, ASCA analysis was carried out at first on data matrices relative to 2017 and 2018 separately, and successively on the joint data. The comparison between continuous irrigation and absence of irrigation was carried out only in 2018.2O5 ha−1), and N dose (0 and 150 kg of N ha−1). ASCA applied to the matrix of experimental data, underlined that the largest part of the variance was due to the effect of harvesting (40.37%), followed by N dose (10.08%) and P dose (6.61%) factors. Permutation testing used to evaluate the significance of each of the design terms (main effects and interactions) showed that only the effect of harvesting period and N were statistically significant (p < 0.05). However, the effect of P fertilization was only slightly above the significance threshold (p = 0.0558). Accordingly, only the effect matrices for harvesting time and N fertilization were subjected to the successive SCA analysis.The 2017 year-data, relative only to irrigated fields, were acquired as a result of three controlled factors, each factor at two levels: harvesting period  and seed maturity stage (B)), P dose , Pro (Proline), Myo-inositol, Malate, TPC, THC, and CBC, whereas stage B samples were richer in Gln (Glutamine), Asp (Aspartate), Asn (Asparagine), Formate, Trigonelline, Succinate, and DPPH, see scores and loadings of ASCA displayed on The scores plot on the SC component shows that stages A and B correspond to negative and positive scores, respectively. This information is fundamental to interpret the corresponding loadings plot, where the variables that significantly contribute to the component are highlighted as red bars. Indeed, all the variables with positive loadings are higher in stage B samples, whereas the ones with negative loadings are higher in stage A.Moreover, an increase of biomass and inflorescences yield was observed from stage A to stage B, −1 dose of N caused an increase of several amino acids, namely Ile (Isoleucine), Phe , Thr (Threonine), Pro, and Trp (Tryptophan). Among these, Ile and Phe were the most statistically significant increased, as shown by scores and loadings plot of the SC component extracted from the SCA analysis regarding the effect of N fertilization, −1 and 150 kg ha−1 correspond to positive and negative scores, respectively. On the other hand, in the loadings plot, the red error bars indicate the 95% confidence interval of the coefficients, estimated by bootstrapping. On the contrary, no statistically significant effect of 0 kg ha−1 N were observed, although an increase of GABA (γ-aminobutyrate), Sucrose, and CBDA was observed.Fertilization. The 150 kg haNote that high N dosed caused an increase of both biomass and inflorescences yield, see 2O5 ha−1), N dose , irrigation (irrigated or not), and harvesting period  and seed maturity stage (B)). ASCA applied to the matrix of experimental data allowed us to observe that the largest part of the variance was due to the effect of irrigation (23.95%), followed by harvesting (21.76%), N fertilization (4.27%) and P dose (3.14%). On the other hand, permutation testing evidenced that all the design terms (main effects and interactions) are statistically significant (p < 0.05).The 2018 year-data were acquired as a result of four controlled factors: P dose , GABA, citrate, Asn, choline, fructose, malate, sucrose, formate, trigonelline, succinate, and glucose.Additionally, the irrigation practice strongly affected the biomass and inflorescences yield. The absence of irrigation caused a strong stress condition in plants, thus reducing biomass and inflorescences at least of three times, see −1 is observed. When this fertilizer was applied, a significant increment of Ile and Thr was observed. Additionally, Val, Pro, GABA, Asp, Myo-inositol, Formate, and GABA also increased, as shown by scores and loadings plot of the SC component extracted from the SCA. On the contrary, high supplementation of N caused a decrease of some cannabinoids, namely THC, THCA, and CBD, and also Phe, Trp, succinate, and TPC. Among these metabolites, THC was subjected to the most statistically significant decrease, see −1 had the same effect of the higher 150 kg ha−1 dose, thus giving helpful information that can be used for reducing fertilizer waste [Fertilization. The inspection of the scores plot A indicater waste .Although P fertilization did not appear to have a significant influence on the inflorescences chemical profile, an increase of Phe was observed when this fertilizer was used.−1) was used. On the contrary, when no irrigation was supplied, the use of high N doses caused a reduction of biomass and inflorescences yield, since high N dosages enhance the plant stress condition in not-irrigated plants. Similarly to what was observed for the chemical profile variations, P fertilization did not appear to influence the biomass and inflorescences yield.N fertilization strongly affected hemp yield, with the behavior being connected with the presence or the absence of irrigation. In particular, in irrigated plants, the use of high N doses caused an increase of both biomass and inflorescences yield. These data were confirmed by literature data where high dosages of N have caused an increase of hemp biomass yield ,17,18. M2O5 ha−1), N dose (0 and 150 kg of N ha−1), harvesting (A and B) and year (2017 and 2018). All data were referred to irrigated crops. ASCA applied to the matrix of experimental data underlined that the largest part of the variance was due to the effect of year (48.59%), followed by that of harvesting (14.16%), N dose (3.79%), and P dose (2.08%). On the other hand, permutation testing evidenced that all the design terms (main effects and interactions) are statistically significant (p < 0.05).The cultivation year represents an important factor that can strongly affect the chemical profile of food and vegetable matrices. Additionally, in this case, comparing the data obtained in 2017 and 2018, it was possible to observe a clear change of the chemical profile of hemp inflorescences. Interestingly some differences were observed in the cannabinoids profile, with THCA and CBG not being detected in inflorescences grown in 2017. The relevant effect of cultivation year was demonstrated by ASCA carried out on the joint data, by considering four factors: P dose  ratio with respect to those calculated in stage A. This data suggested that the biomass increase observed in stage B was mainly due to a major inflorescences production, as also demonstrated by the inf/bio increase.The reported results underlined that the agronomical practices as well as the cultivation year can strongly influence the chemical profile and the yield of hemp inflorescences. In particular, cultivation year and harvesting period and N fertilization significantly affected the inflorescences chemical profile. It is also important to note that irrigation and harvesting during the full female flowering period cause an increase of THC and CBD . On the other hand, the absence of irrigation caused an increase of several amino acids, sugars, and organic acids, as well as a reduction of biomass and inflorescences yield, by triggering a stress condition in the plant. Regarding the harvesting period, some metabolites such as glutamate, asparagine, aspartate, and formate increased during the time in both 2017 and 2018, as observed previously . Among fC. sativa plants; so it is auspicious to apply proper practices that do not cause an increase of this cannabinoid. Moreover, in the perspective of using hemp inflorescences as a novel food, the possibility of using proper agronomic practices to obtain a specific chemical profile can strongly contribute to enhance the potential nutritional value of this matrix.Up to now, the studies on the inflorescences reported in the literature are often not comparable since they were carried out in different conditions or more simply since the agronomical conditions are not reported. For this reason, it is important to improve the research in this field since, as demonstrated in this study, the agronomic practices can influence the chemical composition of hemp inflorescences. This issue can be firstly a notable factor if, for instance, THC concentration is considered. Indeed, its amount has to be below the legal limits to avoid the cultivation and the industrial uses of Considering that the food application of hemp inflorescences is a debated topic, this study can contribute to further underline the potential use of this matrix as a food or food ingredient, since several metabolites important from a nutritional and biological point of view were measured. Of course, these kinds of studies, based on chemical characterization, also have to be supported by safety, toxicity, and biological studies to further improve the total eligibility of hemp inflorescences as a novel food. The here reported results suggest the importance to choose the proper cultivation conditions to obtain products with a determined chemical composition, thus offering the possibility to have potential foods or food ingredients with a variable profile based on specific applications. The here reported investigation that involves chemical and agronomic aspects aims to propose a methodological approach that could be useful for future applications in this sector and also for the design of the experimental phase.


Therefore, a set of thirteen of them, randomly selected from the same screen-printing deposition, were laboratory tested, and the outcomes were statistically analyzed in order to assess their consistency. At first, the working temperature that maximized both the sensor sensitivity and response repeatability was identified. Then, the sensors were subjected to different gas concentrations and humidities at this optimal working temperature. It resulted in the (SnTiNb)O2 nanosensors detecting and discriminating CO concentrations as low as 1 ppm and at high humidity degrees (up to 40%) with high repeatability since the response relative standard error ranged from 0.8 to 3.3% for CO and from 3.6 to 5.4% for water vapor.Nowadays, most medical-diagnostic, environmental monitoring, etc. devices employ sensors whose fabrication reproducibility and response repeatability assessment are crucial. The former consists of large-scale sensor manufacture through a standardized process with almost identical morphology and behavior, while the latter consists of giving the same response upon repeating the same stimulus. The thermo-activated chemoresistive sensors, which change their conductance by interacting with the molecules composing the surrounding gas, are currently employed in many devices: in particular, thick-film (SnTiNb)O Metal-oxide (MOX) sensors are widely used for gas detection and employed in many application fields, such as automotive, environmental monitoring, agri-food, and diagnostic, thanks to their cheapness, high reliability, and great versatility. They present many advantages with respect to other gas sensor types  because of their ease-of-use, high sensitivity , low detection limit, low recovery and response times (up to some minutes), and long duration  without any maintenance. However, their poor selectivity and their performance worsening in the presence of interfering humidity are their highest limits. Nevertheless, the former can be overcome by combining them in an array, and the latter by mixing different MOX materials in order to minimize the water vapor influence with the sensor active material ,2. In or2 sensors (named STN in the following), whose sensing material was obtained through a standardized sol-gel synthesis and applied on the sensor substrate by using a screen printer  [In this paper, thirteen thermo-activated thick-film (SnTiNb)OMethods) , were raMethods) ,13). In Methods) ,15,16,17Methods) ,19. ThenMethods) , in partMethods)  and tumo2 solid-solution (Sn:Ti = 7:3) was obtained by the sol-gel technique from stoichiometric solutions of their metal-alkoxides precursors (Sn(II)-ethyl hexanoate 95% Aldrich, Ti-butoxide 97% Aldrich) [3 solution to the (Sn0.7Ti0.3)O2 one, while gently stirring the solution with a magnetic anchor; the appearance of a pale yellow color indicated that the colloid solution was ready to work as a solid solution precursor. After the filtration of the colloid material, the resulting xerogel was transformed into a binary oxide by calcining at 550 °C for 2 h under air in a muffle oven. This process lead to an almost (84.5%) pure white (Sn0.7Ti0.3)O2.O0.7Ti0.3)O2 by dropping NbBr5 (Sn:Nb = 20:1), previously dissolved in a minimal amount of methanol, in the Sn(II)-Ti(IV) stoichiometric solution. The further hydrolysis of all these materials (by using diluted HNO3) led to a white colloidal precursor; the following filtration and calcination (550 °C for 2 h in air) of the precursor held to a high purity, 85.5%, O2, (STN in the following).By using the sol-gel technique, Nb(V) was inserted into (SnThe STN paste was then deposited on the sensor substrate in form of a thick film (~30 μm) by using a screen-printing machine , Italy); the printing process is largely employed because of its low cost, fast preparation time, and deposition homogeneity ,22.Finally, the thick films were hardened, and all the impurities were removed by firing the deposited paste at 650 °C in an oven.The grain size homogeneity and the porosity of the sensor sensing film was assessed employing the scanning electron microscope (SEM) technique  up to 20.000× of magnitude.The STN sensors produced for this work were entirely assembled at the Sensor Laboratory of the Department of Physics and Earth Sciences of the University of Ferrara. Sensor active material consists of a nanostructured semiconductor thick (~30 μm) film in the shape of nanograins, capable of detecting gaseous chemicals at low concentrations . Sensors are typically composed of three independent parts: (i) the substrate, (ii) the sensing film, (iii) and the heater .The substrate  has the double function of an insulating layer and a mechanical support for the sensor. On its top, it hosts two interdigitated comb-shaped gold contacts, connecting the sensor to its signal transduction circuit. The sensing film (1 mm × 1 mm) was screen-printed on the substrate top see , electri2 and 20% O2) and ultra-pure CO (99.99%) technical gas were withdrawn from gas cylinders and diffused uniformly starting from the chamber center , where the temperature, humidity and air composition were kept constant.A computer-controlled mass-flow regulator, by means of a dedicated software, routed the desired gas at the chosen concentrations (from 0 to 100 ppm) to the sensor chamber through Teflon pipelines.In order to investigate the homogeneity, the porosity and the grain interconnection of the sensor film surface, the thirteen sensors were imaged by means of a scanning electron microscope (SEM) , followiAs first, it is necessary to find out the optimal WT for the thirteen sensors, at which nd so on . With thme graph , and theOnce ensured the strong similarity of the Arrhenius plots, the thirteen sensors were concurrently tested by exposing them to 10 ppm of CO. The sensors were heated to different increasing temperatures , and for each temperature, they were exposed to CO thrice.The average value and the standard errors of all the STN sensor responses were calculated for all the WTs tested . The optThe sensors, heated at the optimal WT of 500 °C , were suSince the MOX sensor responses are, in general, rather affected by humidity ,26,27,28To further calibrate the sensors, it is crucial to assess their behavior in the presence of a progressively increasing concentration of water vapor added to dry air. Each one of the thirteen sensors was subjected to a fixed RH  for three times (to assess the measurement repeatability) and these three responses, after subtracting the baseline , were averaged for each sensor. The latter was then averaged on the thirteen sensors for each one of the ten RHs and reported in the data .n = 39) was found to be To assess the suitability of STN sensors to be employed in biological applications, a sample of 10 cc of Dulbecco Modified Eagle Medium, was measured three times in a row by the thirteen sensors. The average response , the data confidence intervals for each RH  were calThe resulting confidence intervals were well separated in presence of dry air only, and they were even more separated in the presence of humidity, making this sensor type particularly suitable for these applications where water vapor is unavoidable . In any case, STN sensor behavior, as all the MOX sensors, is significantly affected by humidity, that, if present in the environment before the gas target injection, leads to a non-negligible sensor response decrease to the latter (here CO). This mechanism is related to an unavoidable adsorption on the sensor film surface of e states ,13.(2)H2O), because of their power law behavior (see below). Therefore, the average responses of the thirteen sensors to different CO concentrations and to absolute humidities  were plotted as a 3D-surface  at all the A, B, C, α, γ can be inferred by fitting the experimental points according to (7). The average STN responses to the diverse CO concentrations at different RH percentages, transformed in absolute humidity degree (AH) with (3), were employed to calculate a 3D fitting surface , diagrams ,32. Humie models . Consideductance  in the g surface .(7)z=a+Since the responses started from zero, then 3) but become less evident at high AH values. In conclusion, STN sensors demonstrate, once again, suitability for application involving environments with a relatively high humidity degree .STN responses to CO is higher at small AH concentration  and a very low CO concentration (up to 0.2 ppm) with almost negligible errors. The confidence intervals  confirmeFinally, these STN sensors can be completely characterized since the calibration surface constructed on the statistical outcomes computed on their responses can be represented by an exact mathematical equation.Given the excellent performance exhibited by STN sensors in the CO detection (despite the presence of high humidity), the future aim is focused on the investigation of the STN behavior subjected to other gas types and interferers. Moreover, the STN nanostructures could be modified (changing the manufacture process parameters) to optimally detect any desired target gas.


Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150–450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management. Capsicum sp.) is an important commercial spice crop cultivated throughout the globe [viz., C. annuum, C. frutescens, and C. chinense [C. annuum is the most widely cultivated chilli across the country. Although the chilli is grown in all the states and union territories of the country, the Northeastern Region (NER) of India, comprising of states of Manipur, Nagaland, Assam, Sikkim, and Mizoram, holds unique importance in terms of the cultivation of one of the hottest chilli landraces known as King Chilli / Umorok / Naga chilli (C. chinense). This, in addition to several other landraces of chilli like C. frutescens and C. annuum, are grown traditionally since for ages, their usage in different ethnic cuisines and ethno-pharmacological practices have contributed significantly to the agricultural economy and livelihood of farmers [Chilli  in the Indian subcontinent [Viral diseases have always been a major constraint to chilli production and productivity worldwide ,8,9,10. ontinent . The NERontinent ,14. A thontinent ,16, peppontinent , chilli ontinent ,14, cucuontinent ,19, chilontinent , and newontinent .Tospovirus in the family Tospoviridae, has emerged as a serious threat to chilli crops in India during the last 10 to 15 years, leading to considerable economic losses [The capsicum chlorosis orthotospovirus, a member of the genus c losses . The segc losses , and arec losses . Pepper c losses ,26 and ic losses  and causc losses .Potyvirus in the family Potyviridae, has a wide host range and causes serious yield losses to chilli production all over the globe. The ChiVMV has a 9.7 kb single-stranded positive-sense RNA genome with a 3′-terminal poly (A) tail, which is prominently distributed in the Asian region [C. chinense and C. annuum grown in the NER of India has been recorded during recent years [Macluravirus in the family Potyviridae, which naturally infects large cardamom was recorded to infect C. annuum , a solanaceous crop, for the first time, exhibiting the cross-species transmission capability of macluraviruses [Rhopalosiphum maidis, Brachycaudus helichrysi, and Myzus persicae in a non-persistent manner [Bemisia tabaci), which is abundant year-round in tropical and subtropical climates where a wide variety of hosts serve as reservoirs [Another important virus, the chilli veinal mottle virus, a member of the genus n region . Its occn region ,30. Its nt years . The Chint years ,32,33, ant years . The CMVnt years ,35,36. Int years ,37. The nt years ,39, withnt years . Large caviruses ,41. The t manner ,43. Its t manner ,44,45. Tt manner . The emet manner . The Chiservoirs .Capsicum spp. leads to the infection of multiple viruses in a single plant species [The increase in global international trade and the commercial movement of plant materials at a domestic and international scale during recent years, coupled with the climate disruptions, has led to the emergence and re-emergence of diverse viruses in chilli crop, which were previously either not reported or not present in the geographical areas under study . This, c species . Synergi species ,51. It i species ,53.This scenario highlights the importance of the development of a robust and proper diagnosis of associated viral agents, which is very crucial in disease management ,55. DiffAlthough many diagnostic assays have been developed, the benchmark detection of plant viruses still relies on the use of PCR based methods. The detection and identification of various viruses or viral strains from a single assay simultaneously reduces the time and cost of the analysis , which iInitially, the virus infected leaf samples of chilli plants were collected from different chilli growing locations in Manipur, India. Infected plant samples as well as healthy chilli plants were maintained in a glasshouse under insect-proof conditions. Leaf samples of both infected and healthy chilli plants were stored at −80 °C for later use. RT-PCR and PCR detection were used for the confirmation of the viruses in these samples.® Plant mini kit  following manufacturer’s protocol. The first-strand cDNA was synthesised using an M-MLV Reverse Transcriptase kit . A DNeasy® Plant Mini Kit  was used for the extraction of the total DNA from healthy and infected chilli leaves according to the product’s manual. The PCR amplified products were purified with a gel extraction kit , ligated into a pGEM-T Easy Vector , and cloned in Escherichia coli DH5-alpha . The positive clones were screened with colony PCR, and the recombinant plasmids were bi-directionally sequenced at a commercial facility .Total RNA was extracted from healthy and infected chilli leaves using an RNeasyhttps://mega.software.informer.com/7.0/; accessed on and before 22 February 2019). The primers were designed to target genes of the respective viruses with distinct amplicon size for easier visualization. Precautions were taken to avoid any non-specific amplification and the self- and cross-complementarity of primer pairs. Multiple sets of specific primers  were designed manually for each virus, and the best primers standardized for multiplex PCR conditions are shown in The specific primers of the nucleocapsid (NP) gene/coat protein (CP) gene of CaCV, ChiVMV, ChiLCV, LCCV, CMV, and PMMoV were designed by aligning the nucleotide sequences downloaded from the NCBI GenBank database using MEGA7 software ; 1 μL dNTP (10 mM); and 1 μL of forward and reverse primer mixtures (10 µM), and the final volume was adjusted to 25 μL with nuclease-free water. The PCR procedure consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 50 s, extension at 72 °C for 1.20 min, and final extension at 72 °C for 10 min. The amplicons were separated on 2% agarose gels (ethidium bromide dyed) in a 1× TAE buffer and visualized using the Gel Doc XR system  and sequenced bi-directionally at a commercial facility . In all the singleplex experiments, plasmid DNA containing a viral gene insert of respective viruses and the cDNA/DNA of an infected sample were used as a positive control, and the cDNA/DNA of a healthy sample was used as a negative control. Water was used as a non-template control.Initially, the virus-specific primers were used to detect the respective virus in singleplex PCR using 50 ng of cDNA from CaCV, ChiVMV, LCCV, CMV, and PMMoV and DNA from ChiLCV positive samples, employing the procedure mentioned in ®, taq polymerase 5 μL of 5× GoTaq® Green Buffer ; 2.5 μL MgCl2 (25 mM); 2 μL dNTP (10 mM); 6.7 μL of total forward and total reverse primer mixtures ; and 50 ng of mixed plasmid DNA containing respective viral gene inserts as template, and the final volume was adjusted to 25 μL with nuclease-free water. In multiplex PCR experiments, a mixture of plasmid DNA-containing viral gene insert of respective viruses (50 ng) was used as a positive control and the cDNA/DNA of a healthy sample was used as negative control. Water was used as a non-template control.Multiplex PCR was optimized to enable the synchronous amplification of six target regions in one reaction. To ensure optimal amplification conditions for the six target sequences in a multiplex PCR assay, a range of PCR-related parameters were evaluated, such as the annealing temperatures of the primers, extension time, and the concentrations of primers. An equal concentration (50 ng) of each of the plasmid DNA containing the gene inserts of CaCV, ChiVMV, ChiLCV, LCCV, CMV, and PMMoV were mixed as templates for multiplex PCR. The optimized factors of the multiplex PCR were the ratio of the 6 pairs of primers with equal and unequal concentrations, annealing temperature , and extension time (1–2.5 min). Multiplex PCR reactions were amplified using 0.25 μL (5 u/µL) of GoTaq−1–10−9) from 50 ng/μL to 0.50 fg/µL, respectively. Then, 1 μL of each dilution was used as a template in a sensitivity evaluation of the multiplex PCR assay. This assay was performed to determine the limit of detection  [Purified plasmid DNA containing the gene inserts of CaCV, ChiVMV, ChiLCV, LCCV, CMV, and PMMoV were serially diluted using 10-fold serial dilution (10 method) .To rule out the possibility of cross-reactions, the multiplex PCR was evaluated by removing a primer pair at a time from the total multiplex reaction mixture of the multiplex PCR. Respective primer pairs of CaCV, ChiVMV, ChiLCV, LCCV, CMV, and PMMoV were removed individually from the total master mix solution while the other five primer pairs were added in the mixtures to assess the multiplex PCR system for stability, and the results were analyzed.C. annum, C. chinense, and C. frutescens) were collected from different fields of Northeast India. Both the DNA and RNA were extracted from all the samples to capture both the virus groups. The total RNAs were extracted with a RNeasy® Plant mini kit , and 50 ng of the total RNA was used for synthesizing the first-strand cDNA using an M-MLV Reverse Transcriptase kit . The total DNA was extracted using a DNeasy® Plant Mini Kit  following manufacturers protocol. An equal concentration of 50 ng each of the cDNA and DNA were taken and mixed properly. Then, 6 μL (50 ng/μL) of cDNA and DNA mixture was used as a template and processed for multiplex PCR protocols. In all the validation experiments, a mixture of plasmid containing viral gene inserts of all six viruses was used as a positive control, the cDNA and DNA mixture of a healthy plant as a negative control, and water as a non-template control.Symptomatic and asymptomatic chilli samples  in multiplex PCR for the simultaneous detection of all six target viruses in a single assay. The specific amplification of respective targets was obtained at annealing temperatures between 48 °C and 56 °C G. Based The extension time for the singleplex and multiplex PCR was also optimized. The optimum extension time was based on the size of the largest amplicon in the mPCR reaction . Extension periods of less than 1 min did not give the amplification of all the six targets, but an increase in the duration to 1.20 min gave the amplification of all six targets, thus it was considered the most suitable extension time for the mPCR set-up.To enable the selection of primer concentrations that gave similar amplification of each target, multiplex PCR was carried out using primers with the same G.Therefore, PCR conditions were optimized using the primer concentrations of 450 nM for CaCVmCF/R, 150 nM for ChiVMVmF/R, 350 nM for LCVmF/R, 150 nM for Chirke-mF/R, 150 nM for CMVmF/R, and 150 nM for PMMoVmCF/R  of CaCV, ChiVMV, ChiLCV, LCCV, CMV, and PMMoV were serially diluted and used as templates. The targeted fragments could be amplified at the lowest concentration of 500 fg/µL for CaCV and ChiVMV, 5.00 fg/µL for ChiLCV and LCCV, and 50.00 fg/µL for CMV and PMMoV A–F. AddiDespite having the mixture of six plasmid DNA (containing gene inserts of six viruses) used as template, only the target specific amplification to the added primer set was observed. When a specific primer pair was removed purposely, there was no visible band of that specific reaction . Primer Sixty-one chilli leaf samples exhibiting the symptoms of mottling, yellow-mosaic, leaf curling, chlorotic, and necrotic spots were collected from different locations of Northeast India, covering the states of Arunachal Pradesh, Manipur, Nagaland, Meghalaya, and Sikkim, and used for validation of developed multiplex PCR assay . The samThe general approaches for plant virus detection are serological and molecular methods. Serological methods, such as ELISA, can handle a large number of samples over a short period of time despite limitations like low sensitivity, numerous steps involved, and ability to detect individual virus at a time. In addition, the benchmark PCR and RT-PCR assays are time consuming and cost expensive. The crop plants are often infected with multiple viruses belonging to different taxonomic groups. The crops like chilli are susceptible to multiple viruses and therefore, mixed infections are a common occurrence. Multiple viruses are responsible for the rapid destruction of chilli crops in India; however, its impact on chilli growth and yield has not been established properly. The diversity in symptoms and overlapping symptomatic expressions caused by different viruses make the symptom-based detection unreliable and tedious. Moreover, singleplex PCR assays require separate amplification of each virus of interest and are considerably resource intensive and expensive; therefore, a mPCR to detect multiple viruses—CaCV, ChiLCV, ChiVMV, CMV, LCCV, and PMMoV—in a single reaction is desirable. Further, it is of paramount importance to have the exact identity of virus infection identified and detected with the highest possible level of sensitivity. The development and optimization of multiplex PCR assays are of utmost importance in crops plants as infection of multiple viruses occurs more often.The development of a multiplex PCR assay is often complex since primers must comply with several similar conditions that all primers can function under the same PCR conditions like compatibility, avoiding cross-binding, competition, and sufficient sensitivity . Multipl


Obesity is a chronic, relapsing disease representing a global epidemic. To date, bariatric surgery is the most effective treatment for morbid obesity in the long-term. Roux-en-Y gastric bypass (RYGB) is one of the most performed bariatric interventions, with excellent long-term outcomes. However, about one-third of patients may experience weight regain over time, as well as dumping syndrome. Both these conditions are challenging to manage and require a multidisciplinary and personalized approach. The dilation of the gastro-jejunal anastomosis is a recognized etiological factor for both weight regain and dumping syndrome. Dietary modifications, behavioral interventions, and medications represent the first therapeutic step. Revisional surgery is the traditional approach when non-invasive treatments fail. However, re-interventions may be technically difficult and are associated with increased morbidity and mortality. Transoral outlet reduction (TORe) is an endoscopic procedure aimed at reducing the size of the anastomosis and is proposed as a minimally invasive treatment of weight regain and/or dumping syndrome refractory to conservative therapies. This review is aimed at providing a narrative overview of the role of TORe as part of the multidisciplinary therapeutic toolkit nowadays available to approach weight regain and dumping syndrome after RYGB. Obesity is a chronic multifactorial condition that has recently become a global epidemic . It reprWeight recidivism after RYGB is an extremely relevant issue since it may lead to comorbidities recurrence and deterioration in the quality of life . This evThe enlargement of the gastro-jejunal anastomosis (GJA) is also associated with the incidence of dumping syndrome, which has been reported to arise in 20–50% of patients after RYGB ,14. DumpWith referral to interventional approaches, revisional surgery for weight regain and/or intractable dumping syndrome can be technically challenging due to modified anatomy and adhesions, and compared to first-time surgery, it is associated with an increased risk of complications, morbidity, and mortality ,25. FurtIn recent years, endoscopic techniques for the reduction of a dilated GJA, such as sclerotherapy and superficial or full-thickness suturing devices, have been proposed as minimally invasive and repeatable therapeutic options ,30. AmonThe aim of this review is to provide a narrative overview of the role of TORe, which plays a pivotal role in the complete therapeutic toolkit nowadays available to approach challenging obesity complications, mainly referring to weight regain and dumping syndrome. Since its first description in 2013 , TORe wip = 0.07). Vargas et al. performed a multi-center retrospective analysis including 130 patients (with a mean weight regain of 24 kg) . Each paIn a retrospective study by Tsai et al., 81 patients (with a mean weight regain of 19.2 kg) showed an absolute weight loss of 8 kg at 12 months after TORe . Better A more recent meta-analysis by Dhindsa et al. including 850 patients from 13 independent cohorts showed an absolute weight loss of 6.14 kg, 10.15 kg, and 7.14 kg at 3, 6, and 12 months, respectively. The mean pooled percentage of the TBWL was 6.69% at 3 months, 11.34% at 6 months, and 8.55% at 12 months .As regards long-term outcomes, a prospective series of 150 patients by Kumar et al. showed an absolute weight loss of 10.5 kg at 1 year and 9.5 ± 2.1 kg at 3-year follow-up . The autFive-year outcomes of TORe have been reported by Callahan et al. in a retrospective analysis of 70 patients with a mean weight regain of 27.5 ± 32.1 kg (42.8 ± 18.7%) after RYGB . This stBetter 5-year outcomes have been described in a retrospective cohort of 331 patients by Jirapinyo et al. . This stAs the diameter of the GJA has a positive linear association with weight regain, the specific goal of the TORe technique is to obtain a durable anastomotic rim restriction ,37. For Furthermore, a modified TORe technique for weight loss has recently been described: it combines full-thickness suturing with anastomotic rim endoscopic submucosal dissection (ESD). The rationale of the dissection before the suturing is to enhance the scarring process and, thus, the durability of the anastomotic restriction . A studyAs reported in With reference to procedural safety, traditional TORe proved to have a good safety profile. According to a recent meta-analysis  includinAlthough most of the available evidence refers to the treatment of weight regain after RYGB, some studies evaluating TORe for dumping syndrome have been recently published. p < 0.001) 4 weeks after TORe [In a small series of 14 patients with late dumping syndrome, Stier et al. reported a significant reduction of Sigstad’s dumping score  at the baseline to 8.6 (0–28) at a mean follow-up of 14.8 (3–32) months after the procedure . Nine pap < 0.0001). They reported nine (3%) failures with a recurrence of dumping syndrome; of these, three patients repeated TORe because of a GJA dilation found at a repeated endoscopy, and three underwent an enteral feeding tube insertion. Parallelly, this interventional study proved an effective TORe-related weight loss (with a mean TBWL of 9.3% and mean absolute WL of 9.3 kg at 3 months). Vargas et al. published a multicenter prospective study including 115 patients with dumping syndrome, defined as a Sigstad’s score of ≥ 7 and non-responsive to medical therapy . After Tp < 0.001) [Relly et al. reported an 85% (11/13) resolution rate of dumping syndrome (Sigstad’s score of < 7) at 6 months after TORe, with a reduction of Sigstad’s dumping score from 19.4 to 5.2 (< 0.001) . Two patAs regards long-term results, a retrospective study by Brown et al. showed the resolution of dumping syndrome in 80% of patients at 2 years after an endoscopic revision . The autFurthermore, in a recent large study by Petchers et al. including 98 patients, the reported resolution rate of dumping syndrome was 88% at one month and 84% at the long-term follow-up after TORe (an average of 3.5 years) . In thisAs for TORe for weight regain, different suture patterns have been proposed also for dumping syndrome treatment, with no evidence supporting the superiority of a particular suturing technique. TORe’s good safety profile was confirmed also with regards to dumping syndrome treatment, as no severe adverse events have been reported, except for Petchers et al. who described acute gastrointestinal bleeding from anastomotic ulceration within 30-days after the procedure ,46,47,48Roux-en-Y gastric bypass has proved excellent long-term results in the treatment of morbid obesity. Nevertheless, weight recidivism may occur over time in about 30% of patients, with a recurrence of obesity-related comorbidities and deterioration in the quality of life ,9,49,50.As such, the management of weight regain is challenging, requiring a dynamic and personalized approach to the patients, guided by a multidisciplinary team . The cooAt first, the correction of any psychological, endocrinological, and nutritional issues is mandatory . HoweverTORe represents an endoscopic procedure aimed at narrowing the GJA to delay gastric emptying and to induce a prolonged satiety. This non-invasive repeatable procedure proved to be safe and effective in several studies, with initial evidence of efficacy up to 5 years ,35,36,43As such, TORe represents a low-risk treatment that should be included in the multidisciplinary decision-making process for weight regain after RYGB. Similarly, dumping syndrome represents another common complication after RYGB . This coAlthough avoiding food ingestion to prevent dumping symptoms may conceptually increase weight loss, some patients suffer from particularly disabling symptoms with a significant impact on their quality of life .Dietary adjustments represent the first therapeutic step in the management of dumping syndrome; it is specifically recommended to reduce the quantity of food consumed at each meal, delay fluid intake until at least 30 min after meals, avoid simple sugars, and increase fiber-rich food intake ,15. WhenSurgical interventions, including stomal revision, pyloric reconstruction, Billroth II to Billroth I anastomoses, jejunal interposition, and Roux-en-Y conversion and enteral nutrition, represent the traditional options for patients with refractory symptoms . HoweverA large systematic review by Tran et al. evaluated the outcomes of several methods of surgical revision after RYGB, including conversion to distal Roux-en-Y gastric bypass (DRYGB), revision of the gastric pouch and anastomosis, revision with a gastric band, and conversion to a biliopancreatic diversion/duodenal switch (BPD/DS) ,61,62,63Endoscopic transoral outlet reduction (TORe) has recently been proposed as a minimally invasive treatment for patients with dumping syndrome refractory to medical therapy after RYGB ,46,47,48TORe demonstrated having a satisfactory safety profile, with a < 1% rate of severe adverse events and no procedure-related deaths reported ,46,47,48Overall, given the advantageous safety profile and the promising efficacy results, TORe can be considered the first minimally invasive interventional step in the case of weight regain and/or dumping syndrome refractory to medical and behavioral treatments . Furthermore, TORe is repeatable, per definition, as all endoscopic procedures are. Some authors reported the successful use of 1–2 additional TORe for both weight regain and dumping syndrome ,45,46,48Given the currently limited evidence on TORe for dumping syndrome, further studies investigating this minimally invasive technique with a larger sample size and long-term follow-up are needed. With referral to the comprehensive therapeutic strategy for both weight regain and dumping syndrome after RYGB, the role of the ancillary multidisciplinary team, including nutritionists, endocrinologists, and psychologists, and TORe should also be investigated by including an integrated therapeutic program in the study protocols. The evaluation of adherence to a multidisciplinary follow-up and its impact on the efficacy of the outcomes also represents an unmet need. Overall, randomized head-to-head studies comparing the efficacy for both weight regain and dumping syndrome and the safety of surgical revision and TORe would be of high scientific value. However, the practical feasibility of such studies may be limited by the need for a large sample size and homogeneous cohorts and by the heterogeneity of surgical and endoscopic techniques among different centers.TORe is a minimally invasive procedure that has proven to be safe and effective in inducing weight loss in patients with weight regain and, more recently, in the treatment of dumping syndrome after RYGB. As with every bariatric procedure, TORe cannot be prescinded from long-term management by a bariatric multidisciplinary team operating on different levels, including dietary and behavioral habits. The therapeutic strategy in such complex conditions should be tailored to each patient and requires dynamic interactions between the several health professionals of the bariatric multidisciplinary team. A multimodal tailored strategy that combines nutritional, psychological, medical, and endoscopic interventions is probably the best option, while revisional surgery should be reserved for patients refractory to conservative and minimally invasive treatments.


Current diagnostics for patients with lingering symptoms categorized as post-treatment Lyme disease syndrome (PTLDS) have their limitations and may be difficult to interpret. The aim of this exploratory study was to evaluate the feasibility of protein biomarker profiling as a diagnostic platform for this category of patients and to compare these results with similarly obtained results from a group of patients with acute neuroborreliosis.Borrelia spp or other TBIs. When comparing the two cohorts, different protein profiles were observed, with 125/148 significantly different proteins in CSF and 93/174 in serum, in patients with laboratory confirmed acute neuroborreliosis, of which 6 in CSF and 6 in serum were significant at the p <0.001 level.Two groups of patient cohorts (Cohort 1 and 2) were analyzed for biomarkers in serum and cerebrospinal fluid (CSF); the results were used for group-level comparison. Cohort 1 comprised 158 adult patients selected from 224 previously diagnosed patients, who between October 2015 and December 2018, after referral, were enrolled and structurally investigated based on defined inclusion criteria. They displayed similar lingering symptoms, with a duration of at least 6 months, after presumed previous tick-borne infection (TBI) and are fully described in a previously published study originating from the Center for Vector-borne Infections (CVI), Uppsala University Hospital, Sweden. Cohort 2, comprised 30 patients diagnosed at Uppsala University Hospital between 2016 and 2019 with laboratory-confirmed acute neuroborreliosis. Their proteomic results, based on serum and CSF analyses, were compared with the 158 patients in Cohort 1. The expression and the concentration of potential biomarkers in each patient’s serum and CSF samples were measured based on two multiplex protein panels enabling simultaneous analysis of 92 inflammatory and neurology biomarkers. The PTLDS patient subgroup showed no nominally significant proteins compared to the other CVI patients in Cohort 1. However, CVI patients with signs of inflammation, which were evenly distributed in Cohort 1, showed 16 significantly (p <0.05) different proteins in both CSF and serum, but no association was seen with laboratory-confirmed exposure to Borrelia or other TBI’s were present. However, the expressed markers differed from those found in patients with confirmed acute neuroborreliosis, which does not support the view that PTLDS reflects an ongoing Borrelia infection. Further studies are needed to understand and assess the usefulness of biosignatures of patients with PTLDS before they can be applied in a clinical setting.In this first comprehensive inflammatory and neurological biomarker profile study no differences in biomarker profiles were detected between patients with PTLDS and patients with similar persisting symptoms but who did not meet the PTLDS criteria, regardless of whether laboratory verified previous exposure to  Borrelia burgdorferi s.l. following a bite from an infected tick [Proteins play key roles in various biological processes, including defense against infection . New multed tick . Symptomted tick , 6. Varited tick –10. Thisted tick .B. burgdorferi infection such as PTLDS [The pathology of LD is the result of complex vector-pathogen-host interactions and the different manifestations of host immune responses to pathogen invasion, dissemination, and persistence . The higas PTLDS . Since 2as PTLDS . The impas PTLDS –19.Diagnosis of chronic LD has often been based on nonstandardized interpretation of serological tests, on nonvalidated or insufficiently validated laboratory tests, or on the clinical presentation alone .Given the limitations of existing diagnostics for early LD or for patients with lingering symptoms such as PTLDS, and the absence of previous comparative data using protein biomarker profiling, the present exploratory study aimed to evaluate the feasibility of protein biomarker profiling as a diagnostic platform for TBI. We used two multiplex protein panels, that have previously been used in studies of fibromyalgia and chronic pain, thus allowing for simultaneous analysis of 92 inflammatory and neurology biomarkers , 22. TheTwo groups of patient cohorts (Cohort 1 and Cohort 2) were analyzed for biomarkers in serum and CSF, and the results were used for group-level comparison.The patients in Cohort 1 were chosen from a pool of 224 patients, from a previously published study originating from the CVI clinic, where referred patients were enrolled and structurally investigated between October 2015 and December 2018 .In these patients, having symptoms lasting longer than 6 months was a mandatory inclusion criterion. The other six inclusion criteria, of which patients should fulfill at least four, were > 18 years of age with suspicion of previous TBI based on: (a) previous tick exposure; (b) symptoms consistent with TBI; (c) laboratory findings , (d) previous treatments for TBI and/or (e) laboratory or clinical suspicion of co-infection with TBI other than LD.Borrelia spp. had previously been tested, including Western Blot analysis as well as antibodies to Anaplasma phagocytophilum, Bartonella henselae and B quintana, Rickettsia spp. and TBEV, Babesia divergens and B. microti and PCR of Rickettsia spp. in CSF and Candidatus Neoerlichia mikurensis in serum. Most tests, including the immunological assays, had been performed using commercial kits as part of routine diagnostics at Uppsala University Hospital, Uppsala, Sweden.Enrolled patients were examined by an infectious disease specialist and, besides a full medical history, underwent a panel of blood and CSF laboratory tests including hematological, biochemical, microbiological, and immunological analyses. The RAND-36 scale was used to allow patients to score their quality of life. Lumbar puncture was performed on patients with no contraindications. Antibodies in serum and CSF to Borrelia burgdorferi s.l. (Bb s.l.) but lacked documented objective signs of LD. Group 1 (n = 31) included patients with antibodies only to Bb s.l.. Group 2 (n = 40) included patients with antibodies to Bb s.l. and any of the other TBIs. Group 3 and 4 were both seronegative for Bb s.l.. Group 3 (n = 32) represented patients with antibodies to any of the other TBIs, but not to Bb s.l.. Group 4 (n = 36) included patients with no antibodies to Borrelia s.l. or TBI , musculoskeletal (79%), neurological (82%) and neurocognitive (57%). Tick bites were recalled by 74% of patients. The RAND-36 score was significantly below that of the general Swedish population. However, approximately 20% of the total group of patients had elevated titers of myositis antibodies, a possible sign of autoimmunity, and 21% had slightly elevated fibrinogen values between 4.3–5.9 g/L, interpreted as a result of increased inflammatory activity. The outcome of that study is reported in a previous publication .Borrelia that was confirmed by western blot (no. 78); Group C—seropositivity (IgG) for other TBIs (no. 64); and Group D—presence of inflammation (fibrinogen level > 4.3 g/l) (no.84). The patient’s expression of biomarker proteins in the respective subgroups A-D were compared group by group with the other patients in the cohort. The division and mutual distribution between groups is shown in From the original pool of 224 patients, the 158 patients who had undergone lumbar puncture and had both frozen serum and CSF samples available for protein biomarker analysis were selected for the current study. Of these 158 patients, hereafter referred to as Cohort 1, 55/85 represented patients from the PTLDS group in the previous study, 25/31 from Group 1, 32/40 from Group 2, 21/32 from Group 3 and 25/36 from Group 4. For statistical processing in the present study, the patients were regrouped based on previous clinical and laboratory findings. The new subgroups were: Group A—patients who met the PTLDS criteria (no. 55); Group B—seropositivity (IgG) for The criteria used for PTLDS were in accordance with the Swiss and US case definitions of PTLDS, i.e., a clinically and/or laboratory documented episode of LD that, despite appropriate antibiotic treatment, leads within 6 months post-treatment to a constellation of disabling symptoms consisting of at least one of the following: fatigue, widespread musculoskeletal pain or cognitive problems . PatientBorrelia spp. including B. burgdorferi, B afzelii and B. garinii and in CSF for anti-B. burgdorferi sensu latu with IDEIA Lyme Neuroborreliosis Kit .The second patient cohort, whose proteomic results, based on serum and CSF analyses from 30 patients with clinical and laboratory-confirmed neuroborreliosis and analyzed as part of routine diagnostics at Uppsala University Hospital Sweden, were compared with the 158 patients in Cohort 1. Between 1–3 weeks prior to diagnosis, those patients had been troubled by fever, fatigue, headache, sometimes also nausea, neck and back pain, or neuralgic pains. Cohort 1 and 2 were analyzed on different occasions. To normalize the cohorts to each other, eight samples from the sample matrix of Cohort 1 were included as bridge samples, i.e. re-run together with the samples from the group of patients with neuroborreliosis in Cohort 2. The samples in Cohort 2 had been previously tested for IgG and IgM antibodies in serum by ELISA  against The present study, which was affiliated with the Department of Medical Sciences, Uppsala University, Sweden, was reviewed and approved by the Swedish Ethical Review Authority, Uppsala, Sweden (reg. no. 2015/249 and 2021/04739). The analyses were performed in accordance with relevant guidelines and regulations, and the participants gave their written informed consent.https://www.olink.com/products/targets/).Two Olink Target Assay Panels, Olink Target 96 Inflammation and Olink Target 96 Neurology, based on proximity extension assay (PEA) techniques were performed in a 96-well plate to evaluate the expression of and measure the concentration of potential biomarkers on the Olink Multiplex platform , in each patient’s serum and CSF samples in the two cohorts. In each case, only 1 μL of the sample was used and treated with a pair of oligonucleotide-labelled antibodies that bind to their DNA target protein. When the two antibodies are in proximity, a new PCR reaction target sequence is formed, which is then detected and quantified using quantitative real-time PCR . These aBorrelia spp. (Group B), other TBI (Group C) or inflammation (Group D). To claim statistical significance, a p-value was calculated using the Welch 2-sample t-test. An adjusted p-value was created using the Benjamini-Hochberg correction, thereby adjusting for multiple testing within each test, where an adjusted p-value less than 0.05 was considered statistically significant.The present study was designed to be exploratory, with given inclusion criteria and an unpredictable outcome, which precludes a power calculation. Instead, an arbitrary target of 158 + 30 patients was set based on the number of patients who could be included during the initial years of the study. After 3 years, that number had been achieved and the analysis of collected data began. The variation in the data between and within runs of the two panels was normalized to an arbitrary “Normalized Protein Expression (NPX) scale using the platform-specific “Olink NPX manager” software, which background corrects, log2 transforms and normalizes all samples. The statistical analyses were performed on normalized data and were expressed in NPX units on log2 scale, where a high value corresponds to high protein concentration and where a difference of one NPX corresponds to a doubling of protein concentration. The difference in mean NPX value for the biomarkers, between the groups compared in Cohort 1, was used for the calculations. T-tests were performed between the patients representing PTLDS (Group A) and the three other subgroups, i.e., with laboratory-confirmed signs of Cohort 1 consisted of 158 patients, of whom 72 (46%) were men and 86 (54%) women. The overall median age was 54 years (range 20–79) and 53 years (range 18–80) for men and women, respectively. The number of patients and the subgroups A-D that were used for statistical processing are presented in Borrelia spp. confirmed by Western blot; 64/158 (41%) patients were IgG positive for any other TBI, and 84/158 (53%) had fibrinogen elevation as a marker for inflammation. Some patients were represented in more than one of the groups; 31/158 (20%) had antibodies for both Borrelia spp. and TBI; 43/158 (27%) were seropositive for Borrelia spp. and had concomitant signs of inflammation: 30/158 (19%) were seropositive for TBI and had signs of inflammation: 18/158 (11%) were seropositive for both Borrelia spp. and TBI and showed signs of inflammation. Six of the patients in the PTLDS group had elevated levels of antibodies in CSF, but no signs of current infection and 16 patients in total had detectable myosit antibodies. The distribution between variables and subgroups is illustrated in Of the 158 patients, 55 represented the PTLDS group. In total, 78/158 (49%) patients were seropositive (IgG) for Proteins with at least 25% detectability were included in the further analysis. As a result, 26/184 (14%) biomarker assays were excluded from the CSF assays and 6/184 (3%) assays from the serum assays because the signals were too low. The results for all remaining samples, 158/184 from CSF and 178/184 from serum, were assessed using a boxplot and unsupervised principal component (PCA) analysis of the NPX distribution, and the samples that differed from others in the box plots regarding low spread of the signal–or low or high signal, or that deviated in the PCA analysis and were assessed as outliers, were excluded from the compilation. These consisted of nine CSF samples in the Olink 96 Target Inflammation and Neurology assays, resulting in 149/184 remaining CSF samples. The corresponding figures for the serum samples were four and three samples, respectively, which differed in the box plot but did not deviate in the PCA analysis, which is why they were retained (178/184) in the continued analysis.Borrelia spp. or other TBIs. When using PCA for CSF and serum, no clear separation or subgroup of samples was seen , but no obvious correspondence to Borrelia spp. other TBIs or inflammation was seen, though some potentially interesting subgroups, which could be investigated further, were revealed. The data used for statistical tests and the results can be found in the supporting information T-test of the PTLDS patients (Group A) for both sample types , showed no nominally significant proteins when comparing the PTLDS group to the other patients in Cohort 1 . In t-tewas seen . In a fuBorrelia spp. and negative for other TBIs; these bridge samples were compared with the group of patients with “ongoing Borrelia” in Cohort 2. It was obvious that the NPX values were much higher in patients with ongoing Borrelia compared to both the bridge samples analyzed on the same sample plate and to all sample results from previous analysis of the entire Cohort 1. The NPX levels for the bridge samples were rather similar between the two cohorts, leading to just a small adjustment between the analysis occasions, mainly for the CSF samples. Boxplots of the distribution after normalization are shown in Cohort 2 consisted of 30 patients (14 men and 16 women) who were sampled between Oct 2016 and April 2019. The median age for men was 40 years (range 3–84 years) and for women 66 years (range 5–78 years). Four of the patients were children, one boy and three girls, between three and eight years of age. Proteins with less than 25% detectability after normalization and combination of datasets were excluded from further analysis. This resulted in 150 proteins for CSF and 179 proteins for serum analysis. After normalization, a subset of samples from Cohort 1 were selected that were serologically positive for The number of proteins that were significantly different between the cohorts and that were mainly expressed in patients with neuroborreliosis were, in CSF, 125 of 148 proteins and, in serum, 93 of 174 proteins. Of the most significant proteins that differed between the cohorts and were expressed in Cohort 2 in CSF at the p<0.001 level, we can note CCL19, CXCL10, CXCL11, CXCL9, GZMA and IL18 and in serum 4E−BP1, AXIN1, CASP−8, CLEC1B, EN−RAGE and TNFSF14. The differences are illustrated in Borrelia exposure could or could not be detected. Moreover, in patients suffering from acute neuroborreliosis, in Cohort 2, several significantly expressed proteins were detected in both CSF and serum compared with Cohort 1.This is the first extensive inflammatory and neurology biomarker profile study that has been conducted on patients with persisting symptoms after presumed tick-bite exposure. The biomarkers that were detected in Cohort 1 had no association with whether Borrelia or TBI. These proteins were MSR1, IL-18R1, IL6, EDA2R, HGF, SCARF2, CTSC, SCARA5, SIGLEC1, NRP2, EFNA4, SCARB2, EZR, LAYN, CLM-1 and CTSS, which are represented in several different disease areas including pulmonary, cardiovascular, neurological, skeletal, hemic, lymphatic, metabolic, and inflammatory diseases such as rheumatic disease, systemic sclerosis, and Sjögren’s syndrome, but also demyelination, neuropathies, and prostate cancer  and after re-grouping of patients in Cohort 1 (A-D). The groups represents both the groups of patients from the previous CVI study (Groups 0–4) and the regrouping of Cohort 1 (Group A-D) in the current study for the statistical calculations based on the outcome of key parameters, i.e., patients who met the criteria for PTLDS, serological outcomes and laboratory signs of inflammation.(XLSX)Click here for additional data file.S2 TableThe table summarize the correlation of the expressed biomarkers to biological process, tissue expression, disease area and associated diseases among CVI patients in Cohort 1.(XLSX)Click here for additional data file.S3 TableThe table summarize the correlation of the expressed biomarkers to biological process, tissue expression, disease area and associated diseases among patients with ongoing neuroborreliosis in Cohort 2.(XLSX)Click here for additional data file.S1 FileStatistical test data and results of t-test, and cluster calculations of CSF and serum.(XLSX)Click here for additional data file.S2 FileStatistical test data and results of t-test of CSF and serum.(XLSX)Click here for additional data file.S3 FileT-test and normalized CSF and serum data.(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.


Unmanned Aerial Vehicle (UAV) photogrammetry, thanks to the development of Structure from Motion (SfM) and Multi-View Stereo (MVS) algorithms, allows the generation of dense point clouds, capable of representing three-dimensional objects and structures in a detailed and accurate manner. In addition, the possibility of associating more semantic information through automatic segmentation and classification models, becomes of fundamental importance in the field of development, protection and maintenance of Cultural Heritage (CH). With the developments in Artificial Intelligence (AI), classification algorithms based on Machine Learning (ML) have been developed. In particular, the Random Forest is used in order to perform a semantic classification of the point cloud generated by UAV photogrammetry and Global Navigation Satellite Systems (GNSS) survey of a structure belonging to CH environment. Indeed, this paper describes the images collected through a UAV survey, for 3D reconstruction of Temple of Hera  based on photogrammetric approach and georeferenced by the use of 8 Ground Control Points (GCPs) acquired by GNSS survey. In addition, the shared dataset contains the point cloud and data for classification using Random Forest algorithm. The images were acquired on 01 February 2020 at 10:00 am  in DGN format and subsequently transformed into TIFF format. The photogrammetric dataset was processed in close range photogrammetry GNSS Survey”, https://data.mendeley.com/datasets/ydpn8xnx24/1) according to a formatting of the type East, North, Ellipsoidal Height.The photogrammetric dataset consists of 57 images acquired with DJI Mavic 2 Pro UAV .In this way, it was possible to obtain a colour dense point cloud consisting of 973125 points , that can be found in the repository .Subsequently, the point cloud was classified into 5 classes, using Random Forest algorithm. In particular, the dataset “22.1The semantic classification of the point cloud can be carried out in two successive steps: the first involves the three-dimensional reconstruction of the site, object or structure taken into consideration starting from images 2.2For image capture, UAV DJI Mavic 2 PRO developed by DJI Company, Shenzhen, China, was used. The DJI Mavic 2 Pro is a popular consumer UAV (Quadcopter) equipped with a high-resolution colour camera. In fact, the Hasselblad L1D-20c camera features a 1" 20MP sensor that offers better low-light shooting than other drone cameras. Before the aerial survey, GCPs realized in wooden material (black and white chequered or rhombus) and in such a size that they are easily recognised in the images, were positioned uniformly within the surveyed area. The flight planning was designed in Map Pilot for DJI app, according to a grid path, using an overlap and sidelap value respectively of 80% and 60%; in addition, 45° oblique images with an overlap of 60% were added in order to ensure complete coverage of the temple's shaft, capital and architrave.In order to georeference the model and evaluate the accuracy of the photogrammetric process, GCPs were surveyed with GNSS technique in fast-static mode, using Leica GS12 dual-frequency receiver and using RINEX data obtained from 2 master stations belonging to HxGN SmartNet CORS (Continuously Operating Reference Station). The post-processing of the GNSS data, was performed using Leica Geo Office (LGO) v. 8.2 software. In this way, the coordinates of the GCPs were determined with a plano-altimetric accuracy of less than 0.001 m.https://data.mendeley.com/datasets/ydpn8xnx24/1).The processing of the photogrammetric data was performed in the 3DF Zephyr, which is a photogrammetric software developed and marketed by the Italian software house 3DFLOW. This software is based on Structure from Motion (SfM) and Multi-View Stereo (MVS) algorithms and allows to automatically perform 3D reconstructions. The images were processed by setting "Highest" in the definition of key point density and an “Accurate” mode was chosen in the matching type. Using a special tool implemented in this software, it was possible to import 3D coordinates of GCPs by approximating them to the nearest point of the selected object; if no object is selected, the points are imported directly. The geometric quality of the 3D model was evaluated on several GCPs. The Root Mean Square Error (RMSE) obtained in photogrammetric process was 0.009 m. Furthermore, this process allowed the construction of a dense point cloud of 973,125 points as shown in 2.3The classification of the point cloud can be performed using different algorithms, such as Hough Transform (HT), Random Sample Consensus (RANSAC) and Random Forest (RF). In particular, the classification of the point cloud of the temple was performed using the RF algorithm https://data.mendeley.com/datasets/ydpn8xnx24/1).The evaluation of the quality of the point cloud was carried out using four performance indices, such as Precision, Recall, F1 measure and Overall Accuracy ,9. ThereThe other parts of the structure were classified with RF algorithm. Therefore, once the manual annotation phase of the data was finished, it was necessary to evaluate which features to select in order to classify the scene adequately. After this operation, the dataset is used as input in the predictive model.It is not applicable to this dataset.Massimiliano Pepe: Conceptualization, Methodology, Software, Data curation, Writing – original draft preparation, Writing- Reviewing and Editing; Vincenzo Saverio Alfio: Conceptualization, Methodology, Software, Data curation, Writing – original draft preparation, Writing – review & editing; Domenica Costantino: Conceptualization, Methodology, Software, Writing – original draft preparation, Validation, Supervision; Daniele Scaringi: Visualization, Investigation, Conceptualization, Methodology, Software.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.