On March 15, 2019, Cyclone Idai made landfall near the port city of Beira in central Mozambique causing significant casualties and serious damage to infrastructure. The Emergency Medical Team Type 2 – Italy Regione Piemonte (EMT2-ITA) was deployed approximately two weeks after the disaster to support the country in need, providing essential medical and surgical care.The EMT2-ITA staff was composed of 77 team members including two rotations and integrating local staff. A total of 1,121 patients  were treated during the 27 days of field hospital activity; among all the admissions, only few cases  were directly or indirectly attributed to the disaster event. Only three cases of cholera were confirmed and transferred to one of the treatment centers set up in Beira. The EMT2-ITA performed a total of 62 surgical operations , of which more than one-half were elective procedures.The objective of this manuscript is to report the mission of the EMT2-ITA in Mozambique, raising interesting points of discussion regarding the impact of timing on the mission outcomes, the operational and clinical activities in the field hospital, and the great importance to integrate local staff into the team. Event Type: CycloneEvent Onset Date: March 15, 2019Location of Event: City of Beira, MozambiqueGeographic Coordinates: lat = -19.84361; lon = 34.83889; elevation above sea level = 14mDates of Observation Reported: March 23 – April 24, 2019Response Type: Medical Relief The cyclone, described by the United Nations (New York USA) as one of the deadliest storms on record in the Southern Hemisphere, caused a total death toll of more than 1,000 people  and left 2.6 million people in need of humanitarian assistance. Moreover, on March 27, the Ministry of Health  declared a cholera outbreak, and through May, the number of cumulative cases increased to 6,766 with eight deaths.In the early hours of March 15, 2019, Cyclone Idai made landfall near the port city of Beira, home to 500,000 people, capital of Sofala Province, in the central region of Mozambique. Approximately 90% of the city of Beira was destroyed. able to assure 24-hour operativity, to provide seven major/fifteen minor surgical operations per day, to manage 100 out-patients and 20 in-patients per day, and to remain totally self-sufficient from a logistical point of view.Under these circumstances, the Emergency Medical Team Type 2 – Italy Regione Piemonte (EMT2-ITA) was deployed approximately two weeks after the cyclone made landfall in Beira to support the country in need providing essential medical and surgical care. The EMT2-ITA is a classified World Health Organization  Emergency Medical Team (EMT) Type 2, which is an “in-patient surgical emergency care” unitThe objective of this manuscript is to report the first mission of the EMT2-ITA after the WHO classification, focusing on the operational and clinical activities in the field.Data regarding the field hospital clinical activities were collected by the field hospital paper patient records and surgical procedures logbook filled by EMT2-ITA personnel during the entire mission. The purpose of this chart review was to depict a detailed picture of the clinical activity of the field hospital: the anonymous data of the patient records were manually entered into a data collection form  following specific coding rules to detect the variables of interest. The data collector was a medical doctor in order to recognize medical jargon and to reduce the risk of misinterpretation of chart entries or notes. The resulting electronic database was reviewed by two other professionals with experience in chart review studies. In case of missing data, these were clearly stated with a “not available” (N/A) value. The database was analyzed with Stata 15.1 .The study protocol was submitted to the Ethics Committee at Ospedale Maggiore della Carità in Novara, Italy and obtained its review approval (protocol number 258/CE). The day after on March 21, the EUCPM sent a request to the Italian Civil Protection Department . According to the needs and the resources available within the European voluntary pool, the EMT2-ITA was dispatched to Mozambique.On March 20, 2019, the European Union Civil Protection Mechanism  was activated following an official request for assistance of the Mozambique National Authorities .pm local time, the EMT2-ITA was the first EMT Type 2 to become operational among the 13 different international EMTs that arrived in the country. Figure On March 30, 2019 at 3:00 Since the seven operating theatres (OTs) of the CHB were damaged and not functional from the day of the cyclone, the objectives of the EMT2-ITA were: (1) to support the CHB in its role as a referral hospital for surgical care within its area of reference; and (2) to become the receiving facility within the referral system established for the other international EMTs deployed in the region.As requested by the MNA, the EMT2-ITA was set up within the courtyard of the Central Hospital of Beira , which is one of the three tertiary-care referral hospitals of the country, serving more than eight million people from Sofala Province and from the central region of Mozambique.A total of 77 team members, including a first group of 56 people and a subsequent rotation of 24 people; three people ensured the continuity of the operations by extending their stay for the whole deployment period (27 days). The second group was integrated by local staff, including medical doctors, surgeons, and nurses for the clinical activities and technicians for the logistics ones. In details, the EMT2-ITA was composed of 58 health care professionals  and 19 operation support personnel . Table A temporary structure of nine tents with a total of 26 beds. As depicted in Figure The EMT2-ITA consisted of: During the 27 days of EMT2-ITA activity in Beira, a total of 1,121 patients were treated. Given the several re-presentations, a total of 1,183 triage admissions were recorded.The ages of the patients were highly variable , ranging from newborn to 93 years. The distribution was 53% (596) of patients were females, within the adult age group (18-64). Among all the admissions, only few cases  were directly or indirectly attributed to the disaster event and they presented to the facility during the first 10 days of activity. Three cases of cholera were confirmed and transferred to one of the three treatment centers set up in Beira by Doctors Without Borders/Médecins Sans Frontières . Almost all the admissions  ended with discharge the same day of admission and more than one-half of them  were due to chronic conditions. Table As shown in Table The mission ended on April 25, 2019 with the last three patients admitted during the night; all the field hospital equipment was handed over to the CHB with a formal donation to the MNA.Describing the first mission of the EMT2-ITA after the WHO classification, this report raises interesting points of discussion regarding the impact of timing on the mission outcomes, the field hospital operativity, and the great importance to integrate local staff into the team. reported only minimal effort in treatment of earthquake-related injured victims (90% of patients with non-traumatic illnesses) when describing the Israeli Defense Forces mission in Duzce, Turkey after the earthquake of 1999. Bar-On, Peleg, and Kreiss, in their recent book Field Hospital: A Comprehensive Guide to Preparation and Operation, analyzed the evolution of health needs during a generic disaster, reporting the decrease of surgical cases in spite of medical cases in the first seven and fourteen days after the event. In addition, the introduction of chronic disease care and rehabilitation in the WHO checklist of the minimum standards for EMTs Type 2 is further proof that EMTs must be ready to cope with daily emergencies and routine activities during their missions.First, the impact of mobilization timing on the clinical presentation: the EMT2-ITA became operational 15 days after the event due to the delayed request for assistance and the long travel time. Only few patients with specific disaster-related injuries had access to the EMT2-ITA, in contrast to many patients coming to the field hospital for routine medical care. Similarly, the surgical activity was predominantly elective. As largely described in the literature, this clinical presentation is very typical in the aftermath of disasters. In 2005, Bar-Dayan and colleaguesAdditionally, the EMT2-ITA was embedded into the CHB, whose entire surgical floor was flooded, the roof damaged, the equipment destroyed, and the power supplies cut off after the cyclone. This meant that all the surgery rooms, except one for C-sections, were not functional. Therefore, apart from the timeline, it is clear the essential role played by the EMT2-ITA was replacing the surgery capacity and facing the routine patient load of the local hospital. shows a significant rise of cases in the first weeks after the cyclone, reaching the peak on April 8, 2019, with a gradual decrease in the following days. From April 3 to April 9, the MoH conducted a mass-vaccination campaign that reached more than 800,000 people (90% of the target population). Moreover, the MNA and international partners immediately set up several treatment centers and established a WASH taskforce, which then quickly implemented interventions to ensure the provision of safe WASH facilities for the local population.Even though the EMT2-ITA became operational just three days after the declaration of the cholera outbreak, only three cases presented to the field hospital and all of them arrived during the first week of activity. The epidemic curve of cholera cases reported by the WHOSecondly, the under-utilization of the in-patient wards: the small number of patients needing hospitalization after treatment were admitted directly inside the CHB . Admitting patients to the local hospital shows again the important complementary role of the EMT2-ITA for the CHB. According to this situation, it is evident the importance of the information provided during the request of assistance that, since it was done one week after the cyclone, should have reported data about the local hospital and the role expected for an EMT Type 2.pm to 8:00am), on-call availability was organized for the surgery room and a medical doctor supervised the ER and the triage tents in case of patients coming.An equally important aspect regarding the field hospital management is that, although the EMT2-ITA is ready to assure field hospital operativity 24/7, as requested by minimum standards established by WHO classification, during the Mozambique mission, no patients presented at night as transportation became much more difficult after dark. To adapt to this night in-activity, the personnel shifts were scheduled only during the day, while at night (from 12:00Thirdly, the integration of the local staff in the team composition during the rotation of the personnel of the EMT2-ITA: this integration was really remarkable because, on one side, it allowed to limit the number of professionals coming from Italy and, on the other side, it ensured an effective training about the correct use and maintenance of the entire field hospital equipment before the final donation of it to the MNA. regarding the Mozambique mission, reporting that the 82% of patients treated by EMTs were not related to the Cyclone Idai.The data collected show once again that the main role of an EMT becoming operative several days after a disaster event is the full commitment for elective activities to support and maintain the ordinary health care capacity of the affected country; this is confirmed by the final report of WHO direct experiences in the field, that must be kept in mind for different levels of the disaster response and management.This is an important conclusion, driven by this and several other


The current work presents an objective overview of the impact of one important heterocyclic structure, the pyrazole ring, in the development of anti-proliferative drugs. A set of 1551 pyrazole derivatives were extracted from the National Cancer Institute (NCI) database, together with their growth inhibition effects (GI%) on the NCI’s panel of 60 cancer cell lines. The structures of these derivatives were analyzed based on the compounds’ averages of GI% values across NCI-60 cell lines and the averages of the values for the outlier cells. The distribution and the architecture of the Bemis–Murcko skeletons were analyzed, highlighting the impact of certain scaffold structures on the anti-proliferative effect’s potency and selectivity. The drug-likeness, chemical reactivity and promiscuity risks of the compounds were predicted using AMDETlab. The pyrazole ring proved to be a versatile scaffold for the design of anticancer drugs if properly substituted and if connected with other cyclic structures. The 1,3-diphenyl-pyrazole emerged as a useful scaffold for potent and targeted anticancer candidates. The notion of the chemical scaffold as an essential structure directly connected with the pharmaceutical profile of drugs is a major tool for medicinal chemistry researchers and is widely applied in the drug design process. The analyses of the structure activity relationships highlighted several scaffolds that are associated with compounds having a specific activity ,2. The cA high number of heterocyclic scaffolds are described as privileged structures in the design of anticancer drugs, being found in the structures of a wide variety of approved anticancer drugs . The thiSome cyclic structures, when properly substituted, have a preferential affinity for specific anticancer targets. Arylthioindole and indolylglyoxyamide scaffolds can generate potent inhibitors of tubulin polymerization and cancer cell growth . ChalconThe protein kinase inhibitors are specific anticancer agents that specifically block oncogenic kinases. The majority of these drugs are ATP competitive inhibitors due to their structural similarity to the ATP molecule. Several heterocyclic scaffolds, like imidazopyridazine , pyrido and aminThe objective of this research was to assess, in a quantitative and qualitative manner, the anti-proliferative potential of the pyrazole scaffold, and to highlight the additional structural requirements.The dataset with the 2D structures of the compounds from the National Cancer Institute (NCI) website  containsFor all the compounds in the PRZ set, the average (A) of all GI% values and the corresponding standard deviation (SD) were calculated. The registered A values range between −86.0 and 139.7 with a mean value of 84.2. A number of 696 compounds have at least a GI% value under 50, and 285 have at least one cell for which the GI% is negative. In the PRZ set are 178 compounds with an A score below 50, representing 11.5% of the total number of compounds. This value is significantly higher than that of 7.3% found in the ALL set indicating that the presence of a pyrazole ring increases significantly the chances of finding an anti-proliferative compound.The search of the ALL set for the isomer imidazole scaffold as sub-structure (SMILES: c1c[nH]cn1) identified 2667 compound with an average of the A value almost equal to that registered for the pyrazole fragment (84.3). This average value for the A scores was also obtained for the 325 derivatives that contain the related isoxazole ring, but in the case of these types of derivatives no compound has an A value below 0. The pyrrole scaffold was found in 1969 compounds for which the mean of the A values is 79.5. These results indicate that the pyrazole and imidazole scaffolds have a similar anti-proliferative potential that is significantly higher than the related isoxazole fragment, and slightly smaller than the pyrrole ring.Based on the values of A and SD, the outlier’s values were identified for each compound . The varA number of 1381 compounds from the PRZ set (89%) presented at least an outlier cell line. The number of outliers varied from 0 to 6, with the majority of the compounds having 1 or 2 outliers. These values indicate that the pyrazole-based derivatives possess selective mechanisms towards a small number of cancer cells.In terms of cancer types, the renal cells registered the highest number of outliers (621), followed by leukemia cells (578) and non-small cell lung cancer (NSCLS) with 425. The prostate cell lines DU-145 and PC-3 had the lowest number of outliers. The highest number of outliers was observed for the renal cell line UO-31 with a number of 316 compounds (20.4%) selective towards it, followed by the HOP-92 line (NSCLS) with 205, SNB-75 (CNS) with 174, SR (leukemia) with 167, and A498  with 123 compounds.DataWarrior 5.2.1 software was used to generate the plain rings (PR) and the Bemis–Murcko (BM) skeletons. Each structure was divided based on all the cycle structures it contains, and for each fragment, the single-bonded substituents were erased, keeping only the double-bonded heteroatoms connected to the ring system generating the PR fragments.BM skeletons consist of all the ring systems of each compound’s structure and the atoms joining them. All the side-chain elements and all the atoms’ labels are removed. All bonds are transformed to single bonds. As an example, the results of the transformation of the compound NSC757926 to its BM and PR fragments are presented in The substitutions on the pyrazole ring can modulate the chemical and pharmacological properties of the compounds. The N-unsubstituted pyrazoles possess amphoteric properties having an acidic pyrrole-like NH group and a basic pyridine-like nitrogen  and the The analysis of the PRZ set indicates that the majority  of the compounds have 3 substituents on the pyrazole moiety. A number of 337 compounds contain a 1,3,4,5-tetrasubstituted pyrazole structure, while 323 compounds include a disubstituted pyrazole, and only 17 compounds are monosubstituted pyrazoles. In There is a clear difference in the number of compounds that have a 1,4 disubstituted pyrazole (7 compounds) and those sharing a 3,5-substituion arrangement (130 compounds). Both are associated with good SP scores; 20.7 for the 1,4-compounds and 24.8 for the 3,5-substituted derivatives. In the case of the trisubstituted pyrazoles, the 1,3,4 pattern is the most frequent (295 compounds) and the most useful in terms of the anti-proliferative potential, with an average SP of 23.9. Overall, only 382 compounds in set PRZ have no substitution at the position 1 nitrogen. There is no significant difference in terms of the average values of A and SP scores when compared with the N-substituted derivatives.The set of compounds generated 267 PR fragments coded as PR01-PR267. The numbering of the PR scaffolds was based on the reversed order of their frequency of occurrence. For all the compounds sharing a certain PR scaffold, the averages of the A and SP scores were calculated to assess their usefulness for the design of anticancer drugs.The benzene cycle (PR01) was the most frequent, appearing at least once in 1194 compounds (77%), while 631 compounds contained at least 2 benzene rings. The next most frequent PR fragment was the unfused pyrazole ring (PR02) found in 825 compounds. Pyridine (PR03) was found in the structure of 195 compounds. For 114 types of PR fragments, there is only one compound that contains them, indicating the high chemical diversity of the working set.H-pyrazolopyrimidine , indazole , pyrazolopyrimidine , 1H-pyrazolopyrimidine , and pyrazolotriazine . The structures of these are presented in A number of 146 types of fused pyrazole rings were identified as PR fragments. The most frequent of them were 1The compounds containing an unfused pyrazole ring have on average better anti-proliferative effects, in both A (78.6) and SP (34.2) scores, as compared to the pyrazolopyrimidines derivatives or the indazole derivatives. The pyrazolotriazine derivatives appear to have better selectivity, with low SP average values and high average A values.The use of just one PR02 scaffold is not sufficient for a good anticancer effect. The number of PR fragments in a compound is also important for the anti-proliferative effect and the compounds’ targeted mechanism. The results denote that a single pyrazole-based ring is not enough for the design of an effective anticancer drug. This type of compounds has, on average, an A value of 99.8 and a SP value of 69.1. The best results are observed for compounds that contain between 2 and 5 PR structures, for which the mean of A values is 81.7 and the mean of the SP values is 36.9.The prevalence of the use of the PR02 fragment and its efficiency correlated with the poor performance of the compounds, with only one PR element focused the research on the analysis of the effect of other PR fragments together with PR02. The presence of a benzene ring (PR01) simultaneously with PR02 slightly improves the anti-proliferative effect as the average of SP scores drops to 29.9. The average of the SP values decreases to 23.6 if there are two PR01 fragments present, and has a lower value (11.5) if there are three benzene rings present. The pyridine fragment (PR03) has also a good impact; the compounds sharing at least a PR02 and a PR03 scaffold have an average SP value of 9.6. The average of the SP score is similar for the compounds with both a benzene and pyridine ring next to the pyrazole fragment. The benzimidazole ring has a small benefic effect, while the related 1,3-benzodioxole ring (PR06) has almost no impact on the average SP value.The unfused or fused pyrazole ring is 1-phenyl substituted in 553 compounds (35.7%) and the SP values indicate that its presence is advantageous. The average of the SP values is even lower for the 476 compounds that share a pyrazole ring 3-phenyl substituted. The 209 compounds that have in their structure a 1,3-diphenyl-pyrazole scaffold have an average of SP scores of 15.6, and the average of the A values is 67.6.The 1551 pyrazole-based compounds generated 422 distinct BM skeletons (BM01-BM422). All the BM structures obtained are presented in the The architectural types of the BM skeletons were classified based on the number of cyclic fragments that they contain, using the roman numbers (I to IX) to code them. The most frequently represented skeleton type is III.1 with 521 (33.6%) compounds, followed by type II with 303 (19.5%) compounds. The schematic representations of the architectural classes I to VI are presented in The highest anti-proliferative effect was observed for the type V.2 compounds for which the mean of the A values was the lowest (62.24). The subsequent type is represented by type VI.2 compounds (66.94), but these scaffolds have a low frequency of appearance. The type IV.1 skeleton has slightly better anti-proliferative effects having a mean of the A values of 74.3, as compared to the related type IV.2 (82.9). Type V.2 has also the best SP mean value (7), followed by types V.1 (23.9) and VI.2 (24.3). The type III.1 compounds have also good SP values, with an average of 34.4, while types IV.1 and IV.2 have approximately the same mean value (close to 39). In For the majority of the architectural types of BM skeletons, the ICh value most frequently observed is 0, indicating no connecting atoms between the cyclic structures. In fact, close to half of the compounds in the working set (49.3%) present structures with no linkers. This type of compounds seems to be popular, but their overall performance is low compared with the structures where the ICh values are above zero. The compounds sharing BM skeletons with three linking atoms (ICh = 3) seem to be better in terms of both A and SP scores. The average of the A values is 61.9, and for the rest of the compounds is 86.6, while the average of the SP values is 11.2 compared to 45.0 for all other derivatives in the set.The SC score is an indication of the dimension of the side-chain elements attached to the ring structures. It varies from 0 to 19, but the majority of the compounds (85.4%) have a SC value between 1 and 8. Only 72 compounds have no side-chain elements in their structure. Of the 1551 compounds in the working set, 629 have at least a halogen atom as substituent on their ring’s structures. These compounds have, on average, better anti-proliferative effects, as indicated by the lower A and SP values. The chlorine atom is the most frequent, being observed in 527 compounds, followed by fluorine (153 compounds) and bromine (39 compounds). For more than half (56.2%) of the compounds having fluorine atoms in their structures, the fluorine exists as a trifluoromethyl group.Type I contains 21 distinct BM skeletons, while type II contains 82 and type III 153 BM skeletons. The IV types of skeletons have 98 members, the V types have 45 BM skeletons, while the VI types have only 19. Hexahydroindane (BM01) represents the scaffold with the highest number of occurrences (128 compounds) and cyclopentane (BM02) the second (41 compounds). The average of the SP scores was calculated for each BM skeleton that appeared in 5 or more compounds. The BM skeletons with the best results are presented in For a number of 10 BM fragments, the average values of the SP scores are under zero, indicating a high anti-proliferative effect targeted to specific cells. With the exception of BM32, BM15, and BM39, all the other 7 fragments have an average value of the A scores below 50. A total of 127 compounds share these 10 BM scaffolds. It is interesting to notice that the four type V.2 BM fragments are very similar, but their anticancer efficacy differ significantly. While the BM7 fragment is associated with an average of the SP values of −56.9, its position isomer BM20 has a value −20.3, and the shorter analogue BM15 (ICh = 2) has a value of −5.7. In the case of BM19 the increase of the ICh value from 2 to 3 (BM20) has the opposite effect, diminishing the anti-proliferative potential. The effect also decreases when BM19 is transformed in BM62 by adding a new 6-atoms ring.The search for similar structures was performed for the 285 pyrazole derivatives that have at least one negative GI% value in their anti-proliferative profile. The search returned 117 non-pyrazole compounds from the ALL set. The pairs of similar compounds were filtered to find the derivatives where only the pyrazole ring was substituted with an analog ring. A series of 4,5-diarylpyrazoles belonging to the III.1 skeleton type was identified coupled with the corresponding isoxazoles and 1,2,3-triazoles analogues. The values of the A scores are nearly equal suggesting that for this series of compounds the pyrazole ring is not essential and it can be replaced with similar rings.The ALL set of compounds was explored based on the BM skeletons. The search’s objective was to find non-pyrazole compounds that have their structure based on one of the 10 BM scaffolds identified to have the best anti-proliferative potential. The investigation returned 25 compounds for BM17, 9 compounds for BM32, and no results for the other 8 BM fragments. The average of the A values was higher for both types, BM17 and BM32, indicating the importance of the pyrazole ring for these types of structures.The online platform ADMETlab 2.0 used to perform the in silico ADMET evaluation of the compounds  was usedThe drug-likeness of the compounds in the PRZ set was assess using the quantitative estimate of drug-likeness (QED) parameter . The obtThe fraction of sp3-hybridized carbons (Fsp3) in the molecule is considered a measure of the compound’s three-dimensionality, complexity and drug-likeness . In the In terms of the risks of chemical reactivity or to be promiscuous compounds , only 57A high majority of the compounds (1176) respect the Lipinski rule, but those that fail the rule have an average of the SP values significantly lower as those obeying the rule. There are many reviews that present and demonstrate the anticancer potential of the pyrazole ring, unfused or fused in structures like indazole or pyrazolopyrimidines, but these works present only on the active derivatives ,36,37,38The use of NCI data has the advantage of offering a big volume of data that are uniformly obtained using the same assay protocol, but it lacks the association with a mechanism of action. The analysis of the pyrazole derivatives extracted from the NCI database indicated a large chemical diversity as demonstrated by the high number of PR and BM fragments. Popularity, synthesis accessibility, and activity performance are the main factors that influence the design of anticancer candidates, but it is difficult to evaluate the importance of each factor individually.A series of factors were shown to influence the anti-proliferative profile of the pyrazole compounds, but no clear quantitative relationships were established. The most probable cause for this is that the analyzed compounds may act through different mechanisms. Another cause is the fact that the study used as scores averages over a high number of cells in order to compare the compounds. These cancer cell lines have different sensibilities to various mechanism of action making difficult to pinpoint a clear structure-activity relationship. These differences prompted the introduction of the SP score, as a measurement of the activity on specific cells. Even if a compound has a high A value, it can be active in a small number of cells, the effect observed in a low SP value.Overall, the results of the study confirmed the anti-proliferative potential of the pyrazole moiety, but only if proper substituted and if is connected to other cyclic scaffolds in particular structural arrangements. The 3,5- or 1,4-disubstituted and the 1,3,4-trisubstituted pyrazoles seem to have a better anti-proliferative potential. This result correlates with the requirement for additional cyclic structures; the presence of two or three aromatic rings like benzene or pyridine improves the anti-proliferative effect. The 1,3-diphenyl-pryrazole scaffold substituted in position 4 emerged as a versatile scaffold for potent and targeted anticancer candidates.The design of pyrazole derivatives needs to incorporate proper side-chain elements beside other cyclic structures. The dimension of the chains connecting the cyclic structures demonstrated to be important also. The BM skeletons with 3 linking atoms seem to be better in terms of both A and SP scores, as those with fewer atoms. The compounds with no connecting atoms between the cyclic structures seem to be popular, probably because of the chemical accessibility, but their performance tends to be lower. This result correlates with the observation on the low Fsp3 values registered in the working set. The introduction of saturated carbons as linking bridges between the cyclic structures could be a good method to boost the anti-proliferative potential of the pyrazole derivatives and to improve their drug-likeness.https://wiki.nci.nih.gov/display/NCIDTPdata/NCI-60+Growth+Inhibition+Data, (accessed on 7 March 2022)), together with the chemical structures ). DataWarrior 5.2.1 ) software was used to prepare the raw dataset and remove the compounds with errors.The anti-proliferative data representing one-dose screening values were collected freely from the DTP website  after 48 h exposure to 10The data set matrix was analyzed in order to assess the performance of each compound. The average (A) of all GI% values registered for a specific compound (i) was calculated as an indicator of anti-proliferative potency:Each compound is tested on a panel of up to 60 cancer cell lines resulting a vector of GI% data. The outliers were defined as any GI% data below the threshold (T) based on the compound’s A value and the standard deviation (SD) of the GI% values, following the formula:The presence of outliers indicates that the corresponding cells are specifically sensitive to the tested compound and that the compound presents a selective anti-proliferative effect. A measure of the selective potency (SP) of the compounds was introduced as the average value of all GI% for each compound’s outliers, if present.DataWarrior 5.2.1 software was used to generate the plain rings (PR) existing in each compound and the Bemis–Murcko (BM) skeletons. Each chemical structure was divided in multiple fragments based on the cycle structures. The single bonded substituents were removed keeping only the double bonded heteroatoms connected directly to the ring system. The BM structures represent the molecular frameworks incorporating only the rings and their interconnecting chains. The atom types are ignored and each bond is considered as single bond .Pi represents the percentage frequency of each element i in the whole set of n elements.The Shannon diversity index (H), also known as the Shannon entropy , was calWe defined as a linker (Lk) the number of nodes in the graph of each BM scaffold that interconnects two ring fragments. The variable interior chain (ICh) was calculated as the sum of all Lk values of each BM scaffold. A similar score, SC, was implemented to measure the dimension of the side-chain elements attached to each compound. It was calculated by counting the number of non-hydrogen atoms in all the side chains of a compound, but not in the chains connecting the ring structures.The ALL set was used to find the compounds with over 0.85 similarity with the compounds in the PRZ set that have at least one GI% value under 0. The search was performed using the DataWarrior 5.2.1 software’s default descriptor FragFp. This descriptor is a binary fingerprint based on dictionary of 512 predefined fragments . The com


The control of inclusions in steel components is essential to guarantee strong performance. The reliable characterization of inclusion populations is essential not only to evaluate the quality of the components but also to allow the use of analytical procedures for the comparison and discrimination of inclusion populations. In this work, inclusion size distributions in wire rod specimens from six plant-scale heats were measured and analyzed. For the measurements, the metallographic procedure specified in the ASTM E2283 standard was used. The population density function (PDF) approach and the extreme value statistical procedure specified in the ASTM E2283 standard were used to analyze the whole size distribution and the upper tail of the size distribution, respectively. The PDF approach allowed us to identify differences among inclusion size distributions and showed that new inclusions were not formed after the liquid steel treatment process. The extreme value statistical procedure led to the prediction of the maximum inclusion length for each heat, which was used for the statistical discrimination of heats. Furthermore, the estimation of the probability of finding an inclusion larger than a given inclusion size using the extreme value theory allowed us to order the heats for different critical inclusion sizes. Control over inclusion cleanliness in steel products is necessary to ensure their performance under specific conditions. Inclusion size distribution is one of the parameters used to evaluate steel quality, and therefore its control during the steelmaking process is required ,2. Hence2O3 inclusions.The size distribution of an inclusion population is frequently estimated from metallography measurements and image analysis and represents an inclusion cleanliness parameter. Higgings  represenmaxL), i.e., the longest inclusion expected to be found in a predetermined area. The fitting of the Gumbel distribution to the size distributions of different specimens enables the statistical comparison and discrimination of maxL. Variations in the procedures used for the application of GEV led to the development of the ASTM E2283 standard “Standard Practice for Extreme Value Analysis of Nonmetallic Inclusions in Steel and other Microstructural Features” [On the other hand, the generalized extreme value (GEV) theory based on Murakami et al.’s pioneering work  has beeneatures”  as a guieatures”  showed teatures”  confirmeeatures” . More reeatures” In this work, size distributions were measured in steel wire rod specimens from six different plant-scale heats following the metallographic procedure indicated in the ASTM E2283 standard. The PDF approach was used to analyze the whole size distribution, whereas the GEV theory was employed to describe the upper tail of the size distributions following the procedure specified in the ASTM E2283 standard and to estimate the survival probability.C is the constant of proportionality, and D is the fractal dimension. The probability density function of the lognormal distribution ism and s are the mean and standard deviation, respectively.The PDF concept, introduced by Higgins , is exprectively . The proThe ASTM E2283 standard  describe2 (control area oA) must be evaluated in four different metallographically prepared planes in each specimen, and the largest measured inclusion in each oA is recorded. The measurements must be made using the correct magnification to ensure that the detected largest inclusion is a minimum of 20 pixels in length. The procedure provides a dataset of the 24 largest inclusions, which are listed in ascending order and can be represented as ix. The cumulative probability iP is calculated by the following equation:N = 24.The ASTM E2283 procedure is based upon quantitative optical metallographic measurements and their analysis via statistical GEV theory. Six specimens per analysis are required for metallographic measurements. An area of 150 mmmaxL is based on the fitting of the Gumbel extreme value distribution to the 24 recorded inclusion lengths. The probability density function of the Gumbel distribution is expressed byx is the random variable (maximum inclusion length), and l and d are the location and scale parameters, respectively. The corresponding cumulative distribution isy:The prediction of x,Solving Equation (8) for y can be expressed in terms of the cumulative function asFrom Equation (7), y is rewritten asEquation (10) can be related to Equation (4), and the expression for T is used to predict how large an inclusion could be expected to be found if a reference area (refA) larger than oA were to be evaluated. That is,On the other hand, the return period refA is chosen to be 1000 times larger than oA.In the ASTM E2283 standard, T is statistically defined asFurthermore, T value of 1000, the corresponding P value is 0.999 (99.9%).For a P = 0.999, the xP=0.99). The calculated xP=0.99) is rewritten asThus, for δ and λ parameters are calculated using the maximum likelihood method ML from the log of the distribution function:The MLλ and MLδ and are used to construct the best-fit line through the data points using Equation (9).The estimated parameters are referenced as SE for any inclusion of length x isThe standard error The approximate 95% confidence interval is given byA and B, is calculated by the approximate 95% confidence interval for max(A)L − max(B)L by the following equation:The comparison of differences in sizes of large inclusions in two steels, denoted CI include 0, then it is concluded that there is no difference in the characteristic sizes of the largest inclusions in heats A and B.If the lower to upper bounds of the 95% Measurements and analyses of inclusion size distributions in wire rod specimens from six plant-scale heats were conducted. The PDF approach was used to analyze the whole size distribution, and GEV theory was employed to analyze the upper tail of the size distributions.Specimens from six different heats of high carbon rod wire produced at the plant scale were studied. The heats were produced by an electric arc furnace-ladle treatment-degassing-continuous casting route, and the liquid steel was deoxidized with Si. The studied steel corresponded to SAE 9254, and 2. Three additional planes were analyzed, ensuring that the space between neighboring planes was at least 0.3 mm, thus avoiding inclusions being counted more than once. The total number of analyzed fields per heat was 600.Inclusion size distributions were estimated using the metallographic procedure detailed in the ASTM E2283 standard, which was rigorously followed. For each heat, six specimens of wire rod in as-rolled conditions of 25 mm diameter and 200 mm length were available. Each specimen was cut into a probe of 25 mm length and sectioned longitudinally as shown in The totality of the analyzed inclusions in each heat was considered to estimate the corresponding PDF using Equation (1). The frequency of inclusions per volume unit specified in that equation was estimated from the two-dimensional inclusion measurements obtained by image analysis, which were transformed into three-dimensional data using a procedure based on the Saltikov method ,17.By applying the ASTM E2283 standard, the measured maximum inclusion size in each of the inspected planes (24) was used for the statistical analyses described in the standard and synthetized in the previous section of this paper.2) and area fraction for the six specimens of each heat. In general, Heats 5 and 6 presented the highest values for both parameters, whereas Heats 1 and 2 presented the lowest values. Furthermore, Heats 5 and 6 showed more variability among specimens of the values of both parameters.The logarithmic representation of the calculated PDFs is shown in 2-Al2O3-MnO inclusions were observed at the beginning of the ladle treatment, which evolved to SiO2-Al2O3-CaO-MgO inclusions during treatment. Of note, SiO2-Al2O3-CaO-MgO inclusions were observed in this work under as-rolling conditions, as shown in 2-Al2O3-MnO inclusions during the Mn-Si deoxidation process and their evolution to SiO2-Al2O3-CaO-MgO inclusions during ladle treatment. Furthermore, the linear behavior indicates that the new inclusions were not formed after liquid steel treatment and that inclusion populations evolved by the growth of inclusions, breakage, and removal of inclusions. In addition, the inclusions found in all samples were embedded in a matrix similar to that presented in Then, the differences between the slopes of the straight lines of each heat can be deduced. The slope of the straight line is associated with the refining process and is specific to each process. Van Ende et al.  showed rλ, δ, and maxL are plotted in λ and δ are, the lower are the values of maxL. Thus, the heats can be listed in increasing order of maxL values as follows: Heat 2, Heat 1, Heat 3, Heat 4, Heat 5, and Heat 6. This behavior is illustrated in λ and δ included in maxL values were statistically significant, maxL values were compared according to the procedure described in the ASTM E2283 standard. The criterion of a 95% C.I. was calculated from the predicted value of maxL for each heat as well as the corresponding standard error. maxL; otherwise, it is denoted in green and indicates that there was no difference. The maxL of Heats 5 and 6 did not differ from each other; in contrast, they differed from the maxL of the rest of the heats. Furthermore, maxL of Heat 2 was different from other maxL, and maxL of Heats 1, 3 and 4 were not different from each other. Thus, using the maxL parameter as the cleanliness index, it can be stated that Heat 2 is the cleanest and significantly different from the rest of the heats; Heats 5 and 6 are the worst and significantly different from the rest of the heats; Heats 1, 3 and 4 are not different from each other, and their inclusion cleanliness is intermediate between Heat 2 and Heats 5 and 6.To determine whether the observed differences in maxL represents a useful indicator that enables the comparison and discrimination of inclusion populations, there is often interest in knowing the probability of finding inclusions greater than a certain “critical” size depending on the specific application of the steel component. For example, in components subjected to fatigue, it is accepted that the size of the inclusion is important because cracks can form at the inclusion–steel interface. In this context, the survival function S(x)  rather than maxL is more convenient. (x) as a function of the inclusion length for all heats. The order of heats previously stated as a function of maxL is confirmed for inclusion lengths greater than 35 μm, where no overlapping of curves is observed. At 35- and 10 μm inclusion lengths, the curves of Heats 3 and 4 and Heats 1 and 2 overlapped, and therefore, the order of heats was modified.Although (x) is plotted in (x) value for each critical value; for example, for an inclusion length shorter than 10 μm, the order is Heat 1, Heat 2, Heat 3, Heat 4, Heat 5 and Heat 6. This order is altered when the critical value of 20 μm is selected, changing the order to Heat 2, Heat 1, Heat 3, Heat 5, Heat 4 and Heat 6. The observed difference is explained by the overlapping probability density curves and S(x) curves for Heats 1 and 2 and for Heats 4 and 5. For example, in Heat 1 and Heat 2, overlapping can be observed in the upper left corner in maxL is considered as the cleanliness parameter.SInclusion size distributions in wire rod specimens from six plant-scale heats were measured and analyzed. The measurements were taken following the metallographic procedure specified in the ASTM E2283 standard. The analysis of size distributions was developed using two approaches: (1) the estimation of PDFs according to the Higgins formalism [Heats were listed in decreasing order of inclusion cleanliness based on the analysis of the linear logarithmic representation of PDFs.No new inclusions were formed after the ladle treatment process, as inferred from the linear behavior of the logarithmic representation of PDFs, a power-law-type with time. Hence, the evolution of inclusion distribution was associated with growth, breakage, and the removal of inclusions.maxL. The use of the extreme value statistics procedure specified in the ASTM E2283 standard led to a statistical comparison of maxL.Heats were listed in decreasing order of inclusion cleanliness using the maximum inclusion length parameter (x) values  estimated using the GEV theory. It was shown that the order can change depending on the critical value.Heats were ordered by considering the survival function S


The role of cholinergic projection systems of the neocortex and hippocampus in memory consolidation in healthy and neuropathological conditions has been subject to intensive research. On the contrary, the significance of cholinergic cortical and hippocampal interneurons in learning has hardly been studied. We aimed to evaluate the role of both cholinergic projection neurons and interneurons of the neocortex and hippocampus at an early stage of spatial memory consolidation (2s1) in normal and chronic brain hypoperfusion conditions. Control rats and rats subjected to permanent two-vessel occlusion were trained with the Morris water maze, and the activity of membrane-bound and water-soluble choline acetyltransferase was evaluated in the sub-fractions of ‘light’ and ‘heavy’ synaptosomes of the neocortex and hippocampus, in which the presynapses of cholinergic projections and interneurons, respectively, are concentrated. Animals were ranked into quartiles according to their performance on stage 2s1. We found: (1) quartile-dependent cholinergic composition of 2s1 function and dynamics of cholinergic synaptic plasticity under cerebral hypoperfusion; (2) cholinergic hippocampal interneurons are necessary for successful 2s1 consolidation; (3) cholinergic neocortical interneurons and projections can be critical for 2s1 consolidation in less learning rats. We conclude that targeted modulation of cholinergic synaptic activity in the hippocampus and neocortex can be effective in reversing the cognitive disturbance of cerebral hypoperfusion. We discuss the possible ways to restore the impaired spatial memory 2s1 in the presence of cerebral hypoperfusion. Neuronal, mediator-specific components of functional neuronal networks and the dynamics of their relationships underlie the study of brain functions in normal and pathological conditions ,2,3. ThiThe cholinergic afferents of the hippocampus and neocortex from the basal forebrain nuclei are widely known as modulators of neuronal activity in these structures and cognitive processes in animals and humans. However, studies on the role of cholinergic projection neurons in memory consolidation have produced ambiguous results, ranging from affirmation to denial of their global significance in the formation of a memory trace or assumptions about the participation of cholinergic systems only in events preceding or accompanying learning ,7,8,9,10First, investigation of the functional properties of cholinergic interneurons of the neocortex and hippocampus along with the properties of cholinergic projections. For example, such studies have proven to be informative at the sub-synaptic level in biochemical research. In both the neocortex and hippocampus, cholinergic influence comes from two main sources, namely sub-cortical projection neurons and interneurons ,12,13,14Our biochemical data on the ratio of cholinergic activity in the ‘light’ and ‘heavy’ fractions of synaptosomes in both brain structures are in good agreement with these data . In addiSecond, there is heterogeneity in the neuronal organisation of functions in individuals with different learning abilities. Thus, in experimental practice, the ratio of laboratory animals with different cognitive abilities in different experimental settings can vary greatly and thereby introduce variability into the results. Hence, the selection of individual cohorts of animals with different learning dynamics could be used as an experimental approach. The feasibility of this approach has been emphasised in some studies ,20. ThisOur first empirical experience of dividing a small group of intact rats according to learning dynamics revealed certain differences in the cholinergic organisation of spatial memory in rats with greater and less capability to learn . The resIn the present study, we used a statistical technique to divide trained rats into quartiles according to their ability to navigate in the Morris water maze (MWM), a behavioural task used to assess spatial learning and memory. To identify stable boundaries between quartiles of rats, we processed a statistically sufficient array of animals see . The resO-acetyl transferase  as a cholinergic marker. Moreover, in vivo experiments have convincingly shown that mChAT and sChAT can also indicate the functional state of cholinergic synapses and quantitative changes in the cholinergic synaptic pool -acetylcholine was moved into scintillation liquid for organic solutions and a radioactively of synthesised acetylcholine was quantified (DPM) with a beta counter.ChAT activity was determined by Fonnum’s radiometric method . The fin14C)-CoA sodium salt was obtained from Amersham Pharmacia Bioscience ; acetyl CoA sodium salt, physostigmine salicylate, tetraphenylborate sodium salt, choline chloride, naphthalene, tris(hydroxymethyl)aminomethane sodium salt and sucrose were obtained from Sigma-Aldrich ; NaCl, MgCl2·H2O, Na2HPO4·2H2O, and ethylene glycol were obtained from Merck; toluol, butyl acetate, EDTA-Na2, PPO, POPOP, and some other reagents were obtained from REACHIM . or 1 month  both 2VO and sham-operated  animals began training in the MWM. Experimental data were obtained in three different years, designated as experimental batches of rats ‘a, b, and c’.The rats underwent surgical procedures and after 6–8 days , 3 days  or 4 days .The rats were trained for 2 days . There were no deviations from normality. Microsoft Excel was used for correlation analysis, calculating Pearson correlation coefficients (r), and correcting for small samples (n ≤ 15) [p < 0.05.STATISTICA 8.0 was used for statistical analysis. The normality of the data was examined by using the Kolmogorov–Smirnov test (parameter d and (n ≤ 15) . The difr means that the ChAT activity higher, the T value greater, that is, the worse the performance of behavioral task 2s1 (early memory consolidation). Accordingly, the sign ‘-’ has the opposite meaning: the higher the ChAT activity, the lower the T value, that is, the 2s1 consolidation is more successful. This contradicts the physiological significance of the dependence of T values on enzyme activity. Therefore, to display physiological significance, we presented the coefficient r of T-ChAT correlations with the opposite sign both in the text and in the illustrations. Thus, in the data on T-ChAT correlations, the sign ‘+’ means a positive relationship between ChAT activity and T values , and the sign ‘−’ means the opposite.An important note on the correlation analysis of T-ChAT. The ‘+’ sign of the coefficient Using Microsoft Excel, the data of each sub-group was divided into quartiles according to the ability to 2s1 performance. It was clear that the boundaries between quartiles are a species-specific attribute (specific for each learning model) and therefore should be more or less constant values, as well as the fact that only natural boundaries will make it possible to distinguish clusters of animals united by a common mechanism that determines their cognitive property. We identified fairly stable, presumably natural boundaries between quartiles (±0%–5%) for laboratory rats when processing data sets from ≥89 rats. Interval values of quartiles and boundaries between them at stages 2s1, 3s1, and 4s1 are presented in Experimental data for each quartile was presented as a sequence of variational samples . ExperimIn the control (sham-operated) rats, the distribution of animals by quartiles according to the indicators of spatial memory consolidation on the second, third and fourth days of training were more or less uniform I. The quIn the control group in each studied sub-synaptic fraction, ChAT activity was similar in all quartiles. At the same time, the quartile correlation analysis of ChAT activity with task performance at the 2s1 stage (with T indicating the escape latency) revealed the uniqueness of the cholinergic composition of the 2s1 performance of each quartile in all the studied groups.In order to demonstrate the stability of some correlations and instability of others within a quartile, we presented the fractional results of correlation analysis as the variational samples consisting of sub-groups of experimental batches a, and the gradual addition of the corresponding sub-groups of experimental batches b and c to them.r values of the significant T-ChAT correlations are presented in r values of significant mChAT-sChAT correlations are presented in In rats with high memory consolidation, mChAT activity of the HD fraction, alone or together with sChAT activity correlated positively with T values in all variational samples under all experimental conditions. For both the 2VO-7d and 2VO-1M groups, ChAT activity of this presynaptic population of the 1st quartile was resistant to hypoperfusion , and if they were observed, there were correlations between sChAT of both cortical fractions and mChAT activity in the HD fraction (p < 0.05) and 29 ± 4% (p < 0.05), respectively (p < 0.025) and 29 ± 9% (p < 0.025), respectively  and, conversely, inhibition of sChAT by 20 ± 27% (p < 0.05) in the CC fraction. There was a T–sChAT correlation in this faction, as was observed in the corresponding control sample 1, a + b + c  and sChAT by 41 ± 6% (p < 0.05), and a negative T–mChAT correlation appeared  in sample II, a + b + c (p > 0.05) and sChAT by 54 ± 14% (p < 0.005)  while a decrease in sChAT activity by 41 ± 1% was not significant (p > 0.05). In sample III, a, only one rat was in the 3rd quartile. The mChAT and sChAT activity was reduced by 65 and 58%, respectively, and their values were lower than all control values in the I, a sample. A drop in the mChAT and sChAT activity in the CD fraction was conjugated in all 2VO samples   , mChAT and/or sChAT activity in the CD fraction was consistently positively associated with T values in the control, 2VO-7d, and 2VO-1M groups. ChAT activity of this presynaptic population of the 4th quartile was resistant to hypoperfusion, and a mChAT-sChAT correlation was in all variation samples of experimental batche a in this fraction (p < 0.05) in the II, a + b sample and by 22 ± 8% (p < 0.05) in the II, a + b + c, 1/II, a + b + c samples . In the III, a sample of the 2VO-1M group, normalization of the mChAT activity  and insignificant sChAT activation  was observed in this fraction, but there was no restoration of T-sChAT association as in the control I, a sample  and conjugated with sChAT insignificant mChAT activation by 8 ± 13% (p > 0.05) in the sample II, a + b + c, 1 or II, a + b + c, and there was a positive T–mChAT correlation in the same samples (p > 0.05), and there was no a T-mChAT correlation in these samples  and restoration of the ability to consolidate in 2VO-1M rats at the 2s1 stage . The phaData on the marked impairment of long-term spatial memory at the critical period of seven days after 2VO at all the studied stages indicate the universality of the observed phenomenon. At the same time, normalisation of 2s1 performance at one month after 2VO has not been reported consistently in the literature. According to a number of authors, delayed death of hippocampal pyramidal neurons may occur in 2VO rats at or after 1M, a phenomenon that exacerbates the pathological consequences of hypoperfusion ,43. AccoIn the present work, we detected the stability of some cholinergic connections and the instability of others, findings consistent with the dynamics of 2VO-initiated plastic changes in the synaptic pool. Identification of these patterns required us to perform a detailed study. Hence, we conducted a fractional, step-by-step analysis of the results obtained in the control and 2VO rats, as far as our experimental data allowed . With thBy employing a biochemical study, we found that in rats of the first quartile, 2s1 performance is stably and positively correlated with cholinergic presynapses of the HD fraction of synaptosomes  inspires confidence in the method we used to divide the experimental rats into quartiles. The method objectively groups animals according to neuronal (and possibly other) mechanisms of the function under study.In rats of both extreme quartiles (first and fourth), a stable correlation appeared with mChAT activity and, in certain experimental groups, with mChAT-associated sChAT activity . TherefoThe identity of cholinergic presynapses of the HD and CD fractions is unknown. We have reason to believe  are distributed topographically, which suggests their functional heterogeneity ,45,46,47It is well known that the presynapses of projection neurons are predominantly small and medium in size and relatively light in density ,13,48, tThe participation of interneurons in the mechanism of information storage seems to be physiological, especially considering their localisation in the hippocampal CA1 and CA3 zones . CorticaIn the extreme quartiles (first and fourth), there were inconsistent T–ChAT correlations with cholinergic presynapses of other synaptosomes; this finding also applied to the corresponding synaptosomes in the middle quartiles. For the first and fourth quartiles, these inconsistencies were different for the T–sChAT and/or T–mChAT correlations of presynapses of projection neurons of the neocortex and hippocampus , as well as presynapses of all fractions of projective neurons and interneurons in the second and third quartiles . We clasOf note, in intact rats of the second and third quartiles, 2s1 performance is stably correlated with the cholinergic presynapses of neocortical projective neurons (CC fraction) and interneurons (CD fraction), respectively . AdditioAlso, the stable presence of some irregular T–ChAT correlations could be traced within the experiment batche. In the experiment batche a in the second quartile , in the Two more findings from the 2VO-1M group indicate the non-randomness of irregular T-ChAT connections: (1) their restoration in the case of loss during normalisation of cholinergic activity  and (2) restoration or the occurrence of new interstructural and interfractional correlations with the restored synaptic populations, including with the key populations of cholinergic interneurons . The functional significance of these irregular connections in memory consolidation is an open question.It should be noted that in our pilot MR study of two rats, neither rat showed ischaemic brain injury. It has been repeatedly shown that cognitive deficit in the 2VO model develops against the background of the absence of ischaemic damage to brain structures, among other factors ,33,41,49In 2VO rats, hypoperfusion initiated: (1) an associated decrease in mChAT and sChAT activity , which may reflect degeneration or elimination of the corresponding synaptic populations; (2) an unassociated decrease in mChAT and sChAT activity or one of them , which may indicate a decrease in the cholinergic effects of one or more independent synaptic populations due to their degeneration/elimination as well as inhibition/dysfunction of mediator activity in one of them (mChAT) and the synthesis of acetylcholine in the others (sChAT); (3) an associated increase in mChAT and sChAT activity (in the second and fourth quartiles CC fraction at stage seven days), which may reflect synaptogenesis (sprouting); and (4) activation of mChAT (in the second quartile CC fraction at stage seven days and the third quartile HD fraction at stage seven days) which suggests cholinergic activation of the mediator function .In models of neurodegenerative diseases, including 2VO, along with degeneration or elimination of fibres and synaptic contacts reflecting dysfunction ,43,50,51According to the ChAT reaction, in the majority of cases, hypoperfusion provoked degeneration/elimination or inhibition/dysfunction of cholinergic synaptic populations and the disappearance of the corresponding presynapses from the neuronal network. Also, the connection with the function could also disappear with an increase in the power of cholinergic influences. In these cases, it is possible that reorganisation at the critical period of 2VO (seven days) could be aimed at allowing the individual to survive under adverse conditions —for examUnder the influence of hypoperfusion at the critical time of 7 days, the 2s1 performance of about 75% of the experimental rats was classified in the fourth quartile . ObviousNevertheless, we were able to trace some possible pathways of cholinergic reorganisation that promote the transition to the lower and lowest level of memory consolidation abilities at the 2s1 stage.In the first quartile  Coherent changes in the sChAT-mChAT activity in the HD-CC fractions occurred in first quartile rats due to a disorder in the key population of hippocampal presynapses that led to a decrease in abilities to 2s1 consolidation to the level of the second quartile. These individuals, when restored, at stage one month, returned to the first quartile.(2) In parallel with cholinergic activation in the sub-sample of rats from the first quartile, in the CC fraction projective presynapses, inhibition of acetylcholine synthesis (a drop in the sChAT activity) occurred in the native population of rats of the second quartile. These changes did not disrupt the coherence of changes in the sChAT-mChAT activity in the HD-CC fractions in samples II, a, and II, a + b  and manifested themselves in a significant and mChAT-independent decrease in the sChAT activity in sample II, a + b + c .At stage 1 month, the sChAT activity returned to normal, and neocortical projective presynapses were restored in the neural network of the second quartile. It should be noted here that the T-sChAT correlation in the CC fraction was also observed in sample II, a + b + c . It is then likely that in native rats of the second quartile, the 2s1 function did not lose the CC fraction at stage seven days and, therefore, neocortical cholinergic projections are key for this quartile.(3) Inhibition of synaptic function and acetylcholine synthesis in the cholinergic projections of HC fraction  also apparently occurred in the native population of rats of the second quartile, selectively in experimental batche a, as we already reported above, in (4) Degeneration of cholinergic presynapses in the HD fraction (associated drop in mChAT-sChAT activity) at stage one month apparently also occurred in the native rat population of the second quartile after stage seven days and also led to the formation of a new, negative T-mChAT association at stage one month.As a result, at stage one month of 2VO, the ability to consolidate 2s1 in the second quartile was realized through a reorganized neural network, with new cholinergic hippocampal components and cross-correlations with them of restored (or key) neocortical cholinergic projections.In the third quartile ((1) In the cholinergic presynapses of cortical projections (CC fraction), there was a suppression of acetylcholine synthesis (decrease in sChAT activity) and the disappearance of this fraction from the 2s1 neuronal network. At stage one month, there was a restoration of the connection between the CC fraction and function, as well as the emergence of interstructural relationships with the cholinergic presynapses of the CD fraction. At the same time, ChAT activity was reduced.Here it should be noted that only one rat entered the third quartile at the 2VO-1M stage, and, accordingly, control values prevail in the correlation analysis of sample III, a. Therefore, the data for this sample are preliminary.(2) There was sustained activation of cholinergic presynapses (mChAT activation) in the HD fraction and degeneration/elimination of cholinergic presynapses in the CD fraction (decrease in mChAT activity associated with sChAT activity). Coherence was found between changes in mChAT activity in the HD fraction with mChAT and sChAT activity in the CD fraction only in sample II, a + b + c, 1, which included the 2VO-7d rats without control rats  There was synaptogenesis of neocortical projections in the samples II, a + b = c, 1 and II, a + b = c (sChAT activation conjugated with mChAT activity in the CC fraction), association with 2s1 function in both samples, which was not observed in the control samples, as well as interfractional mChAT-mChAT association with the HC fraction, which is key for the fourth quartile. It is possible that the potentiation of neocortical projections was a summation multidirectional response to hypoperfusion in individuals from all quartiles that were in the fourth quartile at stage seven days. At the same time, only in the fourth quartile was a relationship observed between the CC and HC fractions, in which mChAT positively correlated with 2s1 performance. Therefore, we suggest that the T-mChAT correlation was inherent in rats of the fourth quartile and emerged as a new connection in the 2s1 neuronal network due to the reorganization of cholinergic presynapses of neocortical projections.(2) There was inhibition of synaptic function in the cholinergic projections of the hippocampus (a decrease in mChAT activity in the HC fraction) in the samples II, a + b, II, a + b = c, 1, and II, a + b = c, similar to the second quartile at the stage one month. At the same time, there was no T-ChAT correlation in the HC fraction, as in the second quartile, as well as the restoration of T-ChAT correlation with normalization of ChAT activity at the stage one month, which was observed in the control sample I, a in rats of the fourth quartile.The apparent absence of T-ChAT correlation of hippocampal projections under conditions of hypoperfusion may be the result of a similar cholinergic response in rats of the second and fourth quartiles and an inclusion of projections in the 2s1 functional network with a negative effect on 2s1 performance in rats from the second quartile and, conversely, with a positive effect on 2s1 performance in rats of the fourth quartile.It is also possible that in rats of one of the quartiles, cholinergic reorganization in the projection system of the hippocampus specifically for the level of the fourth quartile eliminated them from the functional 2s1 consolidation network.In any case, the fourth quartile of 2VO rats includes at least two sub-samples of rats, since changes in the CC and HC fractions did not conjugate in the samples III, a + b + c, 1, and III, a + b + c.(3) Pays attention that the predominance of non-native rats in the fourth quartile did not disrupt the association of the CD fraction with the 2s1 function. We assume the transition to the fourth quartile of a sufficient number of rats from the first quartile with a positive T-ChAT correlation in the functional 2s1 network  and its preservation against the background of disturbances in the HD fraction .(4) In the interneurons of the hippocampus (fraction HD), changes in the activity of ChAT could not be manifested, apparently due to multidirectional deviations in the activity of enzymes in sub-samples of rats from the first quartile, similar to those observed in the middle quartiles.Apparently, for the same reason, the degeneration/elimination of synapses of the HD fraction, similarly observed in the second quartile at the stage one month, did not manifest itself. At the same time, we assume that changes of this type in hippocampal interneurons in a sub-sample of rats from the second quartile accompanied their transition to the fourth quartile. The presence of these rats in the fourth quartile appeared on day seven in sample II, a + b + c, 1 as a negative T-sChAT correlation in the HD fraction and an interstructural association of HD and CC fractions similar to those in the second quartile at stage one month. A disorder in this sub-sample of cholinergic function or network connections of the presynapses of the CC fraction could be a reason for this transition, if our assumption is correct and the neocortical projections are key for the second quartile.Obviously, the fourth quartile included individuals from the higher quartiles in which the neuronal reorganization of the 2s1 function developed according to the most dramatic scenario. According to our data, this can be indicated by events in the HC and HD fractions, which in certain sub-samples of rats in the fourth quartile were observed earlier than in the middle quartiles at the critical stage 2VO-7d. It is also possible that the changes in other fractions were more profound, i.e., the differences with the middle quartiles were quantitative, or these cohorts of rats had critical changes in the other neuronal populations of functional network 2s1.In general, based on the specifics of the quartile cholinergic organization of 2s1 performance, there does not seem to be a universal cholinergic regulation of spatial learning in normal conditions or its maintenance/restoration under hypoperfusion conditions.Under normal conditions, we do not see obvious ways in cholinergic organization for potentiation of 2s1 execution to move into a higher quartile. Apparently, in the 2s1 functional network, a component of another mediator specificity is such binder. For example, this role can perform the dopaminergic projections to the hippocampus, critical for the successful consolidation of spatial memory in the early stages of learning . AccordiUnder 2VO conditions, reorganization of the cholinergic synaptic pool, individual for each quartile, provided information on possible ways to restore the ability to consolidate 2s1. It should be noted the high plasticity of hippocampal cholinergic interneurons and the prospects for using this plasticity. Correction of impaired function by modulating the efficiency of these interneurons could bring learning dynamics to a high level. At stage seven days, this can be a directed effect (1) on the cholinergic interneurons of hippocampus or projections of the neocortex in the sub-sample of animals that fell into the second quartile, (2) on the cholinergic interneurons of the hippocampus or neocortex in the sub-sample of animals that fell into the third quartile. It follows from our data that it is necessary to know the native abilities of an individual in order to choose a recipe for correcting impaired function.Also, of note is that the fourth quartile is the most representative at the critical stage of hypoperfusion of seven days and, thus, the most popular in the study of mechanisms of vascular dementia. Unfortunately, the complex composition of the fourth quartile and the low information content of obtained data made our conclusions on this quartile the most rough. The study of this issue may require additional methodological approaches.It should be taken into account that in individuals with chronic hypoperfusion, the neuronal organisation of functions would not be completely identical to that in the intact brain: the brain is being ‘rewired’ in accordance with the changed environmental conditions . This isFinally, the distribution of early consolidation abilities occurred in our experimental sample well consistent with the distribution in the actual array of 2VO rats II. ThereNeuroplasticity, the ability of the brain to change and adapt as a result of the interaction of the body with the environment, underlies the restoration of impaired functions, primarily learning and memory under pathological conditions affecting the brain . SynaptiQuartile-dependent cholinergic composition of the studied function and pathways of synaptic cholinergic plasticity seven days and one month after onset of chronic cerebral hypoperfusion;Key and non-key cholinergic elements of the neural network of the function;Poorly studied synapses of cholinergic interneurons of the hippocampus and neocortex (presynapses of heavy synaptosomes) play a key role in 2s1 consolidation;Cholinergic hippocampal interneurons are critical for successful 2s1 consolidation and correction of dysfunction provoked by cerebral hypoperfusion;Cholinergic neocortical interneurons and projections can be critical for 2s1 function in normal and chronic brain hypoperfusion conditions in rats with middle and low memory consolidation abilities;Multifactorial nature of potentiation of memory consolidation in the norm and correction under cerebral hypoperfusion conditions.The quartile method for data analysis is used in a number of psychological and clinical studies. At the same time, our work seems to be the first or one of the first in the neurobiological experiments. In the present study, we employed the quartile method to combine experimental rats into cohorts according to the 2s1 performance of the MWM, a spatial learning and memory task. We obtained new data on the synaptic cholinergic components of the neuronal organisation of this function and made the following findings:We put forward a working hypothesis that delayed cognitive dysfunction is perhaps the most common natural way of brain adaptation in conditions of chronic cerebral hypoperfusion, aimed at maintaining the energy resources necessary for the viability of the organism. Therefore, under hypoperfusion, it is necessary to study the temporal dynamics of the physiological status and compensatory processes to determine the phase that is optimal for the restoration of cognitive functions. It also seems promising to study the dynamics of restoration or reorganisation of the quartile-dependent receptor signalling of the memory consolidation function under conditions of hypoperfusion. This knowledge may reveal the way to correct cognitive dysfunctions.


In intensive fruit growing systems, the recovery and maintenance of soil fertility play a crucial role in both environmental protection and sustainable support to plant productivity. The circular economy approach adopted at the EU level strongly promotes the use of organic products instead of mineral fertilizers. This work focuses on two different soil improvers, compost from the organic fraction of municipal solid waste digestate (CO) and “matured” manure, produced after a fast and controlled aerobic treatment in an aerated pile (MM), which were applied in three apple orchards with different soil tillage. The soil improvers have been characterized for amendment and fertilizing properties. After the amendment, the soils were sampled twice a year (Spring and Autumn) for three years. Each sample has been characterized for texture, pH, cation exchange capacity, nutrients, soil organic matter, and micronutrients. The amendments obtained differed on C, N, P, and K contents, but had similar biological stability. The main effects on soils were the increasing of N and soil organic matter after compost application, while the use of matured manure mainly act on available P and exchangeable K. The treatments showed significant effects among fields with a linear increasing trend only for compost. Matured manure showed more effects in earlier times. The data collected aim to improve the knowledge about sustainable management of soil organic matter and organic nutrients in intensive fruit-growing agriculture by using local products. In the last years, the intensification of agriculture has led to an increase in the use of chemical fertilizers to sustain crop yield and meets the demands of food and feed for the growth of the world population. This results in soil degradation, with loss of organic matter, greenhouse gas emissions increasing erosion, contamination, acidification, salinization, and loss of biodiversity . In addiThe recent EU goals (Farm to Fork strategy) aim to reduce the use of chemical pesticides 50%), fertilizers (20%), and nutrient losses (at least 50%) by 2030. Achieving sustainable soil management in the EU is a crucial action contributing to the 2030 Agenda and Sustainable Developments Goals 0%, ferti,7. MoreoLivestock is historically the main nutrient provider for sustaining crop growth ,10. A stThe compost (CO) application in apple orchards has positive effects on both soil quality and plant productivity . The CO The two processes lead to products with a different amendment and fertilizing properties, but they are currently being used in the same manner in managing the SOM in apple orchards. The knowledge of the main and specific effects on soil, considering both soil organic matter content and nutrient dynamics is very important to reach a better use in intensive agriculture through efficient fertilization strategy and to avoid pollution phenomena (loss of nutrients and GHG emissions) due to surplus of nutrients, which would be difficult to relocatable. Moreover, the recovery of local nutrients could contribute to creating a positive synergy among sectors, particularly in the mountain region, where often, livestock, fruit growing, and urban area coexist. The aims of this work were (a) to define the quality of amendments suitable to use in apple orchards, obtained from local biomasses from agricultural and urban sources in mountain regions, from a circular economy point of view, and (b) to study the effects on soil properties after long-term monitoring of two different amendments. The matured manure (MM) has been obtained after a fast and simple aeration process at the farm level, located in the Non-Valley . The straw previously used as litter for animal housing at the recommended dose of about 4 kg/head*day, was added as a bulking agent to guarantee good aeration and C/N balance of the pile. The manure has been stored directly in a pile for at least three months, then covered with sheets of geotextile. The adequate provision of oxygen for the process has been guaranteed by at least 7 turnings during the storage time, by using a tractor-pulled compost turner.The compost from digestate (CO) has been provided from a full-scale AD plant, located near Trento , which treats about 40,000 tons of OFMSW and 14,000 tons of green waste, source separately collected on all the provincial territory. The AD digestate was produced using Kompogas technology, which permits the double exploitation of organic fraction of municipal solid waste and lignocellulosic waste and residues. The thermophilic process is carried out for 21 days at 51 °C in two horizontal mixed continuously stirred horizontal tank reactors (CSTR) fed continuously. One part of the digestate is anaerobically recirculated as inoculum, the other part is sent to the subsequent composting phase after addition with yard waste to balance the C/N ratio of the starting mixture. The mix ratio for digestate composting was 50% digestate, 33% screened waste, and 17% green waste. The plant produces about 14,000 Mg/y of CO. The composting plant has eight aerated biocells and a followed maturation section in forced aerated piles. The aerobic treatment lasts about 20 days. The flowchart of amendments productions is provided in 4, P, and total organic carbon (TOC) according to APHA methods (2005) [2/kgVS*h) was determined with an AIR-NL dynamic respirometer using 1 kg of the sample following the aerobic process. The final value of DRI has been expressed as a 24-hour moving mean. The piles of digestate from OFMSW composting were sampled for biological stability evolution assessment (DRI measurement) at the beginning of the aerobic phase, after the intensive phase , and at the end of the process (after 20 days). The piles of manure maturation were sampled at the beginning of the aerobic process, after 42 days, and at the end of the process (85 days).The soil improvers have been characterized regarding pH, EC, TS, and VS according to standard methods UNI (1998)  NTK, NNHs (2005) . The dynN/ha; 35–40 kgP/ha; 80–90 kgK/ha).Long-term monitoring has been carried out in three different apple orchards chosen by different soil tillage, located in the Non-Valley . The soils were labeled as D1, D2, and N1. The amendments were applied in the Spring of 2015 before flowering at different doses, 20 Mg/ha for CO and 40 Mg/ha for MM. Two orchards were replanted orchards (D1 and D2), while the third one was a productive apple orchard (5 years) (N1). All of them cultivated the Red Delicious apple variety, grafted on the M9 rootstock. D1 is located 330 m above sea level and was a replanted orchard. Before planting, in Autumn, the soil of D1 has undergone deep work with the removal of the fertile layer, which was again placed above the fill material after the processing. D2 is located at the same altitude as D1 and was a replanted orchard. The soil was the same as D1. Before planting, in Spring, the soil has been subjected to light processing by mechanical digging of the fertile layer. N1 is located 550 m above sea level and was a productive orchard. It has been subjected to important remediation work with the total removal of the fertile layer in 2011. The three experimental sites have been chosen to study the effect of amendments in different conditions (fields): in replanted orchards with different soil tillage; and in a productive orchard. The orchards have been fertilized each Spring with NPK chemical fertilizers as recommended by the local quality protocol in force  for each treatment in each soil were monitored. The soils were sampled in the fertile layer (about 20 cm depth) in each treatment area considered . Four technical replicates (subsampling) in each area were collected by using an Edelmann auger . The sampling for chemical analysis of amended  soil has been made each year for three years (2015-2016-2017) in two seasons (Spring and Autumn). The overall sampling point times were six . T0 corresponds to the soil data of each treatment area, before amending. Detailed descriptions of the sampling points were provided in the Moreover, weather data  from the three years of monitoring are provided In the 2 and TEA, pH 8.1, ISO 13536:1995), soluble B (ICP-OES quantification after MgCl2 extraction), available Cu, Fe, Mn and Zn , available P (Olsen-P) by the colorimetric method after sodium bicarbonate extraction using an ONDA V10 spectrophotometer  and following ISO 11263:1994, exchangeable K by ammonium acetate 1M pH 7 extraction and quantification with an Optima 8300 ICP-OES , total inorganic C by the volumetric method after acidification using a calcimeter (ISO 10693:1995), total C and N by dry combustion using a Vario Macro CN analyzer  and following ISO 10694:1995 and ISO 13878:1998 respectively. Organic C was calculated as the difference between total C and total inorganic C and then multiplied by 1.724 to obtain SOM. Particle-size analysis (by sieving and with a hydrometer), cation exchange capacity  were anThe data were analyzed by one-way ANOVA and multiple pairwise comparisons with Tukey formulation (level of significance α = 0.05). The comparisons were performed to study the effect of MM and CO compared to the Control during the three years of monitoring (from T0 to T5 of sampling points). The difference among fields and between treatments (CO vs. MM vs Control) for each orchard has been performed by multivariate chemometric PCA analysis. Inferential statistics were performed through STATISTICA 9.0 software  and the PCA analysis and the graphs were through R-studio.The amendment and fertilizing properties of the two tested products were verified by means of the chemical analysis and the characterization of the biological stability through the determination of the dynamic respirometric index (DRI).The chemical characterization and the DRI values of the final products are reported in pH and EC are similar for CO and MM. CO had a higher content of DM (+29%) and for this reason, the TOC content, when expressed on fresh weight, was about 112 g/kg TOC for MM and 186 g/kg TOC for CO. The nitrogen content was different, but the organic nitrogen fraction was prevalent, while the ammonia content was negligible for both the amendments considered. The same difference was found for P and K content. The C/N ratio differs among the two products and was higher for MM. Both the products were in compliance with the limits and expected values for good quality soil improvers, reported in the Italian Law and the recent EU Regulations . The heaSV for both the products, MM and CO, highlighting the reaching of good biological stability [Also as regards the biological stability of the amendments the results were comparable . The oxytability . Manure The recommended doses used for apple orchard application and used in this experimental work determined similar application doses of both TOC and the main nutrients. 10.7 Mg/ha DM (+/−2.3) and 12.67 Mg/ha DM (+/−1.1) and 4.48 Mg/ha TOC and 3.73 Mg/ha TOC were applied for MM and CO respectively. The nitrogen and phosphorus contents were 0.25 Mg/ha N and 0.25 Mg/ha  and 0.09 Mg/ha P and 0.08 Mg/ha P . The K content was the main different nutrient compared to the two products, considering the doses applied: 0.12 Mg/ha K for MM and 0.05 Mg/ha K for CO.The soil characterizations are reported in The preliminary characterization of the apple orchards showed similar conditions as regards the texture, pH, CEC, and micronutrients . The data elaboration aims to understand both the effect of different amendments and different fields, also considering different soil management (tillage). The data collected were provided in the following Figures .The increase in the nutrient content was more evident in D1, as well as SOM. The tendency to increase was observed also in N1, while in D2 the main difference was in the first years, comparing the first sampling point (Spring) and the second one (Autumn). In D1, the MM application determined a slight increase of N and SOM content (about +33% N and +50% SOM) , but a sIn D2, the MM application determined a less increase compared to D1 data  and confirmed the increase of P and K content (about + 11% P and + 164% K) in the first monitoring year (T0 and T1) . N1 regiThe CO application determined different conditions. In all the fields the effect on P and K was negligible, also considering the increase of the Control, while the effect on N and SOM was greater considering the comparison to T0 and T5 for D1 (+37% N and +83% SOM)  and N1 , even ifThe PCA analysis of the data obtained after three years of monitoring is reported in The principal component 1 (PC1) explains 68.14% of the variance, while PC2 explains 20.69% of the variance, all explaining 88.83% of the total variability. The PC1 clusters clearly D1 from D2 and N1. The D1 showed a very different response to the treatments compared to other fields; the amending effect is greater. In this regard, it is important to consider the starting conditions of the three fields, which are different. Before the amendment application, the SOM content of D1 (12 g/kg) was significantly lower than others , as well as the N content . The same difference was found for P and K content; D1  differed for the starting conditions from D2  and N1 . The vertical axis clusters D2 from N1. The correlations among the variables (biplot) showed a positive correlation in PC1 for all the variables considered, while PC2 showed correlations with P and K.The data of the available P and K fractions and total P and K fractions are reported in The analysis of these two fractions has been performed for a better understanding of the strong increase of available P (P-Olsen) fraction and K exchangeable in the MM treatment areas. The MM application determined a significant increase of available P  and more lightly total P (+16%). The next year the available P decreased , and total P decreased slightly (−5%). The same trend showed the K fractions. Exchangeable K and total K increased in the first year , while in the next year, exchangeable K significantly decreased , and total K too  a,b. CO aThe work aims to study both the quality of different kinds of soil improvers available locally for fruit-growing sectors and the specific effects on soil. The matured manure and compost from digestate application have been made in the apple orchard with the provision of recommended NPK mineral fertilizers for apple growing too. Normally, the farmers use alternatively MM and CO for SOM management in their orchards. The study of the amendment and fertilizers properties of the products could provide information to reduce the mineral fertilizers use and at the same take care and improve the management of the soil quality and biodiversity in intensive agriculture.Compost from digestate and mature manure are two products available locally in rural areas. Both products may use for SOM management. The chemical characterization showed good environmental and agronomic quality, and both are suitable for orchard application. The MM has less content of DM, but a greater content of TOC, P, and K compared to CO. N content is similar for MM and CO, as well as the biological stability determined after DRI measurement, showing the aerobic transformation of the OM and mineral nutrients. The compliance of all the parameters, including heavy metals contents with Italian Law and EU Regulations has been verified ,24. The The intensive livestock sectors and the use of raw manure directly on soil could have a depleting effect on the environment considering the increase of GHG, soil, and water pollution by leaching nutrients and phytotoxic elements. The concentration of many animals in a restricted area requires the introduction of treatments, which permit manure delocalizing ,28,29. TThe digestate obtained from OFMSW anaerobic digestion contains mineral nitrogen, and microelements could have a role as fertilizers for plant growth. Its composting allows for overcoming the issues regards the health risks for animals and humans, removing the presence of phytotoxic biological compounds and improving its handling . The treThe considerations regarding the quality and the properties of the soil improvers are a preliminary step and the study of the effect on soil and plant is crucial for better identifying the proper use. In general, the use of matured and stabilized products avoids the environmental issue occurring due to the use of fresh products . MoreoveCO and MM applications determine different effects, even though both are derived from an aerobic process. The characterization data  and the The different contributions of SOM in soils confirm the importance not only of the quantity but also of the quality of OM applied, as detected in long-term monitoring of soils amended with products with low C/N . Soil or2 emission, and the manure rate had a great impact on GHG emissions [Significant differences were found also in comparing the orchards monitored. The D1 and D2 were both replant apple orchards; D1 has been subjected to deep work with the removal of the fertile layer, D2, instead has been subjected to more light processing by mechanical digging of the fertile layer, and N1 was a 5-year productive orchard and has been subjected to a remediation work with the total removal of the fertile layer, before planting. The different processing of the soil could explain the different starting conditions, particularly as regards the SOM and N content. The texture analysis demonstrated the very similarity of the soil, and the difference does not due to the physical conditions. Based on previous work, the accumulation of nutrients was greater in silt sites than in sandy sites, where the leaching of organic compounds may have occurred . Moreovemissions ,38. ProbThe work demonstrated the crucial role of the specific knowledge of soil and soil improvers used to obtain the expected fertilizing and amendment effects. The data on P and K dynamics pose a crucial question regarding the possibility of drastically reducing phosphate and potassium fertilization in apple orchards with proper management of SOM. The work aims to study the properties of different kinds of soil improvers available locally for fruit-growing sectors. Normally, the farmers use alternatively MM and CO for SOM management in their orchards, but the data collected showed different effects of the two products. Both, MM and CO have characteristics compatible with high-quality amendments, and the aerobic process (fast maturation of manure and OFMSW digestate composting) determined the increase of biological stability and reduction of mineral nutrients. The study of the amendment and fertilizing properties of the products provide useful information to limit the use of mineral fertilizers and at the same time to take care and improve the management of the soil (particularly as regards SOM) in intensive agriculture. Moreover, the work demonstrated the crucial role of the specific knowledge of soil and soil improvers used to obtain the expected fertilizing and amendment effects. The work reports data about the long-term monitoring of two different amendments, highlighting the relevance of different factors on nutrients and SOM, such as soil tillage, soil management, and the kind of soil improvers used. The work suggests interesting future studies on the fertilizing effect, to reduce mineral fertilization. The data on P and K dynamics pose a crucial question regarding the possibility of drastically reducing phosphate and potassium fertilization in apple orchards with proper management of SOM. In line with the circular economy strategy, the exploitation of organic matter–related nutrients available at the local level promotes the reuse of bioresources, the reduction of fossil fertilizers, and the implementation of sustainable and efficient synergies between the livestock sector and urban-waste recovery systems with other agricultural sectors.


The structure of the resulting oxidized pullulans (PO) was carefully characterized by titration, intrinsic viscosity, FTIR, 13C-NMR, and zeta potential. The content of carboxyl groups in PO was dependent on reaction time and varied accordingly. Furthermore, a fast reaction rate was found in the first 2–3 h of the reaction, followed by a decreased rate in the subsequent hours. FTIR and 13C-NMR proved that the selective oxidation of the primary alcohol groups of pullulan was achieved. The oxidation also caused the glycoside linkages in the pullulan chain to break, and the viscosity of the pullulan itself went down.The C-6 in the maltotriose unit of pullulan was oxidized in an alkaline medium (pH = 10), utilizing a green method that included hydrogen peroxide (H In. In13C-N-4g) ppm , a new p2O2 in the absence of any cocatalyst that yields the PINO radical was tested using the water-soluble polysaccharide pullulan. However, H2O2 was still involved in the production of the PINO radical from NHPI, as shown through UV/Vis measurements. The content of the carboxylic groups introduced in the pullulan backbone depended significantly on the reaction time and pH value, which was adjusted to 10. The oxidized pullulan samples were characterized by FTIR and 13C-NMR techniques. A potential issue when H2O2 was employed for the oxidation was represented by the degradation of the polymer chains in the first hours of reaction. Therefore, the yield of oxidized pullulan decreased while the time of oxidation increased. Decreasing the reaction time and the amount of H2O2 could lead to a decrease in the degradation of the macromolecules.In this study, the ability of an oxidation system based on NHPI and H