The complexity of Neolithic population movements and their interpretation through material culture have been the subject of archaeological research for decades. One of the dominant narratives proposes that groups from the Starčevo-Körös-Criş complex spread from the central towards the northern Balkans in the Early Neolithic and eventually brought the Neolithic lifestyle into present-day Hungary. Broad geographical migrations were considered to shape the continuous expansion of Neolithic groups and individuals. However, recent archaeological research, aDNA, and isotope analyses challenged the synchronous appearance of specific material culture distributions and human movement dynamics through emphasizing communication networks and socio-cultural transformation processes. This paper seeks to retrace the complexity of Neolithic mobility patterns across Hungary by means of strontium and oxygen stable isotope analyses, which were performed on a total of 718 human dental enamel samples from 55 Neolithic sites spanning the period from the Starčevo to the Balaton-Lasinja culture in Transdanubia and from the Körös to the Tiszapolgár cultural groups on the Great Hungarian Plain (Alföld). This study presents the largest strontium and oxygen isotope sample size for the Neolithic Carpathian Basin and discusses human mobility patterns on various geographical scales and throughout archaeological cultures, chronological periods, and sex and gender categories in a multiproxy analysis. Based on our results, we discuss the main stages of the Neolithisation processes and particularly trace individual movement behaviour such as exogamy patterns within extensive social networks. Furthermore, this paper presents an innovative differentiation between mobility patterns on small, micro-regional, and supra-regional scales, which provides new insights into the complex organisation of Neolithic communities. Population movements on different geographical, temporal, and demographic scales during the Neolithic have since long been posited through the interpretation of the archaeological material culture. One of the dominant narratives proposes that groups from the Starčevo-Körös-Criş complex, which spread from the central towards the northern Balkans in the Early Neolithic, moved farther north and brought sedentism to present-day Hungary –4. AccorBy integrating scientific methods such as anthropology, molecular genetics and biogeochemistry, and comparing them with the chronological sequence and archaeological material types in the project, important aspects of the nature and dynamics of settlement and population development in the Carpathian Basin could be depicted in their temporal depth over the entire Neolithic and the first half of the Chalcolithic. Within the framework of population genetic analyses, we divided the Carpathian Basin into two main regions: Transdanubia and the Great Hungarian Plain (Alföld). The archaeological and demographic data required for the evaluation of the sites were provided by staff members of the Institute of Archaeology of the Hungarian Academy of Sciences and by many other Hungarian archaeologists and anthropologists. The molecular genetic data and conclusions of two dissertations written by Anna Szécsényi-Nagy and Victoria Keerl have in part been already published –8 and arth–5th millennia BC in Transdanubia and in the Alföld. The main questions are as follows: Are there any disparities in mobility patterns between geographical areas, archaeological cultures, chronological periods, and/or between sex and age categories? What kind of mobility patterns can be identified for the Neolithic period? How can the results of isotope analyses be compared to the archaeological records and the results of aDNA-analyses in this research area?The determination of strontium isotope baselines for this research area, which are based on the interpretation of faunal dental enamel, human bone and shell strontium isotope data, and on their integration with multivariate environmental analyses, have been published in a separate paper . In eachThe shift to sedentary life and food production is certainly one of the major developments in human history. The Carpathian Basin appears to have been culturally divided into two main regions with somewhat different traditions, although both shared roots in the northern Balkans , 13. TheThe Körös groups reached the heartland of the Carpathian Basin around 6000 cal BC: after crossing the River Maros, they gradually occupied the floodplain of the three branches of the Körös rivers on the southern Alföld plain, a region abounding in water, advancing from south to north , 15. TheThe long-lasting process of gradual change suggests that the genuine change in subsistence strategies did not occur in the initial phases of the western Transdanubian Neolithic, but one phase later, at the time when longhouses appeared, in the period hallmarked by the Transdanubian Linearbandkeramik (TLBK). This assumption is confirmed by the observation that the shift to food-production did take place, although not in the earliest TLBK phase, but some three or four generations later , 16. SimA contact area lies along the boundary between the lowland and the mountains, where strong genetic relations between the early Alföld Linear Pottery (ALP) and the Körös culture can be demonstrated . Local fth millennium BC. The Late Neolithic of the Tisza region was determined by the emergence of tell settlements surrounded by an extensive network of satellite settlements, whose significance is now somewhat better understood in the wake of extensive field prospections  and, to a lesser extent, at the CSAT site (under 30%) in Fig 10. This can perhaps be taken to indicate that the South-East European impacts observed in the material record of the Lengyel culture can probably be ascribed to trade rather than to the arrival of new population groups. In contrast, the increased proportion of site outliers during the Balaton-Lasinja period (nearly 40%)  correlates well with the archaeological record, according to which a new Balkanic influx can be attested . The widespread homogeneity of the material culture further suggests that these large-scale social networks were probably linked to considerable economic fluxes ).When juxtaposing the results of the strontium and oxygen isotope analyses with the archaeological record, no apparent patterns emerge relating to mobility and burial practices. Three examples can be cited to illustrate this assertion. The first concerns the Tiszadob/Bükk site at Tiszadob-Ókenéz (TISO), where two of the three sampled individuals that were buried in single graves  had non-18O value of modern precipitation data at each site, it is realistic to assume that the ranges could be lower or higher as suggested in this study. As shown by the OIPC European map that presents annual average δ18O data (accessible at http://wateriso.utah.edu/waterisotopes/pages/data_access/figures.html), the local isotope composition is generally homogeneous across present-day Hungary. This study confirmed that despite small regional disparities, the baseline ranges are consistent over large geographical areas . This also means that it was not possible to distinguish between a site-specific and a micro-regional baseline. Consequently, individuals with a late-mineralising tooth with non-local δ18O data can also be regarded as “non-regional”.The first requirement for the interpretation of isotope data is the determination of a reliable baseline. Concerning strontium isotopes, Kempf and colleagues  showed t). Consequently, if an individual exhibits an isotope composition close to the baseline range limits, the interpretation depends on the comparison with the isotope data of the rest of the group. At the Budakeszi Site 8 Szőlőskert-Tangazdaság (BUD) site for example , the first permanent molar of a female (BUD2) and the third molar of an adolescent (BUD7) have strontium isotope ratios close to the lower baseline range limit. But because the differences compared to the rest of the site-specific group do not exceed 0.00015, they are not considered site outliers. However, the isotope composition of another adolescent’s (BUD9) first molar is right on the upper limit of the baseline range, but less consistent with the rest of the site-specific group and is therefore classified a site outlier.Nevertheless, using these strontium and oxygen isotope baselines implies that modern data are comparable with Neolithic conditions. Even so, it remains difficult to determine the actual extent of the activity ranges of Neolithic groups around their sites , or to m87Sr/86Sr ratio. The other four POPI individuals are site outliers that could nevertheless have originated from the micro-region around the site. This surprisingly high number of site outliers is confirmed by the oxygen isotope analyses. However, the δ18O data suggest that these non-local individuals probably did not originate from the direct surroundings of the site. One male (POPI5) in particular could have originated from a more distant place. Another example can be found at Ebes-Zsong-völgy (EBVÖ). The teeth of the three EBVÖ individuals show consistent site-specific strontium isotope compositions. Nevertheless, the only local δ18O value is found in a third permanent molar of the female (EBVÖ4), while the values of the two male individuals exhibit a non-local δ18O origin. Given that geographically distant areas can have similar strontium isotope values [The combination of strontium and oxygen isotope data, if available, provides more precise results than a single type of analysis would deliver. At the ALBK site of Polgár-Piócási-Dűlő (POPI), for example, only the first permanent molar of a female (POPI1) exhibits a site-specific e values , 70, 77,This study presents the strontium and oxygen stable isotope data of one of the largest Neolithic samples in Europe, whose assessment reveals the development of various mobility patterns over approximately 2000 years in both Transdanubia and the Great Hungarian Plain. Despite the sometimes small sample size for each site, it is nevertheless possible to interpret the data on different scales, from the individual and the burial community to the archaeological cultural group.In Hungary, the Neolithic was very diversified in terms of the history of individual and group mobility. The migration and mobility models constructed within the framework of strontium and oxygen stable isotope analyses suggest that Neolithic communities were mostly organised on a local or at least a micro-regional scale, especially in the Great Hungarian Plain, which suggests a relatively homogeneous population structure. Small-scale mobility patterns involving both males and females to varying degrees within the local or micro-regional area around the site could be observed for nearly every Neolithic cultural group. For example, there are indications of patrilocality in the Starčevo, TLBK, Vinča, Lengyel and Balaton-Lasinja groups, while the Sopot and Esztár cultural groups can rather be considered matrilocal communities. Strontium and oxygen stable isotope analyses also highlighted large-scale mobility patterns, especially in Transdanubia. The particularly high number of non-local individuals in the Körös and Starčevo groups would only confirm the currently accepted Neolithisation process that was accompanied by extensive population movements if the sampled individuals corresponded to the first generation of settlers, which is not possible to attest. This influx was already over during the Hungarian LBK, while the Vinča and Balaton-Lasinja cultural groups in Transdanubia again showed higher proportions of non-local individuals, reflecting considerable new inputs from more distant regions. The archaeological record would suggest that they most likely moved along the Danube valley from the northern Balkanic area, although this could not be verified by this sample. In the Alföld, the Esztár culture also shows an increased proportion of supra-regional inputs. According to the archaeological evidence, the Esztár culture had received impulses from the neighbouring Partium area of western Romania.However, it must also be borne in mind that large tracts of the Hungarian lowlands are covered with loess soils, meaning that their strontium isotope composition can be very similar despite the greater geographical distance between particular areas. Therefore, it can be assumed that non-local individuals are not recognised when the place of origin and destination have similar strontium isotope ratios. In this context, oxygen isotope values are a good complement to the strontium isotope values, although the data from early mineralised teeth cannot be used because of the breastfeeding effect. In sum, the results of the strontium and oxygen stable isotope analyses presented in this paper revealed further insights into the complex social and economic organisation of Neolithic groups that could be put into perspective with archaeological and aDNA data. The disparities between adult males and females regarding mobility patterns and the fact that it is not possible to identify settler generations in the sample suggest that high proportion of non-local individuals could be in some cases rather related to extended marriage, social, and economic networks than to Neolithisation processes, which also explains the widespread homogeneity in material culture of the corresponding Neolithic groups. Furthermore, no significant correlation between the strontium and oxygen isotope data and the grave goods or burial context is attested in this sample, suggesting that the variability of isotope ratios cannot be explained by social differences, and that local and non-local individuals did not receive differential mortuary treatment. Finally, the large total sample considered in this study not only provided outstanding results regarding small- and large-scale mobility patterns, but also delivered invaluable information on the organisation and dynamics of the various Neolithic communities discussed here.S1 TableTable A, Description of the samples; Table B, Details on the samples for strontium isotope analyses; Table C, Details on the samples for oxygen stable isotope analyses; Table D, Standards for oxygen stable isotope analyses; Table E, Comparative data for strontium isotope baselines in Hungary; Table F, Comparative data for oxygen isotope baselines at the studies sites.(XLSX)Click here for additional data file.S2 TableThe sampling process was permitted by the museums or scientific institutions whose collection included the sampled anthropological material.(XLSX)Click here for additional data file.S3 TableInterpretation of the strontium and oxygen stable isotope data.(XLSX)Click here for additional data file.S1 FigTransdanubia (A), Croatia (C), and Alföld (C).(TIF)Click here for additional data file.S2 FigTransdanubia and Croatia (A), Alföld (B).(TIF)Click here for additional data file.S3 FigTransdanubia (A), Croatia (B), and Alföld (C).(TIF)Click here for additional data file.S4 FigTransdanubia and Croatia (A), Alföld (B).(TIF)Click here for additional data file.S5 Fig(A) Green lines represent the site-specific strontium isotope baseline range. The green dashed lines represent the baseline range that includes baseline outlier samples. (B) Light green lines represent the site-specific strontium isotope baseline range. The green dashed lines represent the baseline range that includes baseline outlier samples. Dark green lines represent the micro-regional baseline range that includes baseline samples from Budapest Békásmegyer . (C) Gre(TIF)Click here for additional data file.S6 FigLight green lines represent the site-specific strontium isotope baseline range at each site. The green dashed lines represent the baseline range that includes baseline outlier samples at each site. Dark green lines represent the micro-regional baseline range that includes the baseline samples of these three sites.(TIF)Click here for additional data file.S7 FigLight green lines represent the site-specific strontium isotope baseline range at each site. The green dashed lines represent the baseline range that includes baseline outlier samples at BÖVÖ. Dark green lines represent the micro-regional baseline range that includes the baseline samples of these four sites.(TIF)Click here for additional data file.S8 Fig87Sr/86Sr ratios of human bones from the RADO, VINJ, VINK and VUKG sites. (B) Dark green lines represent the micro-regional baseline range suggested by C. Gerling for the same spatial area [Light green lines represent the site-specific strontium isotope baseline range at each site. (A) Brown dots represent the additional ial area . (C) Das(TIF)Click here for additional data file.S9 Fig(A and C) Green lines represent the site-specific and micro-regional strontium isotope baseline range for this site complex. (B) Light green lines represent the site-specific strontium isotope baseline range at each site. Dashed green lines represent the baseline range that includes outlier baseline samples at HELI. Dashed violet lines represent the micro-regional range at POPI. (C) Dashed green lines represent the baseline range that includes outlier baseline samples.(TIF)Click here for additional data file.S10 FigLight green lines represent the site-specific strontium isotope baseline range at each site. (A) Dashed green lines represent the baseline range that includes outlier baseline samples at MEKÖ. (B) Dashed green lines represent the baseline range that includes outlier baseline samples at MECE. Violet dashed lines represent at MECE the baseline range of Mezőkövesd-Moscolyas and at KOKI the baseline range of Füzesabony-Gubakut suggested by A. Whittle and colleagues . (C) Das(TIF)Click here for additional data file.S11 Fig18O value of modern precipitation data [18O value of the modern river Danube in Hungary [The green dashed lines represent the local oxygen isotope baseline range calculated from the long-term annual average δion data –40 from  Hungary .(TIF)Click here for additional data file.S12 Fig18O value of modern precipitation data [The green dashed lines represent the local oxygen isotope baseline range calculated from the long-term annual average δion data –40 from ion data  in the s(TIF)Click here for additional data file.S13 Fig(A) At the Kóny 85 Enese (KON) site. The green lines represent the local/site-specific oxygen and isotope baseline at the site. Samples from a same individual are related by a grey line. (B) At the Tolna-Mözs (TOLM) site. The green lines represent the local/site-specific oxygen and isotope baseline at the site. Samples from a same individual are related by a grey line. (C) At the Csabdi Télizöldes (CSAT) site. The green lines represent the local/site-specific oxygen and isotope baseline at the site. Samples from a same individual are related by a grey line.(TIF)Click here for additional data file.S14 Fig(A) At the Budakeszi 8. lh. Szőlőskert-Tangazdaság (BUD) site. The green lines represent the local/site-specific oxygen and isotope baseline at the site. The violet line represents the extension of the micro-regional strontium isotope baseline when including the baseline samples from Budapest Békásmegyer . Samples(TIF)Click here for additional data file.S15 Fig(A) At the Alsónyék elkerülő (ALE) site. (B) At the Bátaszék-Lajvér (BAL) site. (C) At the Bátaszék-Mérnöki telep (BAM) site. The light green lines represent the site-specific isotope baseline ranges at each site. The dark green lines represent the micro-regional baseline range. Samples from a same individual are related by a grey line.(TIF)Click here for additional data file.S16 Fig(A) the Mórágy Tűzkődomb (MORT) site. The green lines represent the site-specific/local oxygen and strontium isotope baseline ranges. (B) At the Lanycsók Csata alja (M6-116) site. The light green lines represent the site-specific/local oxygen and strontium isotope baseline ranges. The dark green lines represent the micro-regional strontium baseline range. (C) At the Versend-Gilencsa (VEGI) site. The light green lines represent the site-specific/local oxygen and strontium isotope baseline ranges. The dark green lines represent the micro-regional strontium baseline range.(TIF)Click here for additional data file.S17 FigVinkovci Jugobanka (VINJ), Vinkovci Nama (VINK), and Vukovar Gimnazija (VUKG). The green lines represent the site-specific/ oxygen and strontium isotope baseline ranges.(TIF)Click here for additional data file.S18 Fig(A) At the Hódmezővásárhely Kotacpart (HOKO) site. The green lines represent the site-specific/local oxygen and strontium isotope baseline ranges. (B) From the Tiszalök Hajnalos (TIHA) and the Sajoszentpeter-vasúti örhaz (SAVÖ) sites. No strontium isotope baseline could be determined at TIHA and at SAVÖ. The oxygen isotope baseline range  is not represented on the diagrams.(TIF)Click here for additional data file.S19 Fig(A) At the Cegléd Ipari Park (CGIP) site. The light green lines represent the site-specific/local oxygen and strontium isotope baseline ranges. The dark green lines represent the micro-regional strontium isotope baseline range. (B) the Berettyóújfalu-nagy-Bócs dűlő (BENA) site. The green lines represent the site-specific/local oxygen and strontium isotope baseline ranges. The violet lines represent the baseline range suggested by C. Gerling  in the s(TIF)Click here for additional data file.S20 Fig(A) At the Polgár-Piócási-Dűlő (POPI) site. The light green lines represent the local oxygen and strontium isotope baseline ranges. The violet lines represent the micro-regional strontium isotope baseline range. (B) At the Tiszabura Bonishat (TIBO) and Tiszaszőlős-Domaháza-puszta, Réti-dűlő (TIDO) sites. The light green lines represent the local oxygen and strontium isotope baseline ranges. (C) Tiszaföldvár Téglagyár (TITE) site. The green lines represent the site-specific/local oxygen and strontium isotope baseline ranges.(TIF)Click here for additional data file.S21 Fig(A) Starčevo and Körös. (B) TLBK, ALBK, and Vinča. (C) Sopot, Esztár, Tiszadob/Bükk, and Szakálhát. (D) Lengyel and Tisza. (E) Balaton-Lasinja and Tiszapolgár.(PDF)Click here for additional data file.


Moreover, mutation N155H and the resistance-related mutation Q148H are mutually exclusive for unknown reasons. In the present study, we use molecular dynamics simulations to understand the impact of the N155H mutation in the HIV-1 integrase structure and dynamics, when alone or in combination with Q148H. Our findings suggest that the Mg2+ ions of the active site adopt different orientations in each of the mutants, causing the catalytic triad residues involved in the ion coordination to adapt their side-chain configurations, completely changing the INSTIs binding site. The change in the ion coordination also seems to affect the flexibility of the terminal viral DNA nucleotide near the active site, potentially impairing the induced-fit mechanism of the drugs. The explanations obtained from our simulations corroborate previous hypotheses drawn from crystallographic studies. The proposed resistance mechanism can also explain the resistance caused by other mutations that take place in the same region of the integrase and help uncover the structural details of other HIV-1 resistance mechanisms.HIV-1 integrase is the enzyme responsible for integrating the viral DNA into the host genome and is one of the main targets for antiretroviral therapy; however, there are documented cases of resistance against all the currently used integrase strand transfer inhibitors (INSTIs). While some resistance-related mutations occur near the inhibitor’s binding site, the mutation N155H occurs on the opposite side of the drug-interacting Mg The current guidelines for AIDS treatment rely mainly on inhibitors of the viral reverse transcriptase and integrase (IN) ,b. The h2+ ion pair at the active site  are the only class of IN inhibitors used in therapy . The INSive site .2+ ions. This observation raises questions about the resistance mechanism behind the N155H variant since the mutated residue does not interact directly with the drug molecules.Despite the effectiveness of the current IN inhibitors, many HIV-1 resistance-related mutations are known to emerge in patients treated with INSTIs . There a2+ in the active site. Moreover, experiments with the N224H mutant of the PFV showed that the mutant only binds one ion in the active site (ive site . The recive site  is a breB9VIC1) was used as reference to build the structure of the cSSC of each system  through comparative modeling. One of the templates used was the HIV-1 strand transfer complex (STC) structure obtained by cryo-EM (PDB code: 5u1c)   . This ste: 5u1c) . Anothere (3OYA) , given te (3OYA) . Here, wler 9.18 . The modler 9.18  and thenler 9.18 .+ ions. In the next step, all systems went through an energy minimization with the steepest descent algorithm, using a maximum of 100000 steps and a maximum force of 0.01 KJ mol–1 nm–1 as convergence criterion. After the minimization step, each of the systems was divided into three replicas. For the heating step, each of the replicas was assigned velocities using different random seeds for atom velocity generation and then heated to 310 K during the first 0.5 ns of a 1 ns MD run in an NVT ensemble. Temperature coupling was accomplished using the v-rescale thermostat  was calculated for each frame of the MD simulations in relation to the starting structures. The snapshots generated after the RMSD of each replica reached equilibrium were compiled for each system and used to calculate the root-mean-square fluctuations (RMSF) and to analyze the non-bonded interactions. The RMSD equilibrium was defined as a contiguous set of frames within a replica where the correlation between time and RMSD is between -0.35 and 0.35. The compiled snapshots were also used to generate a set of clusters based on the RMSD of the protein backbone and another set of clusters using the vDNA RMSD, both using a 2 Å RMSD cutoff. The distances between N1 of the cytosine and N9 of the terminal adenine were calculated through the whole simulations in chain A and chain C, in order to investigate the dynamics of the terminal nucleotide near the active site.2+ ion pairs, according to its known spatial configuration  is different from the WT enzyme  to 1.2 KJ/mol (±4.1) , these o2+  in the active site. Possibly, the altered position of the ions in the presence of the inhibitors can cause the site to lose one of the Mg2+, assuming that the PFV goes through a similar change in the presence of the equivalent mutation. Hare et al. also suggest that the lack of one of the ions in the crystals was likely caused by many subtle changes rather than by one major one. It is possible, however, that the addition of histidine in position 148 along with the mutation N155H destabilizes the coordination site beyond the minimum configuration required for catalysis.In a study with crystals of the PFV IN soaked with INSTIs in a mutant equivalent to N155H, INSTIs are known for binding through an induced-fit mechanism, where the terminal nucleotide of the vDNA chain in the active site must rotate away from its position to enable binding . In the In the crystal structure of the PFV bond to Raltegravir, the N1-N9 distance is of ≈8.6 Å, ≈9.8 Å in the SIV structure bond to Dolutegravir  and ≈9.4This opening motion is evidence that the vDNA ends are flexible near the active site, and the presence of the INSTI is not required for the opening event to occur, but it probably stabilizes the terminal. The lack of conformational change in the terminal adenine of the N155H variant is consistent with predictions made by 2+ coordination in the active sites of the HIV-1 IN, the N155H variant, and the double mutant N155H+Q148H. The differences in structure and energetics help explain observations from previous studies carried out with the PFV IN (2+ ion pair disruption, for instance, in the mutants T66KI. The different coordination of the ions can also explain the deficient catalysis (In the current study, our simulations suggest different Mge PFV IN . We belie PFV IN . Possiblatalysis  since noMoreover, the dynamics of the outer chains are similar to previous MD studies that used only the IN CCD, and the so-called flexible loop and neighbor regions show different RMSF values in WT and the mutants.Large conformational changes in the terminal nucleotide near one of the chains of the WT IN suggests that its vDNA terminal may be flexible, while no large conformational changes were observed in the terminal nucleotide of the N155H variant and the double mutant. Moreover, H155 seems to have more favorable interactions with the terminal adenine than N155. This is evidence for the hypothesis that claims N155H has stronger vDNA-IN interactions . We beliThe datasets generated for this study are available on request to the corresponding author.LM and AG were responsible for designing the research and writing the manuscript. LM carried out the modeling, simulation, and analysis steps. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


They were identified from a computational virtual screening approach using the Mpro as the drug target, and their enzymatic inhibition against SARS-CoV-2 Mpro was validated in the fluorescence resonance energy transfer (FRET)-based enzymatic assay (inhibitory constant Ki = 0.04 to 3.27 µM).Li et al.  recentlypro but also five other cysteine proteases, and the inhibition was only observed in the absence of reducing reagent, 1,4-dithiothreitol (DTT) , is a weak inhibitor of SARS-CoV-2 Mpro with an IC50 value of 29.4 ± 3.2 µM    A and B. ng assay C. Third,e of DTT A. Howeveft assay C or natift assay H. This s >50 µM) A and B, ng assay C. Candesng assay G.pro. A GST-tagged Mpro was used in their enzymatic assay; however, it is known that Mpro requires a native N terminus to form the enzymatic active dimer (Overall, our data suggest that there might be a significant flaw with the enzymatic assay inhibition results presented in this study. None of the identified hits was confirmed to inhibit or bind to SARS-Co-2 Mve dimer . DTT wasve dimer B.


NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB , ammonia-oxidizing archaea , and a nitrite-oxidizing bacterium , all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC50 = 0.3–0.7 μM) compared to EQ (EC50 = 1–1.4 μM) and EQNL (EC50 = 26.6–129.5 μM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC50 = 221.9–248.7 μM vs EC50 = 0.6–2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC50s of 0.8–2.1 and 1.0–6.7 μM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO.Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agr NOB subsequently transform NO2– to nitrate (NO3–) using nitrite oxidoreductase . AOB oxidize ammonia to hydroxylamine  is limited. The use of inhibition assays with pure cultures of a diverse range of soil-derived strains is a necessary benchmarking step to define the exact spectrum of activity of NIs destined for use in agriculture. Most culture inhibition assays have focused on AOB e.g.,  or testegrestis” , “Candidnaterra” , and Nitennensis . In addiennensis , hence tennensis , impliesIn previous soil microcosm studies we showed that ethoxyquin , an antioxidant used as preservative in fruit-packaging plants, and its derivative 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), strongly inhibited the activity of AOB and AOA . EQ in sNitrosomonas europaea and Nitrosospira multiformis, belonging to AOB clusters 7 and 3, respectively, . We expanded our liquid inhibition assays to NOB to gain insights on the impact of NIs on a microbial group functionally associated with ammonia oxidation, and directly linked to nitrogen loss from disturbed agricultural ecosystems in the form of nitrate production. Specifically, we used (i) AOB strains ctively, , with clctively, ; (ii) AOklandus”  and “Caninensis” , occupyiinensis” , and 2SO4] and phenol red (0.5 mg L–1) as a pH indicator. AOA “Ca. N. franklandus” C13 and “Ca. N. sinensis” ND2, were incubated at 35°C in a medium supplemented with 1 mM NH4+ (NH4Cl). The former was cultured in HEPES-buffered modified freshwater medium .All strains were grown aerobically in the dark without shaking. AOB s medium  containi pH 7.5; , while t pH 7.5; . Nitroba pH 5.2;  supplemeAnalytical standards of DCD (99% purity), NP (≥98%), and EQ (95%) were purchased from Sigma-Aldrich (Germany), while DMPP (99.1%) analytical standard was provided by BASF Hellas. The oxidation derivatives of EQ, QI, and EQNL were synthesized as described by 2– production) dictated the range of NI concentrations that will allow calculation of inhibition thresholds. Cultures were established in triplicate for each strain × NI × concentration combination in 100-mL Duran bottles containing 50 mL of growth medium and inoculated with a 1 or 2% (v/v) transfer of exponentially growing cultures of AOB or AOA/NOB, respectively. EQ, QI, EQNL, and NP were added to the cultures as filter sterilized dimethyl sulfoxide (DMSO) solutions due to their low water solubility (≤60 mg L–1 at 20°C). The final concentration of DMSO in all cultures was 0.1% (v/v), which did not exert a significant inhibitory effect to any of the isolates tested (data not shown), in line with previous studies with the same isolates (2O before addition of 25 μl (0.5% v/v). All NIs were added to batch cultures at the beginning of the exponential growth phase. For all assays, triplicate cultures with the same inoculum not amended with NIs were included. Upon inoculation all liquid batch cultures were sampled at regular time intervals to determine the effect of NIs on the activity and growth of nitrifying microorganisms by measuring changes in nitrite concentrations and the abundance of amoA (AO) or nxrB (NOB) genes, respectively.The activity of all NIs was determined in liquid batch cultures over a range of concentrations to establish relevant inhibition thresholds per strain and compound. Preliminary assays with a broad range of concentrations for each NI and isolate . The amoA genes of AOB and AOA was amplified with primers amoA-1F/amoA-2R  showed recoveries of >80% for all compounds studied.Ethoxyquin, QI, EQNL, and NP residues were extracted from liquid media by mixing 0.3 mL liquid culture with 0.7 mL of acetonitrile. Residues of DCD and DMPP were extracted by mixing 0.1 mL liquid culture with 0.9 mL of ddH–1 for DCD and at 1 mL min–1 for all other NIs. Column temperature was set at 40°C for DCD and DMPP, and at 25°C for all the other NIs. Mixtures of acetonitrile and ammonia [0.25% (vol/vol)] or ortho-phosphoric acid [0.1% (vol/vol)] were used at a ratio of 70:30 (vol/vol) for mobile phases in the analyses of EQ, QI, EQNL, and NP, respectively, and detection was achieved at 225, 245, 230, and 269 nm, respectively. Similarly, chromatographic separation of DCD and DMPP was achieved using ddH2O (100%) and a mixture of methanol and ortho-phosphoric acid [0.1% (vol/vol)] solution 50:50 by volume, respectively. DCD and DMPP residues were detected at 218 nm and 225 nm, respectively.High performance liquid chromatography (HPLC) analyses were performed in a Shimadzu LC-20ADHPLC system equipped with an UV/VIS PDA detector. A Shimadzu GVP-ODs  pre-column, connected to a RP Shimadzu VP-ODs  column, was used for NI separation. The injection volume was 20 μl. The flow rate of the mobile phase was set at 0.8 mL min50 describes the concentration of the inhibitor that reduces half of the activity (nitrite accumulation or consumption) of AO or NOB. Dose-response modeling was performed using normalized data whereby nitrite concentration values were divided by the mean value of the matching control. Analyses were carried out using the dose response curves (drc) v3.0-1 package . Variance between the EC50 values of the different NIs for one strain and between different strains for a given NI was analyzed by one-way ANOVA, and Duncan post hoc test (P < 0.05). The four kinetic models proposed by the FOCUS working group on pesticide degradation kinetics . Curve fitting was performed with the mkin v0.9.47.1 package , respectively, while complete inhibition of “Ca. N. sinensis” was evident at levels ≥4.6 μM. Growth inhibition profiles of AO isolates corresponded with NO2– production (p ≤ 0.05) in the growth of Nitrobacter sp. NHB1 was observed at the end of the incubation period for all EQ concentration levels.Ethoxyquin fully inhibited the activity of f 460 μM . In contoduction . In contN. europaea and N. multiformis at concentrations ≥270 μM and ≥135 μM, respectively, (p ≤ 0.05) at all QI concentrations, with little or no activity at concentrations ≥2.7 μM, and a gradual recovery observed only for “Ca. N. franklandus” at the lowest concentration level (0.27 μM). Nitrite consumption by Nitrobacter sp. NHB1 was significantly suppressed at concentrations ≥135 μM (p ≤ 0.05), though a persistent inhibitory effect was evident only at 540 μM. The inhibition of QI on AO growth concurred with the NO2– production patterns, unlike NOB where QI persistently inhibited the growth of Nitrobacter sp. NHB1 at concentrations ≥135 μM , respectively, and complete inhibition occurred at 500 μM and ≥125 μM, respectively. Nitrite oxidation by Nitrobacter sp. NHB1 was completely inhibited by EQNL only at the highest tested concentrations of 500 μM , probably due to the decreased number of living cells at EQNL concentrations ≤25 μM. Variations in the growth inhibition pattern of EQNL was observed also for Nitrobacter sp. NHB1 which was not significantly (p = 0.063) affected by EQNL even at the highest tested concentration  for the sum of EQ + QI + EQNL in cultures supplemented with 460 μM of EQ ranged from 2.1 days for Nitrobacter sp. NHB1 to 60.1 days for N. multiformis . QI showed limited persistence and a weak dose-dependent degradation pattern with DT50 = 0.05–1.52 days at the lowest concentration level (2.7 μM), and 2.23–5.65 days at the highest concentration level  the activity of both AOB strains at concentrations of 250 μM and 500 μM, with complete inhibition observed only at 500 μM (Ca. N. franklandus” and “Ca. N. sinensis” being significantly inhibited (p < 0.05) at concentrations ≥1 mM and ≥0.5 mM, respectively. However, persistent inhibition was evident only at concentrations ≥2.5 mM and ≥1 mM, respectively, (Nitrobacter sp. was significantly inhibited (p < 0.05) by DCD only at the highest concentration tested (100 mM). The growth inhibition patterns of AOB and NOB were congruent with the NO2– production patterns. This was not the case for AOA where “Ca. N. franklandus” growth was significantly reduced at 0.5 mM , with complete inhibition observed at ≥5 μM and ≥25 μM, respectively,  but significant (p < 0.05) inhibition of N. multiformis growth at 0.1 μM compared to the control. Conversely, DMPP significantly inhibited the activity of both AOA isolates at higher concentrations of ≥0.5 mM (p ≤ 0.05), with complete inhibition of “Ca. N. franklandus” and Ca. N. sinensis” occurring only at 5 mM and ≥1 mM, respectively, . DMPP and NP were the most potent inhibitors of N. europaea, followed by EQ, QI, and DCD which were not significantly different, with EQNL being the weakest inhibitor (EC50 = 181.4 ± 23.3 μM). For N. multiformis, DMPP, NP, and QI were equally effective inhibitors, followed by EQ, DCD, and EQNL. The two AOA strains exhibited contrasting responses, with “Ca. N. franklandus” having decreased sensitivity to DCD and DMPP compared to “Ca. N. sinensis.” EQ, its derivatives and NP were equally effective inhibitors of both AOA isolates, with QI having the lowest EC50 values (0.3 ± 0.0–0.7 ± 0.4 μM), while DCD and DMPP were the weakest AOA inhibitors  probably reached or exceeded its inhibition threshold levels for both AO groups.This study is the first to investigate the inhibitory effect of EQ, a novel NI of potential agricultural relevance, and its oxidation derivatives, QI and EQNL, on soil nitrifiers grown in pure cultures and demonstrate greater inhibition of all three compounds on AOA compared to AOB isolates. In all cultures, EQ was rapidly transformed to QI and EQNL, with the former being the major but least persistent derivative, while the latter being the minor but more persistent derivative, and was consistent with previous studies in soil . ConsideN. multiformis compared to the AOA N. viennensis. Of these two NIs, DMPP showed greater inhibitory activity toward both AOB isolates. Data on the inhibitory activity of DMPP toward soil-derived cultures of AOB and AOA strains are scarce. 50 = 448 μM) compared to DCD (EC50 = 947.1 μM) to “Ca. Nitrosocosmicus agrestis,” a soil strain closely related to “Ca. N. franklandus.” Unlike DMPP, there are several reports on the inhibitory activity of DCD on soil AOA and AOB cultures with DCD strongly inhibiting Nitrososphaera sp. JG1  and AOA strains  at levels varying from 0.86 μM for AOB (Ca. N. devanaterra ND1” at concentrations ≥10 μM compared to ≥5 μM for “Ca. N. sinensis (ND2)” in our study, while 50 of 0.6 μM for “Ca. N. agrestis” compared to 1 μM for “Ca. N. franklandus” in our study. However, in contrast to our findings for NP inhibition of N. multiformis (EC50 0.8 ± 0.3 μM), 50 > 173 μM). In addition to the minor differences in cultivation conditions between the two studies, Shen et al. added solid NP directly into the cultures to achieve concentrations in the range of 40–173 μM, with the highest level corresponding to the upper limit of NP water solubility at 20°C (40 mg L–1), entailing a risk for precipitation of the active compound.Nitrapyrin was the only tested NI that showed an equivalent and strong inhibitory effect toward both AOB and AOA isolates, suppressing their activity at concentrations ≥0.5–5 μM and ≥1–5 μM, respectively. This in line with previous studies which showed inhibition of AOB , we also observed differences in the sensitivity between the two AOA or two AOB strains examined. For QI and EQNL, and EQNL . Alternahemicals .Ca. N. sinensis” to both simvastatin (Ca. N. franklandus” to DCD and DMPP might be associated with its capacity to produce extracellular polymeric substances (EPS) leading to aggregate formation that block the hydrophilic NIs DCD and DMPP of accessing the surface of cells engulfed into hydrophobic EPS  could have a universal inhibitory effect on both AOA and AOB, while 0.06 mg Kg–1 (<0.1 Kg ha–1) would be required to achieve effective inhibition AOA only. This is consistent with earlier studies that showed that EQ, when applied in soil at concentration levels (50 mg Kg–1) simulating a wastewater disposal scenario, resulted in inhibition of both AOA and AOB  and a large fraction of European agricultural soils , and low to moderate persistence in the AOB cultures (DT50 = 8.7–60.1 days), a difference most likely attributed to abiotic factors such as medium pH  rather than an enzymatic transformation, considering the autotrophic lifestyle of the tested isolates (50s = 44.5 to >1000 days) with the lowest DT50 values observed in the liquid cultures of Nitrobacter sp. NHB1, suggesting a potential interaction with this strain. Microbial mineralization of DCD by pure cultures of soil isolated bacteria has been previously reported  and urease-negative  AOs were observed. Abiotic parameters such as temperature or pH could potentially influence the stability of DCD in soil  on DCD stability in our liquid culture conditions. NP degraded rapidly (DT50 = 0.12–12.5 days) in all liquid cultures. In contrast, DMPP showed a high persistence in all liquid cultures, except of Nitrobacter where a great variation in its persistence was evident. Considering that Nitrobacter sp. NHB1 and AOA were cultured in media of similar content and pH, the above variation was most probably driven by interaction between DMPP and the bacterium. Genomic analysis suggested limited catabolic capacity of aromatic compounds by Nitrobacter strains  and the effects observed. The total residues of EQ showed limited persistence in the AOA and NOB cultures  introduces a novel potential NI, EQ, which possesses desirable characteristics, including transformation into a highly potent NI (QI) characterized by high inhibitory activity against AOA compared to currently registered NIs in Europe, and (iii) demonstrates the different sensitivity of AOA and AOB to NIs, which indicates that novel strategies for effective nitrification inhibition should rely on new broad-range NIs, or more likely, mixtures of NIs with complementary activity against different nitrifier groups. Future work will focus on the elucidation of EQ and QI inhibitory mechanisms, and on the evaluation of their environmental and agronomic performance under diverse edaphic and climatic conditions and on soils with different microbial communities.We compared the inhibition potential of EQ, a novel NI, and those currently used in agricultural practice, on the activity and growth of soil-derived AOA, AOB, and NOB isolates grown in liquid culture. EQ, and primarily its major derivative QI, showed high potency against AOA, in contrast to DCD and DMPP (the only NIs currently registered for use in Europe) which were inhibitory to AOB only. Conversely, NP showed an equally high inhibitory activity against both AOA and AOB isolates. EQ, QI, and NP were the most potent AOA and NOB inhibitors, unlike DCD and DMPP, which demonstrated no activity. DMPP and NP were the most potent AOB inhibitors, with EQ, QI, and DCD showing lower but still appreciable inhibitory activity. Our study (i) offers benchmarking knowledge of the activity range of currently used in agriculture and potentially new NIs to soil AO and The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.50 and DT50 values, constructed and edited data figures, and reviewed the manuscript. CT planned and participated in part of the culture inhibition assays. UM-S supervised the chemical synthesis of EQ derivatives and reviewed the manuscript. GN provided nitrifying cultures, supervised part of the culture inhibition assays, and reviewed the manuscript. DK conceived the experimental rationale, co-supervised the experiments, and reviewed the manuscript. All authors contributed to the article and approved the submitted version.EP planned, performed, and supervised the experiments, carried out data analysis, drafted and revised the final manuscript. EB, EL, EA, and AK participated in parts of the culture inhibition assays. SV performed the modeling for the calculation of ECThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Two parallel cannulated screws along with an anterior wire to construct a tension band is a popular approach in transverse patellar fractures. However, the optimal screw proximity, either deep or superficial screw placements, remains controversial. Hence, a new concept of the addition of a third screw to form a triangular configuration along with the original two parallel screws was proposed in this study. Therefore, the biomechanical effect of the additional third screw on the stability of the fractured patella was investigated with finite element (FE) simulation.An FE knee model including the distal femur, proximal tibia, and fractured patella (type AT/OTA 34-C) was developed in this study. Four different screw configurations, including two parallel cannulated screws with superficial (5-mm proximity) and deep (10-mm proximity) placements and two parallel superficial screws plus a third deep screw, and two parallel deep screws plus a third superficial screw, with or without the anterior wire, were considered for the simulation.Results indicated that the addition of a third screw increased stability by reducing the dorsal gap opening when two parallel screws were deeply placed, particularly on the fractured patella without an anterior wire. However, the third screw was of little value when two parallel screws were superficially placed. In the existence of two deep parallel screws and the anterior wire, the third screw reduced the gap opening by 23.5% (from 1.15 mm to 0.88 mm) and 53.6% (from 1.21 mm to 0.61 mm) in knee flexion 45° and full extension, respectively. Furthermore, in the absence of the anterior wire, the third screw reduced the gap opening by 73.5% (from 2 mm to 0.53 mm) and 72.2% (from 1.33 mm to 0.37 mm) in knee flexion 45° and full extension, respectively.Based on the results, a third cannulated screw superficially placed (5-mm proximity) is recommended to increase stability and maintain contact of the fractured patella, fixed with two parallel cannulated screws deeply placed (10-mm proximity), particularly when an anterior wire was not used. Furthermore, the third screw deeply placed is not recommended in a fractured patella with two parallel superficial screws. Patellar fracture is a common scenario in orthopaedic clinics, and it usually occurs due to a sudden impact to the patella, such as a fall or traffic accidents , 2. The To date, many surgical approaches, such as tension band, modified tension band, cannulated screws with anterior wire, and even locking plate, have been developed for the fixation of patellar fractures –12. AmonTo strike a balance between superficial and deep screw proximities, a new concept of the addition of a third screw to form a triangular configuration along with the original two parallel screws was proposed in this study. Therefore, the first aim of this study was to investigate the biomechanical effect of the additional third screw on the fractured patella in different knee positions by using the finite element (FE) method. The second aim was to compare the mechanical differences of the fractured patella between the use and nonuse of the anterior wire with the cannulated screws. FE analysis is a numerical method for obtaining an approximate solution in engineering problems, and this method has been used in many orthopaedic studies , 20.Firstly, a knee model with a fracture patella fixed with the traditional fixation approaches, namely two parallel cannulated screws and the anterior wire, was developed and validated. After the validation succeeded, the proposed triangular screw configuration and anterior wire were applied on the fractured patella model and then analysed.An intact knee model including the distal femur, proximal tibia, and patella was developed based on the computed tomography images of the open source from the US National Library of Medicine’s Visible Human Project. Images were taken of the knee full extension with a 1-mm interval, and bony contours in each image were retrieved by the higher gray value of the bone than that of the soft tissue in the Avizo software, Version 6.0 . The 3D model was developed by the lamination of obtained contours. Then, the 3D model was imported into a CAD software Solidworks 2019  to modify the knee position to flexion 45° and create the fracture pattern of the patella and screw implantations. A transverse fracture at the middle of the patella was created according to the type AT/OTA 34-C. Because the type is one of the indications of cannulated screw and tension band technique. The fracture was created with a virtual plane, and no gap existed between fragments. Thereafter, parallel cannulated screws with an anterior wire were used to construct a tension band to stabilize the fractured patella.At first, different traditional approaches including two parallel superficial screws (at 5-mm depth from the anterior surface of the patella) at an 18-mm interval with (Model A) and without (Model B) the anterior wire, and two parallel deep screws (at 10-mm depth from the anterior surface of the patella) at an 18-mm interval with (Model C) and without (Model D) the anterior wire were used for validation  and without (Model F) the anterior wire, and two parallel deep screws at a 22-mm interval plus a third superficial screw in the middle of the two parallel screws with (Model G) and without (Model H) the anterior wire  for mesh and simulation. The maximum edge lengths of the element for the wire, screw, patellar bone, patellar cartilage, and distal femur and the proximal tibia were set to 0.25 mm, 0.5 mm, 1 mm, 2 mm, and 5 mm, respectively, with the command “Sizing” in ANSYS. The contact areas of the patella with the wire and the screw and the distal femur with the patella were also locally refined with the commend “Sizing,” and the maximum length of the element edge was set to 0.5 mm and 1 mm, respectively. Overall, 519,421 nodes and 322,223 elements were used to mesh the model with two screws and the anterior wire. The other models were meshed using an identical setting.The stiffness of the patellar ligament was about 2826 N/mm in the literature . In the The material properties of the bone and cartilage were defined based on the literature Table  24–27].–27.24–27For validation, a 400 N force  was applied on the base of the fractured patella with the anterior wire (Models A and C) in knee flexion 45°. The bilateral ends of the knee were totally fixed. The displacement of the loading site on the proximal fragment under 400 N was compared with that in the Bryant’s study . In addiFor the comparison of the biomechanical effect between the traditional and the proposed approaches, two different degrees of force, namely 800 N and 400 N, were used in the simulation. The 800 N tension force parallel to the long axis of the femur was applied to the base of the fractured patella with the anterior wire , in knee flexion 45° Fig. c and fulThe maximum displacement of the fragment and the maximum gap opening distance were used as indices to assess the stability of the fractured patella with various screw configurations. The contact area maintained between the fragments was used to judge the ability of screw configurations to stabilize the fractured patella. The fractured patellar fragments were initially in contact with each other without any gap and separated from each other after addition of the load. Greater the contact area maintained after the static balance, better was the stability achieved by the screws.The linear stiffness and load with 2-mm deformation of the present FE model with 10-mm screws placement without the anterior wire were similar to that in Dargel’s study, whereas the results with 5-mm screw proximity were larger than that in Dargel’s study  after using the additional third screw in two parallel deep screws and wire in flexion 45°under 800 N.The additional third screw also maintained the contact area between the patellar fragments with deep screws in knee flexion 45° Fig. a–c, wherThe optimal fixation approach for transverse patellar fracture is still unknown, because the mechanical responses of the patella are complicated during knee motions. Each of the existing approaches have advantages and disadvantages , 31, 32.The effect of the additional third screw on increasing the stability was especially obvious in the two parallel deep screws, particularly without the anterior wire, because the added third screw increased the ability to resist torsional loading on the fractured patella. In two parallel deep screws, the loadings on the patella finally developed a torsion load after static balance both in knee flexion 45° and full extension. The superficial (leading) surface is on the tension side, whereas the articular surface is on the compression side. Therefore, no screw was in place to resist torsion when screws were deeply placed without the anterior wire , 18. TheNevertheless, using the additional third screw with two parallel deep screws could maintain the contact between proximal and distal fragments, both in knee full extension and flexion 45°, because the loading finally developed a torsional load in the original deep screw placement. Initially, the patellar fragments should be in contact with each other without any gap after fracture reduction, and the contact area should decrease with increasing applied load. A proper fixation should be able to maintain the contact of the fractured patella to promote direct bone healing  of the fracture , 34. DirThe role of the additional third screw is similar to that of the anterior wire when the original two parallel screws were deeply placed, because they were all on the same side. Although the location of the anterior wire is more superficial than the third screw, the diameter of the third screw (4 mm) was much larger than the anterior wire (1.25 mm). The increased outer diameter increased the ability to resist the external force, particularly to the torsional load. From a mechanical view, moment of inertia of a cylinder is proportional to the third power of the outer diameter. Therefore, increase the outer diameter is very helpful to increase the stiffness and resistance ability to the loading. Hence, the effect of the additional third screw was more obvious than that of the anterior wire in the present simulation, in spite of shorter distance from the articular surface than the wire. Hence, the additional third screw reduced the gap opening but increased the contact area more than the anterior wire in the fractured patella with two deep parallel screws under identical (400 N) force.In addition to the improved stability, the use of an additional third screw has another advantage –the feasibility of minimally invasive surgery over the wire. The insertion of cannulated screw can be executed via minimally invasive techniques, whereas the wiring of the encircling of the wire often required open manners . FurtherThe aim of the surgical fixation of the patellar fracture is to construct a rigid fixation for early rehabilitation soon after the surgical intervention , 36. AltThis study has some unavoidable limitations. First, the distal femur and proximal tibia were fixed at different knee flexion degrees, and the meniscus and the articulation of the distal femur and the proximal tibia were not considered in the simulation. Furthermore, the alignment of the knee joint and patella in flexion 45° was manually adjusted and was not based on the images in flexion. Second, in the present simulation, only a worst-case scenario was considered and the force magnitudes in flexion and extension were uniform. The alerting of the force and moment at the beginning and mid-stage of knee flexion were ignored. Third, the material properties of the cortical and cancellous bones were simplified as linear elastic, isotropic, and homogeneous. The trabecular architecture was not modelled. Finally, the manipulated tension of the wire was not considered.The effect of implementing an additional third screw was highly related to the original screw proximity in the fixation of patellar fractures with cannulated screws. Based on the present results, a third cannulated screw superficially placed (5 mm from the anterior surface of the patella) with two parallel cannulated screws, deeply placed (10 mm from the anterior surface of the patella) is recommended in the management of patellar fracture when the anterior wire was not used. While a third screw deeply placed is not recommended for addition to the fractured patella along with two superficial parallel cannulated screws whether the anterior wire was used or not.


The ability to sense and move within an environment are complex functions necessary for the survival of nearly all species. The spinal cord is both the initial entry site for peripheral information and the final output site for motor response, placing spinal circuits as paramount in mediating sensory responses and coordinating movement. This is partly accomplished through the activation of complex spinal microcircuits that gate afferent signals to filter extraneous stimuli from various sensory modalities and determine which signals are transmitted to higher order structures in the CNS and to spinal motor pathways. A mechanistic understanding of how inhibitory interneurons are organized and employed within the spinal cord will provide potential access points for therapeutics targeting inhibitory deficits underlying various pathologies including sensory and movement disorders. Recent studies using transgenic manipulations, neurochemical profiling, and single-cell transcriptomics have identified distinct populations of inhibitory interneurons which express an array of genetic and/or neurochemical markers that constitute functional microcircuits. In this review, we provide an overview of identified neural components that make up inhibitory microcircuits within the dorsal and ventral spinal cord and highlight the importance of inhibitory control of sensorimotor pathways at the spinal level. The spinal cord is capable of generating a range of motor behaviors in response to various interoceptive and environmental cues. Somatosensory pathways span the entirety of the neuraxis and recruit a unique combination of neuronal networks to elicit appropriate behavioral responses ,2,3. DesA variety of experimental approaches, including classic electrophysiology and recent complex genetic and transcriptomic techniques, have been used to dissect inhibitory microcircuits ,18,19,20Primary sensory neurons are excitatory, pseudo-unipolar neurons with cell bodies located in the dorsal root ganglion (DRG), peripheral receptors in the skin and muscles, and central terminals that are concentrated in various layers of the dorsal horn and ventral horn ,21,22. Fi) but none of the neurochemical markers [i-IV have been identified and described using a combination of molecular profiling [More recently, afferent fiber types that convey unique sensory input towards spinal effector neurons and higher order centers have been separated using various cell-type specific markers . Unmyeli markers ,38. Low-rofiling ,39,40, irofiling ,42 in mirofiling ,44,45. Trofiling ,47.Spinal INs are primary targets for many types of afferent fibers ,9,48,49.In addition to INs which may be more specialized in function, there are several populations of spinal neurons serving a more integrative function. These tend to be located within the deep dorsal horn (III-VI) and include neurons that receive polymodal input from various sensory pathways ,61,62. TVentral horn INs have been largely studied in the context of locomotion, with distinct populations contributing to rhythm and pattern ,67,68,69There are multiple potential points of control including the afferent and excitatory INs involved in specific pathways, excitatory integrator neurons, locomotor circuit neurons, ascending tract neurons, and motor neurons. Inhibitory neurons reduce the activity in these pathways by postsynaptic and presynaptic inhibitory mechanisms.Inhibition at the level of the spinal cord maintains appropriate levels of activity within neuronal circuits by regulating sensory information en route to ascending and motor output pathways. Inhibitory INs regulate activity of both excitatory and inhibitory neurons postsynaptically through axo-somatic and/or axo-dendritic synapses ,13,76. EA receptors on the terminals results in chloride efflux, producing depolarization. This primary afferent depolarization (PAD) retrogradely invades the afferent fibers, inactivating voltage-gated sodium and/or calcium channels, shunting subsequent afferent signals [Inhibitory synapses onto afferent terminals provide an additional mode of inhibitory gating of sensory information which decreases the excitatory drive to postsynaptic neurons rather than directly depressing the level of activity at the postsynaptic neuron ,76. Inhi signals ,89. Deta signals ,14,86,89Developmental genetic studies have determined the transcriptional regulatory framework that underlies inhibitory cell-type diversification throughout the spinal cord ,91. All Inhibitory neuronal populations in laminae I–III of the dorsal horn have the potential to ameliorate nociceptive signaling at the spinal level prior to ascending to result in pain perception ,98. Theri/III [Parvalbumin (PV) INs in the superficial dorsal horn are primarily located in laminae IIi-III ,101,102,i/III ,105,106,i/III .Genetic removal or synaptic silencing of the entire PV IN population leads to allodynia of previously innocuous stimuli . At leaso with fewer cells in laminae IIi–III [i [There is extensive co-expression of galanin and dynorphin in the superficial dorsal horn and these neurons constitute a subset of inhibitory INs distinct from PV ,109,110. IIi–III ,109. Gal IIi–III ; howeveri–III [i . Immaturi–III [i ,114. Axoi–III [i ,109, tari–III [i  and gasti–III [i ,115, as i–III [i ,114.Several additional lines of evidence indicate a role for galanin and inhibitory dynorphin INs in gating LTMR afferent input by suppressing the activity of excitatory output neurons in pathways, signaling mechanical pain and itch. Chemogenetic activation of dynorphin INs attenuates responses to pruritogens . FurtherInterestingly, genetic ablation of dynorphin INs does not affect itch but instead facilitates Aβ-evoked input to superficial pain pathways and leads to the development of mechanical allodynia . The disAnother grouping of inhibitory INs in the superficial dorsal horn primarily expresses nitric oxide synthase (nNOS) ,18. TherExpression of NPY distinguishes an additional population of dorsal inhibitory INs in both rat and mouse ,109,121 Calretinin INs represent a large portion of inhibitory INs in I-II that were not labeled by any of the previously labeled neurochemical markers ,126. TheThe inhibitory populations in the deep dorsal horn receive multimodal afferent input from a wide array of LTMR afferents ,48. ImpoA population of neurons in the laminae III-V expresses receptor tyrosine kinase Ret (RET) during neonatal development and defines a predominantly inhibitory subpopulation that has interesting circuit properties . Early RGene expression profiling and single-cell RNA sequencing have converged on cell-specific markers that define a subset of neurons in the dorsal horn expressing the retinoid-related orphan receptor-β (Rorβ) ,19. RorβExpression of the nuclear organization factor Satb2 regulates sensorimotor circuit organization in the spinal cord and labels a unique population of neurons that are concentrated in laminae V-VI ,48,135. Tfap2b INs are largely GABAergic neurons found medially in laminae IV, V, and VI . These nMotor circuitry, and minimally motor neurons, must be recruited for sensory-evoked action. Motor neurons are the final output of the nervous system  and receInhibitory INs in the ventral horn are often divided into four cardinal classes (V0–V3) based on the developmental expression of transcription factors unique to INs derived from the same progenitor domain ,143. LesD) and ventral (V0V) populations based on the absence and presence of Evx1, respectively. The V0V neurons are mainly excitatory and the V0D INs are inhibitory, distinguished by presence of Dbx1 in the absence of Evx1 or the co-expression of Dbx1 and Pax2 [D neurons are glycinergic and about half are GABAergic [D neurons receive dense input from vGluT1-expressing terminals, suggesting that these neurons receive monosynaptic input from primary afferents [V INs, inhibitory V0D INs form monosynaptic contacts with contralateral motor pools and are directly involved in alternation at low locomotor speeds [The V0 INs are commissural INs defined by the expression of the transcription factor Dbx1 ,145. Theand Pax2 . The majABAergic ,147. Medr speeds ,148, corr speeds ,150,151.The V1 INs, defined by the expression of Engrailed 1 (En1), are exclusively ipsilaterally projecting inhibitory INs . These nRenshaw cells make up <10% of the V1 population . They arIa inhibitory INs express PV but not calbindin and receive input from proprioceptive Ia afferents and input from Renshaw cells ,159. Ia Approximately 75% of the V1 INs are not Renshaw cells or Ia inhibitory INs . At leasSimilar to the V1 INs, V2b INs are inhibitory, ipsilaterally projecting INs located primarily in lamina VII . A smalldI6 INs are identified by the transcription factors Wt1 and/or Dmrt3 ,168,169.The current description of INs across the dorsal and ventral horns has become increasingly complex as functionally distinct populations are identified and interrogated. The populations described above are unlikely to constitute all spinal inhibitory INs, as there are other inhibitory markers that define candidate populations that have been shown to be distinct but have not been studied in detail to date ,172,173.The true test of population identity should be function. Genetic access to molecularly distinct populations has served to complement previous classification schemes and enable targeted manipulations. Even so, this approach may not capture the full extent of effect from a given population. Further, the idea that molecularly defined neurons will delineate functional pathways, although attractive, has been more recently called into question. Instead, synaptic connectivity and population coding considered together with cell-specific markers is more likely to be required to define functional circuits ,57,139. Inhibitory microcircuits involved in afferent gating represent robust and highly conserved mechanisms employed by the central nervous system to regulate neurotransmission. Dissecting spinal circuits will be one key step towards unraveling the intricacies of neural circuit organization and signaling. Reflexive responses and locomotion are highly stereotypic behaviors that are amenable to observation, measurement, and association with individual spinal IN populations, and more complex responses are underexplored to date. The cumulative findings from targeted genetic manipulations offer several candidate populations for novel therapeutic interventions and functional recovery after injury ,175, how


The development of allergic disease involves the production of IgE antibodies upon allergen exposure in a process called sensitization. IgE binds to receptors on the surface of mast cells and basophils, and subsequent allergen exposure leads to cross-linking of IgE antibodies and release of cell mediators that cause allergy symptoms. Although this process is quite well-understood, very little is known about the epitopes on the allergen recognized by IgE, despite the importance of the allergen-antibody interaction for the allergic response to occur. This review discusses efforts to analyze allergen-antibody interactions, from the original epitope mapping studies using linear peptides or recombinant allergen fragments, to more sophisticated technologies, such as X-ray crystallography and nuclear magnetic resonance. These state-of-the-art approaches, combined with site-directed mutagenesis, have led to the identification of conformational IgE epitopes. The first structures of an allergen (egg lysozyme) in complex with Fab fragments from IgG antibodies were determined in the 1980s. Since then, IgG has been used as surrogate for IgE, due to the difficulty of obtaining monoclonal IgE antibodies. Technical developments including phage display libraries have contributed to progress in epitope mapping thanks to the isolation of IgE antibody constructs from combinatorial libraries made from peripheral blood mononuclear cells of allergic donors. Most recently, single B cell antibody sequencing and human hybridomas are new breakthrough technologies for finally obtaining human IgE monoclonal antibodies, ideal for epitope mapping. The information on antigenic determinants will facilitate the design of hypoallergens for immunotherapy and the investigation of the fundamental mechanisms of the IgE response. The interaction between allergens and IgE antibodies is at the core of the allergic response. Epitopes could potentially be located on any part of the allergen surface. However, evidence shows that antibodies are very specific about the epitopes that they recognize and certain areas on the allergen seem to be preferential for antibody binding. The identification of epitopes recognized by IgE is valuable for the design of hypoallergens or other therapeutics. However, allergen-epitope information has been difficult to obtain. This review will discuss various methods to probe epitopes and the knowledge that has been gained from available studies on allergens.Since the 1980s, efforts to identify antigenic determinants on allergens have been pursued, but progress in the area has been slow due to technical limitations. Original epitope mapping studies were based on the synthesis of overlapping peptides covering the full sequence of the allergen, and the selection of the peptides that bound IgE . This apin vitro expression of either allergen fragments, mutants, or allergen chimeras, and their subsequent testing for IgE antibody binding. The development of microarrays or bead-based epitope assays facilitated the investigation of the relevance of linear epitopes, using large sets of linear peptides  . They wepeptides , 6. Micrpeptides . Severalpeptides , 11. Anopeptides –14. Patcpeptides . It is bpeptides , 17. A khttp://www.meduniwien.ac.at/allfam/). Once the allergen molecular surface is defined, certain amino acids can be selected for site-directed mutagenesis to analyze allergen-antibody interactions  were determined using X-ray crystallography . This te1H, 13C, or 15N) and secondarily by the chemical environment of particular atoms. These data provide a rich source of atomic structure when the resonant frequencies can be specifically attributed to individual atoms  can be readily observed, while much larger complexes  require specific labeling of certain chemical groups that provide high sensitivity. It is important to realize that, in contrast to crystallography that directly determines the structure of the complex, the NMR data on epitopes requires a careful comparison of the atomic frequencies or intensities in the allergen before and after complexation. Therefore, the NMR results are potentially subject to interpretation in the context of previously known structures or epitope mapping data.Another emerging methodology that may become applicable to epitope mapping is cryo-electron microscopy (Cryo-EM) . Due to There have been several papers on epitopes mapped by Cryo-EM, which are worth noting. For example, Fab fragments from monoclonal antibodies were localized on the spike protein of SARS-CoV-2, and on Zika virus particles , 31. Mor1H) can be exchanged for deuterium (2H). The rate of the 1H-2H exchange (HDX) depends on solvent accessibility and dynamics of a particular protein fragment. Generally, hydrogens that are buried within the protein core or shielded from the solvent, such as hydrogens buried in an antigen-antibody interface will have a low rate of 1H-2H exchange. After the incubation in heavy water the complex undergoes enzymatic cleavage and the resulting peptides are identified by the change in mass, using MS. It is expected that surface residues forming epitopes and paratopes will have a relatively low level of incorporated 2H. This information combined with the molecular models of the antigen and the antibody allows for mapping of the interacting molecular surfaces. Therefore, HDX-MS became a very successful technique that not only has found application in analysis of antigen-antibody complexes, but also in mapping of other protein-ligand interactions 2 -that was reduced to F(ab′)- or Fab, respectively. These antibody fragments contain the paratope and were purified and combined with the allergen to form a complex, which was purified for crystallography.The X-ray crystallographic structures of allergen-antibody complexes were first determined for egg lysozyme with fragments of murine IgG monoclonal antibodies (mAb) –47. SubsL) followed by a constant domain. Similarly, heavy chains have a variable N-terminal domain (VH), but it is followed by either 3 (in IgG) or 4 (in IgE) constant domains. The central part of these domains is made of anti-parallel β-sheets, in which β-strands are linked to form the so-called Greek-key motifs. For example, IgG VH domain is made of anti-parallel β-sheets composed of nine β-strands that are linked by eight loops (www.imgt.org) utilizes a consensus for their estimation.Although there are <20 different allergens that have their structures determined in complexes with antibodies  their anht loops  65). Of. OfL) foht loops . A similht loops , althought loops . In a feht loops –70, and www.allergen.org). Three X-ray crystal structures of Der p 1 have been determined in complexes with three different murine IgG mAb , from which mAb 4C1 is a cross-reacting antibody that also binds to Der f 1 . Bl Der f 1 , 72. Two Der f 1 , 52. The Der f 1 . The mur Der f 1 , 67, 73.The antibodies in the majority of allergen-antibody complexes that have their structures determined are IgG1. However, three of the structures reported in H domains, are relatively easy to produce and their biophysical, as well as structural properties, allow for easy application in biotechnology and therapeutics  or single domain antibodies, which have a single antigen binding domain –76. Heavapeutics , 79. Sin Gal d 4  61)..H domainA new type of allergen-antibody interaction was recently reported thanks to the determination of a Phl p 7-antibody crystal structure  62). An. An62). The allergens that we have described are proteins, and their interactions with antibodies are the same as for other proteinaceous antigens. It also has to be stressed that X-ray crystallography provides generally a static picture of the interacting molecules. However, both antigens and specifically antibodies display a great level of conformational flexibility , 81. It 2 range  with one antibody, and an additional interface (~380 Å2) responsible for the non-standard interaction with the light chain from the second antibody , the allergen-antibody interface areas are quite large , perhaps to compensate that the paratope is formed only by the heavy chain.An analysis of 16 allergen-antibody structures selected from antibody . In mostantibody . The ligantibody , 50. HowAnalysis of the allergen-antibody interfaces at the amino acid level shows that paratopes are formed by 18–28 residues that interact with epitopes composed of a similar number of amino acids  , Table 2The chemical interactions that drive allergen-antibody formation include covalent (H-bonds) as well as non-covalent binding interactions . Hydrophobic and electrostatic interactions are most important for a primary contact between antigens and antibodies . HoweverAmino acids with charged side chains also play an important role in mediation of epitope-paratope interactions . For exaE. coli, which do not add carbohydrates to expressed proteins, may lead to a lack of proper recognition by antibodies from allergic individuals raised against the glycosylated natural allergen. Nevertheless, the allergen recognition might still occur, if the antibody recognizes the protein part of the epitope.Besides protein-protein contacts at the allergen-antibody interface, other chemical moieties can form contacts between the two molecules. One of them is carbohydrates. An epitope on Bla g 2 that is recognized by mAb 4C3 includes a glycan  52). Th. Th52). During the process of antigen-antibody recognition many water molecules that were on the surface of the interreacting molecules are displaced. However, this process does not always lead to their complete displacement. In fact, water molecules can play an important role in allergen-antibody interactions, in purely protein-protein complexes, and the aforementioned protein-carbohydrate complex . Very of−7 to 7 × 10−6) . B cells expressing IgE circulate in low frequency in peripheral blood (3 × 10 × 10−6) , which mOne approach to study the human IgE repertoire is to isolate IgE antibody constructs from phage display combinatorial libraries prepared using peripheral blood mononuclear cells (PBMC) of allergic subjects –113. BasThe structure of the Bos d 5-antibody complex illustrates an additional important phenomenon, namely the importance of the oligomerization state or quaternary structure of the allergen. Bos d 5 is a dimer in the reported structure . DimerizOther studies addressed allergen epitope mapping using indirect approaches. A cluster of several IgE antibody binding epitopes was located on the C-terminal domain of Phl p 1 using human IgE obtained by phage display technology. In combination with site-directed mutagenesis, the authors designed a hypoallergenic group 1 grass pollen allergen fragment . Two othRecently, a house dust mite Der p 2-specific IgG mAb overlapping with IgE was mapped by X-ray crystallography and site-directed mutagenesis analysis. A Der p 2-specific IgE construct isolated from a single-chain variable fragment (scFv)-encoding phagemid library recognized the same main residues as the IgG, further confirming the relevance of this epitope to human health . These sAlternative approaches to isolate antibodies are based on sorting single B cells for amplification of mRNA that encodes for the antibody. They have proven effective for identifying the exact pairing of IgG heavy and light chains, but not for B cells expressing IgE due to their low frequency in blood . A studyA new approach to isolate human IgE monoclonal antibodies has emerged using hybridoma technology . IndividBecause of the limitations in the size of proteins for which NMR can determine structures, NMR experiments to determine allergen epitopes necessarily involve clever experimental design and accurate interpretation. In well-designed experiments, the data provides atom-specific information on the epitope region, which can be readily understood in the context of the allergen structure. The following section describes the design and range of applicability of various NMR designed experiments.1H frequencies in the antigen. In the Paterson design, the antibody was covalently linked to beads to make an affinity column. Subsequently, the antigen in solution was added to the column and allowed to bind the antibody. The buffer was then easily changed from 1H2O to 2H2O, so the 1H-amide protons on the antigen surface could be exchanged for deuterons, except in the epitope that was protected by the antibody. Finally, the antigen was eluted at low pH to quench or stop further amide exchange. The cleverness of this design is that the antigen  retains information about the protection. Paterson applied this method to the model antigen cytochrome c, and it was subsequently adapted for the allergens hen-egg lysozyme  and Der p 2  or scFv (~25 kDa) is sometimes possible. These amide detection techniques using antibody fragments have been successfully applied to the allergen Blo t 5 in complex with a Fab. A discontinuous epitope was identified by comparing the 1H-15N chemical shift perturbations of the bound and free allergen  . The aut antigen . This sa2H labeled proteins (2H, 1H,13C-methyl Val, Leu, Ile] Der p 2 bound to an scFv fragment of mAb 7A1 (1H antibody causes broadening or a disappearance of signal, and distal residues can also experience chemical shift perturbations (Instead of looking at fragments of the antibody-allergen complex, or the free allergen in exchange with complex, it is also possible to utilize whole antibodies, but this requires another compromise in the labeling scheme. At the very high molecular weights of allergen-antibody complexes, usually only methyl groups are still observable in a background of otherwise proteins . Focusin mAb 7A1 . The dat mAb 7A1 . This isrbations .Methyl labeled Der p 2 as described above was successfully applied to study 4 human IgE mAb epitopes and 3 murine IgG mAb epitopes . Similar1H-15N labeling of Art v 1 and Bet v 1 was combined with either pooled allergic sera, or individual allergic sera, respectively, but the NMR results were nebulous (While these data are an impressive first mapping of human IgE epitopes, three important drawbacks of the technique need to be mentioned. First, it requires monoclonal antibodies, of which human IgE are very difficult to clone from patients. Although this technique may be applicable to polyclonal antibodies, its effectiveness remains to be demonstrated in this case. nebulous –141. SecIn summary, a variety of NMR techniques and labeling schemes have been applied for allergen epitope mapping. In each case, atoms or residues specific to the epitope were successfully identified.Experimental epitope mapping of IgE antibodies on allergens originated ~30 years ago with the identification of mostly linear epitopes. Several breakthroughs have allowed the identification of conformational epitopes. These epitopes are the most common on allergens, especially on allergens for which exposure occurs by inhalation. Techniques such as recombinant technology, X-ray crystallography and nuclear magnetic resonance were developed and used for the determination of structures of allergen-antibody complexes. These advances required preparation of pure and homogeneous allergens and monoclonal antibodies. Initially, mostly IgG antibodies that inhibit IgE antibody binding were used as surrogates of IgE and fragmented for epitope mapping. Another approach led to the isolation of IgE antibody constructs using phage display technology. Only more recently, single cell antibody sequencing and human hybridoma technology are opening a new era of epitope mapping that will allow direct visualization of allergen-IgE antibody interactions in detail. Other technologies such as cryo-electron microscopy and labeling with mass spectrometry will also contribute to epitope mapping with less demanding protein amounts. Moreover, the experimental results allow for a significant development of many computational approaches to identify and/or analyze paratopes and epitopes . For exaAP designed, wrote, and edited the review. She especially wrote the sections about IgE epitope mapping. GM wrote the sections associated with NMR and cryo-EM. MC analyzed crystal structures of allergen-antibody complexes and wrote sections about X-ray crystallography and mass spectrometry. GM and MC also contributed to editing the manuscript. All authors contributed to the article and approved the submitted version.AP was employed by the company Indoor Biotechnologies, Inc. She is the contact PI of the R01 Award that funded this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors AP.


The results obtained in this study contributed new data on the internal and external load required by professional basketball players in weeks with different number of games and showed that different coaching strategies may demand a different external and internal workload in consecutive seasons. Furthermore, the results highlighted the need to carry out an adequate load management program.The weekly training management and competition loads are important aspects to optimize the performance of professional basketball players. The objectives of the study were (a) to describe the weekly external load (EL), as well as the internal response (IR), of elite basketball players over two consecutive seasons with a different head coach and (b) to compare weekly loads of different competitive densities. The data were collected from 27 elite players from the same team competing in the Spanish first division league (ACB) and EuroLeague during 2017–2018 and 2018–2019 seasons. EL was measured using microsensor technology to determine PlayerLoad values, expressed in arbitrary units (AU). Session rating of perceived exertion (sRPE) was used for IR quantification. Comparisons between the two seasons and of weeks with different competitive densities were made. The inter-week load variability was moderate-high for both seasons. The highest EL values were measured during the weeks with three games (W3) (W3 > W0 > W2 > W1), while the most demanding week for players’ IR was observed during weeks with no competition (W0). Additionally, higher EL ( Leading European teams usually participate simultaneously in two competitions, a national or regional one  and an international one  during the season. This means that during nine months, a team reaching the play-offs in EuroLeague and domestic finals could play around 80 games in one season. This, together with the frequent travelling all over Europe, could negatively affect the individual and team performance during the games. Sometimes teams have to play at congested periods of four or five games in ten days  and therThe number of games played in a week largely determines the accumulation of load on players in team sports during the competitive period  and seemThere are numerous methods to quantify training load. To date, the most common ones were focused on the internal load derived from the heart rate, recovery-stress status, immuno-endocrine response or subjective perception of effort ,10. The The present study aims to describe the weekly external demands and the internal response from the effort made by players to address two goals: (a) to compare two different training strategies carried out on the same team during two consecutive seasons and (b) to compare weeks with different competitive densities . Our main hypothesis states that a higher competitive density will result in a higher external demand and an increased effort perception by the players.A total of 27 players from the same team participated in the study  during two consecutive seasons (2017–2018 (S17–18) and 2018–2019 (S18–19)) and a time gap of two months between them (off-season period from June to August). The team participated simultaneously in the EuroLeague and the Spanish first division league (ACB) and it had a different head coach in each season. A total of 1041 training sessions and matches were recorded (528 in S17–18 and 513 in S18–19). All the players were familiar with the external and internal load monitoring tools. Players who did not complete 75% of the weekly training duration with the team during a given week were excluded from the analysis. All the players were notified of the purpose, investigation procedures and requirements of the study, as well as the benefits and risks before consenting, in accordance with the Declaration of Helsinki . All then = 43 and n = 41 for seasons 2017–2018 and 2018–2019, respectively). Each week was classified according to the number of matches played by the team .An observational and longitudinal study was carried out in this investigation and the researchers did not interfere at any time in the weekly training plan. The recording of the external load and internal response of the players was monitored every week, including during pre-season and in-season, in two consecutive years . Second coach had an opposite strategy that focused on the use of small sided games  together with regular 5 × 5 format of play performed on half court with a single transition. Only team practices and matches were included in the analysis. The load derived from strength training (performed in the weight room), individual training, recovery or injury treatment were discarded from the analysis.®  variable, which determines the mechanical load and has previously been validated in basketball [The external load was measured by the PlayerLoadsketball , using tRegarding the internal response, the subjective scale of perception of effort CR-10 was used . This schttps://jasp-stats.org/, Amsterdam, The Netherlands). The level of significance was set at p < 0.05.All the data are presented using the mean and standard deviation (SD). The differences between weeks with different competitive densities  and between seasons (2017–2018 and 2018–2019) were analyzed using one-way ANOVA and the size of the differences was calculated using the standard differences of the means (Cohen’s d), with its respective limits at 90% of the confidence interval. The interpretation of the effect size (ES) was followed according to Hopkins et al. (2009) [The mean PL values measured during seasons S17–18 and S18–19 were 3023 ± 855 AU and 3323 ± 1119 AU, respectively, while the average sRPE values observed were 2703 ± 887 AU and 3096 ± 1227 AU, respectively. The variability of the external load and internal response in different weeks, expressed as the CV, was moderate-high for PL (16% and 15%) and sRPE (24% and 19%) in seasons 2017–2018 and 2018–2019, respectively. The average external load by the number of games played within a week is presented in However, the players perceived a greater internal load in W1, although this difference was only significant compared to W3 (ES = 1.09) . Weeks wThe results obtained in this study showed that the external load and internal response were higher based on the weekly competitive density, although the external load did not increase proportionally when the competitive density was higher. Additionally, the distribution of the external load during training and competition was different for seasons S17–18 and S18–19. The external load measured in S18–19 was significantly higher than in S17–18 and, consequently, the players perceived it as more demanding.Coaches in all kind of team sports adopt different training strategies regarding quantity , quality  or tactical strategies to promote short-term team adaptations and achieve the best possible performance . In spitThe relationship between external and internal loads has been previously studied in elite basketball showing a positive moderate-high correlation between them ,24. The Elite teams in collective sports  usually participate in a minimum of two competitions , which involves playing two games per week. However, this competitive density is even higher in basketball, where the number of games within a week, in many cases, reaches up to three matches in the same microcycle . It is worth to highlight that to play just a single game per week in elite basketball is rather unusual (14% of the weeks in the monitored seasons). Due to the high competitive density of elite basketball, coaches and trainers should design different strategies to optimize the physical condition of the players so that they are able to perform at their highest level in every game .The weekly accumulated internal load values observed for the team in the present study ~3150 AU) were higher than the 2520 AU of the Lithuanian female players 0 AU were and loweIn relation to the comparison of the internal load in weeks with different competitive densities, the results obtained in the present study for the season 2018–2019 were similar to those reported by Manzi et al.  and ClemThis study is not without its limitations, which should be considered when extrapolating the data to other teams or different competitive levels. Firstly, although the team monitored may be representative of a top-level club in the European arena, only one team and the training strategies of two coaches during two consecutive seasons were analyzed. As a greater number of in-depth studies on load management are available, the training and competition load values measured in this work could be used as a guideline for professional basketball teams. Secondly, the monitoring of the training load was only performed based on global indicators of external load (PL) and internal response (sRPE). These indicators only provide a general idea of training and competition, so for future studies it would be interesting to include representative variables of movement  and inertia , as well as internal adaptation variables of the players, such as heart rate and hormonal markers . FinallyThis study indicates that the external load and internal response values of the weekly workload could be used as a guideline in elite basketball during a complete competitive season. Additionally, different head coaches could manage weekly loads with different magnitudes regardless of the number of games per week. The distribution of the workload over the weeks is based on the competitive density of a microcycle, which should be considered by coaching staff in order to (a) implement recovery strategies, mostly in periods with a greater number of games per week , (b) man